Anvisa Ba Be Guideline II

General Manager of Inspection and Control of Drugs and ProductsAntônio Carlos da Costa Bezerra

Inspection Coordination in Bioequivalence CentersCláudia Franklin de Oliveira

E-mail: [email protected]

Brazilian Sanitary Surveillance Agency

Manual for Good Bioavailability andBioequivalence

Practices

Volume II

Brasília2002

Rights reserved for Brazilian Sanitary Surveillance AgencySEPN 515, Edifício Ômega, Bloco B, Brasília (DF), CEP 70770-502.Internet: www.anvisa.gov.br

Copyright © 2002. Brazilian Sanitary Surveillance Agency.The total or partial reproduction of this work is permitted provided that the source is mentioned.

First edition - 2002

ISBN: 85-88233-06-1

Brazilian Sanitary Surveillance AgencyPerformed by: Inspection Coordination in Bioequivalence Centers -General Office of Inspection and Control of Drugs and ProductsGeneral Coordination: Cláudia Franklin de Oliveira / Inspection Coordination inBioequivalence CentersRevision: Karla de Araújo Ferreira / Inspection Coordination in Bioequivalence CentersDivulgation: Divulgation UnitCovers: João Carlos de Souza Machado / Multimedia Communication Office

Graphic Art, composition and printing: Dupligráfica Editora Ltda./DF

Printed in Brazil

Manual de boas práticas em biodisponibilidade: bioequivalência/AgênciaNacional de Vigilância Sanitária. Gerência-Geral de Inspeção eControle de Medicamentos e Produtos. Brasília: ANVISA, 2002.

2 v.QV38

1. Equivalência terapêutica. 2. Bioequivalência. 3. Disponibilidadebiológica. 4 . Medicamentos. I. Agência Nacional de Vigilância Sanitária.Gerência-Geral de Inspeção e Controle de Medicamentos e Produtos.

PRESENTATION

The Brazilian Sanitary Surveillance Agency, embedded of its institucional mission, elaborated thepresent work with the intention to foment discussion, education and the conduction of clinicalstudies in Brazil.

Concerning the bioavailability/bioequivalence studies, this compendium is part of a bigger contextwhich is intended to enclose the clinical research in its amplest aspect: non clinical studies andclinical studies of phase I, phase II, phase III and IV.

In presenting this work , The Brazilian Sanitary Surveillance Agency would thank the contributionof all the professionals directly and indirectly involved in its consolidation, and it is available forcritics and suggestions that may contribute for future editions, as well as, for the subsequent modules.

Dr. Gonzalo Vecina Neto

PREFACE

A number of clinical trials are being presently conducted in Brazil aiming to evaluate theBioavailability/Bioequivalence of pharmaceutical products. As of June 2001, Anvisa, through theInspection Coordination in Bioequivalence Centers – bound to the General Office of Inspectionand Control of Drugs and Products – started evaluating such centers by means of periodicalinspections in order to assure the quality of the trials.

During the inspection activities, originally for orientation purposes, the coordination observed theneed to clarify some aspects that still posed technical questions to the centers, especially concerningthe standardization of analytical methods, statistical analysis of the trials, storage of biologicalsamples, confinement of volunteers and stability studies of drugs and others.

When such requirement was identified and in an attempt to avoid impairing the quality of the workscarried out, the coordination stimulated the creation of discussion nuclei intended to wear out theexplanation of all aspects related to the conduction of the studies and the integration of theirphases. Such nuclei counted on the participation of 40 experts from the Pharmaceuticals, Medicine,Statistics and Chemistry fields.

This “Manual for Good Bioavailability/Bioequivalence Practices” was created under such contextand comprises six main topics presented in a didactic manner, trying to overwhelm the centers’difficulties and, as a result, complement the guidelines provided for in the Brazilian health legislationfor conducting trials.

The working nuclei started the discussions in September 2001. Each nucleus was dedicated to oneof the three phases of the process – Clinical, Analytical and Statistical Phases – under the coordinationof Dra. Cláudia Franklin de Oliveira and the inestimable cooperation of professor Sílvia Storpirts.During the subsequent months, several meetings were held so as to promote technical debates andreach a consensus concerning theses questions. Consequently, each group prepared a set of relevanttopics to be included in the manual. All such topics were then subjected to careful researches andthe results reported so as to allow a good understanding by the target population. The completemanual took 11 months to be finished. This process involved the participation of about 50professionals in total from several fields of knowledge, including researchers from public universities,technicians from Anvisa and manufacturers of laboratory instruments and equipment. Anvisa isthankful to all such associates for having contributed to such an excellent job.

The final structure comprises two volumes with three modules each. The first volume technicallydetails each phase of the Bioavailability/Bioequivalence studies in the natural sequence of conduction:Module 1: Clinical Phase, Module 2: Analytical Phase and Module 3: Statistical Phase. The secondvolume covers the important aspects concerning laboratory instrumentation and equipment usedto carry out the analytical phase and regarded as critical for the process. In that volume, Module 1relates to the Principles and Operation of Micropipettes, Module 2 refers to Water for Chemical

Instrumental Analyses and the contents of Module 3 relates to the Ultraviolet VisibleSpectrophotometry, Liquid Chromatography (LC), Gas Chromatography (GC), ChromatographySystems Connected to Mass Detectors and Checking of Analytical Instruments Performance.

It is worth emphasizing the international novelty character of gathering information from severalsubjects in a single compendium aiming at synthesizing all the aspects involving the GoodBioavailability/Bioequivalence Practices.

It is evident that the major purpose of this work is to improve the quality of the Bioavailability/Bioequivalence assays carried out in Brazil and, as a result, contribute to the quality of generic drugsavailable in the market by providing widely studied and carefully prepared technical subsides. Withthis in view, we hope to contribute to capacitating the Bioavailability/Bioequivalence Centers, inaddition to develop study monitors for the domestic pharmaceutical industry and help in theformation of knowledge multiplier agents at the Brazilian universities.

The preparation of this manual was only possible thanks to the crucial work of several persons. Iwould like to apologize in advance for possibly forgetting any name. The editors José PedrazzoliJúnior (USF/Unifag) and João Antônio Saraiva Fittipaldi (Pfizer) were essential, as well as thecooperators Fernanda Maria Villaça Boueri (Anvisa), Eliana Regina Marques Zlochevsky (Anvisa),Cláudia Simone Costa da Cunha (Ministry of Health) and Beatriz Helena Carvalho Tess (Ministryof Health), for the module Clinical Phase; the editors Cláudia Franklin de Oliveira (GGIMP/Anvisa),Rui Oliveira Macedo (UFPA), Flávio Leite (T&E Analítica) and Pedro Eduardo Froehlich (UFRGS),and the cooperators Pedro de Lima Filho (GGMEG/SP), Davi Pereira de Santana (UFPE), RafaelEliseo Barrientos Astigarraga (Cartesius), Silvana Calafatti de Castro (Unifag), Thaís Reis Machado,Jaime Oliveira Ilha (Cartesius), Itapuan Abimael Silva (Anvisa), Karen Noffs Brisolla (Anvisa), MarceloCláudio Pereira (Anvisa), for the module Analytical Phase; the editors Arminda Lucia Siqueira(UFMG), Chang Chiann (GGMEG/SP), Cicilia Yuko Wada (Unicamp), Karla de Araújo Ferreira(Anvisa) and Gilberto Bernasconi (USF/Unifag), and the cooperators Reinaldo Charnet (Unicamp)and Renato Almeida Lopes (Anvisa), for the module Statistical Phase; the editors Melissa M. Silva(Nova Analítica) and Walter Pereira (Nova Analítica), Principles and Operation of Micropipettes;the editor José Muradian Filho (Millipore), Water for Clinical Analyses; the editors Ivan Jonaitis(Agilent), Renato Garcia Peres (Flowscience), Ricardo Lira (Flowscience), Renato Gouveia, JoséAparecido Soares (Varian), Josué D.M. Neto (Sync Brazil) Juarez Araújo Filho (Sync Brazil), AlexandreRosolia (Waters), Adauto Silva (Varian) and Reinaldo Castanheira (Agilent), AnalyticalInstrumentation; and the coordination team composed by Cláudia Franklin de Oliveira (GGIMP/Anvisa), Marcelo Cláudio Pereira (GGIMP/Anvisa), Max Weber Marques Pereira (GGIMP/Anvisa),Karla de Araújo Ferreira (GGIMP/Anvisa), Karen Noffs Brisolla (GGIMP/Anvisa), Itapuan Abimaelda Silva (GGIMP/ Anvisa) and Renato Almeida Lopes (GGIMP/Anvisa).

Dr. Gonzalo Vecina Neto

Volume II – Module 1 – Micropipettes: Principles and Operation

Editors:• Melissa M. Silva – Nova Analítica• Valter A. Pereira - Nova Analítica

Coordination:• Cláudia Franklin de Oliveira – ANVISA• Itapuan Abimael da Silva - ANVISA• Karen de Aquino Noffs Brisolla - ANVISA• Karla de Araújo Ferreira - ANVISA• Marcelo Cláudio Pereira - ANVISA• Max Weber Marques Pereira - ANVISA• Renato Almeida Lopes – ANVISA

TECHNICAL STAFF

SUMMARY

1. DEFINITION ................................................................................................................................... 51.1. Types of pipettes ........................................................................................................................... 5

2. OPERATION PRINCIPLE .............................................................................................................. 62.1. Air displacement pipettes ............................................................................................................. 62.2. Positive displacement pipettes ..................................................................................................... 8

3. OPERATION TECHNIQUE......................................................................................................... 113.1. How to store the pipette ........................................................................................................... 113.2. Volume adjustment ..................................................................................................................... 113.3. Tip-fitting ..................................................................................................................................... 123.4. Pre-rinsing of the tip .................................................................................................................. 133.5. Aspiration ..................................................................................................................................... 133.6. Dispensing.................................................................................................................................... 143.7. Tips ejection ................................................................................................................................. 14

4. TIPS ...................................................................................................................................................... 154.1. Tips for air displacement pipettes ............................................................................................. 154.2. Tips for positive displacement pipettes .................................................................................... 15

5. MAINTENANCE ............................................................................................................................. 165.1. Cleaning ........................................................................................................................................ 165.2. Parts replacement ........................................................................................................................ 175.3. Performance checking ................................................................................................................ 20

6. SUGGESTIONS OF PROCEDURE ............................................................................................ 21

7. ATTACHMENT I: EXAMPLE OF A CLEANING PROCEDURE ...................................... 227.1. Disassembling .............................................................................................................................. 227.2. Cleaning ........................................................................................................................................ 237.3. Assembling ................................................................................................................................... 23

8. ATTACHMENT II: PERFORMANCE STANDARD VERIFICATION .............................. 248.1. General ......................................................................................................................................... 248.2. Testing room conditions ............................................................................................................ 248.3. Operator ....................................................................................................................................... 258.4. Tips ............................................................................................................................................... 258.5. Equipment used in the test ........................................................................................................ 258.6. The checking procedure ............................................................................................................. 27

9. ATTACHMENT III: DECONTAMINATION PROCEDURES ............................................ 389.1. Chemistry ..................................................................................................................................... 399.2. Microbiology and cell culture .................................................................................................... 409.3. Molecular biology ....................................................................................................................... 419.4. Methods spectrum of action ..................................................................................................... 449.5. Advantages and disadvantages .................................................................................................. 459.6. Autoclaving .................................................................................................................................. 469.7. UV irradiation .............................................................................................................................. 469.8. Chemical solutions ...................................................................................................................... 46

10. ATTACHMENT IV: DEFINITIONS ........................................................................................... 50

11. GLOSSARY AND ABBREVIATIONS ........................................................................................ 51

12. REFERENCES .................................................................................................................................. 52

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Volume II / Module 1 - Micropipettes: Principles and Operation

1. DEFINITION

Piston-operated pipettes are equipment used to aspirate and dispense specific volumes of liquids.The single-channel pipettes have only one piston. The multichannel pipettes have simultaneouslyseveral receptacles. The pipettes can be factory-set to dispense a certain dado volume or volumesselected by the user within a specific volume range, for example, between 10 µL and 100 µL. Piston-operated pipettes can be of two types: air displacement and positive displacement.

1.1. Types of pipettes

The pipettes can be designed as follows:

Concerning the volume:

– Fixed volume, designed by the manufacturer to dispense only its nominal volume, for example,100µL.

– Varying volume, designed by the manufacturer to dispense volumes selected by the user withina specific volume range.

Concerning the piston:

– There may be an air layer between the piston and the liquid surface (air displacement pipettes).– The piston is in direct contact with the liquid (positive displacement or direct displacement

pipettes). The capillary and the piston may be reusable or disposable.

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2. OPERATION PRINCIPLE

The plastic or glass tip is coupled to the pipette. With the piston at the lower limit (lower aspirationposition), dip the tip into the liquid to be transferred. When the piston moves up to the upperaspiration position, the liquid is drawn. The volume of liquid is dispensed when the piston is pushedagain to the lower position.

The operation principle is detailed below according to the type of pipette (air displacement orpositive displacement).

2.1. Air displacement pipettes

When the button of an air displacement pipette is pressed, the piston located inside the equipmentmoves down and displaces the air in contact with it out of the pipette (the air volume expelled isequal to the volume set in the pipette). The air volume remaining inside the pipette is inverselyproportional to the sample volume: the lower is the air volume inside the pipette, the greater is thevolume of liquid to be drawn.

a) Volume adjustment (valid for the varying volume pipettes only)

The user adjusts the desired volume. The piston moves to the appropriate position.

Remarks: In fixed volume pipettes, this positioning is provided at the factory.

Selected volume,for example, 100 mL

Zone filledwith air

Tip

Piston

Piston position after volumeadjustment

Cone holder

b) Preparation for aspiration

The button is pressed. The piston move down and expels an air volume equal to the selectedvolume (in fixed volume pipettes, the volume of air expelled is equal to the nominal volume of thepipette).

c) Liquid aspiration

Dip the tip into the liquid until the bore is submerged. When releasing the button, the piston returnsto the initial position and a partial vacuum is formed inside the pipette. The atmospheric pressureforces the entrance of liquid through the tip bore.

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The piston moves to thelower limit

Volume of air expelled(equal to the selected volume)

Tip bore submergedinto the liquid

The piston returns to theinitial position

Partial vacuumis formed

Volume ofliquid drawn

d) Liquid dispensing

The button is pressed again. The piston moves down displacing the air and increases the pipetteinner pressure. The compressed air expels the liquid out of the tip.

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e) Second stage

In some air displacement pipettes, the piston can be displaced just below the lower limit (thusdisplacing an additional amount of air), allowing the expulsion of the last drops of liquid. The mostcommon term employed by users for this additional displacement is “second stage”.

2.2. Positive displacement pipettes

The positive displacement pipettes work as a syringe. There is no air volume between the piston andthe liquid. Since there is no air to contract or expand, the aspiration force is always constant eremains unchanged by the physical properties of the liquid to be handled. This kind of equipmentis ideal for handling viscous or high-density liquids.

Destination container

b) Preparation for aspiration

The button is pressed. The piston moves down to the end of the capillary.

a) Volume adjustment

The user adjusts the desired volume. The piston moves down to the initial position.

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Selected volume,for example, 100 µL Cone holder

Capillary

Piston

Initial position

c) Liquid aspiration

The capillary bore is submerged into the liquid. When releasing the button, the piston moves upand the room pressure forces the entrance of the liquid through the capillary bore.

d) Liquid dispensing

The button is pressed again. The piston moves down and expels the liquid out of the capillary.

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Submerged capillary

Piston at the initial position

Volume drawn

Destination container

Dispensed volume

3. OPERATION TECHNIQUE

A good pipetting technique is essential to assure good results. A script on some aspects of thetechnique to be considered during the operation is showed next.

3.1. How to store the pipette

The pipette should be stored in the upright position for two reasons:

– Prevents it from contacting the surfaces that can be contaminated, for example, the workingbench (when laying the pipette on the bench or in a chest of drawers, contaminants can betransferred to the body of the instrument and subsequently transferred to the experiment).

– If the tips are not ejected and if a residual amount of liquid remains inside the tips, this liquidmay flow into the instrument when the pipette is laid down, thus contaminating and damagingthe inner parts and/or the cone holder.

Examples of supports:

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3.2. Volume adjustment

– In order to avoid a parallax error, the volume should be adjusted with the pipette in the horizontalposition. If the operator prefers to adjust the volume with the pipette in the upright position,one of the eyes should be closed.

– When reducing the volume, carefully reach the desired amount and do not surpass the mark.– When increasing the volume, surpass the desired amount by 1/3 of a cycle and then carefully

reduced the volume until the desired amount is reached. Do not surpass the mark.

3.3. Tip-fitting

– The tip is fitted according to the manufacturer’s instructions, thus assuring an adequate sealbetween the pipette tip and the cone holder.

Remark: Seal means an opaque ring formed around the entire tip collar, indicating a perfect connectionbetween the cone holder and the tip. An incomplete seal will lead to imprecise results.

Some suggestions for fitting the tips are given below:

– Loose tips:

Hold the tip by its collar (be careful not to touch the end) and fit it to the cone holder. Press the tipagainst the cone holder by following a rotational movement, as indicated in the figure below. Thisassures a perfect connection between the tip and the cone holder (the above mentioned seal isformed).

– Tips on racks:

Fit the cone holder on the tip and press the pipette downwards by following a rotational movementas indicated in the figure.

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– Multichannel fitting:

For a simultaneous fit on all channels, position the cone holders on the tip collars. Make a to-and-from movement as showed in the figure, thus assuring an adequate connection of all tipson all channels.

3.4. Pre-rinsing of the tip

– When a new tip is placed (or when increasing the volume to be drawn), it is necessary to pre-rinse the tip. To do so, just drawn and dispense the liquid a couple of times.

– The pre-rinsing of the new tip assures accuracy and precision of the volume to be subsequentlytransferred. This is because a film is formed on the tip inner wall when drawing a liquid. Thenature of this film, that causes an error in the first measurement, depends on the liquid beingtransferred. However, this film remains relatively constant after some pipetting operations usingthe same tip. It is necessary to pre-rinse the tip in order to maximize the pipette performance.

3.5. Aspiration

The aspiration should be slow and constant, always keeping the same immersion depth of the tip(during the aspiration and between the samples). It is recommended that the immersion depth isaround 2 to 3 mm below the liquid surface. Some manufacturers suggest that the immersion depthshould vary according to the working volume, as described in the table below:

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– The pipette should be kept in the upright position during the complete aspiration. Wait for 1 to 2seconds before removing the tip from the liquid. If liquid droplets still remain outside the tip, cleanthem using soft paper (if the methodology allows). Be careful not to touch the tip bore.

3.6. Dispensing

– Touch the tip end on the inner wall of the container and tilt the pipette approximately 30° to45°;

– Continuously press the button until the end of the first stage. Wait some seconds (from 1 to 3 –depending on the liquid viscosity) and then press the button until the second stage (purging) toeliminate droplets that may have remained on the tip;

– Keep the button pressed until the end. Remove the pipette from the container keeping the tip intouch with the container inner wall (“scratch” the tip end on the inner wall of the container).

3.7. Tips ejection

– Dispose the tip by pressing the tips ejector button;– Take all required precautions when working with contaminated and/or radioactive materials.

The appropriate procedure should be defined by the laboratory according to the kind of materialpipetted, taking the safety of the users and the working place into consideration.

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4. TIPS

The quality of the results is directly affected by the quality of the tip used. Good results require notonly good quality tips, but also tips that are more appropriate to the model and brand of the pipetteused.

4.1. Tips for air displacement pipettes

– The manufacturer should indicate which brand of tip the user should employ to comply withthe specifications indicated in the pipette operation manual.

– The plastic tips for air displacement pipettes should be used disposed after use.Such tips should not be cleaned or reused, since their metrological features cannot beassured.

4.2. Tips for positive displacement pipettes

– The manufacturer should indicate which brand of tip the user should employ to comply withthe specifications indicated in the pipette operation manual.

– The fitting between the piston and the capillary (perfect sealing between the capillary and thepiston), as well as the piston displacement inside the capillary, should be optimum to assure aneven dispensation of the liquid drawn.

– These tips can be reused or disposed (when changing the tip, the capillary and the piston shouldbe simultaneously changed).

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5. MAINTENANCE

This chapter will cover three essential steps of the maintenance procedure: cleaning, change ofparts and performance checking.

REMARK

It is necessary to adapt the information of this chapter to the manufacturer’s instructions manual.Particularly, the following aspects should be regarded:

– Checkout whether the parts listed herein can be washed;– Assembling and disassembling procedure of the pipette;– How to change damaged parts;– Checkout whether any of these procedures alter the pipette calibration.

If they do change, it is necessary to verify the instrument performance (see attachment II) eoccasionally adjust the equipment (according to the manufacturer’s instructions).

5.1. Cleaning

It is necessary to create a cleaning and inspection routine of the pipette parts. So that the bestresults are always reached, the pipette parts have to be in good operation conditions. There are twodifferent situations requiring different cleaning procedures:

a) “Standard” cleaning

The aerosols formed during pipetting settle inside the pipette, thus impairing its normal operation.In addition to clog the cone holder bore and impair the air displacement inside the pipette, thissettlement may damage internal parts, for example, the piston (leading to scratches or corrosion). Inboth cases, the pipette will not work in compliance with the specifications.

In order to avoid this situation, the pipette internal parts (cone holder, ejector, piston, seal ando’ring) should be cleaned. Follow the procedure recommended by the manufacturer. Attachment Idescribes an example of a cleaning procedure.

Cleaning is the main tool to allow users to extent the pipette life and assure reliable results.There is no cleaning schedule to be adopted by the laboratory (this will depend on the typeof material handled, the routine to which the pipette is subjected, number of users of thesame pipette and acceptable errors for the procedure). So as to assure reliable results, thepipette should be subjected to performance testing in addition to be frequently cleaned(attachment II).

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b) Pipette used for handling acid or corrosive materials

The piston conditions directly interfere with the pipette specifications. If the piston is scratched orcorroded, the accuracy and precision standards are not met. Acid materials (for example, sulfuricacid, hydrochloric acid, nitric acid, phenol) or corrosive materials (for example sodium hydroxide,chlorinated solvents) may damage the piston. A damaged piston leads to unreliable results.

In order to avoid piston damage, it is recommended to clean the pipette parts right after handlingthis kind of liquid.

Contrary to the procedure described in item “a”, this cleaning does not require the use of a chemicalagent. Distilled water should be enough.

• Disassemble the pipette according to the manufacturer’s instructions.• Pour distilled water on the following parts: ejector, cone holder, piston and piston seals.• Let it dry at room temperature or in an oven up to 40°.• Assemble the pipette according to the manufacturer’s instructions.

REMARKS– Certify that said parts can be washed and whether this procedure will affect its calibration (that

is, if it is necessary to follow the performance checking procedure right after cleaning).– The procedure described in item “b” above is particularly indicated for pipettes with metallic

piston. For pistons made of other materials (glass, plastic, ceramic) the use of silicone grease forsealing purposes is common. Thus, it is recommended to verify and follow the specific procedurescontained in the manual.

5.2. Parts replacement

Some parts of the pipette have to be periodically replaced, since they wear out and directly interferewith the pipette performance.

5.2.1. Piston seals

This part or parts provide a sealing between the piston and the cone holder (upper portion). Withuse, microvilli are formed on the surface of these parts and the sealing becomes imperfect, thusimpairing the instrument precision. Such parts should be replaced once a year (see the replacementprocedure in the manufacturer’s manual).

5.2.2. Cone holder

Another important sealing point is the contact zone between the cone holder and the tip. With theuse, the cone holder surface wears out on the tip-fitting portion and the contact between thembecomes inadequate and impairs the measurement (loss of precision). Replacement is required

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whenever the tip-fitting portion is worn out or in case of part damage (broken, scraped). Verify inthe manufacturer’s manual whether the procedure affects the instrument calibration.

5.2.3. Other parts

Other parts interfere with the performance pipette. A very simple checking procedure is describedbelow to help recognizing the damaged parts. Some procedures should be followed according tothe manufacturer’s manual (for example: disassembling, assembling). We emphasize again theimportance of certifying whether it is necessary to recalibrate the pipette after one of these procedures(this information can be found in the manufacturer’s manual).

1st STEP: GENERAL ASPECT

• Verify that there is no apparent defect.

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Operations bar

Volumeter

Cone holder

Button

Ejector button

Tips ejector

Tip

2nd STEP: FUNCTIONS TESTING

• Turn the volumeter fully so as to go through the complete volumetric range of the pipette.Verify whether the minimum and maximum adjustable volumes match with the actual volumetricrange of the pipette.

Problems and causes:

Adjustment is not possible Autoclaved pipette *Incorrect volume adjustment Incorrect adjustment or assemble of the volumeter

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* Some pipettes can be autoclaved, others can’t. See the manufacturer’s manual.

• With the volumeter set at the maximum value specified for the pipette model, press the buttonfully to verify the easiness of movement, feel occasional obstructions or friction variations thatmay cause damages, such as piston corrosion or crooked operations bar. Listen to the springnoise that may indicate its incorrect positioning.

Problems and causes:

Uneven movement Damage of the friction ringLack of displacement The operations bar is crookedInappropriate movement Corroded, dirty or scratched piston

• Fit one tip on the pipette and press the tips ejector button to verify the ejection efficiency.

Lack of movement Damage of the ejection mechanismUndue fit of the tip Ejector misplacementDifficult removal Corrosion

3rd STEP: LEAKAGE TESTING

For pipettes having a maximum volume above 200 µl, set the volume at the maximum value anddraw some water to fill the pipette tip. Observe the tip for 20 seconds. A drop at the end of the tipmeans the occurrence of leakage. This is also true for pipettes with volume ranges below 200 µL;for this volume range, however, the following procedure should be adopted: after observing thepresence of a drop for 20 seconds, dip the tip end into the water in the container. The level of waterinside the tip should not be changed. The presence of leakages is confirmed if the level decreases.

The cause of leakages can be:• Damaged piston seals;• Scratched or corroded piston;

• Chemical or mechanical damages on the cone holder at the tip-fitting portion;• Use of tips not recommended by the manufacturer (see chapter 4).

4th STEP: DISASSEMBLING

This process should be adopted in case of any leakage in step 3. When disassembling the pipette,one can evaluate the internal parts and find out the cause of the problem (damaged piston, lack orimpairment of the seals, etc.) and define which parts should be replaced. An example of disassemblingroutine adopted for certain models pipettes is showed below. Be sure to follow the manufacturer’sinstructions to disassemble and assemble the pipette.

– Pull down and remove the tips ejector (in some models, the ejector is not detachable);– Remove the connection nut;– Carefully remove the piston;– Verify the piston surface (whether it is corroded and/or scratched) as well as the seals.

5th STEP: ASSEMBLING

Assemble the pipette according to the manufacturer’s instructions.

REMARK

We recommend steps 1 to 3 to be carried out daily before starting the work and recorded for thelaboratory control. If abnormalities are found, carry out steps 4 and 5.

Some parts can be changed by the user itself without compromising the instrument calibration.After the change of some other parts, it is necessary to check the instrument calibration andoccasionally adjust it. This information is contained in the pipette instructions manual.

5.3. Performance checking

In addition to the parts cleaning and replacement procedures, the user has to verify the pipetteperformance in order to assure reliable measurements. Such checking analyses both the accuracy(systematic error) and the precision (random error).

The performance checking, in fact, evaluates the set pipette + tip + operator (pipetting technique).So as to reach reliable testing results, the operator should be trained and skilled (pipetting techniquedescribed in chapter 3) and the tips should be those recommended by the pipette manufacturer.

The user should define a schedule for testing the pipettes based on: requirements in terms ofaccuracy and precision, frequency of use, number of users of a same pipette, number of cycles inwhich the pipette is used and the nature of the liquids pipetted. In attachment II, we described thegravimetric method for evaluating the pipettes performance (reference: rule ISO/FDIS 8655- Piston-operated volumetric apparatus - Part 6 - Gravimetric test methods).

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6. SUGGESTIONS OF PROCEDURE

• Piston-operated pipettes are equipment for drawing and dispensing specific volumes of liquids.• In order to assure the reliability of the results, it is necessary to implement the following routine:

– Checking of the general aspect, functions testing and leakage testing (steps 1, 2 and 3)described in item 5.2.3. herein. If abnormalities are found out, follow steps 4 and 5 of thesame procedure.

– Periodic cleaning of the pipette partsThe cleaning periodicity should be established according to the frequency of use, numberof users of a same pipette, number of cycles in which the pipette is used and the nature ofthe liquids pipetted. We recommend laboratories to follow the cleaning procedureevery three months (if dirt accumulates within these three months, reduce the intervalsbetween each cleaning procedure). It is very important for the laboratory to record thecleaning procedures; said records should contain the pipette serial number, cleaning dateand procedure used.

– Parts replacementThe piston seals should be replaced annually (if the number of pipetting cycles exceed100,000, the seals should be replaced within a shorter period of time to be estimated by thelaboratory routine). The cone holder should be replaced every 2 years or when the tip-fitting portion is worn out. Such replacements should be recorded (recording should containthe pipette serial number, replacement date and information on the new part - lot or bill ofsale number). Parts affecting the pipette performance (for example: piston, volumeter, rod)requiring replacement or repair should be forwarded to specialized technical assistance; therepair work should be duly recorded, in addition to the new calibration of the pipette.

– Periodically check the pipette performance (accuracy and precision) as described in attachmentII. Such verification should be made every 3 months and duly recorded (recording examplein attachment II item 8.6.5. herein). The maximum errors found can be up to twice higherthan the errors defined in tables 1 and 2 herein (attachment II, item 8.6.4.). When replacingparts that affect the pipette calibration, the checking procedure should be followed (see item“parts replacement” above).

– Operator testingSince the operation technique is crucial for obtaining good results, it is necessary to test andrecord the operator’s pipetting technique. For that, using an equipment operating within themanufacturer’s specifications, carry out 10 measurements with the maximum e minimumvolume specified for that pipette model. Assess the accuracy and precision of themeasurements obtained. The maximum errors found should be up to twice the values specifiedin tables 1 and 2 herein (attachment II, item 8.6.4.). Record data in a report (an example isprovided in attachment II item 8.6.5.) identifying the operator tested.

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7. ATTACHMENT I: EXAMPLE OF A CLEANING PROCEDURE

The example below can be used for various pipette models, particularly those having a metallicpiston with no grease; for other models, an adaptation is required. Follow the manufacturer’sinstructions contained in the user’s manual. Be sure to certify that the steps described herein do notimpair the pipette calibration.

Warning! Do not mix parts of a pipette with parts of another pipette

7.1. Disassembling

It is necessary to disassemble the pipette for cleaning (wear gloves when contamination risk isinvolved). A general scheme of the pipettes parts is showed below:

LIST OF PARTSFigures 1 and 2

A ButtonB HandleC Cone holderD Tips ejectorE TipF Connection nut

G and H Piston seals

Remarks: This scheme may have variations according to the model and brand of the pipette. Forfurther details, see the manufacturer’s manual.

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– Remove the tips ejector (D) by pulling it;– Disconnect the connection nut (F) thus separating the pipette body (B) from the cone holder

(C);– Remove the piston set carefully and separate the piston seals (G and H), keeping the cone

holder with the finest portion downwards.

The piston seals are frequently captured inside the cone holder. To remove them, place the pistonagain and press it against the cone holder as if you are pipetting. When removing the piston, theseals will be attached to it.

7.2. Cleaning

– Place the cone holder, the piston set, the tips ejector, the seals (do not place the handle) in abeaker and complete to volume with 4% to 8% neutral detergent solution with warm water (±50°C);

– Place the beaker in an ultrasound bath for about 15 minutes (if an ultrasound is not available,keep the beaker in the solution for 40 minutes);

– Dispose the detergent solution used;– Wash each part with running water;– Place the parts in a clean container and wash for the last time with distilled water;– Dry it in an oven at approximately 50°C (do not exceed this temperature) for at most 2 hours, or

allow it to dry at room temperature.

REMARKS

– Use a 4% neutral detergent solution for cleaning slightly dirty pipettes and a 8% solution forheavily dirty pipettes.

– For those pipettes still dirty after washing, use cotton wetted with isopropyl alcohol. Be carefulnot to damage the piston.

– After using the pipette in acid and/or corrosive solutions, always wash all parts with distilledwater or ethanol.

7.3. Assembling

– Place the seal and the o’ring on the piston by first fitting the seal;– Using one of the hands, hold the pipette body in the upright position and place the piston;– Fit the cone holder on the pipette body;– Next, place and thread the connection nut;– Place the tips ejector;– The pipette is ready for use.

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8. ATTACHMENT II: PERFORMANCE STANDARD VERIFICATION

8.1. General

This attachment describes the pipettes performance checking procedure using the gravimetric method(according to rule ISO/ FDIS 8655 - Piston-operated volumetric apparatus - Part 6: Gravimetrictests). The use of the procedures described in this method assures compliance with the internationalspecifications for accuracy and precision.

The compliance test for checking accuracy and precision is applicable to any pipette (definitions inchapter 1). This test is in according to international standards, analyzes the pipetting system as awhole: pipette, tip and operator.

The method described herein includes a procedure for correcting the evaporation losses for smallvolumes (below 50 µL). Further, when converting to the volume of masses obtained in the balance,corrections are made concerning the temperature and pressure at the time of testing.

The user should define a routine for testing the pipettes based on: requirements in terms of accuracyand precision, frequency of use, number of users of a same pipette, number of cycles in which thepipette is used and the nature of the liquids pipetted. There is no standardized interval betweeneach checking. It is the user responsibility to define such an interval. Rule ISO/ FDIS 8655defines a minimum periodicity of once a year.

The pipette should be disassembled and assembled (according to the manufacturer’s manualinstructions) at least once a year before the checking test. The pipette should be handled accordingto the manufacturer’s manual.

8.2. Testing room conditions

The testing room (laboratory) should be cleaned and the temperature and humidity controlled sothat the conditions in the room where verification will take place and the equipment temperatureare stable and homogeneous both before and throughout the procedure.

The pipette and water used in the gravimetric test should be stabilized in the checking roomtemperature at least 2 hours before starting the procedure. Ideally, the verification should take placeunder the following conditions:

1) Stable temperature (water, pipette and room) between 15° and 30°C;2) Relative Humidity above 50%.

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8.3. Operator

8.3.1. Pipette operation

An adequate pipetting procedure significantly contributes for the reproducibility of the verificationtest results. Inexperienced operators may have significant fluctuations in the results. For reliableresults, the operator should be duly trained and qualified. The pipetting technique aspects werediscussed in chapter 3.

8.3.2. Training

A qualified operator should take the compliance test. If required, contact a specialized company torequest the operator’s training program.

8.4. Tips

The pipette to be tested should be handled according to the instructions provided in the operation’smanual. Again, based on the pipette’s operation manual, certify that the pipette is clean, correctlyassembled and that the tips recommended by the manufacturer are used before starting the test.

See chapter 4 for further details.

8.5. Equipment used in the test

To assure the checking procedure integrity, all equipment used during the procedure should beregularly verified.

8.5.1. Balance

The balances should be calibrated and certified by skilled personnel using weights certified by domesticauthorities (Companies/Institutions accredited by INMETRO and members of Rede Brasileira deCalibração - RBC).

The balance legibility should be chosen according to the volume selected in the pipette being tested.

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In practice, the nominal volume (highest volume selectable by the user) can be used for choosing thebalance .

8.5.2. Other equipment

– Thermometer: maximum measurement uncertainty of 0.2°C– Hygrometer: maximum measurement uncertainty of 10%– Barometer: maximum measurement uncertainty of 0.5 kPa

8.5.3. Containers

Containers used during the procedure:

– Origin containerThe container holding the water to be used in measurements (the volume of such a containershould be enough for ALL measurements).

– Weighing containerThe container to be used for measuring (to be placed on the balance weighing plate).

– Disposition containerA third container for collecting the aliquots not weighted.

Especially for testing the lowest volume of the pipette, we recommend an average ratio of 3:1 tothe container diameter and height for weighing purposes or that the container is provided with a lid.Some companies supply container kits appropriate for the gravimetric test.

8.5.4. Water

The test requires distilled or deionized water. In both cases, the water should be degasified (grade 3water according to Rule ISO 3696) at the temperature of the room where verification will takeplace. In order to avoid water temperature fluctuations, use an origin container big enough to holdthe water for all measurements.

8.6. The checking procedure

The performance checking analyzes both the accuracy (systematic error) and precision (randomerror). The conditions, procedures and qualifications previously described herein should beimplemented in order to assure the validity of the tests results. After rinsing the tip, perform 10individual weighing runs* for each selected volume. For the varying volume pipettes, three differentvolumes should be selected according to the volume range of the model being evaluated. Such

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* O user can select the number of measurements according to the accuracy and precision specifications accepted byits methodology.

volumes should be the nominal volume (maximum), 50% of the nominal volume (intermediatevolume) and the minimum volume of the model volume range (these volumes are defined in thepipette’s manual). For the fixed volume pipettes, only the nominal volume is employed.

1. Adjust the testing volume in the pipette (this volume should be kept throughout the procedure).2. Estimate the evaporation rate (for small volumes, below 50µL). See the procedure below.3. Run the checking test: record the masses on the Performance Checking Report.4. Calculate: record the results on the Performance Checking Report.5. Compare the results with the accuracy and precision specifications showed in tables 1 and 2

(item 8.6.4).

These steps will be detailed later.

8.6.1. Estimating the evaporation rate (average mass evaporated per weighing cycle)

For volumes below 50µL, it is necessary to use a lidded container. The purpose is to minimize,control and quantify the evaporation loss during the weighing cycle. The use of clamps for handlingthis container is also advisable.

The evaporation can be estimated by a sequence of four weighing simulations where the weighingcycle is repeated without dispensing water into the weighing container. The total difference attributedto the evaporation is calculated and divided by 4 so as to obtain the average. The range is expressedin mg/cycle (in case of a single cycle, it should be expressed in mg). Excessive handling of thecontainer should be avoided. The use of clamps for handling the weighing container is advisable.

The evaporation rates are usually between 0.010 mg and 0.025 mg per weighing cycle. Recalculatethe evaporation rate whenever the conditions are changed (temperature, pressure e humidity).

1. Add water to the weighing container up to 1/3 of its volume.2. Place the capped container on the balance plate.3. Using the pipette, draw the aliquot from the origin container.4. Tare the balance and remove the weighing container from the plate (the use of clamps for

removal is advisable).5. Dispense the aliquot into the disposal container or return it to the origin container. Do not

dispense into the weighing container.6. Record the resulted e1.7. Repeat steps 3 to 8 three times so as to obtain e2, e3 and e4.8. Calculate the loss/cycle: e = (e1 + e2 + e3 + e4)/4 (mg).9. The evaporation per cycle e (mg) should be added to the average weight before calculating the

average volume.

* O usuário pode selecionar o número de medidas de acordo com as especificações de exatidão e precisão aceitaspela sua metodologia.

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An example calculation of the evaporation rate is showed at the end of this chapter.

8.6.2. Gravimetric test

The aspiration and dispensing techniques are described in chapter 3. Although several alternativepipetting methods are possible, always follow the normal mode (direct mode, do not use the reversemode). For all pipettes, avoid holding or heating the cone holder using your hand during the test.

For multichannel pipettes, fit the tips in every channel; however, test each channel individually.Record the results for each channel.

1. Preparation

1.1. Transfer the water from the origin container to the weighing container at a depth of at least 3mm.

1.2. Measure and record the origin container water temperature (t1), the air pressure and relativehumidity of the room. If the weighing container has a lid, place it.

1.3. Fit the new tip.1.4. Select and adjust the volume to be tested (this volume cannot be changed during the test).1.5. Pre-rinse the tip by drawing an aliquot from the origin container and dispensing it into the

disposition container 5 times to balance the air humidity inside the pipette atmosphere.1.6. Place the weighing container with water on the balance plate and tare (m0 = 0).

2. Testing cycle

Each testing cycle should take less than 1 minute. However, a constant rhythm during the weightingoperation should be kept (the cycle rhythm and the rhythm between the cycles).

2.1. Place a new tip in the pipette.2.2. Pre-rinse the tip by drawing and dispensing into the disposition container once.2.3. Draw the volume to be tested as specified in chapter 3.2.4. If the weighing container has a lid, remove it.2.5. Dispense the volume tested in the weighing container and replace the lid with the aid of the

clamps.2.6. Record the mass m1 of the volume tested.2.7. Tare the balance.

3. Repeat the test described above (from item 2.1 to 2.7) up to 10 measurements (m1 tom10).

4. After the last measurement, check and record the origin container water temperature(t2), the atmospheric pressure and the relative humidity of the room.

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8.6.3. Calculations

1. Average temperature calculation

Calculate the average temperature (t) of the distilledwater (maximum fluctuation between t1 and t2: 0.2°C)

2. Average pressure calculation

Use the average barometric pressure (B) and theaverage temperature (t) to determine thecorresponding Zfactor in the table.

3. Volume calculation from the mass

After the evaporation losses corrections (for volumesbelow 50 µL), multiply the masses (mg) by the Z factorto obtain the volumes (µL).

4. Average volume calculation

After calculating the weightings individual volume,calculate the average volume (result expressed in µL).


B = (B1 + B2)/ 2

Vi= Z (mi + e)

Vi = individual volumes (µL)mi = individual masses (mg) e = evaporation rate (mg)Z = Z factor (µL/ mg)

t = (t1 + t2)/ 2

5. Accuracy (systematic error)

The difference between the test average volume andthe volume set in the pipette. For fixed volumepipettes, replace Vs with Vo = nominal volume. Theaccuracy can be expressed in mL or ...

...as percentage:

es = V - Vs

es = average error‘V = average volumeVs = adjusted volume

es = 100% (V - Vs)/Vs

Vi= individual volumesV = average volumen= number of weightings

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6. Precision (random error)

Dispersion of the volumes dispensed around thedispensed volumes average. Also known (accordingto the context) as standard deviation, reproducibilityor repeatability.

Vi = individual volumes

V = average volume (calculated asin the previous item)

n = number of measurementsSD = standard deviation

CV = (SD / V)x 100%As percentage, also known as coefficient of variation(CV).

SD

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Calculations: Z factorZ = conversion factor (mL/ mg), t (°C) = temperature average * , B = atmospheric pressure (hPa)

* this average is obtained from the origin container water temperature measured at the beginningof the test (t1) and the origin container water temperature measured at the end of the test (t2).

8.6.4. Specifications

TABLE 1

– This specifications table applies to the air displacement fixed volume pipettes and positivedisplacement pipettes using reusable capillaries and pistons.

– This table can be also used for the air displacement varying volume pipettes. The nominalvolume in these pipettes is the greatest volume selectable by the user and specified by themanufacturer. For example: a pipette with a specified volume range from 10 µL to 100 µL, thenominal volume is 100 µL. The maximum acceptable errors for the nominal volume apply to allthe volumes within the specified range: if we consider a pipette with a volume range from 10 µLto 100 µL, the maximum acceptable systematic error for any selected volume is 0,8 µL and themaximum acceptable random error for any selected volume is 0,3 µL.

For intermediate volumes to those listed in the table, the immediately greater absolute value of thevolume maximum acceptable errors should apply. For example: to determine the maximumacceptable errors for 25 µL, the values specified for 50 µL are used.

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TABLE 2

This specifications table applies to the positive displacement pipettes using reusable capillaries andpistons and/or fixed volume pipettes.

For intermediate volumes to those listed in the table, the immediately greater absolute value of thevolume maximum acceptable errors should apply.

REMARKS

– The user may define the maximum acceptable errors (systematic and random) based on itsmethodology.

– The user may regularly test the equipment, for example, every three months. Other time intervalsbetween the tests can be established (provided that they do not exceed one year) based on thefollowing aspects: frequency of use, number of users of the pipette, aggressive nature of thetransferred material, maximum errors accepted by the user.

– When the test is carried out after the equipment maintenance or repair, the maximum acceptableerrors (systematic and random) should follow the tables listed herein (tables 1 and 2).

8.6.5. Checking procedure report

After the test, information has to be recorded. We attached a report template, however, the usermay prepare his own template provided that it contains the following information:

– Pipette identification;– Date;– Test conditions (temperature, pressure, relative humidity);– Measurements obtained for each volume tested;– The calculations’ results;– Result from the comparison with the specification table.

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TEMPLATE:

Pipette identificationModel Serial number Tips used in the no test

Technician identificationCompany Name Phone/Email

Technician Name Phone/Email

ConditionsDate/ Test time Location

Temperature (°C) Humidity (%) Pressure (hPa)

BalanceModel Serial number Sensitivity

Test results

COMPARING TO THE SPECIFICATIONS: APPROVEDFAILED

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8.6.6. Performance checking numeric example

We will now give an example of the calculations for the performance checking. We will be based ona hypothetic verification of 10 µL in an air displacement varying volume pipette.

1. Since the tested volume is lower than 50 µL, the evaporation rate should be determined (item8.6.1.).

Number of measurements: 04 (e1, e2, e3, e4)

Average evaporation rate: e

2. The test initial temperature and pressure are recorded:Ti = 21.5°CPi = 1013

3. Ten measurements (item 8.6.2.) are performed and the results recorded.

e1 = 0.016 e3 = 0.021

e2= 0.018 e4 = 0.017

e = (e1 + e2 + e3 + e4)/4 (mg)

e = (0.016 + 0.018 + 0.021 + 0.017)/4

e = 0.018 mg/ per cycle

W1 = 9,84W2 = 9,90W3 = 9,91W4 = 9,86W5 = 9,87

W6 = 9,90W7 = 9,92W8 = 9,93W9 = 9,95W10 = 9,92

4. The test final temperature and pressure are recorded:Tf = 21,5°CPf = 1013

5. Average pressure and temperature calculation:

T = (Ti + Tf) / 2T = (21,5 + 21,5)/ 2T = 21,5°C

P = (Pi + Pf) /2P = (1013 + 1013) / 2P = 1013

These temperature and pressure average results will be used for determining the conversionfactor Z.

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6. Conversion of mass to volume:Vi = (Wi +) x Z

For a temperature of 21.5°C and an atmospheric pressure of 1013 hPa, the Z factor is 1.0032 µL/mg.

7. Average volume calculation

V = (9.89 + 9.95 + 9.96 + 9.91 + 9.92 + 9.95 + 9.97 + 9.98 + 10.00 + 9.97)/10V = 9.95 µL

8. Accuracy analysis– Systematic error - EE = V - VoVo is the value set in the pipette (nominal volume). In this example, it is 10µL.E = 9.95 - 10E = - 0.05 µL

– Relative error - E%E% = (V

- Vo) x 100/ Vo

E% = (-0.05*100)/ 10E% = - 0.50 %

9. Precision analysis (repeatability - random error)– Standard deviation - SD

SD SD

SD2v = 1/9 x [(9,89 - 9,95)2 + (9,95- 9,95)2 + (9,96 - 9,95)2 + (9,91 - 9,95)2 + (9,92 - 9,95)2

+ (9,95 - 9,95)2 + (9,97 - 9,95)2 + (9,98 - 9,95)2 + (10,00 - 9,95)2 + (9,97 - 9,95)2 +]

SDv = 0,03 µL

– Coefficient of variation – CVCV = (DP/ V)x 100%CV= (0,03/9,95) x 100%CV = 0,34 %

10. Results are transferred to the report (item 8.6.5.)

The following specifications for 10 µL are showed in table 1 (item 8.6.4.):

Simply compare the results obtained (report) with those specified in the table. In this case, thetested pipette is approved.

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9. ATTACHMENT III: DECONTAMINATION PROCEDURES

The procedures described next can be used for some pipette models, particularly those having ametallic piston. Thus, it is essential to read the manufacturer’s instructions before applying any ofthe methods described herein.

INTRODUCTION

This document describes a number of methods for pipette decontamination, especially those havinga metallic piston. Such methods can be physical (autoclaving or UV irradiation) or chemical. Accordingto the type of application, the most efficient decontamination method should be chosen.

However, considering the action spectrum as well as the advantages and disadvantages of thesemethods, we conclude that the chemical is the most effective. Commercial chemical solutionscontaining detergents such as disinfectants, assure an efficient decontamination.

The chemical decontamination method is recommend for routine decontamination proceduressince it is effective, fast and easy.

Decontamination procedure

The types of contamination include: from sample to operator, from one sample to another or fromthe sample to the pipette. This can affect the safety of the operator, the sample and consequentlythe experiment result or eventually the pipette.

There are several methods for eliminating contaminants; however, there are some doubts concerningits efficiency and compatibility with the pipettes.

This document intends to solve doubt related to the decontamination process, taking the variousapplications of the pipettes into consideration. Thus, different fields of application will be commented(chemistry, microbiology and molecular biology) and the chemical or physical methods discussed.The efficiency of a method can be explained by means of its mechanism of action. When morethan one method is proposed, there will be a comparison to help choosing the most appropriate.Once the decontamination method is chosen, read the protocols at the end of this chapter.

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Field of application

9.1. Chemistry

By simply washing the pipettes with distilled water eliminates contaminations by acids, alkalis, buffersolutions or organic solvents.

It may be necessary to remove other products such as oils and greases. Then, the use of detergentsis required. Detergents (anionic surfactants) are organic molecules having a polar hydrophilic endand a non-polar hydrophobic end. Due to its structure, they convert insoluble residues (grease, forexample) into soluble residues, therefore, becoming effective cleaning agents.

9.1.1. Standard cleaning procedure (for more details, see attachment I)

This procedure should be used to eliminate contaminants resulting from the use of the pipette (forexample: proteins and buffer solutions) and that do not require special treatment for elimination,such as microorganisms, radioactivity and other that will be discussed later.

– Remove the tips ejector by pulling it;– Disconnect the connection nut thus separating the pipette body from the cone holder;– Remove the piston set carefully and separate the seal(s), keeping the cone holder with the finest

portion downwards;– Place the cone holder, the piston set, the tips ejector, the seal(s) in a beaker (do not place the

pipette body) and complete to volume with 4% to 8% detergent solution in warm water (±50°C);

– Place the beaker in an ultrasound bath for about 15 minutes (if an ultrasound is not available,keep the beaker in the solution for 40 minutes);

– Dispose the detergent solution used;

The seals are frequently captured inside the cone holder. To remove them, place the piston againand press it against the cone holder as if you are pipetting. When removing the piston, the seals willbe attached to it.

– Place the parts in a clean container and wash for the last time with distilled water;– Dry it in an oven at approximately 50°C (do not exceed this temperature) for at most 2 hours, or

allow it to dry at room temperature.

REMARKS

– Use a 4% detergent solution for cleaning slightly dirty pipettes and a 8% solution for heavilydirty pipettes.

– For those pipettes still dirty after washing, use cotton wetted with isopropyl alcohol. Be carefulnot to damage the piston.

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– After using the pipette in acid and/or corrosive solutions, always wash all parts with distilledwater or ethanol.

– Assemble the pipette.

9.2. Microbiology and cell culture

In these fields it is essential to eliminate several microorganisms (for example: virus, bacteria and/orfungi, such as yeasts).

Several techniques with different spectra and mechanisms of action can be used.

9.2.1. Autoclaving

The characteristics of the sterilization cycle depend on the initial amount of microorganisms (theso-called microbial load). It is generally accepted that the possibility of a viable organism to bepresent in an autoclaved item is below 1 in 1 million (Sterility Assurance Level 10-6).

9.2.2. UV irradiation

The UV wavelength is between 100 nm and 400 nm. There are 3 types of UV radiation, accordingto the wavelength: UVC (200 nm to 290 nm), UVB (290 nm to 320 nm) and UVA (320 nm to 400nm).

Aims Lethal for most of the microorganisms 19 (Table §2.1)Action Photochemical changes of cell enzymes (UVA) and DNA (UVC) - see details

in table § 1.3.2.

9.2.3. Chemical agents

A chemical solution should be chosen taking into the consideration the type of microorganism tobe eliminated.

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Aims Any vegetative cell or endospore 19.The kinetics leading to virus inactivation is still unclear 9.

Action Heat degrades nucleic acids, enzymes and other essential proteins.The cell membrane may be damaged.

Level of activity: +++: strong, ++: medium, +: weak, -: no activity.

The mechanisms of action of such chemicals are described in the following table:

9.3. Molecular biology

Biochemists and molecular biologists are familiar with the contamination sources such ascontaminating proteases, nucleic acid (DNA, RNA), nucleases (RNases, DNases) and radioactivity.

9.3.1. Proteases

Proteases are enzymes that degrade proteins. Several decontamination methods and their mechanismsof action are detailed in the table below:

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The following table lists the main microorganisms and some common chemical agents. When specialstrains are handled, consideration should be taken regarding the specific commercial products usedto destroy them and also certify the chemical compatibility of the pipette and the correspondingchemical (see § 3.3).

9.3.2. Nucleic acids (DNA and RNA)

The specificity and sensitivity of the PCR and RT-PCR techniques involve a disadvantage: easycontamination of reagents and samples, thus leading to systematic errors 7.

Other method:

Glycine/HCl buffer (pH=2). The action of this solution is not documented (see the protocol later)

Note: all such methods degrade the nucleic acids into molecules having lower molecular weightsthat are not normally amplified; however, such methods do not clean.

9.3.3. Nucleases

Nucleases are enzymes that degrade nucleic acids. They have a wide distribution and are found inprokaryotic and eukaryotic cells, vegetal and animals (skin, water). DNases are not very resistantand are not essential for most of the applications in molecular biology, while the RNases are moredurable and resistant to heat.

DNases need metal ions for its activity and can be easily inactivated using some appropriate buffersolutions (such as chelating agents, for example, EDTA). RNases do not require metal ions, as theyuse the 2-hydroxyl group as reagent; therefore, they are not easy to inactivate.

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Method/Chemical agent Action

Autoclaving Not always enough to inactivate RNases that may recovertheir activity after several treatment methods(boiling, for example)

UV irradiation Lack of data when reading.DEPC Alkylates residues of histidine from the catalytic sites of

(diethyl pyrocarbonate) RNases A, thus inactivating them.This is a strong agent; however, it is not an absolute inhibitor.

9.3.4. Comments on nucleic acids and nucleases

All methods described partially degrade nucleic acids and nucleases, however, none of them showed100% effectiveness.

Precaution is the best way to avoid contamination in molecular biology:

– Separate the areas and sets of pipettes to prepare the samples and carry out the assays.– Use tips provided with filters so as to avoid contamination by aerosols.– Or use positive displacement pipettes provided with disposable capillaries and pistons (total

protection against cross contamination). For critical PCR protocols, the use of positivedisplacement pipettes is required when no contamination is involved.

9.3.5. Radioactivity

In case of weak radioactive contamination, specific detergents containing complexing agents canbe used.

Comparing the methods

The methods are compared by first considering its efficiency in terms of contaminants and then theadvantages and disadvantages.

9.4. Methods spectrum of action

Consider two approaches:

1. I have the materials required for this method in my lab. How effective are they (read the tablevertically)?

2. I need to eliminate this contaminant. What can I do (read the table horizontally)?

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Level of activity: +++: strong, ++: medium, +: weak, -: no activity, SI: not informed.

*: specific detergents (with complexing agents).

For UV irradiation: +: some activity, -: no activity.

The UV irradiation activity limit is difficult to evaluate since its action do not depend on the wavelengthonly (between 100 nm and 400 nm), but also other factors such as: time of exposure, distance ofthe lamp, emission density, angle of exposure and atmospheric temperature.

9.5. Advantages and disadvantages

See the table below. The method should comply with the following requirements:

– Compatibility with the pipette manufacturing materials.– Fast action.– Easy and safe to apply.

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* Those made of PVDF. UV radiation resistant cone holders (made of PBT) are available for P200and P1000.

Thus, the chemical procedures can be considered the most appropriate for pipettes decontamination.

Protocols

According to the method used, different parts of the pipette can be decontaminated.

Method Portion of the pipette

Autoclaving Cone holder, connection nut, tips ejectorUV irradiation External portionsChemical solutions Different levels, depending on the solution used

(external portion, complete immersion)

Note: Wear disposable gloves throughout the decontamination procedure.

9.6. Autoclaving

Remove the tips ejector and unthread the connection nut. Clean the cone holder, the connectionnut and the tips ejector using detergent and then autoclave for 20 minutes at 121°C, 0.1 Mpa. Allowthe parts to dry or place them in an oven at 50 - 60°C for about 30 minutes.

Note: – Do not autoclave such parts at 134°C as they will be damaged.– Do not autoclave parts other than those listed above.

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9.7. UV irradiation

UV lamps can be installed on the top of the bench or in a biosafety hood where handling is allowed.The combination of 254 nm and 300 nm bulbs for 20 minutes can destroy double strand DNA(long sequences are easier to inactivate) 7.

9.8. Chemical solutions

Before using a chemical solution, be sure of the compatibility of the materials used to manufacturethe pipettes. The manufacturer should provide a list of the materials used to manufacture the pipette.Some examples are showed below:

PVDF, PBT, PC, PE, POM, nitrile rubber, stainless steel, ABS, PEI and PMP. See “Glossary andAbbreviations”.

Note: it is not advisable to soak the seal and the o’ring.

9.8.1. Cone holder, tips ejector and connection nut decontamination

Two examples of chemical solutions and their protocols are showed below.

Sodium hypochlorite

Sodium hypochlorite has a wide antimicrobial activity, fast antibacterial action e also denaturesproteases, DNA and RNA. In addition, it is an easy-to-use and low-cost chemical agent atoxic at theconcentrations used.

It is very important to clean the pipette (using a detergent, for example, Mucapur) before disinfectingusing sodium hypochlorite, since the method may lose efficiency in the presence of highconcentrations of organic material.

• Cleaning

– Remove the tips ejector, unthread the connection nut and remove the cone holder piston.– Dilute the detergent with warm water at 50-60°C in:

a) A ultrasound bath or inb) A beaker.

– Place the cone holder, the tips ejector and the connection nut:a) In an ultrasound bath for 15 minutes.

orb) In the beaker – the parts should be brushed.

– Remove the parts and rinse well.

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Before disposing the cleaning solution, add 10% sodium hypochlorite to the solution and allow it toreact for 10 minutes. This procedure will decontaminate the cleaning solution before entering thelocal water network.

• Disinfection

– Dilute the sodium hypochlorite in distilled water at a concentration of 10% and place it in alarge beaker.

– Place the cone holder, the tips ejector and the connection nut in the beaker and allow it toreact for 30 minutes.

– Rinse well with tap running water first and then with distilled water.– Allow them to dry at 50ºC - 60°C for about 30 minutes.– Keep them at room temperature for 15 minutes before assembling the pipette (so that the

parts cool down before assembling the pipette).

Note: the hypochlorite solution should be discarded after 3 days.

Glycine /HCl Buffer (pH2=2)

Prepare the solution as follows:

10x buffer: NaCl 30.6 g; glycine 39.2 g, complete with distilled water to the volume of 523 ml.Add 1N HCl in a sufficient amount to reach a final volume of 1000 mL.

– Remove o tips ejector, unthread the connection nut and remove the cone holder piston.– Place the cone holder, the connection nut and the tips ejector into the 1x diluted buffer (1/10

dilution of the previously mentioned buffer) at 95°C for 30 minutes.– Rinse well with tap running water first and then with distilled water.– Allow them to dry at 50ºC - 60°C for 30 minutes.– Keep them at room temperature for 15 minutes before assembling the pipette.

9.8.2. Decontamination of the pipette lower portion and body

For most of the applications, decontamination of the pipette lower portion and body suffices.

A mixture of chemical agents is recommended (detergent and disinfectant in a single product).

The pipette body should be cleaned using a compatible solution (see item 3.3) on the surface only(do not immerse the pipette body on the decontaminating solution). The lower portion of thepipette, including the piston, the tips ejector, the cone holder and the connection nut (except for thepiston seals) should be also cleaned (using small brushes to clean the cone holder interior) or can beimmersed as per the following procedures:

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– Remove the tips ejector, unthread the connection nut and remove the cone holder piston.– Dilute the chemical agent chosen (according to the manufacturer’s instructions) in a beaker.– When disassembled, place the lower portions (piston, tips ejector, cone holder and connection

nut) in the beaker.– Rinse well with tap running water first and then with distilled water.– Dry at 50 °C - 60 °C for 30 minutes.– Keep the parts at room temperature for 15 minutes before assembling the pipette.

Conclusion

The pipette decontamination method is selected according to the type of application. For most ofthe applications, just thoroughly decontaminate the lower portions and clean the pipette body usinga chosen solution (see § 3.3.2).

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Appendix 1

What about the disposable materials (tips, capillaries, pistons and Distritips)?Methods used for the sterilization of consumable plastics of the pipettes

At the laboratory, autoclaving is the most common method for sterilizing the tips. However, thisprocess takes time: the autoclaving time itself, the drying cycle and stabilization at room temperaturefor some hours.

Note: the autoclaving process should be carried out at 121°C (20 minutes, 1 MPa). It is notrecommended to autoclave at 134 °C as plastics may be damaged.

Depending on the type of material, other methods are used by the manufacturer to sterilizeconsumables: ethylene oxide or more often irradiation (gamma rays: 60Co, or beta rays: electronsbeam).

As this manufacturing process is automated, including the final step, “clean” tips are generallyproduced with no manual contact.

Method Action

Ethylene oxide Alkylates the amino terminus of amino acids, thus resulting in thecellular death 11.

Irradiation Ionization of important cell components (including amino acids) 11.Inactivation of DNA templates through free radicals.

Comments

Ethylene oxide is highly toxic and mutagenic. Therefore, health and environmental control authoritiesin several countries have questioned its use. The irradiation used in the industrial sterilization processesdoes not render materials radioactive. The dosage is chosen based on the regular experimentaldeterminations.

Appendix 2

Pyrogenic materials

Contamination by pyrogenic materials can be troublesome in some pharmaceutical procedures inwhich medical devices are used in parenteral preparations (mentioned in various methods of thePharmacopeia) 4.

Pyrogens are endotoxins produced by Gram negative bacteria and are chemically and physicallystable. Very little has been published on how to eliminate them.

The use of gamma rays at high levels of energy (minimum of 25 kGy) seems to eliminate the risksof pyrogen contamination. In other words, a product has to be manufactured and controlled as amedical device so as to be certified as ‘pyrogen-free’.

10. ATTACHMENT IV: DEFINITIONS

Accuracy (average error or systematic error): the difference between the volume dispensedand the nominal volume or the volume selected in the pipette.Calibration: set of operations that establish the relation between the volume dispensed and thenominal volume or the corresponding selected volume in the pipette.Dead air volume: in air displacement pipettes, the volume of air between the lower portion of thepiston and the liquid surface.Evaporation rate: estimated water loss caused by evaporation during the weighting process.

Gravimetric analysis: general procedure based on the determination of the mass of the wateraliquots dispensed by a pipette. The values are corrected concerning the evaporation loss; then, theactual mass and volume are calculated based on the water density at specific temperatures withcorrections to the room atmospheric pressure (Z factor).

Nominal volume: the greatest volume selectable by the user and specified by the manufacturer.Note: therefore, for a varying volume pipette with a useful volume range from 10 µl to 100 µl, itsnominal volume is 100µl.Selected volume: the volume set by the user for dispensing the chosen volume within the usefulvolume range.Note: for the fixed volume pipettes, the selected volume corresponds to the nominal volume.

Precision (random error): dispersion of the volumes dispensed around the dispensed volumesaverage. Also known (according to the context) as standard deviation, reproducibility or repeatability.

Repeatability: the dispersion between the results of successive measurements (carried out within ashort period of time) with no change of parameters or conditions.

Reproducibility: the dispersion between the results of measurements made under different conditions(different location or operator), the parameters being constant.

Volumetric specifications (maximum acceptable errors): the allowed limits (upper and lower)for deviation between the dispensed volume and the nominal volume or the selected volume.

Useful volume range: part of the nominal volume that can be dispensed within the maximumerror specified in the International Rule ISO/DIS 8655. The upper value of the useful volumerange is always the nominal volume. The lower valor is 10% of the nominal volume if not otherwisespecified by the manufacturer.Recalibration (operator-adjusted): a procedure defined by the manufacturer that can be followedby the final user to assure that the pipette works according to the published specifications.Weighting: weigh means to determine the mass of an aliquot.

Z factor: conversion factor (µL/mg) considering the density of the water in contact with the air asa function of temperature and pressure.

Ex: Designation (abbreviation) of an instrument designed to dispense volumes.

In: Designation (abbreviation) of an instrument designed to draw volumes.

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11. GLOSSARY AND ABREVIATIONS

Glossary

Detergent: organic molecules that convert insoluble residues into soluble residues (oils and fats).They are effective cleaning agents (most of them do not kill microorganisms).

Decontamination: disinfection of infected articles, thus allowing their handling /use (reduction ofmicroorganisms to an acceptable level).

Note: chemical decontamination requires a previous cleaning process using a detergent so that thedecontamination process can be effective.

Disinfection: selective elimination of some kinds of microorganisms so as to avoid transmission(reduction of the number of infecting microorganisms below the level required to cause an infection).

Sterilization: complete elimination of all organisms (for example: cells, spores and virus).

Abbreviations

DNA: Deoxyribonucleic AcidEDTA: Ethylene Diamine Tetraacetic AcidRNA: Ribonucleic Acid

Materials

ABS: Styrene Butadiene AcrylonitrilePBT: PolyButylene TerephthalatePC: PolyCarbonatePE: PolyEthylenePEI: PolyEther ImidePMP: PolyMethylPentenePOM: PolyOxyMethylenePP: PolyPropylenePVDF: PolyVinyliDene Fluoride

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12. REFERENCES

Cimino, G.D., Metchette, K.C., Tessman, J.W., Hearst, J.E. and Isaacs, S.T. 1990. ‘Post PCR sterilization:a method to control carryover contamination for the polymerase chain reaction’. Nucleic AcidsResearch, 19 (1): 99-107.

Deragon, J.M., Sinnet, D., Mitchell, G., Potier, M., Labuda, D. 1990. ‘Use of ã irradiation to eliminateDNA contamination for PCR’. Nucleic acids research, 18 (20): 6149.

Ferron, A.. 1992. ‘Spectre d’activité des antiseptiques et désinfectants’. Bactériologie médicale (àl’usage des étudiants en médecine), Edition C et R.

Gilson Guide to Pipetting 800353A

prEN ISO/ FDIS 8655-1 - Piston-operated volumetric apparatus

Guyomard, S., Goury, V., Laizier, J., Darbord, J.C. ‘Defining of the pyrogenic assurances level (PAL)of irradiated medical devices’. 1987. International Journal of Pharmaceutics, 40: 173-174.

Hanne, A., Krupp, G.. ‘Removing DNA contamination from pipettes’. Bionews, Eppendorf, No.8.

Hayatsu, H., Pan, S.K, and Ukita, T. 1971. ‘Reaction of sodium hypochlorite with nucleic acids andtheir constituents’. Chem. Pharm. Bull, 19 (10) 2189-2192.

Heinrich, M. 1991. ‘PCR carry-over’. BFE, 8 (10): 594-597.

Jette, L.P., Ringuette, L., Ishak, M., Limmer, M. and Saint-Antoine, P. 1995. ‘Evaluation of threeglutaraldehyde-based disinfectants used in endoscopy’. J Hosp Infect, 30 (4): 295-303.

Jürgen, H. and Kaiser, K. 1996. ‘Avoiding viral contamination in biotechnological and pharmaceuticalprocesses’. Nature Biotechnology, 16: 1077-1079.

Kwok, S. and Higuchi, R. 1989. ‘Avoiding false positives with PCR’. Nature, 339: 237-238.

Leuci, C. 1998. ‘Intérêt des EVC dans le secteur biomédical’. Caoutchoucs et plastiques. 769.

Manual de operação da Pipetman P - marca Gilson LT801117

Mifftin, T.E. ‘Control of contamination’. Molecular Bio-Products.

Ou, Chin-Yih, Moore, J.L, and Schochetman, G. 1991. ‘Use of UV irradiation to reduce false positivityin Polymerase Chain Reaction’. BioTechniques, 10 (4): 442.

– Part 1: terminology, general *


– Part 2: pipettes *


– Part 6: Gravimetric test methods *

Prince, A.M. and Andrus, L. 1992. ‘PCR: how to kill unwanted DNA’. BioTechniques, 12 (3): 358-360.

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Sambrook, Fritsch, and Maniatis. 1989. ‘In Vitro Amplification of DNA by the Polymerase ChainReaction’. In Molecular Cloning: A Laboratory Manual, 2nd ed. 14:14.

Taibi, C. ‘L’infection existe, sa prévention aussi’. Guide pratique d’hygiène hospitalière.

Tritt, C.S. 1997. ‘Sterilization methods’. http://www.msoe.edu/~tritt/be4xx/stermeth.html.

Van Bueren, J., Simpson, R.A., Salman, H., Farelly, H.D. and Cookson, B.D. 1995. ‘Inactivation ofHIV-1 by chemical disinfectants: sodium hypochlorite’. Epidemiol Infect, 115 (3): 567-579.

‘Le contrôle des micro-organismes par les agents physiques et chimiques’. Partie 4, chapitre 5: lecontrôle des micro-organismes.

‘Extraction and purification of RNA’. Extraction, purification, and analysis of messenger RNAfrom eukaryotic cells (7.3-7.5).

Sterilization and sterility assurance of compendial articles, USPXXI. Sterilization and sterilityassurance, general information

‘Avoiding Ribonuclease contamination’. ‘neb.com’.

TECHNICAL STAFF

Volume II – Module 2 – Water for Instrumental Chemical Analyses

Editor:• José Muradian Filho - Millipore

Coordination:• Cláudia Franklin de Oliveira – ANVISA• Itapuan Abimael da Silva – ANVISA• Karen de Aquino Noffs Brisolla – ANVISA• Karla de Araújo Ferreira – ANVISA• Marcelo Cláudio Pereira – ANVISA• Max Weber Marques Pereira – ANVISA• Renato Almeida Lopes - ANVISA

SUMMARY

1. WATER FOR INSTRUMENTAL CHEMICAL ANALYSES ..................................................... 51.1. Introduction ................................................................................................................................... 51.2. Water contaminants ...................................................................................................................... 5

1.2.1. Dissolved inorganic compounds ...................................................................................... 61.2.2. Dissolved organic compounds ......................................................................................... 61.2.3. Particles and colloids .......................................................................................................... 61.2.4. Microorganisms .................................................................................................................. 71.2.5. Dissolved gases ................................................................................................................... 7

1.3. Monitoring and control of the purified water quality .............................................................. 71.3.1. Monitoring of inorganic ionic contaminants – conductivity and resistivity .............. 81.3.2. Monitoring of organic contaminants – TOC – total oxidizable carbon .................. 10

1.4. Quality specifications .................................................................................................................. 111.5. Water purification technologies for laboratories .................................................................... 12

1.5.1. Distillation ......................................................................................................................... 121.5.2. Deionization ...................................................................................................................... 131.5.3. Reverse osmosis ................................................................................................................ 141.5.4. Continuous electrodeionization ...................................................................................... 161.5.5. Ultrafiltration ..................................................................................................................... 181.5.6. Membrane microfiltration ............................................................................................... 191.5.7. Activated charcoal ............................................................................................................ 201.5.8. Ultraviolet radiation (UV) ............................................................................................... 20

2. PURIFIED WATER SPECIFICATIONS ...................................................................................... 232.1. Purpose ......................................................................................................................................... 232.2. Monitoring of the quality of several types of water .............................................................. 242.3. Calibration and qualification...................................................................................................... 242.4. Storage recommendations ......................................................................................................... 252.5. Water purification equipment maintenance ............................................................................ 252.6. Control forms .............................................................................................................................. 252.7. Reference documents ................................................................................................................. 25

3. REFERENCES ................................................................................................................................... 29

1. WATER FOR INSTRUMENTAL CHEMICAL ANALYSES

1.1. Introduction

Water, as found in nature, contains several substances other than the H2O molecule: salts, organiccompounds, microorganisms, particles, dissolved gases. This is basically attributed to a very knownfeature: water is a universal solvent.

At the laboratory, the several usages of water require a higher or lower grade of purity. This gradeis defined as a function of the application, analysis method, interferers and its sensitivity and limitof detection. That is why the purified water and its quality are essential factors for the preparationof solutions in general, buffer solutions, mobile phases in chromatography, blanks e also othercommon, but not less important applications, such as glassware washing (final rinsing).

Defining, knowing, removing and controlling water contaminants are critical steps to effectivelyand economically obtain purified water in the laboratory, particularly complying with the applicationrequirements.

Information and concepts will be presented next in four topics to help attaining such purposes anddecide what kind of water is to be used in a specific instrumental analysis:

• Water contaminants• Monitoring and control of the purified water quality• Quality specifications• Technologies that can be used for purifying water in laboratories

1.2. Water contaminants

As mentioned, contaminants have a direct relationship with the analysis in progress, as well as thesensitivity and limit of detection of the method used. For example, in HPLC – High PerformanceLiquid Chromatography – the analyses using ultraviolet detector are sensitive to organic compoundspresent in the water (mobile phase component) and that absorb in the wavelength used. Thiscompromises the results and the calibration curve itself. Further, organic compounds not detectedin this wavelength can be retained and accumulate in the HPLC column, thus impairing its efficiencyand functionality, leading to the so-called “ghost peaks” in a chromatogram.

For didactics purposes, we divided the water contaminants in five categories:

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Volume II / Module 2 - Water for Instrumental Chemical Analyses

1.2.1. Dissolved inorganic compounds

Basically consist in water-dissolved salts, the inorganic ions present. Can be present as cations oranions.

Anions:

Chloride ion (Cl-), hypochlorite ion (HClO-), Nitrates, nitrites, carbonates, Sulfates, Silicates, etc...

Cations:

Sodium (Na+) , Calcium and Magnesium (Ca++, Mg++, Iron (Fe++), Aluminum (Al+++), Heavy metals(Pb, Ni, Cr, Hg), etc...

1.2.2. Dissolved organic compounds

This class of contaminants can be found in nature or result from pollution or the water potabilityprocess itself for human consumption.

Natural origin

Include organic materials resulting from vegetal and animal decomposition. Vegetal decompositionis very common in open fountains (dams, reservoirs) where the water is captured for subsequenttreatment. Among other examples, we can mention tannins, lignins, phenols, humic acids (complexmixture of macromolecules having phenolic polymer structure). Also include proteins, enzymes(nucleases, for example), amino acids e their derivatives.

Unnatural origin

Includes organic compounds originated from human activities. It can be emphasized all kinds ofwater-soluble pesticides (fungicides, insecticides, herbicides), polyaromatic hydrocarbons (PAHs),polychlorinated biphenyls (PCBs), EDTA (chelating agent always present in soap, detergents andcosmetics formulations), citrates (in regions next to citric crops).

1.2.3. Particles and colloids

Particles can be rigid (sand, stones, earth) or deformable (vegetal wastes). It is characterized as acontaminant for being a protector shield for microorganisms against the action of UV rays anddisinfecting agents. They are also a vehicle for such microorganisms, since bacteria, for example,may adhere to the particles.

In instrumental analyses, the particles may also cause direct serious problems. In HPLC, for example,there is always a risk of column clogging and damage to the injection pump piston. Further, particlesimmobilized at the inlet of the column may become another stationary phase, thus unexpectedlyimpairing the column retention features and the separation itself.

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Colloids are stable suspensions of organic or inorganic particles with size ranging from 0,1 to 0,001mm. All particles have the same charge and signal. The resulting repulsion keeps the suspensionstable. They are difficult to remove by microporous filtration, but can be easily retained by reverseosmosis or ultrafiltration.

1.2.4. Microorganisms

Microorganisms – bacteria, fungi (mold and yeasts) and virus – are commonly present in drinkingwater distributed in a city. Although treatment companies process the water so as to removemicroorganisms hazardous to the human health, the supplied water is not sterile at all. The othermicroorganisms remaining after water drinkability process may impair the analyses.

1.2.5. Dissolved gases

We can find all gases existing in the atmosphere dissolved in water.

The most important gas dissolved in water is carbon dioxide (CO2). It is balanced with carbonicacid that, in turn, is balanced with the bicarbonate ion:

This balance generates an important ion, bicarbonate, which may interfere with the analyses andimpair the purification processes.

CO2 dissolution rate and its concentration in water depend on the temperature (the lower is thetemperature, the higher is the dissolution and absorption rate) and on the CO2 partial pressure inthe environment to which the water is exposed.

Further effects of other gases in the air:

• Oxygen: causes corrosion in tanks, pipings, etc.• Ammonia: contaminant resulting from agricultural fertilization.• SO2 – contained in automobiles escape gases and industrial emissions.

1.3. Monitoring and control of the purified water quality

As mentioned above, the contaminants contained in water may significantly interfere with analyticaldeterminations. Therefore, it is important to quantify them and be sure they comply with the qualityspecifications. Besides, this quantification is a measure of the efficiency of the water purificationprocess.

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This topic will basically consider the detection of inorganic (ions) and organic contaminants.

There is a wide range of inorganic contaminants and analyzing them individually would be extremelyhard and laborious. It is possible to determine the total ionic contamination total of an aqueoussolution using only one parameter: conductivity that is the specific conductance or its reciprocal,the resistivity (specific resistance).

Equally, there are several organic contaminants contained in the purified water. Here, also, onesingle parameter can be used to quantify them: Total Organic / Oxidizable Carbon (or TOC – TotalOxidizable Carbon).

Bacteria and other microorganisms are quantified using methods employing filtration in microporousmembranes and subsequent incubation using culture mediums or, more recently, chemiluminescence.The results are expressed in cfu/mL (colonies forming units per mL).

The amount of particles can be controlled by means of specific removal technologies (microfiltration,ultrafiltration and reverse osmosis). In water purification processes, this is enough and generallythere are no concerns on the quantification of particles using appropriate technologies and assuringthat all particles above a certain size are removed.

1.3.1. Monitoring of inorganic ionic contaminants – conductivity and resistivity

The conduction of electric current depends on the ions contained in water. The purer is the water,the lower is the concentration of such ions and, therefore, the lower is conductivity. For lowconductivity, the reciprocal applies, high resistivity, as they are different expressions for the samephenomena.

Therefore: C= 1/R or R= 1/C

Measurement units:Conductivity – microhm/cm or microSiemens/cmResistivity – Megohm.cm

The water conductivity at the purification theoretical limit, that is, when almost all ions have beenremoved and only H+ and OH- remains, is 0.055 microSiemens/cm or 18.2 Megohm.cm, at 25º C.This is called type I ultrapure water (ASTM).

Graph 1 shows that temperature is an important aspect when measuring conductivity. The lower isthe temperature, the higher is the resistivity (therefore, the lower is conductivity). This is because atlow temperatures, a lower mobility of ions in solution takes place and such mobility is inverselyproportional to the resistivity or directly proportional to conductivity.

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Graph 1. Resistivity and Conductivity Variation as a function of temperature

That is why water temperature should be known when comparing these measurements. Conductivitymeter and resistivity meters installed in water purification equipment generally have a temperatureoffset circuit for 25ºC, thus allowing a direct reading.

As water becomes purer, that is, as its conductivity decreases or resistivity increases, it is more proneand faster to aggregate contaminants from the environment, thus returning to its natural state. Forthat reason, it is crucial that ultrapure water is not stored and obtained right before usage.

Graph 2 shows the highly purified water tendency to reuptake contaminants, evidenced by a sharpdecrease in resistivity some minutes after purification. This decrease in resistivity is due to theatmospheric carbon dioxide contamination that is balanced with the bicarbonate ion, the presenceof which leads to an increase in conductivity (or decrease in resistivity).

Graph 2. Contamination of ultrapure water by atmospheric gases - CO2 (20º C)

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At 25ºC, the ultrapure water resistivity is18.2 MΩ.cm

Resistivity(Megohm.cm)

Temperature (degrees C)

Resistivity (M Ω .cm)

1.3.2. Monitoring of organic contaminants – TOC – total oxidizable carbon

There are currently two kinds of TOC analyzers. Analyzers using physicochemical oxidation andanalyzers using a technology patented by Anatel Corporation.

TOC determination by physicochemical methods first involve the removal of CO2 from the waterto be tested by purging with nitrogen. This leads to a problem: it is difficult to remove all the CO2.

The second step involves the addition of reagents (peroxides) and the action of a catalyst (UV orheat) to start the oxidation reaction. Once this oxidation is complete, the carbon dioxide producedis removed from the water sample by bubbling with nitrogen and collected by adsorption in acolumn. Next, the column is desorbed by increasing the temperature and carryover using a flow ofpure nitrogen. The presence of CO2 is detected by infrared spectrometry and concentrationdetermined by peak integration.

This method has several limitations when measuring TOC in ultrapure water:

– First, there is a risk of contamination during sampling and the method cannot be easily adaptedto on-line measurements.

– As mentioned above, it is difficult to remover the last traces of CO2 in the purging stage.– Volatile organic compounds can be also carried over by nitrogen and, therefore, are not accounted

in the total TOC.– The oxidation reagents used are kept in a normal room and, therefore, subject to CO2

contamination.– Organic materials that are adsorbed and uncontrollably released slowly and cumulatively

contaminated the instrument piping and the column.– Finally, the technique requires the use of a great amount of high purity nitrogen, what is extremely

expensive.

The second technique, developed by Anatel Corporation, is simpler and involves the organiccompounds oxidized by UV radiation. The UV radiation at a wavelength of 185 nm converts O2

into ozone (O3) that is a strong oxidizing agent. With the aid of UV radiation at 254 nm, ozone, inturn, will react with water to form free radicals hydroxyl that will then react with organic compounds,thus oxidizing them into carbon dioxide. The resulting decrease in resistivity (or an increase inconductivity) is then measured and correlated with the TOC reading.

The main advantage is that such a method allows quick e automatic on-line measurements, beingvery sensitive (< 1 ppb TOC), reproducible, does not use reagents and define all kinds of organiccompounds, inclusive the volatile ones.

However, this method is appropriate for waters having a resistivity above 3 Megohm.cm.

The TOC concentration is expressed as ppb – parts per billion.

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1.4. Quality specifications

A number of standardization authorities have quality specifications intended to cover specific activitiesto which they are dedicated. The ASTM specifications – American Society for Testing and Materials– are intended to cover analytical instrumentation and the electronics industry. The United StatesPharmacopeia (USP) specifications cover the production of drugs and ISO – InternationalOrganization for Standardization. Further, the NCCLS – National Committee for Clinical LaboratoryStandards, in liaison with CAP – College of American Patologists, specifies the kinds of water usedin clinical laboratories.

ASTM rule, reference D-1193-99, is the most appropriate for the specification of the kinds ofwater used in chemistry laboratories e, especially, instrumental analysis.

Historically, ASTM was the first entity to propose rules for laboratory water, dividing water intofour grades, according to the applications:

Type I - as known as ultrapure water or reagent grade water, used in critical laboratoryapplications, including the HPLC instrumental analyses.

Type II - for less demanding applications, such as glassware rinsing, qualitative analysis ororganic synthesis.

Type III - for general laboratory applications: preparation of culture mediums and finalglassware rinsing in non-critical applications.

Type IV - supply of reagent grade water producing systems.

Microbiologic Contamination – when bacteria level requires control, the type of grade should beprovided as follows:

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1.5. Water purification technologies for laboratories

In order to purify water to the levels specified in ASTM and other authorities, several technologiesare available:

• Distillation• Deionization• Reverse osmosis• Continuous Electrodeionization (EDI)• Ultrafiltration• Membrane microfiltration• Activated Charcoal• Ultraviolet Radiation

1.5.1. Distillation

Distillation is a classic water purification process that applies water change from the liquid to the gasstate, with liquid phase condensation. By using the distillation process, several contaminants areremoved, however it is still not possible to obtain type I ultrapure water.

Organic substances having a low boiling point are also converted into distilled water. Azeotropicmixtures are also formed and even high molecular weight compounds are carried over with thevapor. Additionally, when chlorine reacts with natural organic compounds at high temperatures,organochlorinated compounds are formed and also carried over by the vapor.

Silica is extracted from glass distillers, as well as other ions in metal distillers.

Further, the distillation process consumes a great amount of electricity and cooling water.

DISTILLATION

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Advantages

• Removes a great percentage of all types ofcontaminants

• Produces water with resistivity between 0.2and 1 Megohm.cm.

• Medium investment• Commonly known and easily operated

process

Disadvantages

• Not all contaminants are removed andmany of them are produced during theprocess.

• The water quality is not controlled.• High operation cost: electricity and water.• Regular and pretreatment maintenance are

essential to assure performance.

1.5.2. Deionization

The deionization process is performed using ion exchange porous globular resins having diametersbetween 0.3 and 0.8 mm. Such resins are manufactured with polymeric chains containing styreneand divinylbenzene cross-links in which charged chemical groups are bonded.

In anionic resins, that is, resins capturing anions, these groups contain quaternary ammonium. Sulfonicgroups are used in cationic resins – resins capturing cations.

At the beginning of a deionization process, when the resin is still unused, ions bonded to negativegroups of cationic resins are H+ protons and ions bonded to anionic resins are hydroxyls OH-.

During the process, cations contained in water (Na+, Ca++, etc.) will bind to the cationic resin edisplace the H+ proton, while anions (Cl-, NO3

-) will bind to the anionic resin and displace hydroxyls.

Deionization is a very efficient process to remove ions and even some ionized organic compounds.However, resins are an excellent support for bacterial growth. As feeding water is not sterile, theresult is that water bacteriological contamination after a deionization column can be 1000 timesgreater than the inlet water.

Figure 1. Scheme of the mixed bed deionization process

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DEIONIZATION

Resins can be regenerated in the laboratory or in contract companies that provided this service bychanging a worn-out column by a regenerated one. This can be a source of residual ioniccontamination as a result of the application of the inlet or environmental water to which these resinare exposed.

Single-use, therefore, non-regenerable, resins are the most indicated so as to avoid all such problems.

1.5.3. Reverse osmosis

Figure 2. Scheme of the osmosis and reverse osmosis principle

Supposed we have a U-shaped tube with both branches separated by an osmosis membrane. In theleft branch, the water molecules are dissolved (represented by dots). According to the laws ofthermodynamics, the molecules would diffuse from the left to the right until their concentrationsare equivalent. However, these molecules cannot cross the membrane, but the water molecules can.Thus, water will cross the membrane from the right toward the left branch to dilute the dissolved

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Advantages

• Effective removal of ions(Resistivity: 1 - 10 Megohm.cm)

• Simple installation.

• Low investment.

• Regenerable.

Disadvantages

• Does not remove particles, organic material ormicroorganisms. Regenerated resins can produceparticles, organic materials or promote the bacterialgrowth.

• Standard ion exchange: the resin origin is unknown.• High operation costs: regeneration/

transportation.• Water quality is variable; damaged globules.

Reverse OsmosisMembrane

(RO)

OsmoticPressure

Osmosis ReverseOsmosis

molecules. This process will continue until a pressure differential (osmotic pressure) is created betweenthe branches, thus balancing the difference in concentrations.

A reverse osmosis is exactly the opposite of this process: water containing ions or other contaminantsis pressurized against a reverse osmosis membrane; the pure water is obtained on the other side ofthe membrane. The pressure exerted should be higher than the osmotic pressure.

Reverse osmosis membranes typically rejected 90% of the monovalent, 95% of the bivalent e 99%of the polyvalent ions. 99% of the organic compounds having a molecular weight above 300, virusand bacteria are also rejected.

In order to obtain a continuous flow of water molecules on this membrane, contaminants have tobe regularly removed. This can be achieved by the so-called tangential flow (figure 3) that allowspart of the water flow to go across the membrane surface, thus promoting an authentic scanningand preventing the accumulation of contaminants.

On equipment, this setting is provided as helically wound membranes, thus forming a cartridgewhere permeate and refuse channels are perfectly separated and tight. This creates a great separationarea in a relatively small volume, thus allowing the construction of compact systems.

Reverse osmosis systems remove a reasonable percentage of all kinds of contaminants; however,since this is a percentile removal, it is not enough to reach even the type II water purity levelsrequired for most of the situations. The table below shows the advantages and disadvantages of thisprocess.

REVERSE OSMOSIS

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Figure 3. Scheme of the tangential flow in a reverse osmosis system

Advantages

• Removes a reasonable percentage of allkinds of water contaminants (ions, organicmatter, pyrogens, virus, bacteria, particlesand colloids).

• Low operation costs as a result of the lowconsumption of electricity.

• Minimum maintenance• Good control of operation parameters.

Disadvantages

• The contaminants are not sufficientlyremoved so as to comply with the type IIwater requirements.

• The Reverse Osmosis Membranes are subjectto long-term encrusting and clogging (if notadequately protected).

• Until recently, the consumption of water wassimilar to distillers.

ReverseOsmosis

Membrane

Permeated

Rejected

WaterFeeding

Electricity is employed in this process to operate the pressurization pump only; therefore, a verylow consumption if compared to the distillation process.

The osmosis membranes can be also incrusted and clogged as a result of salts precipitation, mainlycalcium carbonate, thus reducing the outflow and damaging the membrane structure. For this reason,controlling the incrusting ions is required so as to avoid this kind of problem.

1.5.4. Continuous electrodeionization

If we submerge two electrodes - anode and cathode – into a NaCl solution, for example, and applya potential difference between the two electrodes, the Na+ and Cl- ions will migrate towards thecathode and the anode respectively.

There are membranes – called ion exchange membranes – selectively permeable to cations or anionsand that allow such migration to be controlled. These membranes are made of ion exchange resinfragments included in a polyethylene matrix. Such fragments of cationic or anionic resins respectivelyallow cations or anions to pass.

If these membranes are introduced in the NaCl and electrodes system described, we are able tocontrol the migration of Na+ and Cl- ions as showed in the figure below.

Figure 4. Control of the migration of ions through ion exchange membranes

In our model, only the chloride ions can cross the anionic membrane. Conversely, only sodium ionscan cross the cationic membrane. The so-called electrodialysis system is created in a system whereboth membranes are alternated and the electrodes maintained (figure 5a).

Figure 5. Electrodialysis system

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Anionic Membrane

Anode Cathode

Cationic Membrane

CathodeAnode

AnodeCathode

ProductA - Anionic MembraneC - Cationic Membrane

In the next cell, continuing on the right, both the chloride and sodium ions can leave. We can clearlynotice a depletion of ions; electroneutrality is also maintained since for each chloride ion there isone sodium leaving the same cell.

Conversely, the ions are cumulating in the next cell, as no sodium or chloride can leave.

As a result, there is a system where the ions are concentrated in some cells (disposal flow) andpractically absent in others (product flow). This process can be used for purifying water; however, itis extremely slow once the ions would have to move from a cell to a membrane and towards theelectrode.

In order to improve the system’s performance, the product cells are filled with mixed bed ionexchange resins (anionic + cationic). This will allow an ionic transference from the cell centertowards the semipermeable membrane instead of moving at a low speed in water where collisionscaused by the Brownian movements would decrease their advance. Thus, the ions jump from oneresin active site to another towards the electrode having an opposite signal. This setting also allowsthe capture of weakly charged organic substances.

The resins are continuously regenerated as H+ and OH- are generated in the electric field created,thus composing microenvironments around the resins.

Nevertheless, the continuous electrodeionization purification process has to be fed with waterpreviously purified by reverse osmosis and having an attenuated concentration of salts.

Figure 6. Ion exchange resins filling the product cells

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Anode Cathode

A - Anionic MembraneC - Cationic Membrane

Product

CONTINUOUS ELECTRODEIONIZATION (EDI)

1.5.5. Ultrafiltration

Figure 7. Representation of the schematic section of a ultrafilter showing the ratio betweenthe active separation layer (1) and the support layer (100).

Ultrafilters are polymeric asymmetric membranes with a very fine active layer (1 micrometer thick)on the top and a thicker support layer (100 micrometers).

The ultrafiltration membranes (UF) operate under pressure. Under these conditions, small moleculeswill be able to cross the active layer, while the bigger molecules are retained. The cut-off is calledNominal Molecular Weight Limit – NMWL – and expressed in daltons. This value is used tocharacterize the UF membranes once the molecular weight is easier to found in literature than themolecules sizes.

The molecular weight is directly related to the molecule size, however other factors also have to beregarded: shape, stereochemistry and pH of the dispersion in which they are. This is essential forproteins.

The table below shows the advantages and disadvantages of UF.

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Advantages

• Effectively removes the dissolved organic material.• Requires minimum maintenance.• Requires no regeneration of the resins.• Low operation cost.• Excellent pretreatment for water ultrapurification

systems

Disadvantages

• Requires pre-purification

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Figure 8. Cross-section representation of a surface filter and microphotograph of theDurapore membrane (Photo: courtesy of Millipore Ind. e Com. Ltda.)

There are three main types of filters: depth, surface and membrane filters.

Depth filters are made of agglomerated fibers and retain contaminants throughout its thickness.Although having high capacity, their limit of detection is not very clear. Their efficiency is around95% and varies as a function of the outflow. Besides, they may release contaminants in the filtrate,generally fibers or retained material.

Surface filters are generally made of multiple non-fibrous material layers, capturing contaminantsmainly on their surface and having an intermediate retention and efficiency capacity (98%).

Screen or membrane filters retain contaminants on their surface by means of a sifting effect. Inspite of their low capacity, such filters retain 100% of the contaminants having a size above theirwell-defined cut-off limit. Typical screen filters are the membrane filters which integrity can beverified by means of specific tests, for example, bubble point or diffusion.

ULTRAFILTRATION - UF

Advantages

• Effective removal (>99%) of all organicmolecules having a molecular weight abovethe NMWL. Very effective removal ofpyrogens and virus, as well as particles.

• No risk of incrustation.• Low consumption of water and electricity.• Low maintenance; well documented/

accepted procedures

Disadvantages

• Almost no removal of ions, gases and lowmolecular weight organic matter (UFmembranes provided with a narrower meshhave a cut-off of 1.000 dalton)

SurfaceFilter

As noticed, these membranes are very efficient when removing high molecular weight organiccompounds.

1.5.6. Membrane microfiltration

Durapore®MembraneMicrophotograph

The main advantage of the membrane filters is the total (100%) removal of all contaminants havinga size greater than their pore diameter. Membranes with a pore diameter of 0.22 µm have been usedin the pharmaceutical industry for several years in the sterilizing filtration of solutions.

1.5.7. Activated charcoal

The activated charcoal is mainly used due to capacity to adsorb organic materials as a result of itswide surface area up to 1000 m2/g.

Another function is to reduce oxidizers, such as free chlorine, contained in the water and that couldaffect reverse osmosis membranes or ion exchange resins. Therefore, this is a technology mainlyaimed at the pretreatment and protection of other steps.

ACTIVATED CHARCOAL

1.5.8. Ultraviolet radiation (UV)

UV radiations have wavelengths between 100 and 400 nm, divided into four fields: ultrashort, short(UV-C), medium (UV-B) and long (UV-A) waves. These radiations can be easily produced by lowmercury vapor pressure lamps emitting radiations at two wavelengths: 185 and 254 nm.

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MEMBRANE MICROFILTRATION

Advantages

• Total (100%) removal of all contaminants (particles,bacteria) greater than the pore size. Integrity testavailable.

• Sterilizing filtration (0.22 mm membrane).• Minimum maintenance: simply replace as required.• High outflows are obtained at low pressures.• Outflow independent efficiency.

Disadvantages

• Minimum effect on othercontaminants.

• Surface retention: may be subjectto clogging or obstruction.

Advantages

• Effective removal of a wide range oforganic substances (even those with lowmolecular weight) by means of non-specificbonds (Van der Waals force).

• Large capacity as a result of the greatsurface area.

Disadvantages

• Very weak effect on other contaminants(except for some particles removed bydepth filtration).

• When all sites are busy, balance is reachedand the organic compounds released.

• Bacteria may develop after some time.• Efficiency depends on the outflow.

Radiations with a wavelength between 200 and 300 nm destroy microorganisms by breaking theDNA chain. This is more intense at 260 nm. Therefore, the wavelength of 254 nm has an efficiencythat is very close (80%) to that regarded as optimum for this purpose.

However, a very ascertained design is required so that such efficiency is really reached, mainly takingthe UV radiation penetration capacity (in the order of 1 cm) into consideration. It is necessary toprovide a sufficient outflow to allow this penetration, respecting the dwelling time of water in theexposure chamber.

Another very critical application of the UV radiation is the reduction of the Total Oxidizable Carbon(TOC) levels of the purified water.

In fact, the UV radiation does not directly destroy organic compounds, but generates ozone thatwill oxidize the substances contained in water.

As it can be seen in the figure below, on the left, two different wavelengths are necessary to generatethe free radicals hydroxyl (OH·) that will subsequently oxidize organic compounds.

Figure 9. On the left: sequence of reactions to form the free radical hydroxyl. On theright: oxidation reactions of organic compounds. Example: methanol.

Both mechanisms described in the literature (Norrish et al., 1965 and Banford et al. 1967) requirethe action of the wavelengths 185 and 254 nm and the presence of oxygen in the water. Bothmechanisms have the same stoichiometry and result in the formation of the same number of hydroxylradicals.

On the right side of the figure, an example of oxidation of an organic compound with no charge(methanol) is showed. This is one of the simplest organic molecules, having only one carbon atom.Under the action of the free radicals hydroxyl, this molecule is successively oxidized in an increasingoxidation scale: starting with the alcohol, and then aldehyde, acid and finally carbon dioxide andwater.

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As previously discussed, carbon dioxide and water are balanced with carbonic acid that, in turn, isbalanced with the bicarbonate and H+ ions. Therefore, the ion exchange resins remove the carbon.

Note that by converting organic carbon into an ion that is easy to remove by deionization, it waspossible to reduce the purified water TOC level.

UV RADIATION (185 + 254 nm)

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Advantages

• Conversion of traces of organiccontaminants into charged specimens and,at the end, into CO2 (185 + 254)

• Limited destruction of microorganisms andvirus (254).

• Low electricity consumption.• Easy operation.

Disadvantages

• Polishing technique only: can be impairedif the concentration of organic matter inthe feeding water is very high.

• Organic compounds are converted and notremoved.

• Limited effect on other contaminants.• The design has to be appropriate to assure

efficiency.

2. PURIFIED WATER SPECIFICATIONS

2.1. Purpose

This specification concerns appropriate requirements for water to be used in chemical analysismethods and physical tests. It has been based on ASTM D1193-99 specifications, as amended. Fourcategories were specified:

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Measurement of pH in type I, II and III reagent grade water is not included in the specifications,since water at these purification grades contain no components in amounts sufficient to lead to apH change.

Methods for the preparation of several types of water determine the impurity limits and shouldcomply with the following specifications:

2.1.1. Type I reagent grade water should be prepared by an appropriate pre-purification process,followed by polishing using a ultrapurification system comprising a mixed bed ion exchangeresin and a 0.22 µm final membrane. The preparation should be always carried out at thetime of use. It is recommended, but not compulsory, to provide the ultrapurification system(final polishing) with a UV lamp with emission at 185 and 254 nm so as to maximally reducethe TOC levels. Additionally, it is also recommended, but not compulsory, to provide theultrapurification equipment (final polishing) with a TOC on-line meter for constant monitoringof organic contaminants. Supply (pretreatment) of the water final step should have aconductivity of at least 10 µS/ cm at 25ºC.

2.1.2. Type II reagent grade water should be prepared by a reverse osmosis process combinedwith electrodeionization, distillation or ion exchange having a conductivity below 1.0 µS/cmat 25ºC. Ion exchange, or reverse osmosis and organic adsorption, can be required andassociated with distillation if the purity is not reached by a single distillation step.

2.1.3. Type III reagent grade water should be prepared by reverse osmosis followed by continuouselectrodeionization, distillation, ion exchange or a combination thereof, followed by a polisherwith a 0.45 µm membrane filter. The use of this filter is required when microbiologiccontamination is required.

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2.1.4. Type IV reagent grade water should be prepared by reverse osmosis, distillation, ion exchange,reverse osmosis followed by continuous electrodeionization, electrolysis or a combinationthereof.

The several types above can be chosen by method or investigation.

This specification is not intended to cover safety concerns associated with the application. It isuser’s responsibility to establish the appropriate safety standard and healthy practices and define theapplicability of the priority use regulation limits.

2.2. Monitoring of the quality of several types of water

2.2.1. Conductivity and resistivity: For measuring the conductivity or resistivity, the conductivityor resistivity meter of the water purification equipment should be used provided that they are dulycalibrated. If a distiller having no meter coupled is used, measurement should be carried out usinga duly calibrated portable conductivity meter.

2.2.2. TOC – Total oxidizable (organic) carbon – Although the TOC values for all types ofwater are specified, only Type I grade water is critical, since this water will be in contact with theanalytical equipment and high TOC levels may lead to impaired results. Therefore, this water graderequires a systematic monitoring of the TOC levels. We recommend that water ultrapurificationequipment (final polishers) is provided with a duly calibrated TOC on-line meter. If this is notpossible, measurement should be made on-line using equipment applying the photooxidationtechnique for resistivities above 3 megohm.cm.

2.2.3. Measurements frequency:2.2.3.1. Resistivity and conductivity:

2.2.3.1.1. Daily for type I, II, III or IV water.2.2.3.1.2. Upon removing water for type I water.

2.2.3.2. TOC – total oxidizable carbon:2.2.3.2.1. When the equipment is not provided with a meter, the TOC levels

should be measured at least once every fifteen days.2.2.3.2.2. When the ultrapurification equipment is provided with a TOC meter,

measurement should be made upon the water removal.

2.3. Calibration and qualification

2.3.1. Each water purification system should have their meters calibrated and qualified according tothe Installation, Operation and Performance aspects.

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2.4. Storage recommendations

2.4.1. Type I grade water – Should not stored due to fast degradation. It should be prepared atthe moment of use.

2.4.2. Type II, III and IV grade water – can be stored in a reservoir protected against externalcontaminations. It is strongly recommended to store such purified water for a period not exceedingtwenty-four hours as of its preparation.

2.5. Water purification equipment maintenance

All the manufacturer’s indications should to be followed mainly concerning the change of occasionallyrequired consumable and cleaning material. It is recommended to establish a preventive maintenanceprogram for each equipment and keep records for consumable material replacements andmaintenances carried out.

2.6. Control forms

Attachments 1 and 2 show control form templates for recording the resistivity or conductivitymeasurements , as well as TOC.

2.7. Reference documents

D1193 Standard Specifications for Reagent WaterD1125 Test Methods for Electrical Conductivity and Resistivity of WaterD1129 Termninology Relating to WaterD1293 Test Methods for pH of WaterD4453 Practice for Handling of Ultra-Pure Water SamplesD4779 Test Method for Total, Organic, and Inorganic Carbon in High Purity Water by Ultraviolet

(UV) or Persulfate Oxidation, or Both, and Infrared DetectionD5391 Test Method for Electrical Conductivity and Resistivity of a Flowing High Purity Water

SampleD5542 Test Method for Trace Anions in High Purity Water by Ion ChromatographyD5997 Test Method for On-Line Monitoring of Total Carbon, Inorganic Carbon in Water by

Ultraviolet, Persulfate Oxidation and Membrane Conductivity Detection

Attachment 1. Control form template for water ultrapurification equipment (finalpolishing) provided with a TOC meter.

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Attachment 2. Control form template for water ultrapurification equipment (finalpolishing) not provided with a TOC meter.

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Attachment 3. Control form template for water purification equipment for obtainingtype II, III or IV grade water.

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3. REFERENCES

American Society for Testing and Materials D1193-99, “Standard Specification for Reagent Water”(1999).

Millipore, Internet site: http//millipore.com/H2O.

Millipore Indústria e Comércio Ltda. – Seminários de Purificação de Água para Laboratórios.

Norrish et al., Proc.Roy.Soc.Ser. A. 288: 316 (1965).

Banford et al., Photochemistry and Reaction Kinetics. P.G. Ashmore et al., Ed. Cambridge (1967).


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TECHNICAL STAFF

Volume II – Module 3 – Analytical Instrumentation

Editors and Cooperators:• Adauto da Silva – VARIAN• Celso Ricardo Camargo - SINC BRAZIL• Demian Rocha Ifa – SINC BRAZIL• Ivan Jonaitis – AGILENTTECHNOLOGIES• José Apareido Soares - VARIAN• Luiz Rinalo Bizaio - VARIAN• Renato Guvêa - AGILENT TECHNLOGIES• Renato eres - FLOWSERVICE• Ricardo Lira - FLOWSERVICE• Robson Sanches Bizi - VARIAN

Coordination:• Cláudia Franklin de Oliveira – ANVISA• Itapuan Abimael da Silva - ANVISA• Karen de Aquino Noffs Brisolla - ANVISA• Karla de Araújo Ferreira - ANVISA• Marcelo Cláudio Pereira - ANVISA• Max Weber Marques Pereira - ANVISA• Renato Almeida Lopes - ANVISA

SUMMARY

1. INTRODUCTION... ...................................................................................................................... 72. ULTRA VIOLET-VISIBLE (UV-VIS) SPECTROPHOTOMETRY. ............................ 8

2.1. Introduction ................................................................................................................................ 82.2. Technique principle ................................................................................................................. 10

2.2.1. Light spectrum ............................................................................................................ 102.2.2. Absorption processes ................................................................................................. 102.2.3. UV - Vis absorption spectrum .................................................................................. 122.2.4. Effect of solvents ....................................................................................................... 14

2.3. Basic description of the system ............................................................................................. 152.3.1. Sources of radiation ................................................................................................... 152.3.2. Monochromator (optical) ........................................................................................... 16

2.3.2.1. Single-beam ................................................................................................... 172.3.2.2. Diodes arrangement .................................................................................... 182.3.2.3. Double-beam ................................................................................................ 18

2.3.3. Sample compartments ................................................................................................ 192.3.3.1. Optical fiber coupler .................................................................................... 192.3.3.2. Sipper ............................................................................................................. 192.3.3.3. Support for solid samples ........................................................................... 192.3.3.4. Reflectance accessories ................................................................................ 20

2.3.4. Data acquisition ........................................................................................................... 202.4. Minimum installation and operation requirements ............................................................. 202.5. Basic precautions ..................................................................................................................... 21

3. GAS CHROMATOGRAPHY (GC). ..................................................................................... 223.1. Introduction .............................................................................................................................. 223.2. Technique principle ................................................................................................................. 223.3. Basic description of the system ............................................................................................. 23

3.3.1. Gases used.................................................................................................................... 243.3.2. Flow and pressure controllers ................................................................................... 253.3.3. Injectors ........................................................................................................................ 25

3.3.3.1. Packed injectors ............................................................................................ 253.3.3.2. Capillary injectors ......................................................................................... 26

3.3.4. Columns ....................................................................................................................... 273.3.4.1. Columns oven ............................................................................................... 28

3.3.5. Detectors ...................................................................................................................... 283.3.5.1. Flame ionization detector (FID) ................................................................ 283.3.5.2. Nitrogen and phosphorus (NPD) or specific thermoionic detectors ......

(TSD) ............................................................................................................ 293.3.5.3. Electrons capture detector – ECD ............................................................ 30

3.3.6. Data acquisition and processing ............................................................................... 31

3.4. Minimum installation and operation requirements ............................................................. 313.5. Basic precautions ..................................................................................................................... 32

4. LIQUID CHROMATOGRAPHY (LC). .............................................................................. 334.1. Introduction .............................................................................................................................. 334.2. Technique principle ................................................................................................................. 33

4.2.1. Separation modes: ....................................................................................................... 344.2.1.1. Normal phase chromatography ................................................................... 344.2.1.2. Reverse phase chromatography ................................................................... 35

4.3. Basic description of the system ............................................................................................. 364.3.1. Mobile phase ................................................................................................................ 374.3.2. Solvents pumping system........................................................................................... 384.3.3. Sample introduction – injector .................................................................................. 38

4.3.3.1. Manual injector .............................................................................................. 384.3.3.2. Automatic injector ......................................................................................... 39

4.3.4. Columns ....................................................................................................................... 394.3.4.1. Silica-based stationary phases ...................................................................... 394.3.4.2. Chemically modified phases......................................................................... 40

4.3.5. Detectors ...................................................................................................................... 404.3.5.1. UV-VIS detectors .......................................................................................... 414.3.5.2. Fluorescence detector ................................................................................... 434.3.5.3. Electrochemical detectors ............................................................................ 43

4.3.6. Data acquisition and processing ............................................................................... 444.3.6.1. Area percentages ........................................................................................... 454.3.6.2. Area normalization ........................................................................................ 454.3.6.3. External standard .......................................................................................... 464.3.6.4. Internal standard............................................................................................ 46

4.4. Minimum installation and operation requirements ............................................................. 474.4.1. Bench requirements .................................................................................................... 474.4.2. Electric network .......................................................................................................... 474.4.3. Environmental conditions ......................................................................................... 48

4.5. Basic precautions ..................................................................................................................... 48

5. CHROMATOGRAPHY SYSTEMS CONNECTED TO MASS DETECTORS .. 505.1. Introduction .............................................................................................................................. 505.2. Technique principle ................................................................................................................. 505.3. Basic description of the system ............................................................................................. 51

5.3.1. Ionization source ......................................................................................................... 515.3.1.1. Electrons impact (EI) ................................................................................... 515.3.1.2. Chemical ionization (CI) .............................................................................. 515.3.1.3. Electrospray (ES)........................................................................................... 52

5.3.2. Mass analyzers ............................................................................................................. 545.3.2.1. Quadrupole mass analyzers.......................................................................... 545.3.2.2. “Ion trap” quadrupole mass analyzers ....................................................... 55

5.3.2.3. Tandem mass spectrometry. ......................................................................... 575.3.3. Detectors ...................................................................................................................... 58

5.3.3.1. Electrons multipliers ..................................................................................... 585.3.3.2. Microchannel plates ...................................................................................... 595.3.3.3. Photomultiplier .............................................................................................. 60

5.3.4. Data acquisition and processing ............................................................................... 605.4. Minimum installation and operation requirements ............................................................. 61

5.4.1. Bench requirements .................................................................................................... 615.4.2. Electric network .......................................................................................................... 615.4.3. Environmental conditions ......................................................................................... 62

5.5. Basic precautions ..................................................................................................................... 625.5.1. Vacuum system ............................................................................................................ 625.5.2. Ions source ................................................................................................................... 635.5.3. Nitrogen system .......................................................................................................... 635.5.4. Basic precautions training .......................................................................................... 635.5.5. Autotune files .............................................................................................................. 63

6. ANALYTICAL INSTRUMENTS PERFORMANCE CHECKING.. ....................... 646.1. Introduction .............................................................................................................................. 64

6.1.1. DQ – Design Qualification ....................................................................................... 656.1.2. IQ –Installation Qualification ................................................................................... 656.1.3. OQ –Operation Qualification ................................................................................... 656.1.4. PQ –Performance Qualification ............................................................................... 66

6.2. Ultra violet – visible (UV-VIS) spectrophotometry ............................................................ 666.2.1. Preventive maintenance ............................................................................................. 666.2.2. Operation Qualification (OQ) .................................................................................. 676.2.3. Performance Qualification (PQ) ............................................................................... 67

6.2.3.1. European pharmacopoeia ............................................................................ 676.2.3.2. United States Pharmacopoeia ...................................................................... 68

6.3. Gas chromatography – GC .................................................................................................... 686.3.1. GC preventive maintenance ...................................................................................... 686.3.2. Operation qualification .............................................................................................. 68

6.3.2.1. Flows control ................................................................................................. 686.3.2.2. Temperatures control .................................................................................... 696.3.2.3. Detector(s) signal precision.......................................................................... 696.3.2.4. Automatic sampler precision ....................................................................... 706.3.2.5. Calculations by the data system ................................................................... 70

6.4. Liquid chromatography - HPLC............................................................................................ 706.4.1. HPLC Preventive maintenance ................................................................................. 706.4.2. Operation qualification .............................................................................................. 71

6.4.2.1. Detector qualification ................................................................................... 726.4.2.2. Pump qualification ........................................................................................ 726.4.2.3. Columns oven qualification ......................................................................... 736.4.2.4. Automatic sampler qualification.................................................................. 73

6.4.2.5. Data system .................................................................................................... 736.5. Chromatography systems connected to mass detectors ..................................................... 73

6.5.1. Mass detector systems preventive maintenance...................................................... 736.5.1.1. Vacuum systems ............................................................................................ 736.5.1.2. Mass detector ................................................................................................. 746.5.1.3. Chromatographer .......................................................................................... 74

6.5.2. Operation qualification .............................................................................................. 746.5.2.1. Injector precision ........................................................................................... 756.5.2.2. Injector linearity ............................................................................................. 756.5.2.3. Carry-over ....................................................................................................... 756.5.2.4. Detector linearity ........................................................................................... 756.5.2.5. Mass accuracy ................................................................................................ 756.5.2.6. Sensitivity ........................................................................................................ 75

7. REFERENCES. ................................................................................................................................. 76

1. INTRODUCTION

The use of analytical instrumentation techniques in laboratories means a requirement not only tocharacterize a substance or assure the quality of a product, but also to increase the analysesproductivity; therefore, there is an increasing number of high automation grade instruments thatcan be remotely operated. However, we have to be aware that for a satisfactory use and performanceof such instruments, some basic rules are to be regarded, such as: the usage purpose, adequateinstallation, performance evidences, trained personnel to operate, compliance with the manufacturer’sinstructions concerning basic precautions to be considered by the operator and routines for replacingconsumable materials, personnel with technique reliable knowledge for an occasional implementationof an analytical method, preventive maintenance and performance check programs compatiblewith the routine used, parameters evaluation to guarantee a continuous quality assurance of resultsand, finally, a detailed planning always including evidences of the current activities. This chapterencompasses the main analytical instrumentation techniques used in a bioequivalence laboratory sothat the professional in this field can start his/her studies and use this material as a reference.Another purpose was to discuss the performance checking of each instrument and validation conceptsapplied to analytical instrumentation.

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Volume II / Module 3 - Analytical Instrumentation

2. ULTRA VIOLET-VISIBLE (UV-VIS) SPECTROPHOTOMETRY

2.1. Introduction

The Ultraviolet – Visible spectrophotometry is an analytical technique based on the property ofseveral ionic or molecular specimens to absorb ultraviolet and visible radiations in solution. Radiationsat these regions involve photons with sufficient energy to cause the transition of valence electrons,as a function of disturbances that start occurring.

The disturbance caused by electric and magnetic fields travels through space, thus creating theexpression electromagnetic radiation. We should have in mind that the electric and magnetic fieldsare perpendicular fields between themselves and that the propagation values where the fields’maximums and minimums are always coincident, that is, the fields are in phase, such as in a wave.We can see in Figure 1 that while propagating to the right at a velocity V, any point fixed on the X-axis successively starts to have wave peaks and troughs. Then, the wavelength (l) is defined as thedistance traveled by the wave so that both maximums reach a fixed observation point. The wavelengthis usually expressed in nanometers (nm).

Figure 1. Wavelength

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The Ultraviolet - Visible (UV-Vis) radiations constitute a relatively small portion of the electromagneticspectrum including other forms of radiations as radio waves, according to figure 2. The limits ofsuch regions are determined by the practical limits of experimental methods for the production anddetection of radiations.

The spectral regions differentiation has an additional meaning for the chemist, once physicalinteractions follow different mechanisms and provide different kinds of information. When a radiationarrives a semitransparent substance, the radiation is only partially transmitted. The remaining radiationis reflected or absorbed at several angles (figure 3), depending on the substance and the radiationwavelength.

The type and amount of absorbed radiation is the most important for analytical purposes.Unfortunately, there is no direct method to determine the absorbed radiation; however, thisinformation can be indirectly obtained by measuring the transmitted radiation.

Figure 2. Electromagnetic spectrum

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AbsorbedRadiation

IncidentRadiation

ReflectedRadiation

TransmittedRadiation

Figure 3. Absorbed, transmitted and reflected radiation

If the intensity of a transmitted light is graphically represented as a function of the wavelength, anabsorption spectrum of the substance is obtained. This is the radiation selective absorption thatprovides the base for the quantitative and qualitative analysis by molecular absorptionspectrophotometer.

2.2. Technique principle

2.2.1. Light spectrum

The visible light is a region of the electromagnetic spectrum, the radiations of which are able tosensitize the retina. The visible radiation comprises the radiations with wavelengths located between400 and 700 nm, although such limits are not fixed as the perception capacity varies according tothe observer. Within this wavelengths range, subgroups can be separated according to the colorproduced in a small spectrum range, as the table below.

Table 1. Wavelengths ranges in the visible region

2.2.2. Absorption processes

Considering that light is a form of energy, the absorption of light photon by a molecule results in anenergy increase, ∆E, of the energy contained in the molecule. The amount of increase is exactlyequal to the photon energy, that is ∆E = hn. The absorption process is schematically represented inthe energy levels diagram simplified in figure 4.

Figure 4. Energy diagram

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If the molecule is initially in a normal or fundamental state, E0, before the interaction, the absorptionprocess increases the energy contained in a higher level or in an excited state, E1. The energy exchangeprocess by the absorbed light is not attenuated, by occurs as multiple energy units called quantum.The quantum (hen) is characteristic for each specimen absorbed. To be absorbed by a molecule, theenergy photon should precisely correspond to the difference, ?E, between the two states of energy.

The total energetic potential total of a molecule, except the nucleus energy, can be considered as thesum of the electronic, vibrational and rotational energies. The electronic energies are associatedwith the transitions of electrons inside the atom or molecule. This sum of energies is represented infigure 5 as a change in orbitals.

Figure 5. Electronic energy

The vibrational and rotational energies are associated with molecular vibrations and rotations. Infigure 6 a simple diatomic molecule is showed following a compression and elongation movement(vibration). A molecular rotation is exemplified in figure 7.

Figure 6. Vibrational energy

Figure 7. Rotational energy

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The difference between the rotational energy states is relatively small, much smaller than thedifferences between the electronic energy states. The difference between the vibrational energystates is intermediate if compared to previous states; then:

• Absorptions associated with transitions among rotational energy levels are generally found inthe low energy or high wavelength region of the electromagnetic spectrum, that is, the remoteinfrared region.

• Absorptions associated with transitions among electronic energy levels are found in the highenergy or low wavelength region, that is, the ultraviolet - visible region of electromagneticspectrum.

• Absorptions associated with transitions among vibrational energy levels are found between thetwo levels mentioned above, in the infrared region.

The several kinds of energy transitions are not independent, but interconnected. Rotational energystates are superposed on the vibrational states, and both are superposed on the electronic states.

2.2.3. UV - Vis absorption spectrum

The presence of unsaturations or multiple bonds are widely known as a characteristic process ofultraviolet absorption while the saturated compound is transparent.

According to the theory of molecular orbitals, the electrons involved in single bonds, such as the C– H bond, are called sigma electrons (σ). Double bonds, such as C = O, involve pi electrons ( π ).In the close infrared region, transitions involving pi electrons allow a better observation of theabsorption bands. Electrons not bonded to the molecule, contained in atoms such as oxygen ornitrogen, are called n electrons, and the interactions between pi and n electrons are responsible fora great number of characteristic ultraviolet absorption bands.

A chromophore is a group that, when having a saturated hydrocarbon introduced, produces acompound having and absorption band between 180 and 1000 nm. For example, n-octane is asaturated hydrocarbon transparent in this region; however, if a nitrile group is introduced in theoctyl radical, the octyl-nitrile compound is produced. The nitrile group is classified as a chromophoreand would show light absorption signs in the ultraviolet range.

Table 2 shows a small group of chromophores simple with their maximum wavelengths and molarabsorptivity. The molar absorptivity is the measure of the absorption band intensity. As it can beseen, the absorption intensity of several chromophores notably varies from one group to another.However, all members of a class usually have equal intensity absorption bands and occur in arelatively narrow spectral range. For example, data showed for acetic acid are typical of saturatedcarboxylic acids resulting from formic acid to stearic acid, C1 to C18, all of them having absorptionbands in the region of 204 to 210 nm and molar absortivities from 40 to 75.

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Table 2. Chromophores Group

If one or more chromophores occur in a molecule, their relative positions determine the effectproduced in the spectrum. 1-hexane has a molar absorptivity of 10000 at 180 nm and 1,5-hexadienehas a molar absorptivity of 20000 at the same wavelength. 2,4-hexadiene, with band in anotherregion, has a molar absorptivity of 25500 at 227 nm.

The position and the intensity of the absorption band of 1,5-hexadiene are approximately what wecan expect from two individual propylene molecules. Obviously, the two chromophore groups arewidely spaced and do not interact. However, when both chromophores are conjugated as in 2,4-hexadiene, the effect is not the expected from two individual molecules.

Similar observations of a great number of compounds have construed the basic argumentation forsome general rules:

• When two chromophores in a same molecule are separated by more than one carbon atom, theabsorption spectrum is a simple sum of the spectrum of each chromophore.

• When two chromophores in a same molecule are adjacent, the maximum absorption is observedat high wavelengths and the intensity is increased.

• When two chromophores in a same molecule are bonded to a same carbon atom, the result isintermediate between those above.

In the ultraviolet-visible spectrophotometry, the most interesting compounds are usually those havingmore than one chromophore, especially when the chromophores are conjugated (conjugated systems).

Compounds with at least two conjugated chromophores absorb within the visible range. Saturatedcarbohydrates, fats, oils, simple ethers, acids, most of the carbohydrates and proteins do not absorblight in the visible range, since their structures have no chromophores. Molecules with the samechromophore and similar electronic structure have similar absorption spectra.

The presence of several neighbor chromophores groups changes the molecular absorption spectrumcharacter. The possible resultants can be divided into 4 groups:

• Bathochromic Effect. Displacement of the absorption bands towards the red range of thespectrum, thus intensifying the color.

• Hypsochromic Effect. Displacement of the absorption bands towards the violet range of thespectrum (of a lower wavelength).

• Hyperchromic Effect. Increase of the absorption bands intensity.• Hypochromic Effect. Decrease of the absorption bands intensity.

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The auxochromes are groups that, when introduced in a system as a chromophore, increase thewavelength of the maximum absorption band, the so-called bathochromic effect. The auxochromeshave no own absorption band at low wavelengths. For example, the hydroxyl group is an auxochrome.Alcohols are transparent and used as solvents at low wavelengths, however, the introduction of ahydroxyl group in a system having a chromophore will lead to a bathochromic effect. The typicalauxochromes are the amine groups and their substituted derivatives, halogens, alkaline group andothers.

2.2.4. Effect of solvents

The spectrophotometric analyses in the UV region are generally performed in solutions. Lowconcentrations of the interest analyte are usually employed; high purity grade solvents have to beused since impurities may impair the results due to its high concentration in relation to the analyte.Further, solvents for UV spectrophotometry should have a high optical stability grade; therefore, itis not recommended to use commercial purity grade solvents that will hardly comply with theabovementioned requirements.

Saturated carbohydrates, such as n-hexane, are the most common solvents in UV spectrophotometryand should be specifically prepared for this purpose. Their electronic absorptions caused by theelectrons transitions occur out of the analyzed range. Solvents with heteroatoms, for example, H3-COH-, are also used. Due to their electronic transitions, such solvents have weak absorption bands.This issue should be also focused based on the solvent degree of polarity.

The absorption bands of several substances are clearer and may have fine structures whenmeasurement is made in low bipolar moment solvents. The solvent interactions – solutes are strongerwhen strong bipolar forces are involved.

Figure 8 shows two spectra obtained using phenol in iso-octane and ethanol. One can observe theinfluence of the organic solvent on the determination of substances.

Figure 8. Phenol in iso-octane (____) and ethanol (———)

A difference in pH may also lead to fluctuations in case of this wavelength displacement.

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2.3. Basic description of the system

The system, figure 9, including a UV-VIS spectrophotometer, comprises:

1. Light source2. Monochromator3. Sample compartment4. Detector5. Reading system

Figure 9. UV-Vis spectrophotometer

2.3.1. Sources of radiation

The UV-Vis spectrophotometry requires sources that can produce continuous radiation in the spectralregions of interest. The radiation energy sources consist in material that can be excited by electricheating or high voltage discharge. When returning to energetic levels, the excited materials emitphotons having energies that correspond to the differences between the energies from excitedstates and energies from fundamental states. Some materials have so numerous and close energeticlevels that the emitted frequencies form a relatively wide range of continuous radiation.

The sources of radiation used in the production of UV-Vis spectrophotometry equipment shouldmeet some requirements:

• The source should provide a beam with sufficient radiating power to allow the detection byappropriate means.

• The source should generate continuous radiation, that is, comprising all wavelengths within thespectral region the equipment will operate.

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Input Slot

Dispersion Element

Output Slot

• The source should be stable, that is, the light beam power generated should remain constantduring the determinations. In single-beam equipment, this condition is critical so that theabsorbance measurements can be reproduced. In double-beam equipment, they are simultaneouslymeasured; therefore, high stability of the source is not so critical.

The most appropriate source for obtaining continuous radiation in the visible region is the glassbulb tungsten incandescent lamp. The filament operates at 2600-3000K, supplying continuousradiation from 350 to 2500 nm. The energy emitted in the visible region approximately varies withthe fourth potential of the operation voltage; therefore, the source requires a strict voltage controlto generate radiation or electronic voltage regulators.

The most used ultraviolet radiation sources are the hydrogen or deuterium discharge lamps withquartz window. When at low pressure and subject to electric discharge, hydrogen produces acontinuous beam in the ultraviolet region. The deuterium lamp produces a continuous spectrumfrom 180 to 380 nm.

When operating, the high frequency xenon lamps produce radiation pulses covering the spectrumfrom 180 to 1100 nm.

2.3.2. Monochromator (optical)

Monochromators are devices based on diffraction networks that disperse complex radiation in theircomponent wavelengths and then isolate the desired spectral range. Monochromators allow the freeisolation of spectrally pure and very narrow wavelength ranges along the close ultraviolet, visibleand infrared regions.

The photometric performance of a spectrophotometer is directly related to the monochromatorquality (optical system).

The monochromator setting should provide high resolution of the analyzed sample wavelengthand, at the same time, a greater amount of transmitted light and a low amount of spurious light(amount of light that was not duly isolated by the optical system). The optical system setting has toallow:

•· High transference efficiency between the source of light and the monochromator.• Adequate monochromator output slot to avoid excessive light loss.• Greater focusing of the light source beam on the sample.

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Figure 10 below shows a monochromator normally used in the manufacture of UV-Visspectrophotometers.

Figure 10. “Czerny Turner” Monochromator

There are additionally other usual settings, such as: Littrow, Ebert and Concave Grating. Thesemonochromators settings can be seen in the following types of UV-VIS spectrophotometers:

• Single-beam UV-VIS Spectrophotometers• Diodes Arrangement UV-VIS Spectrophotometers• Double-beam UV-VIS Spectrophotometers

2.3.2.1. Single-beam

In this type of spectrophotometer, the monochromator’s light beam dispersion is totally focused onthe sample compartment and, as a result, the unabsorbed light is directed to the detection systemcomprising a detector; it is usually of the photodiode type.

The manufacture of single-beam devices requires high quality stable precision components. Thedirect reading spectrophotometers operate with moderate precision around ± 1% transmittance;these are relatively cheap and easy-and-fast-to-operate devices with easy maintenance. The single-beam device requires the incorporation of zero circuit to measure transmittance in order to improvethe work accuracy.

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2.3.2.2. Diodes arrangement

This kind of spectrophotometer has a simple monochromator configuration, figure 11, using asingle diffraction grid and a detection system comprising several photodiodes; a polychromatic lightbeam initially reaches the sample compartment and right after the transmitted light is directed to themonochromator; after dispersion, the light reaches the detection system.

Figure 11. Polychromatic monochromator

2.3.2.3. Double-beam

The double-beam spectrophotometers separate the original radiation in space (by means of a mirror)or in time (by means of a rotary sectorial mirror). This kind of spectrophotometer is typically usedin analyses requiring greater analytical precision. The light beam dispersed on the monochromator(figure 12) is synchronized by an optical device that will conduct the beam both to a samplecompartment (also called analytical beam) and another reference compartment (also called referencebeam). The light transmitted on both compartments is synchronized again with the detection system;as a result, interferences can be eliminated, such as: energy source fluctuations, electric voltagevariations, as well as the solvent used in preparing the sample.

Thus, the source power fluctuations, the detector response and the amplifier gain are offset by thedifference in signs. The double-beam spectrophotometers are mechanically and electronically morecomplicated than the single-beam devices and, as a result, construction and maintenance are moreexpensive.

Figure 12. Double-beam monochromator

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Diffraction Grid

Input Slot

Sample Compartment

SynchronismOptical Device

2.3.3. Sample compartments

The UV-Vis spectrophotometer samples compartment has a wide range of connections andaccessories that allow us to expand the instrument’s possible applications. The basic samplescompartment is equipped with a rectangular sample support for 10 mm quartz cuvettes. Accordingto the application, it is possible to introduce sample supports for cuvettes varying from 1 to 100 mmlong.

2.3.3.1. Optical fiber coupler

In several instances, the spectrophotometer is classically used to measure solutions, that is, usingcuvettes. However, some samples are difficult to analyze due to their type or size. In order tofacilitate the analysis of such samples, an optical fiber coupler system can be used; some benefits arereached:

• Handling of samples that will be at high temperatures and/or pressures or radiated is avoided.• Measurement of very small samples, such as crystal, or very large samples, such as metal structure

windows that, otherwise, could never be made using devices with normal settings.• Monitoring of chemical processes.• Determinations in closed systems (reactors).

A series of optical fiber sensors can be used to reach the objective above, such as reflectance,absorbance, glass transmittance and fluorescence sensors.

2.3.3.2. Sipper

A system comprising a peristaltic pump can be used to pump the sample solution to a flow cuvettewithout requiring manual transference of the sample to the cuvette, thus reducing the sample handling.Such flow cuvettes can be positioned on special supports that can heat the sample.

2.3.3.3. Support for solid samples

This kind of support is intended to provide analytical results in transparent solid samples, such asglasses, plastics and lenses. In addition to the easy instrument connection, this support can also havea version in which the transparent sample travels in front of the optical beam so as to obtain thesample homogeneity profile.

A rotary device can be used to check the behavior of this solid and transparent sample, but havingdifferent incident radiation angles.

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2.3.3.4. Reflectance accessories

Solid non-transparent samples can be analyzed and the sample profile (reflectance spectrum) can beobtained. There are several kinds of reflectance accessories available and that can be coupled to thespectrophotometers, for example: mirror reflectance, diffused reflectance, controlled temperatureand pressure reflectance, powders and creams reflectance and solid sample color comparisons.

2.3.4. Data acquisition

The photosensitive devices used in transmittance measuring devices convert the radiating energyinto an electric signal. The photosensitive detectors should respond to the radiating energy on awide spectral range. They should be sensitive at low light levels e quickly respond to the incidentradiation. It is crucial that the electric signal generated is directly proportional to the incident beampower, that is,

R = kP + k’

Wherein R is the detector electric response in current units, resistance or e.m.f. The constant k is ameasure of the detector sensitivity in terms of electric response per unit of radiating power. Certaindetectors display a small k’ response called dark current, even when they are not receiving incidentradiation. The radiating energy detectors currently used in the manufacture of measurementequipment are photodiodes or photomultipliers.

The signs detected by the photomultiplier or photodiodes after amplification generate a wavelengthX transmittance or absorbance intensity graph called ultraviolet-visible spectrum. The identificationis possible by comparing this spectrum to the spectrum of a standard substance. The quantificationis also possible by fixing a wavelength and obtaining the corresponding transmittance or absorbancevalue. This value will be proportional to the sample component concentration. The actualconcentration is determined by using a standard with a known concentration.

2.4. Minimum installation and operation requirements

In order to assure reliable results in compliance with the Installation Qualification (IQ) requirements,the installation and operation requirements have to be met; in this case, the following can bementioned:

• Electrical network: that meets the manufacturer’s specifications, with special concern to theelectrical network stabilization.

• Instrument located under appropriate humidity and temperature conditions.• Operational knowledge to use the equipment correctly.

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2.5. Basic precautions

Some precautions should be taken to keep the equipment life and performance. Such precautionsare related to two kinds of action: user actions and preventive maintenance actions usually taken bythe manufacturer. This section discusses the user actions. The manufacturer actions are described inthe performance checking section.

Daily actions to be performed by the user are crucial to reduce damages that can be caused to theinstrument. Such actions are usually simple and can be summarized as follows:

• Equipment external cleaning.• Sample compartments cleaning.• Cuvettes washing.• Consult the manuals / equipment dealer to clarify operation/maintenance doubts.• Cleaning of the room windows interfacing the light beams and the sample compartments.• The use of safety material when handling samples is critical for the operator’s safety. The personal

protection equipment required includes: safety apron, surgical gloves and wide-view goggles.

The equipment should have consumables so that, is required, the operator himself can take actionssuch as changing lamps. The main consumable items required include:

• Deuterium and tungsten lamps, or xenon lamp.• Quartz cuvette.

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3. GAS CHROMATOGRAPHY (GC)

3.1. Introduction

Gas chromatography is an analytical technique used to separate mixtures of chemical substances.The purpose of the technique is to allow a separation that helps in identifying and quantifying thesubstances contained in the mixture.

As the main element used in the gas chromatography (GC) technique, the first chromatographiccolumns were packed in a typical tube between 1 to 5 m long and 2 to 4 mm diameter. As theanalytical instrumentation advanced, open tubes of melted silica (capillaries) are currently used, thelength of which range from 10 to 100 m and the internal diameters from 0,1 to 0,8 mm , thusproviding better results.


The sample is introduced in the system using a syringe or an injection valve. The sample is volatilizedright after being introduced in the chromatography system and dissolved in an inert carrier gas(mobile phase). When dissolved in the gas, this sample will be carried to the chromatographiccolumn containing the liquid or solid phase as a stationary phase, thus separating the compounds inthis column. Separation is accomplished because the sample components have different affinities tothe stationary phase, resulting in different elution speeds of the components by the column, that is,the higher is the affinity of the component to the stationary phase, the slower it will travel throughthe column and vice-versa.

Figure 1. Basic scheme of a chromatographic separation process

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Injection Migration Elution

After separation, the sample components reach the detector that generates an electrical signal bymeans of a physicochemical phenomena (for example, difference in thermal conductivity, flameionization). These detectors can be selective to a certain chemical group (for example,organochlorinated compounds) and more sensitive for the analysis. As the signal is usually at a verylow intensity, it is amplified and transmitted to a data station where it can be viewed in a signalintensity X time graph. This graph is called a chromatogram. The signal is proportional to theconcentration or mass of each compound; there should be different responses from a compoundto another (the most abundant compound not always generate the higher signal). For this reason,the sample can be quantified.

The GC allows the analysis of several substances, including some inorganic gases such as O2, CO,CO2, NO2, etc., as well as thousands of organic substances of several functional groups, such as,alcohols, ketones, amines, aromatics and others. By using highly selective detectors, it is possible todetermine very small amounts of components contained in the sample, for example, in the order ofpicograms (traces); as well as greater amounts, for example, in the order of percentage.


A basic system includes the following modules:

1. System of gases: The most common gases used as mobile phase (carrier gas) are helium, hydrogen,argon and nitrogen. Other gases can be also used. The use of auxiliary gases is required dependingof the detector to be employed.

2. Flow and pressure controllers: Used to maintain the mobile phase outflow uniformity. They arefurther used to measure the sample splitting ratio (splitter) when injectors for capillary columnsare used and also to control the outflow of auxiliary gases of the detectors.

3. Injectors: Place where a sample is effectively introduced in the system. The injectors can bedesigned for packed or capillary columns; there are different techniques for using them (on-column, flash vaporization, split, splitless, etc). The sample can be also introduced using samplingvalves, mainly for gases. Automation can be used to increase productivity and reproducibility.

4. Columns: The substances contained in the sample are separated inside the columns. The columnscan be made of glass, stainless steel, nickel, teflon or melted silica; they can be packed or capillary.The capillary columns allow better separations.

5. Detectors: Devices that monitor the exit of eluted substances from the column, generating anelectric signal that is proportional to the substance mass or the concentration. Detectors can beof the universal- or selective-type. These detectors are connected to signal amplifiers, calledelectrometers, which amplify the signal and transmit it to the output data devices.

6. Data acquisition and processing system: A system that collects data generated in thechromatographer, performs the necessary calculations for quantifying the substances andgenerates the chromatogram. This system can be integrators or integration software (PC).

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Figure 2. Gas chromatography (GC) system

3.3.1. Gases used

Two classes of gas can be used in the chromatographic system: carrier gases (mobile phase) andauxiliary gases.

The most common carrier gases are nitrogen, argon, hydrogen and helium. The most indicated gasis helium, due to the better chromatographic performance when compared to other gases; however,some decisive aspects when choosing the carrier gas are to be considered:

• Detector compatibility: it is critical to determine that the carrier gas is compatible with thedetector and lead to a loss of sensitivity or excessive noise. For example, when analyzing hydrogengas in TCD, nitrogen and argon impart greater sensitivity than helium, as the thermal conductivitydifference between the two first gases and hydrogen is greater than the thermal conductivitydifference between hydrogen and helium;

• Costs;• Availability;• Performance;• Purity: The purity of the gases should be respected, depending on the application. The

manufacturers’ manuals contain all the required information.

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CarrierGas

PressureController

GCPneumatics

Injector

Column

Detector

Column Oven

Chromatogram

Workstation

3.3.2. Flow and pressure controllers

The carrier gas flow or pressure is an important parameter that depends on the column diameterand length. It should be controlled during the analysis. This control is performed using flow orpressure controlling valves. This parameter can be adjusted manually, with the aid of a flow meteror a bubble meter, or electronically using a software; the purpose is to obtain a better chromatographicefficiency.

For auxiliary gases, control is required since the flow adjustment is critical to reach the bestperformance.

3.3.3. Injectors

The basic function of an injector is to introduce the sample into the chromatographic system. Atthe injector, the sample contacts the carrier gas and is dissolved within it. Therefore, in case ofliquid samples, the injector has to be heated so as to assure a total volatilization of the sample.

There are two categories of injectors: injectors for packed columns and injectors for capillary columns.Gaseous samples can be injected with the aid of a gas sampling valve provided or not with aninjector in series with the valve. For each class of injectors, some options are available depending onthe application.

3.3.3.1. Packed injectors

These injectors are used with packed columns. They can be used in megabore-type capillary columns(internal diameter of column: > 0,53 mm). All the sample is directly injected on the head of thechromatographic column. This is to eliminate any and all loss when transferring the sample to thecolumn. This injector is indicated to clean and diluted samples, in addition to samples having a greatvolatility variation of compounds.

Figure 3. Packed injector

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InjectorBody

Septum

GasInlet

Column

3.3.3.2. Capillary injectors

Injectors designed for use in capillary columns. The main injectors include:

• Split/Splitless Injector: The most commonly used injectors. Allows samples to be divided (split)so as to allow the use of capillary columns (as they are able to receive a small amount ofsample). There are two operation modes:

• Split mode – The injected sample is split between the column and the injector disposal outlet(vent). The valve in charge of this splitting action is called splitter and is used with concentratedsamples (in the order of mg/mL).

Splitless mode – Used with diluted samples. In this case, the splitter will remain closed during thesolute transference to the column; next, the splitter is opened to dispose the solvent.

Figure 4. Split/splitless injector

• On-column and/or programmable temperature injector: Allows two operation modes:• On-column injection - For samples with wide molecular weight range, eliminating the mass

discrimination effect.• On-column injection with programmable temperature – For low boiling temperature or low

concentration thermolabile samples. Known in the market as PTV or SPI Injector.

Figure 5. Programmable temperature injector

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CarrierGasInlet

SeptumPurging

InjectorDisposal

InjectorDisposal

Column

SeptumSeptum PurgingGlass LinnerCooling Inlet

CoolingOutlet

InjectorBody

Heater CarrierGas Inlet

NutWasherColumn

3.3.4. Columns

The column is the main part of the chromatographic system where the separation of the samplecomponents takes place as a result of the different affinities of the substances with the stationaryphase, migrating at different speeds through the column. The columns can be classified into twogroups:

• Packed columns: The oldest chromatographic columns. Usually made of stainless steel or glasstubes, however, other materials can be used. A stuffing (an adsorbent or a solid supportimpregnated with a film of a substance having a low vapor pressure) is introduced in this tube.Presently, these columns are not frequently used due to the lower performance when comparedto capillary columns. Packed columns are more commonly used in the analysis of inorganicgases.

Capillary Columns: The most commonly used columns presently. They are usually made of meltedsilica tubes externally coated with a polyamide film, thus making the column very flexible. Thestationary phase (a film or a solid adsorbent) is deposited on the inner wall. This kind of column hasgreat separation capacity and a wide range of substances can be separated by each kind of column.The phase choice determines the column selectivity. Hundreds of stationary phases are available inthe market with several trade names. They are usually chosen according to the polarity of thesubstances to be analyzed. Polar stationary phases are typically more reactive, thus having a workinglimit temperature lower than the non-polar columns.

Figure 6. Types of capillary columns

To choose the most appropriate column, the following parameters should be assessed:

– Columns internal length and diameter– Film thickness (capillary);– Liquid stationary phase;– Solid Supports;– Solid Stationary Phases.

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POLYAMIDEFILM

MELTEDSILICATUBE

STATIONARYPHASE WCOT

WALL FILMOPEN TUBE

SCOTWALL SUPPORT

OPEN TUBE

PLOTPOROUS BEDOPEN TUBE

3.3.4.1. Columns oven

The chromatographic column is stored in the oven. For each kind of analysis, the oven should beset at a working temperature; at this temperature, the sample components of interest are separatedand having the shortest elution time.

A good oven has measurement precision and a wide ramp programming range. The use of cryogenicsallows the analysis of more volatile compounds.

3.3.5. Detectors

There are several kinds of detectors: universal detectors (Thermal Conductivity (TCD), MassSpectrometry (MS)), and selective or specific detectors (Flame Ionization (FID), Electrons Capture

Table 1. Types of detectors and their use

The most commonly used detectors in gas chromatography (GC) will be discussed.

3.3.5.1. Flame ionization detector (FID)

The most used detector. Some features include: easy operation, high sensitivity and incomparablelinearity. The figure below shows a scheme of the FID detector.

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Figure 7. FID Detector

The detector operation involves a mixture of gases, air and hydrogen, thus forming a flame havinga temperature of about 2000ºC. The sample is burned on this flame. A pair of electrodes is positionedclose to the flame to capture the signal generated by ionized specimens and electrons resulting fromthe substances burned on the flame.

This detector generates a signal corresponding to the compounds having carbon and hydrogen intheir molecule. Rare exceptions include CS2.

The detector should be operated at a temperature of about 400º C. However, the chosen temperatureshould assure that substances eluted in the column do not condense.

The typical concentrations to be analyzed include the range from low (ppb) to high values (%).

3.3.5.2. Nitrogen and phosphorus (NPD) or specific thermoionic detectors (TSD)

The detector has selective response to compounds containing nitrogen or phosphorus atoms.Internally, the detector has a rubidium salt incorporated to a refractory pearl subject to the passageof gases, air and hydrogen. When electrically heated, the pearl produces a gaseous plasma at 600 to900ºC.

The plasma thus produced is enough to make the N- and P-containing substances respond to thedetector.

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Ceramicinsulator

Collector

Signal probe

Ignition probeIgnition spring

Flame tip

Figure 8. TSD or NPD detector

3.3.5.3. Electrons capture detector – ECD

This detector is sensitive to molecules having high electronic affinity atoms and has inside a radioactivesource, typically 63Ni, which emits beta particles. These particles ionize the carrier gas, usually nitrogen,and generate a cloud of electrons inside the ECD cell. The electrons produce a stable backgroundcurrent through the ECD cell electrodes. This signal is amplified by the detector electrometer.When an electrons-absorbent specimen passes through the cell, the current is decreased as aresult of the electrons capture by the absorbent specimen, thus decreasing the number of electronsin the cell.

Figure 9. ECD detector

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Collector

CeramicPearl

H2 and Make-upAir

Column

Flame Tip

Ceramic Insulator

Signal Probe

Pearl Probe

Nickel Source

Cloud ofElectrons

Cell

CollectorElectrode

Make-up

Column

Insulation

Ceramic InsulatorsSignal Probe

Cell Probe

3.3.6. Data acquisition and processing

Data acquisition and processing were discussed in item 4.6 of HPLC technique.


In order to assure reliable and reproducible results in compliance with the Installation Qualification(IQ) requirements, the conditions of the installation room should be verified. For that purpose, thefollowing conditions are to be mentioned:

• Electrical network: it is necessary to check that electrical network voltage is in accordance withthe equipment specification. Network stabilization and the outlet plugs position (inverted phase)should have special attention, as they can interfere with the equipment operation and life.

• Environmental conditions: The instrument should be installed only if local humidity andtemperature conditions are appropriate for the operation. In addition, safety conditions, such asthe proximity to flammable and corrosive agents should be assessed.

• Purity of Gases: Should be in accordance with the system chromatographic requirements, mainlyconcerning the detectors.

• Gases Filter: Should be used to assure the quality of the gases used and the system gas lines.Should be replaced on a periodic basis.

• Pressure Regulators: Should assure the minimum working pressures specified for the equipment.Should be also provided with safety devices, such as double-stage and metal diaphragms.

• Operational knowledge of the equipment: For the correct use of the equipment.

Table 2. Purity of gases

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In order to assure good results, some precautions are required when working with GC. These areusually simple, but important procedures so that the equipment can operate properly.

One of the most common precautions is changing the injector septum typically between 30 and100 injections depending on the injection technique (manual or automatic). The injector glass inserteris also a very important item for the good performance of the equipment. Thus, replacement isrequired according to the application and usage.

Another relevant precaution concerns the use of the columns that should be conditioned andsubject to thermal cleaning whenever required. When heating the column, care should be taken notto exceed the stationary phase thermal limit.

Concerning the gases, in addition to the purity precautions discussed above, it is important toreplace the gases filters when required and also regularly check the cylinder pressure to avoid completeuse, thus preventing occasional contamination by the environmental air admitted by diffusion.

It is recommended to clean the detectors every six months or whenever performance decreaseoccurs. The cleaning procedures are described in the equipment operation manuals.

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4. LIQUID CHROMATOGRAPHY (LC)

4.1. Introduction

High performance liquid chromatography (HPLC) was created when liquid chromatography wasapplied to theories and instrumentations originally developed for gas chromatography.

The indispensable high performance liquid chromatography (HPLC) is currently highlighted interms of modern separation methods. The difference between liquid chromatography and highperformance liquid chromatography (HPLC) rests on the use of stationary phases preferably withspheric microparticles (10.5 or 3 mm). As they are much less permeable, such phases are requiredfor the use of pumps to elute the mobile phase.


The chromatographic separation is based on the differential migration of components from a mixturedue to the differences between two immiscible phases, the mobile and stationary phases, and whenwidening the bands depending on physical processes and not the difference in balance. Theseinteractions can take place by hydrogen bond interactions, electrostatic interactions and hydrophobicand Van der Waals forces, and others.

The differential migration results from the analytes balance difference between both immisciblephases, being determined by factors affecting this balance: the mobile and stationary phasescomposition and the separation temperature. Changes of any of these factors lead to changes in thedifferential migration.

The liquid chromatography classification according to the stationary phase led to a wide variety.The first great division was the adsorption chromatography and the partition chromatography,referring to the solid and liquid stationary phases, respectively. Taking the nature of the interactionsand the above mentioned phenomena into consideration, the separation modes can be classified asfollows: Reverse phase chromatography, normal phase chromatography, ion pairing chromatography(ion exchange) and exclusion chromatography.

If liquid, the stationary phases can be simply adsorbed on a solid support or immobilized on it. Inthe first case, chromatography is referred as partition chromatography.

The partition chromatography was replaced by the chemically bonded-phases chromatography byvirtue of the greater stability imparted when compared to the adsorbed liquid phases. These phases,having modified supports, are considered separately by differing from the other two modes in termsof separation mechanism.

The great chromatographic development obtained from the chemically bounded liquid phases ledthese phases to be mainly used in analytical HPLC.

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4.2.1. Separation modes:

There is always dependence between the solute-mobile phase, solute-stationary phase and mobilephase-stationary phase interactions. Then, the choice of the separation mode depends on the choiceof the stationary and mobile phases for each class of solute.

Two retention modes in liquid chromatography were proposed. The first by Scott and Kucera,interaction-solvent, and the second by Snyder, competition-solvent. The two models are equivalent,since both consider that in a given separation, the interaction of the solute with the stationary phaseremains constant. Therefore, retention is determined by the mobile phase composition.

4.2.1.1. Normal phase chromatography

The stationary phase is more polar than the mobile phase; the opposite occurs in reverse modechromatography. The solvents used are usually a mixture of organic solvents without the additionof water. The stationary phases are organic adsorbents (silica, alumina) or chemically boundedpolar phases (cyano, diol, phenyl, amino).

Both retention models, interaction-solvent and competition-solvent have been successfully used todescribe the effect of the mobile phase in normal mode liquid chromatography. Regardless themodel used, retention in normal phase increases as the mobile phase polarity decreases.

This chromatography mode is mainly applied to neutral molecules, although it can be also used toseparate ionizing or ionic molecules.

The order of elution respects the following sequence: hydrophobic molecules (less polar) are elutedfirst, while the hydrophilic molecules (more polar) are retained.

When the sample dissolution has problems in polar solvents, the reverse phase injection is difficult;the normal phase separation is then recommended.

The elution solvent in the normal mode is selected by choosing a weak solvent and mixing it with astrong solvent so as to reach the desired strength.

The presence of water traces in the mobile phase is the main cause of poor retention reproducibilitywhen working on a normal phase, especially when unmodified silica is used as stationary phase.This problem has been solved by the use of anhydrous solvents with a known volume of water,methanol or acetic acid to deactivate the most reactive silanol groups of the stationary phase. Besidesimproving reproducibility, it also improves the peak shape. This same effect can be obtained byadding triethylamine, an essential compound in the separation of amines in silica gel.

The use chemically modified silicas in normal mode elution has been preferred, since they offerinteraction sites with the solute, in addition to a more homogenous surface when compared to silicagel that has a variety of silanol groups with different polarities. The chemically bounded phases are

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useful for the chromatography of moderately polar compounds. However, these solutes can be alsoefficiently resolved in reverse mode elution. The choice between normal or reverse mode elution isusually more dependent of the matrix than the solute.

4.2.1.2. Reverse phase chromatography

While in the normal phase chromatography a stationary phase is more polar than the mobile phase,in the reverse mode the mobile phase is more polar that the stationary. The reverse phasechromatography is more commonly used in HPLC, since it allows the separation of a wide varietyof solutes and the use of aqueous mobile phases. The most used mobile phase is a mixture ofacetonitrile/water; when required, acetonitrile is replaced with methanol and tetrahydrofuran (THF).The use of only three solvents is a result of the small amount of water-miscible organic solvents. Inthe normal mode, a wider range of solvents is available.

The principle of the reverse phase separation is the hydrophobia, being mainly attributed to theinteractions between the non-polar portion of the solute and the stationary phase, that is, the repulsionof this portion of the solute by the aqueous mobile phase.

It is worth emphasizing that the solvent strength in reverse phase increases as the solvent polaritydecreases. Thus, the water strength (weaker solvent) is lower than the methanol, acetonitrile,tetrahydrofuran and dichloromethane strength, considering the crescent order (see table 1). As awater-insoluble substance, dichloromethane is not used in the reverse phase; however, asdichloromethane is a very strong solvent, it is sometimes used to clean the reverse phase columnscontaminated by strongly retained solutes.

Table 1. Comparison among strengths of different mobile phases

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Besides being used at a low absorption range in the ultraviolet, acetonitrile results in aqueous solutionswith low viscosity (as desired). Therefore, together with methanol and THF, these are the mostcommonly used solvents to control the selectivity and the elution reverse mode separation.

When for some reason water is not used in the mobile phase and apolar stationary phases are used,the chromatography is called non-aqueous reverse phase chromatography. This chromatographywill be used when working with highly hydrophobic solutes, such as lipids and polymers; the solventusually consists in a mixture of polar solvents, such as acetonitrile or methanol (solvent A), with aweaker solvent (B), such as THF, chloroform, dichloromethane, acetone, methyl-t-butyl ether. Inthis case, retention is also changed by the percentage of solvent B.

There are other modalities of ionic compounds chromatography or size exclusion chromatography(gel permeation and filtration, applied to the molecules size separations, for example, polymers,biopolymers, proteins, peptides, etc). As they are barely applied in the separation of drugs andmedicines, these modalities will not be discussed herein.

Another important aspect involving the HPLC chromatographic separations is the separation mode:isocratic or gradient. In the isocratic mode, the separation conditions remain unchanged throughoutthe chromatographic run, being restricted to a single solvent composition. In the gradient mode,there is a mobile phase and/or flow composition variation during the chromatographic run.

There is a number of relevant parameters to be considered during the development of achromatographic method, such as the chromatographic resolution, selectivity, retention factor, etc.These parameters are extremely important when developing, validating and using HPLCchromatographic methods.


A HPLC system basically consists in modules having specific functions and carefully designed toprovide intended analyses with versatility, fastness, reproducibility and high sensitivity.

Current HPLC equipment in the market may range from very simple, where samples are manuallyinjected, to more complex systems provided with an automatic sampling module and controlled bycomputer software able to control the system’s functions, acquire, process and print data, store andorganize them for future reference.

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For a better understanding, the HPLC system can be divided into six main modules:

1. Mobile phase reservoir2. Solvents pumping system (Pump)3. Sample introduction (Injector)4. Column compartment5. Detection system6. Data system

4.3.1. Mobile phase

The mobile phase used in liquid chromatography is extremely important for obtaining the desiredseparation, as it contacts the analyte being separated, as well as a chromatographic column and thesystem as a whole.A wide range of solvents is available; however, there are some desirable properties for their use inHPLC:

• High purity;• Non-decomposition of the analyte and the stationary phase;• Compatibility with the detection system;• Low viscosity;• Sample dissolution;• Low cost.

Among the solvents used in liquid chromatography, it is worthy emphasizing the characteristics andpurity of water; the ultrapure water it is strongly recommended as highly reliable results are obtained.Its resistivity (typically 18.2 MO/cm at 25ºC) is an excellent quality indication. However, the organiccompounds should be monitored since they can interfere with the sensitivity of UV detectors

Figure 1: Schematic representation of a HPLC system:

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commonly used in HPLC analyses. It is important to remember that the ultrapure water should bealways immediately used as the storage modifies its conditions.

4.3.2. Solvents pumping system

The main function of the Pump is to push the mobile phase through the column.

Once they have compacted padding consisting in very small diameter particles, in the order of 3 to10 um, the columns highly resist to the passage of the mobile phase; therefore, the pumping systemshould be able to overpass this barrier and provide a constant, reproductive flow with no pulsations.

Most of the pumps used are of the reciprocating type, also called piston or diaphragm pumps. Theoperation is based on an electric motor connected to gears that move a pistons system.

As this kind of pump produce pulsating flows due to the “to-and-forward” movement of thepiston(s), some resources were developed to overcome this problem ; these mechanisms are calledpulse mufflers.

Analytical pumps are designed to operate at high pressures and flow rates varying from 0.01 to 10mL/min. They are usually made of inert material, such as stainless steel (pumping heads, piping andconnections), sapphire, quartz, ceramics and titanium (pistons), ruby (check valves) and inert materials(seals).

4.3.3. Sample introduction – injector

The injector is the module in which the samples are introduced in the HPLC system so as to allowseparation in the column.

The injector can be manual (the user introduces the sample with the aid of a straight tip microsyringe)or automatic, also known as auto-injector or automatic sampler, that is able to automatically inject agreat number of samples and even perform the dilution, derivation or reagents addition operations.

4.3.3.1. Manual injector

The most commonly used manual injectors are basically the valve-type injectors having an externalsampling loop, defined and precise volume piping that can be replaced to allow the injection ofdifferent sample volumes.

Figure 2 shows the valve schematic diagram. The six small circles represent the valve internal bores,while the large circle represents the injection syringe needle inlet.

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Figure 2. Schematic diagram of an injection valve

The Valve has two positions: LOAD and INJECT. At the LOAD position, the pump flow is deviateddirectly to the column (enters at 2 and exits at 3). Meanwhile, the sample is introduced in thesampling loop (LOOP) through the needle bore. The excess is immediately disposed through exit 6,that is, the injection precision is determined by the volume of sample at the LOOP.

At the INJECT position, the mobile phase now travels through the LOOP dragging the sample tothe column (travel 2-1-4-3).

4.3.3.2. Automatic injector

The automatic injectors are devices widely used in laboratories aiming high productivity, as theyoperate in an unassisted mode. They further provide better reproducibility when compared to themanual injectors. Some models are able to work as a thermostat for the samples, thus increasing thechromatographic system versatility.

4.3.4. Columns

4.3.4.1. Silica-based stationary phases

Silica is undoubtedly the most important material used in stationary phases for HPLC. Silica is aversatile material and its surface can be modified by chemical derivation, then giving raise to severalinteresting materials such as stationary phases for liquid chromatography. Silica also allows workwith different separation mechanisms.

Silica is an amorphous, highly porous and partially hydrated material usually prepared by acid hydrolysisof sodium silicate, followed by emulsification in a mixture of alcohol/water and subsequentcondensation, it is then washed and dried for use.

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Pump

Column

Syringe

Column

Pump

Silica surface results from its preparation conditions; silicas having a high amount of free silanolsare more acid than silicas with bounded hydroxylated groups.

Silica is widely used in normal phase chromatography; it is not recommended in reverse phasechromatography.

4.3.4.2. Chemically modified phases

Currently, the most commonly used phases. Their application to separate polar compounds waswidely disseminated as a result of the difficulties when using silica-based columns.

With the advent of the chemically bounded phases, this gap was filled up and we can currently statethat about 90% of the chromatographic separation is performed using such phases. They have aninteresting mechanism of solutes retention by means of interactions not exclusively base on polarity.

The use of octadecyl groups lead to the formation of the octadecylsilane phase, known as ODS orC18. This group imparts the phase a polar character compared to the unmodified silica.

Retention on these phases depends on the amount of carbon present, usually expressed as percentage.The quality of the separation to which they are applied will depend on this percentage and theamount of residual silanols.

For sterical reasons, this derivation does not affect all silanol groups. In some instances, the remaininggroups have a problem with the peaks tail when interacting with the solute. This problem can besolved by reacting silica with trimethylchlorosilane after the derivation; as the trimethylchlorosilaneis smaller, it can reach some of these groups, then forming trimethylsilanes. Although it is notpossible to derive all the silanol groups, this process is called end capping.

Some analyses require the column temperature to be stable and, for that purpose, there are devicesavailable in the market; such devices are known as oven or column compartment.

4.3.5. Detectors

Detectors are transducing devices connected right after the column outlet. They are responsible forgenerating electrical signals proportional to the compounds passing through them. Several kinds ofdetectors have been used in HPLC depending on the physical or physicochemical features of thesample and the mobile phase. An ideal detector should have the following characteristics:

• High sensitivity and low limit of detection;• Fast response to all solutes;• Insensitivity to temperature and mobile phase outflow changes;• Independent response of the mobile phase;• Small contribution for the peak widening by the extra volume of the detector cell;• Response increasing linearly with the amount of solute;

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•· Non destruction of the solute;• Safety and usage convenience;• Qualitative information of the desired peak;• Low cost.

All characteristics can be hardly found in a single detector; therefore, the most appropriate detectorshould be chosen according to the sample and having as many features as possible.

The most commonly used detectors available in the market include:

• Ultraviolet and visible (UV-VIS) absorbance detectors;• Fluorescence detectors;• Electrochemical detectors;• Mass detectors.

Other detectors:

• Refractive index detectors;• Electric conductivity detectors.

4.3.5.1. UV-VIS detectors

These detectors operate based on the amount of light absorbed by the solute at a certain characteristicwavelength. They can be basically divided into three types:

• Fixed wavelength;• Variable wavelength;• Diodes arrangement.

4.3.5.1.1. Fixed wavelength detectors

The simplest of all detectors. It is not very common nowadays, as they are sensitive to variations ofthe mobile phase composition, thus impairing its use in gradient systems. The wavelength is selectedthrough specific wavelength optical filters.

4.3.5.1.2. Variable wavelength detectors

The most commonly used detectors nowadays, as they can be used both in isocratic and gradientsystems. Used by most of the laboratories in which the sample and its corresponding wavelengthare known. Figure 3 shows the schematic diagram of a variable wavelength detector.

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Figure 3. Schematic diagram of a variable wavelength detector

4.3.5.1.3. Diodes arrangement detectors

Detectors having the capacity to generate absorbance spectra at a speed compatible with the eluantflow. Provides tridimensional data: absorbance x time x wavelength. They are used in research anddevelopment when the best wavelength is not known, when the spectrum is desired forcharacterization purposes (library) and to obtain the purity percentage of the peaks.

The schematic diagram is showed in figure 4.

Figure 4. Schematic diagram of a diodes arrangement detector

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Diffraction net

Lamp Cell

Photodiode

Diffraction net (fixed)

Lamp

Cell Diodes arrangement

4.3.5.2. Fluorescence detector

The fluorescence detectors are specific, as the solute has to fluoresce. It is one of the most sensitiveamong the most used detectors in HPLC.

Figure 5 shows a schematic diagram of this kind of detector.

Figure 5. Scheme of the fluorescence detector

Some fluorescence detectors in the market have the capacity to generate excitation and emissionfluorescence spectra during the chromatographic run.

4.3.5.3. Electrochemical detectors

The most sensitive of all detectors and also the most delicate to work with. The molecules to beanalyzed should have characteristics that allow oxidation or reduction. Detection is performed bymeans of electrodes as showed in figure 6.

Figure 6. Schematic diagram of an electrochemical detector

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Light source Sample cell Detector

Absorption

Detector

Emission

Auxiliaryelectrode

Mobilephase

Workingelectrode

Potentiometer Recorder


As part of the chromatographer, the data acquisition and processing system will allow the user toverify and document the obtained results.

The system will basically receive the electric signal sent by the detector(s), convert them into graphicscalled chromatograms, integrate the peaks using the predetermined parameters and, from a calibrationcurve, quantify the samples.

This signal x retention time graph has two important features. First, the retention time is characteristicand repetitive for each substance; second, the peak area or height is differently proportional to thesubstance concentration or mass.See the figure below:

A file is initially created and contains the information required for the analysis of the interestsubstances, such as:

• Text describing the chromatographic conditions, such as, the mobile phase and column used;• Programming of parameters for the chromatograms integration.

When the system stabilizes, standards are injected in order to check out the performance.

When all parameters are set and correct, a standard containing all components to be quantified isinjected so that a table is created; in this the table, the system is “informed” about the peaks to beanalyzed through the corresponding retention times. This table is required to create a calibrationcurve.

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Baseline

Retention Time

Peak Height

Peak Area

Minutes

Next, the standards for creating this curve are injected and stored, ending the creation of thisanalysis file.

When injecting the samples, care should be taken to inject the control standards at regular intervals,thus allowing the system stability verification.

Next, the four methods that may be used for calibration purposes will be discussed:

• Area Percentage.• Area Normalization.• External Standard.• Internal Standard.

4.3.6.1. Area percentages

The area percentage is the simplest calculation method, as no standard is required.

It is based on the principle that the detector response is proportional to the amount of substancepassing through its cell.

Can be used for substances having the same response to the detector, that is, if equal amounts areinjected, equal areas should be obtained for all peaks. The sample calculation is:

Component A concentration (%) = Component A area x 100

Σ Areas of all components

4.3.6.2. Area normalization

The principle is the same of the percentage area, however the detector response for each substanceis corrected using one of the peaks as a calculation reference. For this method, it is necessary toinject a standard for calculating the response factor of each peak, as showed in the formula below:

Component A = Component A area x Reference component concentrationresponse factor Component A concentration Reference component area

When the response factor of each component is available, one can analyze the sample using thefollowing equation:

Component A = Component A area x Component A response factorconcentration (%) Σ (Areas of all components x Response factor of all components)

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4.3.6.3. External standard

The external standard is the most common calibration method.

It consists in quantifying the sample by injecting standards with known concentrations.

The standard area is corresponded to the concentration and the response factor is obtained for eachcompound, according to the equation below:

Component A = Component A arearesponse factor Component A concentration

With this response factor generated after injecting the standard, the sample is injected.The area generated in the sample is divided by the response factor generated by the standard, thesample concentration is obtained by the equation:

Component A = Component A areaconcentration Component A response factor

4.3.6.4. Internal standard

The internal standard is the most precise calibration method, since it corrects volume fluctuationsby using a reference standard added in the same amount both to the standard and the sample.

The choice of this reference standard should meet the following conditions:

• Should not be present in the sample;• The sample should not have any component with a retention time very close to the standard;• It should be pure, non-reactive and preferably of the same functional group of the component

to be determined.

Many times this method cannot be used as a result of such conditions; however, when feasible, thismethod provides very precise results even when using manual injection.

The calculation consists in determining the response factor related to each component by injectingthe standard and using the equation below:

Component A = Component A area x Internal standard concentrationresponse factor Component A concentration Internal standard area

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When the response factors are calculated, the sample is injected with the internal standard and, withthe areas so obtained, the component concentration is calculated using the equation:

Component A = Component A Internal standard Component Aconcentration area x concentration x response factor

Internal standard area

There are presently two options for acquiring and processing data: the integrator and the personalcomputer with a dedicated software. Both allow the user to perform the same tasks. The basicdifference between them is the operating system.

It is worth emphasizing the importance of transferring obtained data (chromatograms) from aplatform to another; for interlaboratory interchange purposes, this cannot be a drawback for resultscomparison and analysis.


In order to assure reliable and reproducible results in compliance with the Installation Qualification(IQ) requirements, the conditions of the installation room should be verified. For that purpose, thefollowing conditions are to be mentioned:

4.4.1. Bench requirements

The bench should be sized so as to properly accommodate the HPLC equipment and make the userfeel comfortable during work.

The size of the HPLC and the data system (integrator or computer), usually two linear meters, isenough to accommodate the complete system.

The current HPLC equipment is usually modular, their modules remaining over each other forminga tower; therefore, the total height (bench plus tower) should be safe so as to avoid accidents mainlywhen the operator is handling the mobile phase.

The structure should be able to firmly bear about 80 kg without vibrations or swings.

The equipment manufacturers provide detailed descriptions of the required bench on thepreinstallation manuals.

4.4.2. Electric network

The electric network should be duly grounded and stabilized. The use of a magnetic key isrecommended in case of constant electric power supply failures.

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The number of outlets should be adequate and in compliance with the system installation standards.One outlet is required for each module, in addition to the outlets for the integrator. If a computeris employed, outlets are required for the CPU, monitor and printer. For a traditional HPLC, havinga pump, automatic injector, oven, detector and computer, eight outlets are enough. There should bea spare outlet for use in technical assistance and/or validations.

Figure 7 shows the standard outlet used in HPLC equipment.

Standard outlet for liquid chromatographer

Figure 7. Standard outlet for HPLC

The HPLC manufacturer should inform the total power consumption as well as the powerconsumption per module (expressed in Watts or VA or KVA). For the sizing and distribution of theelectric system capacity, an electric professional should be contacted.

HPLC equipment does not tolerate electric network fluctuations; therefore, it is critical to know thevoltage stability and determine that voltage is compatible with the required specifications of eachmodel.

4.4.3. Environmental conditions

Temperature and humidity are the basic environmental conditions to be regarded.

The temperature of the room where the HPLC equipment will be installed should be controlledwith no wide fluctuations during use. The typical temperature required by manufacturers is 25ºC,fluctuations of +/– 2ºC are allowed.

Humidity should be less than 95%; no condensation is allowed.


Maintenance by the user is critical for the good operation and durability of a chromatographer.

After using the equipment, a cleaning procedure should be followed (that will be dependent on thekind of mobile phase used); this is also true for the chromatographic column; as the main componentof the system, the chromatographic column should be given special attention.

For cleaning the chromatographic column, carefully follow the manual’s instructions.

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Systems working with buffer solution as the mobile phase require special care. After use, it is essentialto use water (100%) for the complete removal of the buffer solution and then a mixture, for example,70% of methanol (or acetonitrile) and 30% of water. This mixture is required as fungi can grow ifthe system is left in water only. The user should be familiar with the replacement of consumableparts of the system. Any maintenance intervention should be reported in a logbook.

The main consumable parts for each module are listed below:

• Pump – Seals, pistons, filters.• Manual injector – Rotor seal, sample loop, connections.• Automatic injector – Filters, septa, rotor seal, syringe unit parts (if any), vials for samples and

reagents, needle, sample loop.• UV-Visible and fluorescence detectors – Lamps, lenses, o-rings.• Electrochemical detector – Electrodes, KCl solution, o-rings.

In addition to the equipment operation, therefore, the user’s training should include these basicprecautions. Due professional qualification is critical for this learning level.

Reading the manuals is also an excellent practice. In addition to provide detailed information on theequipment operation, the manual clarifies several doubts of the user. It is important to observe thesafety rules when handling the equipment. Wearing apron, gloves and goggles avoids severe injuriessuch as blindness or sample contamination, for example, plasma.

The system decontamination is always mandatory in order to protect the health of the person incharge of maintenance.

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5. CHROMATOGRAPHY SYSTEMS CONNECTEDTO MASS DETECTORS

5.1. Introduction

Chromatography is an essential separation technique in life sciences and related chemistry fields.Traditional detectors such as ultraviolet-visible, electrochemical, refractive index (HPLC), flameionization, thermal conductivity, etc (GC) are widely used to quantify compounds as bidimensionaldata are produced (response x time).

The mass detectors are characterized by generating tridimensional data (response x time x ionicspecimen), that is, mass spectrum that can provide very important information on the samplemolecular weight, its molecular structure, identity, quantity and purity. Data from the mass spectraadd specificity to both quantitative and qualitative analyses.

For most of the compounds, the mass detectors are more sensitive and much more specific thantraditional detectors. They can analyze several compounds and identify components in non-separatedchromatograms, thus reducing the need of a perfect chromatography. The mass spectral data maysupplement data from other detectors. Although two compounds can have similar UV or Massspectra, such as in LC-MS, this phenomenon is hardly simultaneous; therefore, both types of datatogether can be used to identify, confirm and quantify compounds with highly correct results.

Some mass spectrometers have the characteristic of performing multiple mass spectrometry stepsin a single sample. They can generate a mass spectrum, select a specific ion and then generate a newspectrum. Some of them are able to repeat this cycle several times until the structure is determined(MS/MS or MSn).


The mass spectrometer basically operates through the ionization and fragmentation of molecules.Afterwards, the resulting ions are identified according to their mass/charge ratio. The three keycomponents of the process include: ions source, analyzer and detector. The purpose of the ionssource is to generate ions. There are several kinds of ions source in the chromatography connectedto mass detectors. Each type is specific for a given class of compounds. There are also several kinasof analyzers for the separation of ions and detectors for the generation of measurable signals.

Each one has advantages and drawbacks depending of the type of information searched. Detailsconcerning the most common sources, analyzers and detectors will be discussed next:

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5.3.1. Ionization source

The molecules ionization and fragmentation will take place in the source. Several ionization techniquesare available, including: Atom bombing - FAB, Laser Desorption - LD, Thermospray - TS, Particlesbeam, etc. However, the most commonly used sources are:

5.3.1.1. Electrons impact (EI)

The Electrons Impact (EI) source uses a filament in charge of emitting electrons with a definedenergy of 70 eV. Once the electrons beam energy is much higher than the first ionization potentialin most of the compounds of the sample, this energy is ionized and then fragmented. This kind ofionization is related to the GC.

5.3.1.2. Chemical ionization (CI)

The Chemical Ionization (CI) source uses liquid or gas agents to react with molecules. Ionizationusually takes place by means of the transference of a proton to the molecule, thus forming specimenscalled molecular pseudo-ions. As this ionization is much “smoother” than the electrons impact, the

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SAMPLINGSYSTEM

IONS SOURCEIS

MASSES FILTERMF

DETECTOREM

DATA SYSTEMDS

spectrum produced contains a few fragments and almost exclusively the molecular pseudo-ion;therefore, it is employed in the determination of the molecular weight and/or quantitative analyses.This kind of ionization is related to GC.

5.3.1.3. Electrospray (ES)

The electrospray ionization has a great impact on the use of mass spectrometry applied to biologicalresearches in the last years. It was the first method to expand the instruments useful mass range toabove 50,000 Da. However introduced in its present model in 1984, the technique returns to theinvestigations of electrically assisted liquid dispersion in the beginning of this century. In fact, themain discovery was almost accidental in 1968 when Malcolm Dole and cooperators were able tobring macromolecules to the gas phase at atmospheric pressure. It was possible by spraying a samplesolution to a small tube with a strong electric field in the presence of a warm nitrogen flow to helpin the dissolvation and then measuring the ions formed. Later, innovative experiences in this fieldled to the introduction of an ES ionization source. Since then, a wide range of biomolecules wasinvestigated by ES. The sample is usually dissolved in a mixture of water and organic solvent,typically methanol, isopropanol and acetonitrile: it can be directly infused or injected in continuousflow, that is, contained in the eluant of a HPLC column and CE capillary column.

The ES source is simples, forming a spray occurring in a high voltage field as showed in Figure 5. Ina proposed mechanism, it is believed that the ion formation is the result of an ionic evaporationprocess, first proposed in 1976. A droplets spray is generated by the electrostatic dispersion of theliquid applied by the capillary end. Favored by a heated gas (usually nitrogen), the droplets aredisaggregated, lose solvent molecules in the process and occasionally produce individual ions. Inanother proposed mechanism, the droplets dissolvation lead to an increasing charge density on thedroplet surface that will cause a coulomb explosion eventually producing individual ions.

Regardless the proposed mechanism, the ions are formed at atmospheric pressure and enter into abore located in the vortex of a cone acting as the first barrier to the vacuum phase. A skimmercollects the ions and guides them to the mass spectrometer.

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DropAtmosphericPressure Droplet Clusters and

ionic specimens

Pump

Pump

Ions

The spray formation is the most import part of the ES technique. It is usually advised to filter allsolvents; high electrolyte concentrations should be avoided as they can cause ionization suppressionsand unstable operating conditions. High flows compatible to those used in HPLC, can be now usedthrough a heated nebulizing gas to help in producing the spray.

For macromolecules, each ion that usually enters the mass spectrometer has a high charge number.As the mass spectrometers measure the mass/charge ratio instead of the mass, it is possible thathigh molecular mass molecules have enough charge to fall within the m/z range of a linearquadrupole, typically m/z 20-4000. As showed in Figure 6A, high molecular mass ions often have awide distribution of charge status. The figure shows the mass spectrum of horse myoglobin (molecularmass: 16951.5) at low resolution having observed charges of +12 to +21. This ionic distributionallows the calculation of the original analyte molecular mass by means of neighbor ions with m/zvalues m1 and m2 , with n1 and n2 charges, respectively. If m1 < m2, and n2 = n1-1, then,

M = n1(m1-mA) = n2 (m2-mA) (1)n2 = (m1-mA)/(m2-m1) (2)

wherein M represents the molecular mass of the uncharged molecule and mA is the mass of thecharged adduct A (for example H+, Na+, NH4+). The equations can be then solved to yield n2and M. The measure of the charges distribution in macromoleules is not always easy or reproducibleonce changes, such as relatively small changes of the analysis conditions can occur, for example:pH; addition of solvents or salts; protein partial denaturation; breakdown of disulfide bonds, etc.

The electrospray source is used in connection to all single mass analyzers.

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5.3.2. Mass analyzers

After the admittance of the molecules into the ions source and subsequent ionization, it is necessaryto determine the corresponding masses of ions formed so as to obtain the mass spectrum. Thefunction of the mass analyzer is to separate ions according to their mass/charge ratios and transmitthem to the detector.

There are several kinds of mass analyzers. The most common and widely used are the “Quadrupole”and “Ion Traps” analyzers.

The quadrupole are scanning analyzers, that is, after the admittance of a mixture of ions withdifferent mass/charge ratios (m/z) and different abundances, electric fields are applied; at a giventime, only ions with a specific mass can leave intact. By varying the electric field applied, one canselect and record different ions.

The “Ion Trap” analyzers, however, are not regarded as pure scanning devices, as the ions are storedbefore the scanning itself.

5.3.2.1. Quadrupole mass analyzers

The instrument is based on four parallel bars in a quadrangular area where the ions beam is focusedon the central axis of these bars, a fixed electric potential (DC) and a radio frequency potential (RF)are applied to these diagonal and opposite bars. For a given RF and DC combination, ions of aspecific mass range m/z have their path changed from the central axis. The mass spectrum isobtained from the DC voltage and RF components in a synchronized fashion, that is, keeping theRF/DC ratio constant. The φo potential applied to the opposite bar pairs is determined as follows:

±±±±± φφφφφo = U + V cos φφφφφt

wherein U is a DC voltage and V cos φt the time-dependent voltage in which V the RF amplitudeand φ the RF frequency.

Figure 2. Schematic representation of a linear quadrupole analyzer

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Ions beam

Ionssource

Detectoroutput

QuadrupoleArchitecture

The quadrupole operation (as well as the ion trap to be discussed later) can be qualitatively discussedthrough the stability diagram corresponding the DC potential amplitude, the RF potential amplitudewith the path of a stable ion (that is, an ion that can remain intact after passing the quadrupole).This will be represented in the equations and in graph 1.

The motion equation of a charged particle can be expressed as a Mathieu equation in which the au

and qu parameters can be defined.

au = ax = -ay = 4zU / mφ 2ro2

qu = qx = -qy = 2zV / mφ 2ro2

wherein m/z is the ion mass/charge ration, and ro is half of the distance between the two oppositebars. There is no parameter for z, since the RF field acts on the x/y plane (z is the main axis of thelinear quadrupole).

Graph 1. Stability diagram in the analyzer

In this case, the ion m2 is the only ion that remains stable (observe it is within the stability region ofthe graph), while m3 and m1 cannot reach the detector. The R=100 2e R=10 straight lines representtwo distinct combinations of the DC/RF ratio; by changing these ratios, it is possible to change themass filter resolution, that is, the capacity to differentiate or filter masses very close to each other.The “a” and “q” parameters are respectively proportional to the DC and RF values.

5.3.2.2. “Ion trap” quadrupole mass analyzers

This mass analyzer has the same mathematic operation principles of the conventional quadrupole,that is, ions stability within a specific path.

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However, the major difference is that in the ion trap the ions do not follow a single path towards thedetector, but are “trapped” in orbits within the trap structure, thus giving raise to the name “iontrap”.

While in the quadrupole the ions are formed in the ions source and then expelled towards the massanalyzer, in the ion trap the ions are formed ions and the mass analyzed in the same space region.This space region where the “trapped” ions are found corresponds to approximately the volume of1 cm-side cube.

The figure below shows a schematic representation of the ion trap analyzer.

Figure 3. Schematic representation of the “ion trap” analyzer

As the quadrupole analyzers, the ion traps analyzers also use electric fields; these electric fields areintended to keep the ions confined and separate the masses (mass analysis).

In this kind of mass analyzer, the electric field used is purely RF (radio frequency consisting in a sinewave with a frequency of about 1 Mhz) applied directly on the central annular electrode.

Thus, depending on the RF amplitude applied, the ions can remain stable inside the trap. By increasingthis amplitude, the ions with greater masses are “ejected” from the confinement region and thenreach the detector.

Another peculiarity of the ion trap analyzers is the need to control the number of ions within thestructure, aiming to avoid reactions of these ions with molecules still present. These interactionscan lead to a slight change in the final spectrum of some compounds, thus making interpretation/identification difficult.

This problem is eliminated in the quadrupole analyzers, as the ions are almost immediately “thrown”from the ions source after the formation and do not have the chance to interact with the moleculespresent.

On the other hand, the final sensitivity in the full scanning mode is greater in trap analyzers since allions formed are necessarily detected once they are confined.

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This is not the situation for the quadrupole analyzers. That is, while the quadrupole is canning themass range aiming to produce a spectrum, the unstable ions get “lost” and, therefore, do not generatea detectable signal. It is possible, however, to significantly increase the quadrupole sensitivity bydefining a fixed mass (or a few masses) of interest; this technique is called SIM (Single Ion Monitoring).

5.3.2.3. Tandem mass spectrometry.

The tandem mass spectrometry, MS/MS or MSn wherein n=2, 3..., uses two or more mass analysissteps, one to preselect an ion and the others to analyze the induced fragments, for example, bycollision (CID) with an inert gas, such as argon or helium. This can be a tandem-in-space or tandem-in-time analysis.

Tandem-in-space means several mass analyzers in series. Various combinations are possible, themost common include: triple quadrupole (Q1qQ2), four sectors and hybrid instruments. Q representsa quadrupole mass filter and q a RF quadrupole only (collision chamber). In the case of the triplequadrupole, an ion of interest generated in the ionization source is selected with the first quadrupoleQ1, dissociated in the collision chamber q with energies up to 300 eV; the fragmentation productsare analyzed with the second quadrupole Q2.

Thus, it is possible to obtain information on the sequence of a peptide by selecting the ioncorresponding to the protoned peptide (called precursor ion) and analyzing the fragments of itsstructure using Q2. This process is called ions-product scanning. Several other types of scanning oranalytical experiments can be performed. For example, the search of all precursors of a givenfragment is called ions-precursors scanning. This can be reached by keeping Q2 steady at themass/charge ratio of the ion concerned and scanning all ions present at Q1, while the collision dissociationsat q take place. In another scanning mode, ions that lose a specific fragment can be identified byscanning Q1 and Q2 simultaneously, keeping the mass difference between the quadrupole analyzersequal to the mass of the neutral lost fragment.

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Ions sourceCollision gas

molecule

Ions-productformation

Detector

Total massspectrum

Massspectrumby C.I.D.

Tandem-in-time can be obtained using “ion trap” devices and ICR mass spectrometers (also calledFTMS). In fact, these devices are not limited to MS/MS experiments, but can reach multiple stages(MS/MS/MS…/MS or MSn). In such devices, the ions are selected by applying specific voltagepulses and dissociations normally occur by collisions with other gases.

5.3.3. Detectors

After selection by the analyzer, the ions are guided towards the detector where they will be convertedinto a measurable signal. Detectors can be divided in three groups. Photosensitive plates and Faradaycages are included in the first group and directly correlate the measured signal to the amount of theanalyzed ion. The second group includes the electrons multipliers, photomultipliers and microchannelplates that amplify the intensity of the received signal. These are the most commonly used detectorsand will be discussed herein. The third group is used in ICR devices (FTMS) and consist in a radiofrequency detector applied to the trapped ions.

5.3.3.1. Electrons multipliers

When reaching the conversion dinode where a negative potential is applied, positive or negativeions emit several secondary electrons. These electrons are accelerated towards an electrons multiplierand hit the walls with sufficient energy to remove some electrons. These electrons will hit the otherside of the wall, thus releasing more electrons. This cascade effect continues until a measurablecurrent is finally created at the end of the electrons multiplier.

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Ions

Electron multiplier

Secondaryelectrons

Electrons outlet

5.3.3.2. Microchannel plates

The microchannel plates consist in a plate containing parallel cylindrical microchannels. The inletside of these microchannels is kept with a negative potential of approximately 1kV when comparedto the outlet side. The electrons multiplication, started with the collision of an ion in these channels,occurs through a semiconductive substance that coats each channel and generates secondary electrons.Curved channels prevent the acceleration of positive ions towards the inlet. The cascade effectinside the channels can multiply the number of electrons in the order of 105 and the use of severalcoupled plates allow an amplification that can reach 108. At the outlet of each channel, a metalanode collects the electrons current and the signal is transmitted to the processor. Anothercharacteristic of these microchannel detectors is an extremely low signals multiplication time, makingthem inadequate for detection in devices such as time-of-flight analyzer.

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Electron multiplierSecondary electrons

ElectronsoutletIon

Inlet of an electron multiplier

Ions with different mass/charge rationsseparated in the space

5.3.3.3. Photomultiplier

This kind of detector comprises two conversion dinodes; a phosphorescent screen and aphotomultiplier. This detector, as the electrons multiplier and the microchannel plates, allows thedetection of positive and negative ions. Upon detection, the ions are accelerated towards the dinodehaving a reverse inverse polarity of that of the ion; the electrons are then released and acceleratedtowards the phosphorescent screen where they are converted into photons. The photons are thendetected by the photomultiplier. The phosphorescent screen surface is coated with a fine conductivealuminum layer so as to avoid the formation of charges that could refrain new electrons fromreaching it. The amplification reaches values from 104 to 105.


Specific computer programs integrally perform data acquisition and processing. These programsare in charge of several tasks, ranging from the control of the monitoring time of an ion to theconstruction of calibration curves where the areas (or heights) of unknown sample peaks areinterpolated, thus producing the desired quantitative datum. Each manufacturer has a data acquisitionand treatment program with different resources and limitations. Therefore, the comprehension ofthese programs and the future preparation of standard operating procedures (SOP) are crucial forconducting any study.

Details on the compounds quantification process are provided in item 4.6 of the HPLC technique.

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Ions

Secondary ions

Emitted photons

Photomultiplier


The purpose of the minimum installation requirements is to assure that the installation room is dulyevaluated and prepared with the consumables and supply items required for a perfect installation.

The manufacturers will provide a checklist to be used before the installation date. This checklist will bespecific for the instrument purchased; however, what should be generally noted is described next.

5.4.1. Bench requirements

The bench should be sized so as to properly accommodate the LC/MS equipment and make theuser feel comfortable during work.

The bench size should be compatible with the LC/MS size plus the data system (computer andprinter). Usually, three linear meters, is enough to accommodate the complete system.

The structure should be able to safely bear the equipment weight; therefore, a margin of 20% abovethe equipment weight should be considered. The structure has to be firm, with no vibration orswings.

Some free space behind the bench should be also allowed (do not place the bench touching the wall)so as to accommodate the items required for the equipment operation, for example, vacuum pumpsand water cooling and circulation systems.

The equipment manufacturers provide detailed descriptions of the required bench on thepreinstallation manuals.

5.4.2. Electric network

The electric network should be duly grounded and stabilized. The use of a magnetic key isrecommended in case of constant electric power supply failures.

The number of outlets should be adequate and in compliance with the system installation standards.In addition to the LC/MS module, outlets for the system HPLC and the data system, including theprinter, are to be considered.

The standard outlet for LC/MS will depend on the required operation voltage. Due to the highconsumption, these systems generally require 220 VAC feeding with capacity for 1.5 to 2 KVA (for theMS module only). It is worth noticing that the equipment requires 220 VAC in single or split phase.

The LC/MS manufacturer should inform the total power consumption as well as the powerconsumption per module (expressed in Watts or VA or KVA). For the sizing and distribution of theelectric system capacity, an electric professional should be contacted.

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The equipment does not tolerate electric network fluctuations; therefore, it is critical to know thevoltage stability and determine that voltage is compatible with the required specifications of eachmodel.

5.4.3. Environmental conditions

Environmental conditions for LC/MS systems do not include temperature and humidity only. Thiskind of equipment also requires gas feeding and some of them require the circulation of cooledwater, in addition to exhaustion.

The temperature of the room where the LC/MS will be installed should be controlled with nomajor fluctuations during use. The typical temperature required by the manufacturers is 25ºC, withfluctuations of +/- 2ºC. A LC/MS system dissipates heat from 6800 to 8000 BTU; this heat shouldbe taken into account when sizing the air conditioner.

Humidity should be below 95%; no condensation is allowed.

Gas (nitrogen) can be fed from cylinders, liquid nitrogen compartments or nitrogen generators (thislatter being most indicated). Typical consumption is in the order of 15 L/min and purity should be99.5% for cylinders and 98.0% for other nitrogen sources. Working pressure should be 80 to 100 psi.

The room should be also provided with an exhaustion system with a minimum capacity of 15 L/min. This system should be connected to the vacuum pump and the ionization source outlet.


Maintenance by the user is critical for the good operation and durability of any equipment.

In addition to the basic precautions of the HPLC system described in chapter 4 above, some essentialitems should be regarded for a LC/MS system:

5.5.1. Vacuum system

High vacuum systems usually have three pumps, one auxiliary (mechanical) and two turbomolecularpumps.

The mechanical pump requires fluid replacement at least every six months, whether it is used ornot. Some of them also have filters that have to be replaced and/or verified at intervals set by themanufacturer (typically six months).

The turbomolecular pumps (high vacuum) are more specific and depend on the model andrecommendations of each manufacturer. Some of them have permanent lubrication, but othersneed lubrication at regular intervals. Some others, yet, require the bearings to be replaced atpredetermined intervals. Therefore, it is critical that the maintenance instructions are obtained from

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the supplier and that the process is documented and followed, since a failure of such systems maydefinitely impair the equipment as a whole.

The vacuum (pre and high vacuum) should be read daily and their values weekly recorded in a logbook.

5.5.2. Ions source

The ions source is a part of the equipment most subject to dirt and contaminations since it receivesthe mobile phase with the sample at atmospheric pressure and converts them into ions for thevacuum system.

Maintenance and cleaning should be made based on the usage background of each equipment andwill depend on the type, concentration and number of samples injected. Once established, thecleaning interval should be followed and documented.

The user should be familiar with the replacement of consumable parts of the system. Anymaintenance intervention should be reported in a logbook.

5.5.3. Nitrogen system

The nitrogen system should be checked at predetermined intervals. A nitrogen generator is normallychosen due to the cost/benefit ratio. Such devices are provided with filters, compressors, etc. thatalso require periodic maintenance.

5.5.4. Basic precautions training

In addition to the equipment operation, therefore, the user’s training should include these basicprecautions. Due professional qualification is critical for this learning level.

It is important to observe the safety rules when handling the equipment. Wearing apron, gloves andgoggles avoids severe injuries such as blindness or sample contamination, for example, plasma.

The system decontamination is always mandatory in order to protect the health of the person incharge of maintenance.

5.5.5. Autotune files

The LC/MS systems are provided with autotune programs to be performed at regular intervals.These programs generate reports that should be filed for the good operation background and tohelp with repair interventions.

Basic equipment precautions, such as performance checking, range from equipment to equipment.Thus, the best procedures to perform such tasks are those suggested by the manufacturer. Theseprocedures should be read and a standard operating procedure (SOP) created to define the frequencyand kind of task to be performed. Any non-compliance with this SOP should be documented andjustified.

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6. ANALYTICAL INSTRUMENTS PERFORMANCE CHECKING

6.1. Introduction

By the middle of 1990’s, instruments performance checking started to be have a major importanceand the first focus was on the HPLC systems as a result of its wide use in pharmaceutical industries;however, this concern covers all kinds of instruments used in a laboratory.

As in data management systems, these services are usually offered by analytical instrumentmanufacturers. There are different terminologies among them sometimes referring to the sameservices rendered. For example, the Performance Checking of an analytical instrument can be alsocalled Qualification, Operation Qualification (OQ) or Recertification.

It is important to note that, regardless of the terminology used by manufacturers, it should correspondto the analytical instrumentation system performance checking following a documented procedureso as to meet the requirements of a quality system and in compliance with the GLP (Good LaboratoryPractices) at appropriate intervals defined for each laboratory according to the usage and/or workperformed.

The instrumentation companies recommend this checking at regular periods every 6 months or 1year and whenever the instrument is subject to service or repair that may directly interfere with itsperformance.

The Validation terminology exists for a long time and has some variations according to the countryand company. The best definition for validation is: the set of documented activities and/orinformation evidencing a high safety degree of the process/system employed, meeting thepredetermined specifications with the proper quality attributes. Therefore, validation is not aperformance checking, qualification, operation qualification (OQ) or recertification process only,but a process that should include the following phases:

• DQ - Design Qualification;• IQ – Installation Qualification;• OQ – Operation Qualification;• PQ – Performance Qualification.

6.1.1. DQ – Design Qualification

Within the instrument design qualification, access should be allowed to the instrument developmentdetails, such as:

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• Use of strict specification methods and design during the system development.• Complete documentation of the quality assurance and quality control procedures.• Use of experienced and qualified personnel.• Comprehensive instrument test plan.• Control data, error reports and corrective procedures applied during the system development

and production.• System development background.

Such information should be provided by the instrument manufacturers, however, aiming to checkthe specifications and proposed use adequacy.

6.1.2. IQ – Installation Qualification

The installation qualification means: checking all documentation and information for installing theinstrument and software according to the system specifications described in the installationprerequisites and local regulations. The IQ defines that the instrument is received as specified,properly installed and under operation conditions. This activity should include:

• Checking the items received according to the appropriate documentation.• Filing of all description details and identification of the system components, including the

instrument connection diagrams.• Copies of the user’s test certificate and declaration of compliance.

The Installation Qualification (IQ) has been discussed in every analytical instrumentation techniqueof this chapter as Minimum Installation Requirements.

6.1.3. OQ –Operation Qualification

The Operation Qualification should be documented, checking that the system meets the operatingspecifications. A follow-up system should be used to verify that:

• Operational training is given after the installation and before starting the routine operation of acertain instrument.

• User commitment to follow all steps required.• Performance tests are conducted according to the instrument manufacturer in order to assure

the instrument is under appropriate operation conditions.

6.1.4. PQ –Performance Qualification

The PQ consists in conducting activities to certify that all the instrument specifications are accordingto the usage requirements. This qualification should be strict and involve the tests needed todemonstrate the instrument functionality. It should include details such as:

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• Specific tests of individual components or the system as a whole.• List of tests.• Frequency of tests.• Expected results.• Acceptance criterion.• How tests are documented.• Operator’s qualification required.• Actions to be taken in case of test failure.

For some analytical instrumentation techniques, there are routines defined in internationally accreditedliterature, such as the EP/BP (European Pharmacopoeia), USP (United States Pharmacopoeia) andothers, setting parameters to be determined to validate an analytical system to be used by the user.They are many times performed by the analytical instruments manufacturers.

We cover procedures normally used for checking the performance of the main analyticalinstrumentation techniques and, when applicable, the main Performance Qualification (PQ) routinesdescribed in the international literature.

6.2. Ultra violet – visible (UV-VIS) spectrophotometry

When checking the performance (Operation Qualification) of an analytical instrument, the firststep to be taken is a Preventive Maintenance aiming to check the instrument operation status inadvance, prevent and repair any possible failure.

6.2.1. Preventive maintenance

Preventive maintenance of a UV-Vis spectrophotometer is crucial for the instrument life and shouldbe conducted at 6-month intervals. This maintenance has to include specific activities carried out byan expert. A preventive maintenance routine should include:

• Checking the instrument safety items.Inspection of the sample compartments.Checking the lids and protection sensors.

• Checking the optical system.Checking and cleaning external optics.Conducting performance tests: wavelength accuracy and precision, baseline correction andphotometric noise.Checking the lamps conditions and alignment.

• Electronic checking.Checking the instrument internal diagnostics.Feeding source and electric network checking.Checking cables and connectors.Cleaning electronic circuits and wearing, corrosion and failures.

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• Checking mechanical parts.Testing motors inside the instrument.Checking operation and communication with accessories.

6.2.2. Operation Qualification (OQ)

The OQ of an Ultraviolet-Visible spectrophotometer consisting in checking the following:

• Wavelength accuracy;• Wavelength precision;• Baseline deviation;• Photometric noise.

6.2.3. Performance Qualification (PQ)

Some of the PQ routines that can be used with UV-Vis spectrophotometers and defined at the EPand USP are described below.

6.2.3.1. European pharmacopoeia

The tests below are listed in the European Pharmacopoeia and British Pharmacopoeia (EP/BP) forthe validation process of instruments, being divided into:

• Wavelengths accuracy. Tests aiming to check the wavelength positioning accuracy in order toassure low deviation of the optical system positioning. EP/BP recommends two routines forchecking this item:Deuterium, xenon and mercury-line emission method.Holmium perchlorate method.

• Monochromator resolution. Test to verify whether the instrument monochromator separatesthe spectrum light and sends a small amount of this light through the sample compartments.This test is conducted using the Toluene / Hexane method.

• Stray Light. Stray Light is defined as the amount of light reaching the detector when no wavelengthhas been selected. Stray light causes deviations in the Beer-Lambert Law, thus reducing thesystem linearity. This test is performed by measuring the transmittance passing through a solution;next, the same reading is made with the source turned off. The method used is KCl at 200 nm.

• Photometric accuracy. The photometric accuracy is determined by measuring known absorbancevalues and calculating the difference between the expected value and the actual value measuredby the instrument. The method used is Potassium Dichromate.

6.2.3.2. United States Pharmacopoeia

The tests below are listed in the United States Pharmacopoeia (USP) for the validation process ofinstruments, being divided into:

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• Tests aiming to check the wavelength positioning accuracy in order to assure low deviation ofthe optical system positioning. USP recommends two routines for checking this item:Deuterium, xenon and mercury-line emission method.Holmium perchlorate method.

• Photometric accuracy. The photometric accuracy is determined by measuring known absorbancevalues and calculating the difference between the expected value and the actual value measuredby the instrument. The methods used include:Potassium Dichromate method.NIST filter method.

6.3. Gas chromatography – GC

6.3.1. GC preventive maintenance

Before the Operation Qualification (OQ) step of a GC, it is necessary to follow the PreventiveMaintenance procedures suggested by the manufacturer.

In a Gas Chromatography System, the Preventive Maintenance routine include:

• Pneumatic System cleaning and checking leakages;• Electronic circuits cleaning and checking the voltages used;• Venting Systems cleaning and checking functionality;• Injector(s) and detector(s) cleaning, checking leakages and voltages used;• Detectors response checking test.

6.3.2. Operation qualification

The Operation Qualification of a gas chromatography system consisting in verifying the following:

• Control of carrier gas and auxiliary gas flows;• Control of the Temperature of Injector(s), Detector(s) and Column Oven;• Detector(s) signal repeatability;• Automatic sampler repeatability, when applicable;• Calculations made by the Data System.

6.3.2.1. Flows control

The purpose of this checking is the repeatability of the analyzed compounds retention times,repeatability of the volume of sample introduced in the chromatographic column, in case of “split”injectors and repeatability of the detector response. Usually, chromatographers can be providedwith electronic flow controllers (EFC) or manual controllers.

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The materials required for this verification in both cases include:

• Calibrated Digital Flow Meter;• Reference or test-specific chromatographic column.

In addition to the carrier gas flow passing through the column, the flow control checking shouldalso include the septum purging flows, the division rate in case of a “split/splitless” injector, the“make-up” flow and auxiliary flows of the detector, hydrogen and air in case of a FID detector.These tests are usually conducted under certain conditions previously defined in documentedprocedures.

6.3.2.2. Temperatures control

The columns oven is the most important part and interferes with a chromatographic analysis; smallfluctuations of the oven temperature may result in significant variations of the retention time and,as a result, affect the analytical repeatability.

The materials required for this verification include:

• Calibrated Temperature Meters:• Reference or test-specific chromatographic column;• Reference standard sample.

The Injector(s) and Detector(s) operating temperatures are checked using temperature meters; inaddition to the temperature checking, the columns oven are subject to retention time repeatabilitytests under previously set conditions and reference sample.

6.3.2.3. Detector(s) signal precision

This checking aims to evaluate the detector repeatability; however, in most of the procedures followed,this can be used as the repeatability check for the system as a whole.

The materials required for this verification include:

• Reference or test-specific chromatographic column;• Reference standard sample.

This checking usually takes place under a certain analytical condition by means of consecutiveinjections of a reference standard sample. The purpose is to calculate the relative standard deviationas percentage of this standard sample areas.

6.3.2.4. Automatic sampler precision

Automatic Samplers are checked under the same conditions of the detectors signal repeatabilitychecking; thus, the results obtained as RSD% of the area values are used twice.

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6.3.2.5. Calculations by the data system

Checkings usually made in Data Systems refer to the calculations used in the workstation and include:

• Retention Times Calculation;• Areas Count Calculation;• Time Events Calculation;• Calculation of the Calibration Curves Coefficients for External and Internal Standardization;• Corrected Area Normalizations Calculation.

These checkings are usually made by protected files supplied by the data system manufacturer;when used, these files allow to check whether calculations are in accordance with the results definedin the reference files.

Once the Operation Qualification of a Gas Chromatography system is completed, all such recordsshould be stored at an easily accessible location.

Manufacturers usually provide Manuals with all the compliance documentation of thechromatography system, including information for the Design and System Development Qualification(DQ), Installation Qualification (IQ) that should contain information on which system was installedand the Operation Qualification (OQ), including all procedures to be followed and storage ofchecking records. Therefore, all interventions should be evidenced in this Manual, used as a systembackground.

6.4. Liquid chromatography - HPLC

6.4.1. HPLC Preventive maintenance

Before starting the HPLC system Operation Qualification (OQ) process, it is necessary to followthe preventive maintenance procedures suggested by the manufacturer.

In a HPLC System, the preventive maintenance routine include:

Electronics: cleaning of electronic parts and checking feeding and internal voltages, check thedetectors’ lamps functionality and evaluate their life.

Hydraulics: Cleaning of the pipings and pneumatic system components, check and eliminate anypossible microleakage and occasional replacement of the seals and filters of the mobile phase andpump, check the pressure transducer functionality and the pulses muffling system when applicable,cleaning and checking the sample introduction system functionality.

Mechanics: Cleaning and lubrication of mechanical parts of the automatic sampler pumping system.

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The Operation Qualification process showed below is schematically represented in Figure 1:

Figure 1. Schematic view of the Performance checking procedure for a HPLC System.

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A – Detector– Wavelength accuracy;– Wavelength precision;– Noise Level and Baseline Deviation;– Detector Linearity (optional);– Detector Signal Precision (optional).

B – Pump Qualification– Flow accuracy;– Ramp and pressure drop test;– Gradient Mixture Accuracy;– Gradient Mixture Linearity (optional).

C – Columns Oven Qualification– Temperature Accuracy

D – Automatic Injector Qualification– Injection Volume Precision;– Injection Volume Accuracy (optional);– Injection Linearity (optional).

E – Data System Qualification– Calculation of the retention times, areas

count and calibration curves coefficients.

As a HPLC System is usually made of several modules, each module should be subject to theOperation Qualification. Thus, the Validation process will be completed when the PerformanceQualification (PQ) is conducted. PQ should be always conducted using the complete HPLC Systemin activities confirming that all the instrument specifications meet the usage requirements.

6.4.2.1. Detector qualification

The most important parameters to be verified in an Absorbance detector are:

• Wavelength accuracy;• The purpose of this test is to check the accuracy when selecting a wavelength so as to assure

lowpositioning deviation of the optical system diffraction grid;• Wavelength precision;• The deuterium or xenon-line emission method can be used (both regarded as a natural standard)

and using a certified chromophoric substance standard;• Noise Level and Baseline Deviation;• This test is intended to assure the detector sensitivity specification, since these factors are directly

related to this measurement. The baseline monitoring and measurement method can be used atestablished time intervals and standard values verification.

6.4.2.2. Pump qualification

6.4.2.2.1. Mobile phase flow accuracy

This test is intended to assure that the programmed flow is the actual pump operation flow. It canbe determined by measuring (volumetrically) the pumped flow at a certain period of time.

6.4.2.2.2. Ramp and pressure drop test

The ramp test checks the inlet and outlet valves, connection leakages and the pump capacity tooperate at high pressure.

6.4.2.2.3. Gradient mixture accuracy

The purpose of this test is to check the accuracy of the static and dynamic composition of mobilephase mixtures during an analysis in gradient systems.

The test of pumping two mobile phases at different ratios through tempo can be used and checkthe actual composition established.

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6.4.2.3. Columns oven qualification

6.4.2.3.1. Oven temperature accuracy

With the purpose of checking the oven temperature accuracy, one can use the method ofprogramming a temperature of the oven and measure it using a calibrated electronic thermometerhaving a flexible tip that can be adapted to the oven internal length.

6.4.2.4. Automatic sampler qualification

6.4.2.4.1. Automatic sampler precision

In order to assure the specified precision of the automatic sampler, one can use the test of injectingsuccessive samples of a standard solution, thus obtaining the RSD% between the peaks areas; avalue below 1% is recommended.

6.4.2.5. Data system

The Data System Qualification for HPLC should be conducted according to the same GC procedures.

Once the Operation Qualification of a Gas Chromatography system is completed, all such recordsshould stores in an easily accessible location.

6.5. Chromatography systems connected to mass detectors

6.5.1. Mass detector systems preventive maintenance

As the previous systems, preventive maintenance of mass detector systems should be made beforethe Operation Qualification (OQ) process.

Each manufacturer provides a checklist of the equipment concerned. This checklist should berequested and completed in every preventive maintenance intervention.

The items to be checked in a preventive maintenance are basically divided into three modules:

• Vacuum system;• Mass detector;• Chromatographer.

6.5.1.1. Vacuum systems

The vacuum system is critical for the good operation of any mass spectrometry system and, therefore,regular maintenances are required. These maintenances should be recorded in the equipmentoperation background.

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The items checked in a vacuum system include:

• Checking and replacing the pumps lubricant fluids (auxiliary and high vacuum pumps);• Checking hoses and vacuum connections;• Measuring and recording high-vacuum and pre-vacuum readings on a regular basis.

6.5.1.2. Mass detector

The mass detectors are provided with an autotune or auto-calibration program to be used forevaluating the equipment operation conditions. This program usually produces a reported (to befiled); this report will be used to follow-up the performance as well as prematurely identify failures,for example, the need of cleaning the ions source. It is recommended that these reports are filed atleast once a week, being kept for the period of the performance checking.

The items observed in this report are: mass accuracy, peaks resolution and response as a function ofthe mass.

The ions source should be cleaned whenever required; however, a minimum cleaning frequencyshould be adopted and will depend on how the equipment is being used (sample type, number ofsamples, etc).

6.5.1.3. Chromatographer

The chromatographer preventive maintenance should be simultaneous with the mass detector. Thisprocedure was described in the corresponding chapters (GC or HPLC).


The Operation Qualification is critical to assure the equipment is operating according to themanufacturer’s specifications and, therefore, at regular intervals. Manufacturers recommend theoperation qualification at least once a year or after a major repair intervention. The expert in chargeof that intervention will have means to recommend whether an Operation Qualification after therepair is required. This evaluation should be documented.

The items evaluated in an Operation Qualification include:

• Injector precision;• Injector linearity;• Contamination test from sample to sample (carry-over);• Detector linearity (optional);• Mass accuracy;• Sensitivity.

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6.5.2.1. Injector precision

This test is conducted by injecting several aliquots of a known standard and by verifying the areareproducibility and/or height. Six to ten measurements are made and the relative standard deviationcalculated (it should be within the predetermined standards).

6.5.2.2. Injector linearity

During this test, several aliquots of a standard with a known concentration will be injected; theinjected volume is also ranged. Then, the linearity as a function of the injected volume will beanalyzed using a calibration curve. A correlation factor is used to evaluate the test. The results arethen compared to the predetermined acceptance values.

6.5.2.3. Carry-over

The “carry-over” test or contamination from sample to sample is conducted by injecting a highconcentration standard and then solvent only. The result is optimum when no residue of the previouslyinjected standard is detected during the solvent injection. The limits are usually established as loweror equal to a certain area or height.

6.5.2.4. Detector linearity

A mass detector becomes non-linear when the sample concentration increase does not lead to aproportional increase in the formation and detection of ions.

The test is conducted by drawing different concentrations and plotting the result in a calibrationcurve. The correlation factor is evaluated by comparison with the predetermined acceptance results.

6.5.2.5. Mass accuracy

The mass accuracy is evaluated by injecting a known standard either using the auto-calibrationsystem or the samples injector.

The results are evaluated by comparing the mass obtained using the equipment to the selectedstandard theoretical value. Each manufacturer has recommended standards and acceptance criteriafor this test.

6.5.2.6. Sensitivity

The test is conducted by injecting a known standard and checking the signal-noise ratio of the peakspecified in the chromatogram thus obtained. Each equipment has different approval values for thistest.

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7. REFERENCES

Apostila do Curso de Espectrofotometria de UV-Vis, Varian Brasil (1992).Apostila do Curso de Espectrofotometria de UV-Vis, Varian Brasil (1998).Apostila do Curso de Treinamento de Espectroscopia, Parte A . Testes de performance, VarianBrasil (1999).Manual de Pré-Instalação de Espectrofotômetros UV-Vis, Varian Brasil (1998).Instrução de Trabalho . Manutenção Preventiva - Cary 1/100/3/300, ITST 02 ATOtto Alcides Ohlweiler - Química Analítica Quantitativa, Livros Técnicos e Científicos Editora,2a.Edição, RJ (1980).Cienfuegos, F., Vaitsman - , D. Análise Instrumental, Interciência, RJ. (2000).Carol II, Collins, Gilberto L. Braga e Pierina S. Bonato - Introdução a Métodos Cromatográficos,Editora da UNICAMP, 6a ed., Campinas- SP (1995).Validation Overview , 85-101741-00, Varian (1999) .Back to Basics. Micromass UK (1996).Varian CP-3800 Gas Chromatograph Regulatory Compliance Documentation, Varian, Walnut Creek.(2000).Techniques of Gas Chromatography, Varian, Walnut Creek (1995).E. D. Hoffmann, J. Charette, V. Stroobant - “Mass spectrometry - Principles and Applications”Wiley, New York (1996).W. Paul, H. Steinwedel, - “A new mass spectrometer without a magnetic field”, Z. Naturforsch., 8a,448-450, (1953).E. Fischer, - “Three-dimensional stabilization of charge carriers in a quadrupole field”, Z. Phys.,156, 1-26, (1959).Snyder, L.R.; Kirkland, J.J. - Introduction to Modern Liquid Chomatography, 2.ed., New York,John Wiley and Sons (1979).Snyder, L.R.; Kirkland, J.J.; Glajch, J.L - Practical HPLC Method Development. 2.ed. New York,John Wiley and Sons (1997).Riley, C.M. - Efficiency, retention, selectivy and resolution in chromatography. In WAINER, I.W.;LOUGH, W.J. eds. High Performance Liquid Chomatography Fundamental Principles and Practice.Glasgow, Blackie Academic & Professional. (1995)Scott, R.P.W - Silica Gel and Bonded Phases: Their Production, Properties and Use in LC. NewYork, John Wiley and Sons. (1993)Grant, D. W. Capilary Gas Chromatography. West Sussex. Jonh Wiley & Sons. (1996).Scott, R. P. W. Introduction to Analytical Gas Chromatography. New York. Marcel Dekker. (1981).McNair, H. M., Bonelli, E. J. Basic Gas Chomatography. Walnut Creek. Varian. (1969).Zweig, G., Sherma, J. CRC Handbook of Chromatography . Volume II. Cleveland. (1977).Walker, John Q.Chromatographic Systems 2nd ed.Academic Press, INC, San Diego, California USA(1977)

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Anvisa Ba Be Guideline II

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