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Issue 3 • 2012 Analytix Polymer Reference Materials Polymer Reference Materials Certified Reference Materials for Genotoxics LC/MS Test Mixtures Rapid Screening for Micro- organisms HPLC Buffers Ionic Liquids in MALDI-MS Applications Water Content in Aldehydes and Ketones
Page 1: Analytix - Sigma-Aldrich

Issue 3 • 2012

AnalytixPolymer Reference Materials

• Polymer Reference Materials • Certified Reference Materials

for Genotoxics • LC/MS Test Mixtures • Rapid Screening for Micro­

organisms • HPLC Buffers • Ionic Liquids in MALDI­MS

Applications • Water Content in Aldehydes

and Ketones

Page 2: Analytix - Sigma-Aldrich


Polymer Reference Materials

Dear Reader, Cotton shirts, laptops, children’s toys, plastic bags, tires, pencils … All of these diverse items in our everyday lives have something in com­mon – they all consist of polymers, either syn­thetic ones such as polyolefin, polyurethane, polypropylene or polyacrylate, or natural ones such as cellulose, starch, or caoutchouc. The molecular weight and the molecular weight distribution of polymers are crucial parameters that determine their specific properties. There­fore, we can assume that most of these poly­mers that surround us have undergone a rigid quality control with special focus on their molecular weight distribution.

The analytical technique of choice is gel perme­ation chromatography (GPC). The diversity of application fields for this technique is almost as huge as the diversity of the polymers that exist. Its use is not only important in the plastic and textile industry, but also in both the food and pharma industries for testing the properties of excipients of pharmaceuticals.

To ensure the accuracy of these measurements, the use of reliable analytical standards or certi­fied reference materials is crucial. This is impor­tant because you can only really trust your analytical results if you can trust the quality of your reference materials. The featured article of this issue of Analytix (page 4) gives an overview of the wide variety of polymer standards for the calibration of GPC instruments available at Sigma­Aldrich.

To gain insight into our comprehensive portfolio of analyti­cal standards and reagents, I recommend that you visit our webpage at sigma-aldrich/analytical and hope you enjoy this issue of Analytix.

Best regards,

Dr. Matthias Nold Product Manager Analytical Standards [email protected]. Matthias Nold

Product Manager Analytical Standards

Analytix is published five times per year by Sigma-Aldrich Chemie GmbH, MarCom Europe, Industriestrasse 25, CH-9471 Buchs SG, Switzerland Publisher: Sigma-Aldrich Marketing Communications Europe Publication director: Ingo Haag, PhD Editor: Isabell Davies-Falch





Page 3: Analytix - Sigma-Aldrich



e of






14 Reversed-phase HPLC Buffers High quality buffers (solutions, solids or


15 High-Purity Headspace Grade N,N-Dimethylacetamide in the Analysis of Residual Solvents

17 New Solvents and Additives Exclusively Designed for UHPLC



18 Second Generation Ionic Liquid Matrices for MALDI-MS


19 Volumetric Concentrates for Titration by Sigma-Aldrich

Prepare Acid, Base and Salt Standard Solutions with FIXANAL® Ampoules

21 Determination of Water Content in Aldehyde and Ketone Samples

HYDRANAL®­K Reagent Line for Karl Fischer Titration

Featured Article

4 Applications and Quality Requirements of Polymer Reference Materials


7 Certified Reference Materials for Genotoxic Sulfonic Acid Esters

8 Prescreened, In-Stock Chemicals Pesticides, PAHs, PCBs, VOCs, and More

8 Quality Antioxidant Standards in Convenient Kit

9 Maximize Sample Recovery with Center Drain (CD™) Vials

10 Is Your LC/MS System All Right? Mass Calibration and System Test Standards


12 PCR & DNA was Yesterday; the Future is RNA HybriScan®, a rapid and innovative screening

method for microorganisms based on the detection of rRNA.

Page 4: Analytix - Sigma-Aldrich







Applications and Quality Requirements of Polymer Reference Materials

The use of polymer reference standards for the calibration of GPC/SEC instruments with concentration detectors or viscometers is a well­known procedure [1]. In addition, many other instances exist where polymer reference stan­dards are used routinely. All these applications require well­ characterized reference materials. In this paper, we will discuss quality requirements for polymer standards as well as their scope of application.

Types of Reference MaterialsIn general, polymers are statistically distributed polymer chains. Controlled polymerization processes minimize the chain length distribution. They cannot be described by a single molar mass but by various molar mass average values.

Polymer reference materials are precisely characterized polymers in terms of molar mass average values, such as the molar mass average number (Mn) and the molar mass average weight (Mw). Besides this, the molar mass on the peak maximum (Mp) is important for narrow molar mass standards. Another significant parameter is the polydisper­sity index PDI (Mw/Mn) which describes the width of the molar mass distribution. We make a differentiation between narrow molar mass distribution (PDI <1.2) and broad molar mass distribution (PDI>1.5).

The reference materials can be based on a single molecule species (homopolymer), on two (copolymer) or on three different monomeric molecules (terpolymer). Copolymers and terpolymers are structured as block copolymers or sta­tistical polymers.

Polymer reference standards can show a well­defined structure, e.g. stars, combs, hyperbranched polymers, or even a precisely defined stereo structure (e.g. tacticity). Specific end groups are also possible.

Polymer reference materials are available as single standards or as calibration kits with well­characterized standards of different molar masses. Due to the different complexity of requirements, reference materials are very often designed and characterized according to the needs of the individual user.

Quality Requirements for Polymer Reference MaterialsEspecially in a regulated environment, particular attention is usually paid to the measurement results. This also applies to the molar mass results from GPC/SEC analysis. If possible, the information in the analysis certificates should be well proven and include the measurement uncertainty. The traceability of certificate information is important, i.e. the question of whether the measurements are traceable to generally accepted reference values or at least to the values of an absolute method. Of key importance is information on shelf life and stability as well as long­term and timely quality control by the manufacturer over the entire sales and shelf life of the materials. The reference materials described meet the challenge of the demand for high­quality reference standards with accurate measurements. Constant quality control of our different polymer batches available for sale is standard practice. In addition to the clas­sic GPC/SEC reference standards (molar mass values from GPC/SEC measurements, calibration results based on light scattering), more elaborate reference materials are also available. The difference here refers to quality criteria in terms of peak purity, polydispersity, number and type of characterization methods used for molecular weight deter­mination and the application area. Table 1 shows the methods used for the characterization of polymer refer­ence materials.

T. Hofe, H.-U. Ehmcke, M. Nold [email protected]

Classification of Polymer Standards

Characterization Methods Available as Individual Standard

Available as Kit

GPC organic and aqueous GPC DIN certified GPC + absolute method (NMR, light scattering, viscometry or

vapour pressure osmometry (VPO)

ERM GPC + scattering + viscosity and physical constants, Round­robin test

LS / Visco Validation GPC+light scattering + viscosity (NMR, light scattering, viscometry or vapour pressure osmometry (VPO)

Maldi Validation GPC + MALDI + absolute method ReadyCal GPC

Table 1 Characterization of polymer reference materials

Page 5: Analytix - Sigma-Aldrich







ReadyCal KitsPSS ReadyCal Standards (Figure 1) are polymer cocktails pre­weighted into autosampler vials. Each vial has three or four polymers of the same type with different molar masses. Carefully selected molar masses are placed into each vial so that the resulting chromatograms are baseline separated (if a proper col­umn combination is used).

A ReadyCal Kit allows you to pre­pare a fast and reproducible eight­ to twelve­point calibration curve

without the inconvenience of weighing samples. Just add solvent directly into the autosampler vial, let it stand for two hours, shake gently, and load into your autosampler for analy­sis. Each kit contains 30 autosampler vials (for at least ten cali­bration curves) that are color coded for your convenience.

DIN (German Institute for Standards) Certified StandardsPolymers characterized in the respective solvent (water or THF) according to GPC/SEC DIN requirements are called the DIN standards. In addition to GPC/SEC measurements (rela­tive method), the molar mass of each polymer is deter­mined by an absolute method such as light scattering detection MALS/MALLS (Mw), NMR, vapour pressure osmometry VPO (Mn) or MALDI (Mw, Mn). The results and the experimental conditions are also specified. DIN standards are available as individual standards or kits.

Round-Robin Tested Reference MaterialsThe premium reference materials are the European Refer­ence Materials (ERM) of the German Federal Institute for Material Research and Testing (BAM) in Berlin. These ERM are round­robin tested. The tests were coordinated by BAM and performed by selected laboratories under standard­

ized conditions. In addition to the GPC/SEC experiments, the round­robin tests also cover viscosity and light­scattering measurements. Thus, the molar mass results of these three methods are independent from laboratory and operator. The results were statistically evalu­ated and besides the average values, the mea­surement uncertainties as well as the standard deviation of the round­robin test results were also quantified. In addition, the report also includes the results from NMR and IR measure­ments. Figure 2 shows the certificate of an ERM.

Validation kitsValidation kits allow the specific testing of complex systems and detectors as well as the determination of specific sys­tem and detector constants.

A light scattering/viscometry validation kit consists of narrow and broad distribution polymer standards (aqueous applications: dextran; organic applications: polystyrene, PMMA). The normal molar mass information, Mp from GPC/SEC and the Mark­Houwink constants K and α from the viscosity measurements, the molar mass average values Mw and Mn from GPC/SEC, MALLS and viscometry are also provided. Thus, online viscosity and light­scattering detec­tors can be checked. With the help of the information pro­vided, complex GPC­MALLS­viscosity detector couplings can be validated and corrected, if necessary. For example, using the K and α constants, it is possible to check and correct the inter­detector offset of the viscosity detector compared to the concentration detector (RI or UV).

MALDI kits are available as homologous polymers of dif­ferent molecular weights or as a mix of different polar polymers. In addition to enabling the measurement of the resolution of the spectrometer, they also help select the appropriate matrix for a particular class of polymers. The certificates contain the comparison of the molar mass information from a relative method with two absolute methods based on MALDI, NMR, VPO or MALS/MALLS. In addition, the MALDI spectrum, and the molecular weight distribution MWD from GPC/SEC, are provided.

Application for Polymer Reference StandardsSome of the practical uses of polymer reference materials are listed below.

Homopolymer Standards with Narrow Molar Mass Distribution • The most widely used application for homopolymer

standards with narrow molar mass distribution is the calibration of a GPC/SEC system by narrow standard calibration. The GPC/SEC calibration is performed to correlate the elution volume to a molar mass. Therefore, solutions of different standards are injected into the chromatograph. To create the calibration curve, the logarithm of the molar mass is plotted against the molar mass [1]. A typical calibration curve is shown in Figure 3.

• Universal calibration allows the conversion of existing calibrations to other types of polymers. The method is based on the assumption that the property which deter­mines the elution behavior in GPC is the hydrodynamic volume Vh [1].

(continued on page 6)

Figure 1 PSS ReadyCal kit

Figure 2 Analysis certificate of European Reference Material (ERM)

Certificate ERM®-FA001 All following pages are an integral part of the certificate.

Page 1 of 4




Molar mass Mw


Weight averaged molar mass Mw

Certified value 3) [g/mol]

Uncertainty 4)


87600 2245

Viscosity 2)

Intrinsic viscosity [η]

Certified value 3) [mL/g]

Uncertainty 4)


42.37 0.83

1) obtained by laser light scattering

2) obtained by viscometry using an UBBELOHDE viscometer according to DIN 51562 – 1

3) Unweighted mean value of the means of accepted sets of data, each set being obtained in a different laboratory. The certified value is traceable to the International System of units (SI).

4) The certified uncertainty is the expanded uncertainty estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) with a coverage factor k = 2, corresponding to a level of confidence of about 95 %. The certified uncertainty value is traceable to the International System of units (SI).

This certificate is valid for five years after purchase. This validity may be extended as further evidence of stability becomes available.

Sales date:

The minimum amount of sample to be used is 10 mg.

Accepted as an ERM®, Berlin,

Latest revision Berlin, 2008-05-29 BAM Berlin Department I Analytical Chemistry; Reference Materials 12200 Berlin, Germany

BAM Berlin Division I.3 Structural Analysis 12200 Berlin, Germany

Prof. Dr. U. Panne Dr. A. Thuenemann (Head of Department) (Head of Division)

Page 6: Analytix - Sigma-Aldrich







• The qualification and validation of instrumentation used for the analysis of natural or synthetic polymers and biopolymers. This includes:

– The GPC/SEC system including all detectors (RI,UV, ELSD, light­scattering detector/viscometer)

– MALD­ToF instruments

– Detectors for dn/dc determination in batch mode

• Normalization of MALLS/MALS (Multiangle {Laser} Light Scattering) detectors to obtain a precise determination of the radius of gyration, which describes the size of the molecule in solution.

• Inverse GPC/SEC allows the determination of the average pore size as well as pore size distribution of a material. The test material is filled in a column, which is installed in a GPC/SEC system, and flushed with eluent. Then the reference standards are injected. The elution volume and the molar mass of the reference material enables the calculation of the pore size parameter mentioned [2].

• To determine structure property relationships.

Homopolymer Standards with Broad Molar Mass Distribution • Broad standard calibration allows the precise analysis

of polymers even if narrow standards are not available for the specific polymer. It requires the existence of a base calibration curve and one or more broad polymer samples with given Mw, Mn or intrinsic viscosity [η] [1].

• Allow the detection of column mismatch. This is a shoul­der or side peak that does not result from the physical reality of a molar mass distribution but from uneven or dislocation of pore size distributions [3].

• Characterization of membranes by sieve curve determina­tion. The membrane to be characterized is used to filter a stock solution of a broad molar mass/molecular size distribution reference standard. The stock solution, the filtrate, and optionally the retained fraction (retentate) are analyzed with a GPC system. The comparison of these results allows the determination of important membrane properties such as average pore size, pore size distribu­tion, molar mass cut­off, membrane selectivity, retention efficiency, prep accessibility, membrane stability or hydro­phobic properties (see Figure 4). The sieve curves can help to identify low­quality membranes and establish quality parameters for working membranes [4].

Polymers with dedicated properties are used for deter­mination of physical properties if precise molar mass distri­bution, structure, or composition distribution is a need.

Summary The versatile structure of polymers, and complex analytical problems, all require a variety of reliable and well­character­ized reference materials to assure the quality of analytical results. This includes not only the analysis itself but also the operational check of the analytical instruments used. Poly­mer reference standards are well accepted in various fields of operation such as QC, R&D and in the world of science, as well as in the world of industry and production. They help to gain a deeper insight into the structure and property relations of macromolecules. Please visit the Sigma­Aldrich website at sigma-aldrich.com/polymerstandards to find the extensive range of polymer standards and certified ref­erence materials.

AuthorsProf. Dr. Thorsten Hofe and Dr. Hans­Ulrich Ehmcke PSS Polymer Standards Service GmbH In der Dalheimer Wiese 5, 55120 Mainz/Germany

References[1] Yau, W.W., Kirkland, J.J., D.D. Bly, Modern Size-Exclusion Liquid

Chromatography, John Wiley & Sons, New York, Chichester, Brisbane, Toronto, 1979.

[2] Dauwe, C., and Marme, S., Quality Assurance with Inverse GPC, GIT Laboratory Journal, 3, 106 (2002).

[3] Hofe, T., Beware of Mismatch, The Column, 4,21 (2008).[4] Kilz, P., Sieve Curves – Membrane Characterization Using

GPC/SEC, PSS Streamliner, 1,1 (2007).

Figure 3 Typical GPC/SEC calibration curve using narrow standards

Figure 4 Comparison of three different sieve curves obtained by a filtration experiment and GPC/SEC measurements. A stock solution has been prepared from a broad polymer standard. The analysis of the filtrate and the stock solution by GPC/SEC allows the creation of sieve curves.

Page 7: Analytix - Sigma-Aldrich






Certified Reference Materials for Genotoxic Sulfonic Acid Esters

Sulfonic acid derivatives, such as methanesulfonic acid (MSA), are frequently used in pharmaceutical chemistry. Mesylate salts of amine­containing drug substances may have advantages over the more commonly used hydro­chloride salts, such as higher solubility and bioavailability and the absence of an ion effect [1].

However, there are severe concerns about contamination with alkyl sulfonate ester traces, either as side products resulting from the crystallization and recrystallization pro­cess using methanol or ethanol as a solvent or as impurities of the MSA starting material. These sulfonic acid esters are known to have genotoxic and carcinogenic effects [2].

Therefore, not only the final pharmaceutical product, but also the alkyl sulfonic acids used as starting material for the salt formation must be meticulously tested for the presence of these compounds.

The new SAFC Pharma® Grade Product line provides GMP­ compliant building blocks and reagents for use in the phar­maceutical production (sigma-aldrich.com/pharmagrade). To support our quality control of these products, TraceCERT® organic CRMs have been developed for the methyl­ and ethyl esters of methanesulfonic acid and ethanesulfonic acid, as well as for other pharma grade products such as L­Methionine sulfoximine and methyl laureate.

The specifications of the methanesulfonic acid pharma grade product include limits of 5ppm (GC) for ethyl meth­anesulfonate as well as for methyl methanesulfonate. For calibration of these GC measurements, the corresponding TraceCERT CRMs have been used. The CRMs we use for our internal quality control are also available for our customers as TraceCERT catalogue products.

Organic TraceCERT products are high­quality certified refer­ence materials (CRMs) manufactured according to ISO/IEC 17025 and ISO Guide 34 with direct traceability to NIST SRM. Traceability is achieved by using high­perfor­mance quantitative NMR (HP­qNMR®) as a relative primary method. The big advantage of this method is that, unlike most analytical techniques, the signals of the integrals are not dependent on the structure of the compound. There­

fore, different compounds can be quantitatively compared with high accuracy. Detailed technical articles about the organic TraceCERT certification concept have been pub­lished in previous issues of Analytix [3].

Currently almost 100 organic TraceCERT products are avail­able, ranging from amino acids, natural products, PAHs, fatty acids/FAME, pesticides to pharmaceuticals. Table 1 shows recent product additions relevant for the quality control of the SAFC Pharma Grade Products. A complete and up­to­date product list can be found on our website sigma-aldrich.com/organiccrm together with technical articles and example certificates.

Cat. No. Brand Product Package Size

30934 Fluka® Ethyl ethanesulfonate 120 mg

72945 Fluka Ethyl methanesulfonate 120 mg

50490 Fluka Methyl ethanesulfonate 120 mg

78697 Fluka Methyl methanesulfonate 120 mg

91016 Fluka L­Methionine sulfoximine 100 mg

07041 Fluka Methyl laurate 100 mg

Table 1 TraceCERT Organic CRMs

Cat. No. Brand Product Package Size

11317 Sigma­Aldrich Methanesulfonic Acid 1 kg

76078 Sigma­Aldrich L­Methionine sulfoximine 1 g; 5 g

39937 Sigma­Aldrich Methyl Laurate 250 g

Table 2 Examples of SAFC Pharma Grade Products (GMP)

References[1] Snodin D.J; Regulatory Toxicology and Pharmacology,

45 (2006); 79 –90.[2] Eder E; Kutt W; Deindinger C; Annals Chem. Biol. Interact.

137 (2001), 89 –99.[3] Analytix 03/2010; 01/2011; 03/2011; 04/2011; 01/2012.

Matthias Nold [email protected] Detterbeck [email protected]



















Figure 2 Chemical structures of the sulfonic acid esters

Figure 1 Chemical structure of L-Methionine sulfoximine and methyl laurate



Page 8: Analytix - Sigma-Aldrich






Prescreened, In-Stock ChemicalsPesticides, PAHs, PCBs, VOCs, and More

Quality Antioxidant Standards in Convenient Kit

What do these compounds have in common?They are examples of the thousands of prescreened, in­stock chemicals available through the Sigma­Aldrich cus­tom standards group. We can formulate, test, and package custom standard solutions to meet your needs for all your chromatographic applications. Our custom standard chem­ists will gladly discuss stability and solubility concerns with you, and make suggestions where needed to improve the quality of your purchase.

You can rely on Sigma-Aldrich custom standard solutions to include: • Raw materials and solvents screened for identity

and purity

• Your choice of gravimetric, qualitative, and quantitative testing

• Packaging choices from ampuls to bottles

• Manufacturing processes following the guidelines of ISO 9001/2000

• Proper handling of light­ and/or oxygen­sensitive chemicals

• Documentation and Material Safety Data Sheets

Antioxidants are added to food and other products to pre­vent rancidity. Although the mechanisms are not clearly understood, the antioxidants react with free radicals and peroxides slowing the rancidity. Certain other additives greatly enhance the effectiveness of antioxidants. Metal scavenger and chelating agents, such as citric acid and citrates, tie up the trace metals and greatly reduce their catalytic activity. Synergism between antioxidants has also been noted, and many commercial antioxidant mixes are formulated to contain mixtures of the antioxidants. The most common of these mixtures contain both butyl­hydroxyanisole (BHA) and butyl­hydroxytoluene (BHT).

In order to ensure consistent product quality, these additives must be monitored. In the past, food analysts had to go to multiple vendors to obtain quality standards of all the anti­oxidants they may be required to monitor. Sigma­Aldrich offers an antioxidant standards kit containing several of the antioxidants listed in Association of Official Analytical Chem­ists Method 983.15: Phenolic Antioxidants, Fats, and Butter

• Free technical support

• Strict adherence to all shipping regulations

If you are interested in a customized standard, please email us at [email protected], or complete the on­line custom standards quote request form, available 24/7, at our website sigma-aldrich.com/standards

Oil plus ethoxyquin, an additional antioxidant commonly used in spices and other food products, and in cosmetics. Each antioxidant has been evaluated for purity, then pack­aged neat under nitrogen. A certificate of analysis for each antioxidant is included with the kit.

Reference[1] Official Methods of Analysis (17th Ed.), Method 983.15.

Association of Official Analytical Chemists, Arlington, VA USA (1996).

Description Cat. No.

Phenolic Antioxidant Kit500 mg each, individually packagedtert­Butylhydroquinone (PG)Ethoxyquin2,3­tert­Butyl­4­hydroxyanisol (BHA)2,6­Di­tert­butyl­4­hydroxymethylphenol (IONOX 100)3,5­Di­tert­butyl­4­hydroxytoluene (BHT)Lauryl gallateNordihydroguaiaretic acid (NDG)Octyl gallatePropyl gallate


Vicki Yearick [email protected]

Page 9: Analytix - Sigma-Aldrich






Maximize Sample Recovery with Center Drain (CD™) Vials

Features and Benefits: • Limited volume injections with less than 4 μL dead


• Eliminates bent needle problem associated with limited volume inserts

• Will withstand temperatures from ­70 °C to 160 °C

• Concentrate samples in the same vial that you use in autosampler

• Solid glass base offers better heat sink for concentrating samples

• Made from Type 1 Borosilicate glass

The patented Center Draining or CD Vial has a unique conical inte­rior bottom that promotes drain­ing of the vial’s contents into the center bottom of the vial, where virtually all of it can be extracted. The production process ensures consistency of quality and preci­sion in the CD Vials. The conical well, located in the exact center of the vial bottom, features a smooth

transition from sidewall into the well, and the bottom thick­ness dimension is the same in each vial.

In comparison tests against other manufacturers’ conical high­recovery vials, the CD Vial allows maximum extraction (<4 μL dead volume), while the competitors’ vials allow the contents to hang up in the transition area, leaving several microliters of liquid still in the vials after extraction. As shown in Figure 1, there is a significant amount of sample remain­ing in the competitor’s vial on the right, while the CD Vial has yielded almost total recovery of the liquid in the vial.

Below is a list of the many applications for which these vials can be used. To learn more, e­mail Technical Service at [email protected]

Applications • Library sample storage

• Lyophilization

• Cetrifugation

• Micro mixing

• Extraction

• Concentrations

• Derivatization

• Hybridization

• Anaerobic reactions

• Homogenization

• Small­scale reactions

• Catalytic hydrogenation

Ordering InformationDescription Cat. No.

Certified Center Drain CD Vials

12 x 32 mm, 1.5 mL, large opening, Pk/100

Screw top vial kits with blue cap and septa, 9 mm thread

Clear glass, PTFE/silicone septa 29307­U

Clear glass, PTFE/silicone/PTFE septa 29308­U

Clear glass, pre­slit PTFE/silicone septa 29309­U

Amber glass, PTFE/silicone/PTFE septa 29313­U

Amber glass, pre­slit PTFE/silicone septa 29314­U

Snap top kits with caps and septa

Clear glass, blue cap with PTFE septa 29301­U

Clear glass, blue cap with PTFE/silicone septa 29302­U

Clear glass, blue cap with pre­slit PTFE/silicone septa 29303­U

Crimp top vials (vial only)

Clear glass 29298­UscriTr

Figure 1 Superior sample extraction from CD Vial compared to competitor vial

Trademark on CD owned by QIS, Inc.

Vicki Yearick [email protected]

Page 10: Analytix - Sigma-Aldrich


163.060 267.089











+MS, 9.8-13.2min #(319-372)














-MS, 15.3-17.0min #(405-432)0













200 400 600 800 1000 1200 m/z





Is Your LC/MS System All Right? Mass Calibration and System Test Standards

System Test StandardsModern HPLC or UHPLC systems are very complex systems and should work properly with less attention during daily routine work. Annual maintenance and IQ/OQ/PQ verifica­tions guarantee compliance with hardware specifications. But this includes only the HPLC and MS instruments them­selves and is not related to the chromatographic application during daily routine work. System test standards help to monitor the present performance of a chromatographic system including different types of detectors and possible performance losses caused by intense usage of a column, high matrix load, long duty time of lamps, etc. Additionally, the system test standards may indicate the cause of a failure and shorten the time for troubleshooting.

Mass Calibration and System Tuning StandardsMost of the mass spectrometers have single quadrupole mass analyzers with an m/z resolution of +/­ 0.5 Da, which is good enough for the differentiation of single isotopes. More complex spectrometers are equipped with additional quadrupole mass selectors and collision cells (triple quadru­poles), ion traps, time­of­flight mass or FT­mass analyzers (including Orbi Traps). These kinds of system need a regular optimization of ion transfer, ion storage, and mass scan cali­bration to guarantee a precise and sensitive detection and fragmentation of analytes. Mass calibration standards are needed depending on the chosen mass range. The deter­mination of molecular formulas using high­resolution and

precision spectrometers particularly depends on an exact mass calibration in a range from 50 –1000 Da.

Going Hand in Hand – Calibrating and Testing LC/UV/CAD/MS SystemsBefore starting LC­MS data acquisitions, most of the mass spectrometers have to be calibrated to insure the mass pre­cision, accuracy, and highest sensitivity of MS/MS isolation (ion traps) and fragmentation. High­resolution time­of­flight spectrometers especially need to be regularly cali­brated several times a day if maximum precision is needed. Salt solutions and polymer solutions generate regular m/z patterns in a broad m/z range. Lithium and sodium formate solutions are especially ideal for the calibration of m/z ranges up to 1500 Da, which is suitable for LC­MS analysis of most small molecules using electrospray ionization. Another convenient feature of alkali formate clusters is the ability to use both ESI modes in one step. Figure 1 shows the mass spectra of lithium formate clusters (ESI +/­).

HPLC Performance and MS Sensitivity TestingAlthough well designed and tested, HPLC/MS and even UHPLC­suitable systems contain several potential error sources. The connection to a mass spectrometer increases the possibility of a malfunction or loss of performance. There are several ways to test each component of the LC­MS system, but this is a time­consuming process primar­ily useful after extensive maintenance.

Rudolf Köhling, Namtso Reichlin [email protected]

Figure 1 ESI LC­MS spectra of lithium formate solution (Fluka®) in positive (upper) and negative ion mode (bottom). The regular pattern of the [Li(x+1)(HCOO)x]+ and [Lix(HCOO)(x+1)]­ cluster can be used to calibrate a mass range up to 1200 m/z, which covers most of the small molecules.

Page 11: Analytix - Sigma-Aldrich






Figure 3 Acquired and calculated mass spectrum of reserpine. The difference of the peak position is only 0.7 ppm. The resolution is 11000 in standard resolution mode. This agrees with the specification of the manufacturer and indicates an optimum setup.

Figure 2 Influence of an aged column on the performance of a separation. 5 µl of a 5 ppm reserpine standard is injected and monitored over a column lifetime. Peak height and symmetry decreases. UHPLC separations especially react sensitively to instrumental errors (extra volume, valve malfunction, clogging, or performance loss in the stationary phase).

0 1 2 3 4 5 6 7 8 9 Time [min]0.0





1.0 5x10Intens.




+MS, 5.4-5.4min #(418-420)




C 33 H 41 N 2 O 9 ,609.280




4 4x10Intens.





4 4x10

609.0 609.5 610.0 610.5 611.0 611.5 612.0 612.5 613.0 m/z

On the other hand, daily routine work only allows for short interruptions to complete short, robust tests to monitor the performance of the UHPLC and mass spectrometer. The main focus lies on the sensitivity of the ion source and the efficiency of the chromatographic separation. Injecting a system test standard can identify a lack of performance of the HPLC system including the column by monitoring peak height, peak width, signal­noise ratio, and symmetry. If these parameters are regularly recorded on a control chart, then it is possible to observe changes immediately. Figure 2 shows the effect of a chromatography malfunction on the peak shape and position of the test compound reserpine. Impurities in the solvent lead to the signal suppression in MS.

Caffeine, reserpine, digoxin or other small molecules, pep­tides and proteins are suitable compounds to test both HPLC and MS. Spectral data gives additional information about the status of a detector. Figure 3 shows the acquired and calculated mass spectrum of reserpine, which is used to check the resolution of the spectrometer and the precision of calibration, e.g. with lithium formate solution. Caffeine is

also used for this purpose and can be applied for MS detec­tion (low mass range) or UV detection. The UV spectrum of caffeine is useful to check the adjustment and resolution of the UV detector and the performance of the UV/VIS lamps [1].

Most of these standards are used to specify the perfor­mance of an instrument, which is useful for comparing the results of the tests with the instrument specifications.

Reference[1] J. Emmert, Labor & More, 5 (2008), 56-57.

Cat. No. Brand Description

01886 Fluka® Lithium Formate Solution, Suitable for LC­MS, 10 mM LiOH in isopropanol/water 1:1 (+0.2% HCOOH)

97574 Fluka Sodium Formate Solution, Suitable for LC­MS, 10 mM NaOH in isopropanol/water 1:1 (+0.2% HCOOH)

43530 Fluka Reserpine Standard for LC­MS, analytical standard, for LC­MS

56396 Fluka Caffeine, certified reference material, TraceCERT®


Sigma ProteoMass Peptide and Protein MALDI­MS Calibration Kit

D6003 Fluka Digoxin, analytical standard

new column used column with corrupted

stationary phase

Page 12: Analytix - Sigma-Aldrich







Food and beverages have to be of the highest quality to survive in a competitive market. Spoilage causing organ­isms or pathogens are problems which can cause huge financial loss and serious image damage. Fast and reliable systems need to be in place in order to avoid microbial problems. Many laboratories still use conventional standard based cultivation methods, which are often very time con­suming and take two to seven days for the product to be released to the market.

HybriScan kits are rapid test systems that identify contami­nants and lead to a faster product release. A variety of applications have been developed for HybriScan including the detection of bacteria and yeast in food and beverages. The robustness of the HybriScan assay enables it, in con­trast to other rapid test systems, to detect contaminants under beneficial microorganisms or in a difficult matrix. Furthermore, the HybriScan test system is a perfect tool for microbiological control of dispensing equipment. Without a pre­enrichment procedure, results within two hours are possible.

Specificity is achieved by targeting conserved or unique rRNA sequences. A biotin­labeled capture probe is used to immobilize the target sequence on a solid support plate (streptavidin­coated microtiter plate). A digoxigenin­labeled detection probe provides an enzyme­linked optical signal read­out. Detection results from application of anti­DIG­horseradish peroxidase Fab fragments. The bound complex is visualized by horseradish peroxidase substrate TMB (3,3’,5,5’­tetramethylbenzidine). Photometric data are measured at 450 nm and compared with standard solutions.

Comparison of HybriScan and other rapid test systemsPerforming quality control using the standard cultivation based method takes a long time. In recent years many companies have developed rapid test systems to hasten this procedure. For quality control of food and beverages, some main technologies are available:

• HybriScan (sandwich hybridization)

• PCR (Polymerase Chain Reaction)

• VIT (Vermicon Identification Technology)

• ELISA (Enzyme­linked Immunosorbent Assay)

PCR & DNA were Yesterday; the Future is RNAHybriScan®, a rapid and innovative screening method for microorganisms based on the detection of rRNA.

Jvo Siegrist, Product Manager Microbiology [email protected]

A comparison of these different technologies is given in Table 1. Comparing HybriScan to PCR, ELISA or VIT­tech­nology demonstrates that the benefits of this rapid test system are:

• Fast and cost­efficient analysis

• Inexpensive read­out technology

• High sensitivity and specificity

• Low cross reactions

By using two different probes for detection of microbial RNA, false­positive results are almost impossible. Compari­son of the HybriScan test with a cultivation­based analytical method displays the equivalent results within the limits of microbiological sample variability (see Figure 2).

Figure 1 Bacterial ribosomal subunit consists of protein and rRNA

Page 13: Analytix - Sigma-Aldrich







1 2 3 4 5 6 7 8 9 10 11 12 13 14



plate count










The HybriScan® system can be used as a versatile microbio­logical detection and identification method. There is a wide variety of applications for using this innovative rapid test system with food, beverages, water and other specimens. It is possible to detect both a single species and a defined group of microorganisms.

Further information about the HybriScan method can be found at sigma-aldrich.com/hybriscan

Cultivation-based method


Method cultivation­based method with optical or microscopic read­out

PCR/real­time PCR Immunological Assay fluorescence microscopy

sandwich hybridization and photometrical signal read out

Sample preparation enrichment enrichment and lysis enrichment enrichment, immobilisation

enrichment and lysis

Time 3 to 7 days 3 hours to 2 days 3 hours to 2 days 2 days 3 hours to 2 days

Costs per test €1 approx. €12 approx. €3 approx. €15 approx. €3 approx.

Detection limit (cfu) 1 1–5 x 103 103 – 105 103 1–5 x 103 bacteria 1–3 x 102 yeasts

Devices None PCR cycler microplate reader fluorescence microscope

microplate reader

Advantages high sensitivity, relatively inexpensive

high sensitivity, quantitative analysis

differentiation of serotypes or subspecies, cost­ efficient analysis

simple detection technology set­up, detects only living cells (RNA)

rapid and sensitive, qualitative and quantitative detection of only living cells, cost­efficient analysis

Disadvantages time consuming, no detection of non­culturable microbes, labor expensive

expensive devices needed, no discrimination between live and dead cells, not officially accepted

low sensitivity, low specificity, higher cross­reactivity

time consuming, low sample throughput, expensive, not automatable, difficult data analysis, not officially accepted

no differentiation of serotypes or subspecies, limited probe design (rRNA target), not officially accepted

Table 1 Comparison of different technologies for detection of bacteria

Figure 2 Comparison of total count of bacteria with the HybriScan test and the plate count agar

Table 2 Range of HybriScan detection and identification kits (for more details see sigma-aldrich.com/hybriscan)

Detection Kits for Applications/Groups Cat. No.

Beer Spoiling Organisms 62533

Beverage Spoiling Organisms 68301

Total Bacterial Count 02349

Waste Water Microthrix parvicella 04447

Waste Water Total Bacteria Count 78436

Yeasts 61397

Detection Kits for Genus & Species

Campylobacter 56917

Cronobacter spp. 12838

E. coli 96343

Lactobacilli 62533

Legionella 16593

Legionella pneumophila 07190

Listeria 55661

Listeria monocytogenes 49699

Salmonella 55662

Identification Kits Cat. No.

Brettanomyces 79742

Candida albicans 19503

Lactobacillus brevis 75724

Lactobacillus buchneri 80065

Lactobacillus lindneri 86827

Legionella pneumophila 49417

Leuconostoc 77007

Listeria monocytogenes 49712

Megasphaera 42875

Pectinatus cerevisiiphilus 33018

Pectinatus frisingensis 73582

Pediococcus damnosus 67289

Page 14: Analytix - Sigma-Aldrich



Buffer’s effect on detection: The choice of buffer is also dependent upon means of detection. For traditional UV detection, the buffer needs to be effectively transparent in this region especially, critical for gradient separations. Buffers listed in Table 1 have low enough absorption below 220 nm.

Phosphoric acid and its sodium or potassium salts are the most common buffer systems for reversed­phase HPLC. Phosphonate buffers can be replaced with sulfonate buf­fers when analyzing organophosphate compounds. With the growth in popularity of LC­MS, volatile buffer systems, such as TFA, acetate, formate, and ammonia, are frequently used due to compatibility with mass spectral (MS) detec­tion. In regard to the issue of suppression of ionization, for­mate and acetate are ideal choices for positive­ion mode detection. TFA, however, can negatively impact detector response even in positive­ion mode [4, 5], while it strongly suppresses ionization with negative ion mode. Acetic acid is good for negative­ion mode. LC­MS applications further limit buffer selection and buffer concentration.

References[1] McMaster, M.C. HPLC A Practical User’s Guide, VCH Publishers,

Inc.: New York, NY, 1994; 85. [2] Poole, C.F. and Poole, S.K. Chromatography Today, Elsevier

Science: Amsterdam, Netherlands, 1991; 431. [3] Analytix, Five-part series on Mobile Phase Additives for LC-

MS, Issue 3, 2006 (sigma-ldrich.com/analytix).[4] Temesi, D., Law, B., 1999, The Effect of LC Eluent Composition

on MS Response Using Electrospray Ionization, LC-GC, 17:626.[5] Apffel, A. et. al. 1995. Enhanced Sensitivity for Peptide

Mapping with Electrospray Liquid Chromatography-Mass Spectrometry in the Presence of Signal Suppression Due to Trifluoroacetic Acid-Containing Mobile Phases, J. Chrom. A. 712:177.

Consideration of the effects of pH on analyte retention, type of buffer to use, and its concentration, solubility in the organic modifier and its effect on detection are important in reversed­phase chromatography (RPC) method develop­ment of ionic analytes. An improper choice of buffer, in terms of buffering species, ionic strength and pH, can result in poor or irreproducible retention and tailing in reverse­phase separation of polar and ionizable compounds.

Problems, such as partial ionization of the analyte and strong interaction between analytes and residual silanoles or other active sites on the stationary phases, can be over­come by proper mobile phase buffering (maintaining the pH within a narrow range) and choosing the right ionic spe­cies and its concentration (ionic strength) in the mobile phase [1–2]. In sensitive LC­MS separations that depend heavily on the correct choice of acid, base, buffering spe­cies and other additives [3], a buffer must be chosen based on its ability to maintain, and not suppress analyte ioniza­tion in the MS interface.

Buffer selectionThe typical pH range for reversed­phase on silica­based packing is pH 2 to 8. Choice of buffer is typically governed by the desired pH. It is important that the buffer has a pKa close to the desired pH since buffers control pH best at their pKa. A rule of thumb is to choose a buffer with a pKa value <2 units of the desired mobile phase pH (see Table 1).

Buffer pKa (25°C) Useful pH Range

TFA 0.5 <1.5

Sulfonate 1.8 <1­2.8

Phosphate 2.1 1.1­3.1

Chloroacetate 2.9 1.9­3.9

Formate 3.8 2.8­4.8

Acetate 4.8 3.8­5.8

Sulfonate 6.9 5.9­7.9

Phosphate 7.2 6.2­8.2

Ammonia 9.2 8.2­10.2

Phosphate 12.3 11.3­13.3

Table 1 HPLC buffers, pKa values and useful pH range

Buffer concentration: Generally, a buffer concentration of 10–50 mM is adequate for small molecules.

Buffer solubility: A general rule is no more than 50% organic should be used with a buffer. This will depend on the specific buffer as well as its concentration.

Reversed-phase HPLC BuffersHigh­quality buffers (solutions, solids or concentrates)






Shyam Verma, Market Segment Manager [email protected]

HPLC-grade buffers and additives from Sigma-Aldrich/Fluka®Cat. No. Description Package Size

17836 Ammonium acetate 50 g, 250 g

17843 Ammonium formate 50 g, 250 g

17837 Ammonium hydroxide solution in water 100 mL, 1 L

17842 Ammonium phosphate monobasic 250 g

17839 Ammonium trifluoroacetate 10 g, 50 g

17835 Potassium phosphate dibasic anhydrous 250 g

17841 Sodium formate 50 g, 250 g

71633 Sodium phosphate dibasic dehydrate 250 g

17844 Sodium phosphate monobasic anhydrous 50 g, 250 g

17840 Sodium trifluoroacetate 10 g

09746 Trifluoroacetic acid:Triethylamine 2M:1M 500 mL

09747 Trifluoroacetic acid:Triethylamine 2M:2M 100 mL

For a complete list of HPLC buffers and additives, please refer to our online product catalog sigma-aldrich.com/hplc

Page 15: Analytix - Sigma-Aldrich







Compound ICH Solvent Class ICH Limit (ppm)

Acetone 3 5000

Acetonitrile 2 410

Dichloromethane 2 600

Diethyl ether 3 5000

Dimethylformamide 2 880

1,4­Dioxane 2 380

Ethanol 3 5000

Ethyl acetate 3 5000

Hexane 2 290

Heptane 3 5000

lsopropanol 3 5000

lsopropyl acetate 3 5000

Methanol 2 3000

Methyl t ­butyl ether 3 5000

Methycyclohexane 2 1180

2­Methyltetrahydrofuran Not Classified Not Classified

Tetrahydrofuran 2 720

Toluene 2 890

Triethylamine Not Classified Not Classified

Table 1 Residual solvents analyzed

ResultsThe comparison of the high­purity GC headspace grade DMA to the conventional grade (high­purity) DMA showed the superior quality of the GC headspace grade solvent. Figure 1 demonstrates that the GC headspace grade DMA is cleaner and free from potentially interfering peaks.

Contributed articleMelissa P Grella, Jessica Hoover, and Mark ShapiroPharmaCore, Inc, High Point, NC 27265, USA

Residual solvents in pharmaceuticals are defined as organic volatile impurities (OVIs) that are remnants of solvents used or produced in the manufacture of drug substances and excipients. Residual solvents are classified as: Class 1 sol­vents (solvents to be avoided – known or suspected human carcinogens and environmental hazards), Class 2 solvents (solvents to be limited – non­genotoxic animal carcinogens and suspected of other significant but reversible toxicities), and Class 3 solvents (solvents with low toxic potential and no health­based exposure limit is needed). The solvents are not completely removed by practical manufacturing tech­niques. Testing is, therefore, done to ensure that these sol­vents are not above concentration limits listed by USP and in the ICH guidelines [1, 2].

Static headspace GC (HS­GC) is a commonly used tech­nique in the analysis of OVIs. This technique concentrates volatile analytes and allows their analysis free from sample matrix. Samples to be analyzed by HS­GC must be dissolved in a suitable solvent. N,N­Dimethylacetamide (DMA) is a useful and commonly used solvent for headspace analysis of OVIs in pharmaceutical products.

ExperimentalFor the comparison of the different purity grades of DMA, blank samples were prepared by pipetting 1 mL of solvent into a 10 mL headspace vial. The blanks were analyzed by GC headspace. For the different analytical methods used, the high­purity GC­HS grade DMA from Sigma­Aldrich (Product no. 44901) was used. Residual solvent stock stan­dards were prepared using a positive displacement pipettor to add each residual solvent being tested. The stock stan­dards were diluted in DMA to prepare working standard solutions. The concentration of each solvent in the working standard was calculated using the known density of each solvent. The working standard solutions were aliquoted into headspace vials and analyzed in the sequence along with blanks of the DMA by GC headspace. Table 1 lists the various residual solvents that were analyzed by the different methods and their ICH limits. The chromatograms for the residual solvents analyzed in each method are shown in various figures.

High-Purity Headspace Grade N,N-Dimethylacetamide in the Analysis of Residual Solvents

Shyam Verma, Market Segment Manager [email protected]

Figure 1 DMA comparison: headspace grade vs synthetic grade

The results for the different methods using the GC­HS grade DMA, comparing the blank and standard solution contain­ing various residual solvents are presented in Figures 2 and 3. The corresponding chromatographic and head­space conditions are listed in Tables 2 and 3.

0 5 10 15 20 25min.

A = Fluka® #44901B = Other High­Quality DMA









Page 16: Analytix - Sigma-Aldrich







Headspace Parameters

Temperatures: Oven 90 °C

Loop 105 °C

Transfer Line 110 °C

Times: Vial Equilibration 15 min

Pressurization 0.2 min

Loop Fill 0.2 min

Loop Equilibration 0.05 min

Pressures: ViaI 14 psi

Loop Volume 1.0 mL

lnject Time 0.5 min

GC Parameters

Column Equivalent to USP G43 phase, 105 m x 0.32 mm I.D. x 1.5 µm

lnjector and Detector Temperatures

250 °C, 300 °C (FID)

Oven Program 35 °C (10 min.), 3 °C/min. to 65 °C, 20 °C/min. to 190 °C (13 min.)

Carrier Helium, 2.1 mL/min. (constant pressure)

lnjection Headspace, split ratio 24.1:1

Table 2 Example Method 1

Headspace Parameters

Temperatures: Oven 90 °C

Loop 105 °C

Transfer Line 110 °C

Times: Vial Equilibration 15 min

Pressurization 0.2 min

Loop Fill 0.2 min

Loop Equilibration 0.05 min

Pressures: ViaI 14 psi

Loop Volume 1.0 mL

lnject Time 0.5 min

GC Parameters

Column Equivalent to USP G43 phase, 105 m x 0.32 mm I.D. x 1.5 µm

lnjector and Detector Temperatures

250 °C, 300 °C (FID)

Oven Program 35 °C (10 min.), 3 °C/min. to 65 °C, 20 °C/min. to 190 °C (12 min.)

Carrier Helium, 2.1 mL/min. (constant pressure)

lnjection Headspace, split ratio 24.1:1

Table 3 Example Method 2

DMA Blank

1 23

4 5

6 7 8 9




5 10 15 20 25 30 35 min

5 10 15 20 25 30 35 min

DMA Blank




8 15


10 1216









5 10 15 20 25 30 35 min

5 10 15 20 25 30 35 min


Figure 2 Example Method 1

Figure 3 Example Method 2

• This conclusion is supported by considering that mul­tiple GC oven ramp rates and column chemistries were evaluated in support of multiple and various residual solvents and their associated levels.

In each case, there were no interference peaks resulting from the DMA irksome solvent peaks that could interfere with proper detection of target analytes.

References[1] United States Pharmacopoeia (USP), 31st Edition (2008), <467>

Residual Solvents.[2] ICH Guidelines for Industry, Q3C Impurities: Residual Solvents,

US Dept. of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), ICH December, 1997.

In each case, the method was used for the residual solvents analysis of an early­stage active pharmaceutical ingredient. The GC­HS grade DMA blank chromatograms were clean and free of interfering peaks. The content of each residual solvent was determined with ease. Particularly, the low­ responding solvents or low ICH limit class solvents were detected with no background interference with the high­purity DMA.

• The data in this presentation clearly demonstrate the superior performance of the high­purity GC headspace grade solvent used (DMA).

1. Diethyl ether2 Acetone3. Isopropanol4. Acetonitrile5. Dichloromethane6. Methyl t­butyl ether

7. n­Hexane8. Ethyl acetate9. n­Heptane10. 1,4­Dioxane11. Toluene12. Dimethylformamide

1. Diethyl ether2 Acetone3. Isopropanol4. Hexane isomer 15. Acetonitrile6. Dichloromethane7. Hexane isomer 28. Methyl t­butyl ether9. Hexane

10. Hexane isomer 311. Ethyl acetate12. Heptane isomer 113. Heptane isomer 214. Heptane isomer 315. Heptane isomer 416. Heptane isomer 517. Heptane isomer 618. Heptane isomer 7

19. 2­Methyl THF/Heptane20. Heptane isomer 821. Heptane isomer 922. Heptane isomer 1023. Heptane isomer1124. 1,4­Dioxane25. Toluene26. Dimethylformamide

DMA Blank

DMA Blank

Page 17: Analytix - Sigma-Aldrich







Michael Jeitziner, Market Segment Manager [email protected] Shyam Verma, Market Segment Manager [email protected]

New Solvents and Additives Exclusively Designed for UHPLCLC­MS Ultra CHROMASOLV®

Recent innovations in HPLC and mass spectrometry (MS) have escalated the limits of speed (throughput), efficiency and sensitivity. UHPLC in combination with very sensitive detection systems sets new limits for the purity of solvents and the additives used in the mobile phase. Solvents used for mobile phase, sample preparation, and sample dissolu­tion are critical components of the system. Their influence

on background noise and baseline stability, column lifetime and system integrity cannot be overlooked. For the UHPLC system to provide reliable data and high performance, and eliminate system down­time, it is critical to use solvents that are as carefully developed, prepared, and tested as other components of the system.

Sigma­Aldrich offers the new LC­MS Ultra CHROMASOLV product line that provides an outstanding quality for the ultra­pure mobile phase. These solvents offer advantages such as:

• Extremely small drift in UHPLC gradient analysis

• Positive and negative ion mode testing for all MS applications

• Tested specific to UHPLC applications

• Most narrow and consistent specifications

UHPLC suitability of these solvents is achieved by consider­ing various parameters of the preparation process far beyond filtration. The new LC­MS Ultra CHROMASOLV grade solvents are exclusively tested for high performance with UHPLC gradient separations plus UV, positive and negative mode MS detection. Fluka’s new LC­MS Ultra CHROMASOLV grade solvents are tested in situ under demanding UHPLC conditions, and various detection modes.

For additional information and to order, visit sigma-aldrich.com/uhplc-ms

Cat. No. Brand Name Description Package Size

14261 Fluka® Acetonitrile LC­MS Ultra CHROMASOLV, ≥99.9%, tested for UHPLC­MS 1 L, 2 L

14262 Fluka Methanol LC­MS Ultra CHROMASOLV, ≥99.9%, tested for UHPLC­MS 1 L, 2 L

14263 Fluka Water LC­MS Ultra CHROMASOLV, tested for UHPLC­MS 1 L, 2 L

14264 Fluka Trifluoroacetic acid LC­MS Ultra eluent additive, ≥ 99.0% suitable for UHPLC­MS 1 mL, 2 mL

14265 Fluka Formic acid LC­MS Ultra eluent additive, ≥ 98% suitable for UHPLC­MS 1 mL, 2 mL

14266 Fluka Ammonium formate LC­MS Ultra eluent additive, suitable for UHPLC­MS 25 g

14267 Fluka Ammonium acetate LC­MS Ultra eluent additive, suitable for UHPLC­MS 25 g

Table 1 New LC-MS Ultra CHROMASOLV solvents and LC-MS Ultra eluent additives

Page 18: Analytix - Sigma-Aldrich



Second Generation Ionic Liquid Matrices for MALDI-MS

Matthias Drexler, Product Manager, Analytical Reagents [email protected]

Salts with a melting point below 100 °C are commonly referred to as ionic liquids. Due to their unique properties, they have increasingly gained interest over the last few decades and have been used successfully in many applica­tions. Examples include their use as solvents for organic reactions and homogeneous catalysis, in liquid­liquid extractions, as ion pair reagents, and as stationary phases for gas chromatography.

In 2001, Armstrong et al. first demonstrated that ionic liquids may also be applied as matrices in matrix­assisted laser desorption/ionization mass spectrometry [1]. These ionic liquid matrices (ILMs) offer advantages over the classic solid matrices, most importantly, the increased homogeneity of the sample and subsequently, a higher shot­to­shot repro­ducibility and improved quantification during the MALDI analysis. The need for a time­consuming search for a “hot spot” on the sample plate is reduced, thus enabling better automated analyses. Other benefits of ILMs include their extremely low vapor pressure and overall easier sample preparation, as acidic additives such as trifluoroacetic acid are not necessary.

In general, the anionic part of the ILM is based on classic MALDI matrix substances, such as α­cyano­4­hydro­xycinnamic acid (CHCA) or ferulic acid, and the cationic part consisting of ammonium or imidazolium moieties com­monly used in ionic liquids. Both ionic parts of an ILM can be varied in order to improve the properties and suitability for certain classes of analytes. Armstrong et al. were able to show in 2009 that the second­generation matrix N,N­diiso­propylethylammonium 4­hydroxy­3­methoxycinnamate (DIEA­F, Fluka® 94155) is especially fit for analyses of carbo­hydrates while N,N­diisopropylethylammonium α­cyano­4­hydroxycinnamate (DIEA­CHCA, Fluka 18211) and N­tert­Butyl­N­isopropyl­N­methylammonium a­cyano­4­hydroxycinnamate (IMTBA­CHCA, Fluka 94190) have proven to be best choices for protein and peptide samples in a considerable mass range of 1000 to 270,000 Da [2, 3]. In comparison with first­generation ILMs (such as BA­CHCA and DEA­CHCA), these new matrices perform even better with respect to analyte signal strength.

Overall, the development of ILMs offers a valuable tool for analyses with MALDI­MS—a method where the success is very much dependent on choosing the right matrix sub­stance suitable for the analyte.




Cat. No. Brand Description Abbrev. Gen. Suitable for Package Sizes

18211 Fluka α­Cyano­4­hydroxycinnamic acid N­ethyl­N,N­diisopropylammonium salt

DIEA­CHCA 2nd Peptides, proteins, biodegradable polymers

10 x 10 mg

94190 Fluka α­Cyano­4­hydroxycinnamic acid N­tert­butyl­N­isopropyl­N­methylammonium salt

IMTBA­CHCA 2nd Peptides, proteins 10 x 10 mg

94155 Fluka Ferulic acid N­ethyl­N,N­diisopropylammonium salt

DIEA­F 2nd Carbohydrates 10 x 10 mg

67336 Fluka α­Cyano­4­hydroxycinnamic acid butylamine salt

BA­CHCA 1st Peptides, proteins, polymers

100 mg, 1 g

55341 Fluka α­Cyano­4­hydroxycinnamic acid diethylammonium salt

DEA­CHCA 1st Peptides, proteins, polymers

1 g

Product Table Ionic liquid matrices (ILMs) for MALDI-MS. Please find the complete product list at sigma-aldrich.com/maldi






































References[1] D. W. Armstrong, L. Zhang, L. He, M. L. Gross, Ionic Liquids as

Matrixes for Matrix-Assisted Laser Desorption/ionization Mass Spectrometry, Anal. Chem. 2001, 73, 3679 –3686.

[2] A. Berthod, J. A. Crank, K. L. Rundlett, D. W. Armstrong, A Second-Generation Ionic Liquid Matrix-Assisted Laser Desorption/Ionization Matrix for Effective Mass Spectrometric

Analysis of Biodegradable Polymers, Rapid Commun. Mass Spectrom. 2009, 23, 3409 –3422.

[3] J. A. Crank, D. W. Armstrong, Towards a Second Generation of Ionic Liquid Matrices (ILMs) for MALDI-MS of Peptides, Proteins, and Carbohydrates, J. Am. Chem. Soc. 2009, 20, 1790 –1800.

Page 19: Analytix - Sigma-Aldrich






Volumetric Concentrates for Titration by Sigma-Aldrich Prepare Acid, Base and Salt Standard Solutions with FIXANAL® Ampoules

Andrea Felgner, Market Segment Manager [email protected]

Our volumetric concentrates, packaged in convenient FIXANAL ampoules, allow our customers to achieve specific concentrations of high accuracy. This is accom­plished with speed and simplicity due to the design features of the innovative FIXANAL ampoules. The volumetric concentrate con­tains an exact amount of the pre­scribed substance. By diluting the entire content of the ampoule by a determined volume, the analyst

has complete control over the final solution concentration. The analyst’s confidence is aided by knowing for certain that the absolute amount of the titration substance has been tested against a certified reference material.

distilled and degassed; otherwise sluggish endpoints or changes to the titer may occur.

Certain concentrates with sensitive chemical properties require black pigmented PE ampoules or brown glass ampoules. All FIXANAL concentrates are calibrated against independently produced reference standards. All chemicals and test equipment are examined according to the rules of DIN EN ISO 9001 and DIN EN ISO 14001.

Quality Assurance, Packaging and Handling • FIXANAL ampoules are subject to strict production and

quality control specifications

• A special ampoule­sealing process guarantees titer to be accurate within specified shelf life

• Ampoules are supplied with self­adhesive labels that can easily be attached to a storage vessel

• Ampoules are made of high­resistance polyethylene (PE), natural or black­pigmented, or glass, depending on the content

• PE ampoules have an integrated rinsing funnel for per­forating the membrane (see Figure 1); the integrated deflector accelerates complete rinsing of the concen­trate from the ampoule

• Glass ampoules do not have the twist­opening mecha­nism; instead, a glass rod is used for piercing both ends of the ampoule (see Figure 2)

• The water used to fill up to volume should be distilled and degassed; otherwise, sluggish endpoints or changes of titer may occur

• Concentrates of iodine, containing iodide­iodate­mix­tures, have to be treated with an equivalent amount of acid (+ 1% excess) before bringing them up to volume, in order to react to free iodine

(continued on page 20)

Product Characteristics: • Contains exact amount of substance (e.g. 1 mol)

• Titer precision 1.000 ± 0.2%

• Economical and space­saving

• Final concentration is user­specified

For convenience, the ampoules are supplied with self­adhesive labels that can easily be attached to a storage vessel by the end­user. Most ampoules are made of high­resistance polyethylene and have an attached funnel. To open a FIXANAL ampoule, simply twist the top of the ampoule to activate flow; a specially developed rinsing funnel facilitates the perforation of the sealing membrane. An integrated deflector accelerates the complete rinsing of the concentrate. The water used for dilution should be

Figure 1 PE ampoulesPlace the ampoule on the volumetric flask. Fix the lower part and rotate the upper part in the direction of the arrow (maximum of 2 turns). Remove the funnel, turn it upside down and place it firmly on the ampoule so that the membrane opens. Hold the ampoule at an angle and rotate it while thoroughly rinsing it. Fill the volumetric flask up to the mark at 20 °C and mix thoroughly.

Figure 2 Glass ampoulesPlace the ampoule on the volumetric flask. Carefully pierce both ends using a glass rod. Rinse and remove glass rod, then rinse ampoule. Fill the volumetric flask up to the mark at 20 °C and mix thoroughly.

Page 20: Analytix - Sigma-Aldrich




Special Offer – The following FIXANAL® volumetric concentrates are available with HUGE SAVINGS OF 35% OFF:

Cat. No. Brand Description38210­1EA Fluka® Sodium hydroxide concentrate, for 1 L standard solution

c(NaOH) = 0.1 mol/L (0.1 N)

38200­1EA Fluka Sodium thiosulfate concentrate, for 1 L standard solution c(Na2S2O3) = 0.1 mol/L (0.1 N)

38217­1EA Fluka Sodium hydroxide concentrate, for 1 L standard solution c(NaOH) = 0.5 mol/L (0.5 N)

38020­1EA Fluka Ammonium thiocyanate concentrate, for 1 L standard solution c(NH4SCN) = 0.1 mol/L (0.1 N)

32043­1EA Fluka Sulfuric acid concentrate, for 1 L standard solution c(H2SO4) = 0.05 mol/L (0.1 N)

38282­1EA Fluka Hydrochloric acid concentrate, for 1 L standard solution c(HCl) = 1.0 mol/L (1.0 N)

38073­1EA Fluka Potassium hydroxide concentrate, for 1 L standard solution c(KOH) = 1.0 mol/L (1.0 N)

32047­1EA Fluka Zinc sulfate concentrate, for 1 L standard solution c(ZnSO4) = 0.1 mol/L

38061­1EA Fluka Iodide Iodate concentrate, for 1 L standard solution c(I2) = 0.05 mol/L (0.1 N)

38040­1EA­R Fluka Bromine concentrate, for 1 L standard solution c(Br2) = 0.05 mol/L

32042­1EA Fluka Nitric acid concentrate, for 1 L standard solution c(HNO3) = 0.1 mol/L (0.1 N)

38285­1EA Fluka Hydrochloric acid concentrate, for 1 L standard solution c(HCl) = 0.5 mol/L (0.5 N)

38215­1EA­R Fluka Sodium hydroxide concentrate, for 1 L standard solution c(NaOH) = 1.0 mol/L (1.0 N)

38070­1EA­R Fluka Potassium hydroxide concentrate, for 1 L standard solution c(KOH) = 0.1 mol/L (0.1 N)

38110­1EA Fluka Potassium iodide concentrate, for 1 L standard solution c(KI) = 0.1 mol/L

38002­1EA Fluka Ammonium iron(II) sulfate concentrate, for 1 L standard solution c((NH4)2Fe(SO4)2) = 0.06 mol/L, for COD determination

38100­1EA­R Fluka Potassium dichromate concentrate, for 1 L standard solution c(K2Cr2O7) = 1/60 mol/L (0.1 N)

32046­1EA Fluka Perchloric acid concentrate, for 1 L standard solution c(HClO4) = 0.1 mol/L (0.1 N), for titration in non­aqueous liquids

38283­1EA Fluka Hydrochloric acid concentrate, for 10 L standard solution c(HCl) = 1 mol/L (1 N)

38065­1EA Fluka Iodide Iodate concentrate, for 1 L standard solution c(I2) = 0.005 mol/L (0.01 N)

38150­1EA­R Fluka Sodium (meta)arsenite concentrate, for 1 L standard solution c(NaAsO2) = 0.05 mol/L

38226­1EA Fluka Sodium hydroxide concentrate, for 1 L standard solution c(NaOH) = 0.025 mol/L (0.025 N)

38214­1EA Fluka Sodium hydroxide concentrate, for 10 L standard solution c(NaOH) = 1 mol/L (1 N)

32044­1EA Fluka Sulfuric acid concentrate, for 10 L standard solution c(H2SO4) = 0.5 mol/L (1.0 N)

32038­1EA Fluka Sodium chloride concentrate, for 1 L standard solution c(NaCl) = 0.1 mol/L (0.1 N)

38294­1EA Fluka Sulfuric acid concentrate, for 1 L standard solution c(H2SO4) = 0.5 mol/L (1.0 N)

38140­1EA Fluka Potassium thiocyanate concentrate, for 1 L standard solution c(KSCN) = 0.1 mol/L

38030­1EA­R Fluka Barium chloride concentrate, for 1 L standard solution c(BaCl2) = 0.05 mol/L (0.1 N)

38295­1EA Fluka Sulfuric acid concentrate, for 1 L standard solution c(H2SO4) = 0.25 mol/L (0.5 N)

32036­1EA Fluka Potassium bromide concentrate, for 1 L standard solution c(KBr) = 0.1 mol/L

To take advantage of this offer, please use Promotion Code SCZ. Offer valid until July 31, 2012.



35% Off


Page 21: Analytix - Sigma-Aldrich







+ 2 CH3OH




OCH3 + H2O




+ SO2 +H2O + NR' CR




(NR' = base)


Determination of Water Content in Aldehyde and Ketone SamplesHYDRANAL®­K Reagent Line for Karl Fischer Titration

Andrea Felgner, Market Segment Manager [email protected] Wendt, HYDRANAL Technical Service [email protected]

The HYDRANAL­K reagent product line is designed to meet the needs of the analytical chemist by providing accurate water content determination using Karl Fischer (KF) titration of aldehyde and ketone samples. HYDRANAL­Composite 5K, as a replacement for methanol­containing titrating agents, prevents side reactions that occur during KF titration of aldehydes or ketones. These side reactions can compro­mise the water content determination. HYDRANAL­Com­posite 5K is a special formulation consisting of imidazole, sulfur dioxide, and iodine dissolved in diethylene glycol monoethyl ether. Three Fluka®­branded working media for use with HYDRANAL­Composite 5K are supplied by Sigma­Aldrich:

(1) HYDRANAL­KetoSolver (free of halogenated hydrocarbons)

(2) HYDRANAL­Medium K(3) HYDRANAL­Working Medium K (very toxic /excellent

dissolving properties)

Suppression of Side Reactions that lead to Erroneous Water Content DeterminationsThe HYDRANAL­K line of reagents does not contain the conventional methanol component. Methanol has been omitted in order to prevent nucleophilic addition with aldehydes and ketones which leads to the formation of water (Figure 1). Water produced by this side reaction would cause elevated water content results and vanishing end points. The HYDRANAL­K reagents are also designed to guard against bisulfite addition to aldehydes (Figure 2).

(continued on page 22)

This second side reaction consumes a portion of the original water content from the sample and results in erroneously low water content measurements. HYDRANAL­Composite 5K is a variant of HYDRANAL­Composite 5 with a slightly slower reaction speed to maximize suppression of the bisulfite addition side reaction.

HYDRANAL­K reagents are also suitable for some amines, siloxanes and other titrations requiring methanol­free working media. HYDRANAL­Medium K, HYDRANAL­Work­ing Medium K, and HYDRANAL­KetoSolver can each act as the solvent for the determination of water in any substance where methanol can interfere with the titration and there­fore must be avoided. The general utility of these reagents renders a universal applicability for KF titrations; they are also suited for easy­to­handle samples that do not interfere with the KF reaction.

Distinctives of the HYDRANAL-K Working MediaAll these Fluka­branded working media are free of methanol, but each possesses unique properties for optimization with particular samples. HYDRANAL­KetoSolver is primarily 1­methoxy­2­propanol and is entirely free of halogenated hydrocarbons. It is used with HYDRANAL­Composite 5K for the KF titration of aldehydes and highly reactive ketones like cyclohexanone, trifluoroacetone, and diacetyl. For certain less reactive ketones, HYDRANAL­Composite 5 can be used as the titrating agent.

Figure 1 Aldehydes and ketones undergo nucleophilic addition of methanol resulting in formation of acetal or ketal and water

Figure 2 Bisulfite addition reaction consuming water

Page 22: Analytix - Sigma-Aldrich





HYDRANAL®­Working Medium K has exceptional solvating power but must be designated as toxic because it contains 2­chloroethanol. This hazardous component is omitted from HYDRANAL­Medium K and replaced with less toxic alcohols in order to lower the toxicity rating; HYDRANAL­Medium K contains a percentage of chloroform. Not only is the toxicity level lowered, but the revised formulation of HYDRANAL­Medium K provides performance advantages with very reactive aldehydes such as propionaldehyde, butyraldehyde and crotonaldehyde. Capacity and accuracy are also improved for other compounds such as salicylalde­hyde, acetylacetone, 2,4­dihydroxyacetophenone, and 2­benzolpyridine.

Replacement of HYDRANAL®-Working Medium K with HYDRANAL-Medium KEssentially, HYDRANAL­Medium K is a full­fledged substi­tute for HYDRANAL­Working Medium K, providing the same sample capacity, speed and accuracy. In addition, HYDRANAL­Medium K offers important application, safety and transportation benefits. It not only improves workplace safety, but also reduces the amount of packaging material required for shipment and the associated handling and disposal of that packaging material.

The Sigma­Aldrich laboratories compared HYDRANAL­Working Medium K and HYDRANAL­Medium K and have reported these findings:

• Comparable sample capacity in 30 mL medium

• Comparable results for water content and standard deviation

• Comparable titration speed

• Comparable accuracy of recovery rate of added water after sample titration

Coulometric KF Titration in KetonesFor the coulometric water determination in ketones, Sigma­Aldrich offers specially designed HYDRANAL­Coulomat reagents: Coulomat AK and Coulomat CG­K.

HYDRANAL­Coulomat AK is an anolytic reagent for the cou­lometric determination of water in ketones. It contains imidazole, sulfur dioxide, and iodide dissolved in a suitable solvent mixture and has a capacity of approximately 100 mg of water per 100 mL. HYDRANAL­Coulomat AK can also be used as a single reagent for coulometry without diaphragm. HYDRANAL­Coulomat CG­K is the corresponding catholytic reagent. It does not contain halogenated hydrocarbons. The water capacity of 5 mL HYDRANAL­Coulomat CG­K is 100 mg.

The total sample volume used with one charge of coulo­metric reagents should not exceed 20 mL (as liquid sample or as dissolved solid sample). Because of ongoing side reac­tions, larger sample amounts can lead to high instrument drift, causing vanishing end points and erroneous results of the titration.

Application Example: Determination of Water in AcetoneThe water content in acetone can be determined using both volumetric and coulometric KF titration techniques:

• Volumetric titration Add 30 mL HYDRANAL­Medium K to the titration vessel and titrate to dryness with HYDRANAL­Composite 5 K. Add approximately 5 mL of the acetone sample, weighed by difference, to the vessel. Titrate the water content with HYDRANAL­Composite 5 K.

• Coulometric titration Add 100 mL HYDRANAL­Coulomat AK to the anolyte compartment of the titration vessel and 5 mL HYDRANAL­Coulomat CG­K to the catholyte compartment. After starting the titrator, the cell is dried automatically. When the drift is stable, add approximately 0.5 g of the sample, weighed by difference, to the cell.

Benefits of HYDRANAL-Medium K over HYDRANAL-Working Medium K • Reduced toxicity for improved workplace safety

while providing equal reactivity

• Performance advantages with very reactive aldehydes

• Improved capacity and accuracy

• Less waste of packaging material

Page 23: Analytix - Sigma-Aldrich






General Recommendations for KF Titration in Aldehydes and Ketones • Aldehydes

Short­chain aldehydes show a strong tendency to form acetals (along with water formation). Aromatic aldehydes tend to undergo the bisulfite addition. To overcome these potential interferences, we recommend using relatively small samples and titrating rapidly to suppress the formation of acetals and bisulfite compounds. Cou­lometry is not advised, but if it is necessary, the sample volume should be very small. For example, for water determination in aromatic aldehydes, the sample volume must be less than 0.5 mL. Aliphatic aldehydes, such as acetaldehyde, are highly reactive and tend to rapidly form acetals. Only volumetric titration with ethanol­based K­reagents is recommended and the amount of sample should be kept to a minimum.

• Ketones Ketones have a tendency to form ketals while at the same time forming water. Cyclohexanone and acetone react rapidly, while long­chain ketones and aromatic­substituted ketones show slower reaction rates. Reactive ketones are titrated with ethanol­based K­reagents.

Substance Application Number

1,2­Cyclohexanedione 306

2­Dimethylaminomethyl cyclohexanone (DMC base) 518

Butanal (N­Butyraldehyde and iso­Butyraldehyde) 248

Crospovidone (Cross­linked homopolymer of 1­vinylpyrrolidone­2­on)


Dexpanthenol 507

Diacetyl (2,3­Butanedione) 496

Ethosuximide (3­Ethyl­3­methyl­2,5­pyrrolidindion) 510

Glutardialdehyde 50% 273

Glyoxal solution 40% 267

Glyoxylic acid methylester methylhemiacetal 261

Hexafluoroacetone sesquihydrate 154

N­Formylmorpholine (4­Morpholine carboxaldehyde) 392

N,N­Dimethyl formamide 424

Table 1 Available KF application reports for selected aldehydes and ketones

Cat. No. Brand Description Package Size

34816 Fluka® HYDRANAL­Composite 5K 500 mL, 1 L, 2.5 L

34805 Fluka HYDRANAL­Composite 5 500 mL, 1 L, 2.5 L

34738 Fluka HYDRANAL­KetoSolver 500 mL, 1 L

34698 Fluka HYDRANAL­Medium K 1 L

34817 Fluka HYDRANAL­Working Medium K 1 L

34820 Fluka HYDRANAL­Coulomat AK 500 mL

34821 Fluka HYDRANAL­Coulomat CG­K 50 mL

Table 2 Selected HYDRANAL products (complete list can be found on Sigma-Aldrich website)

To obtain application reports and more information on HYDRANAL­K reagents as well as on our other high­quality HYDRANAL reagents for pyridine­free water determination by KF titration, please visit our website sigma-aldrich.com/hydranal or contact our HYDRANAL laboratories:

Europe and Global MarketMr. Thomas WendtTechnical Service HYDRANALWunstorfer Straße 40D­30926 Seelze, GermanyTel.: +49 (0) 5137 8238­353Fax: +49 (0) 5137 8238­698E­Mail: [email protected]

USA and CanadaMr. Doug ClarkHYDRANAL Technical Center545 S. Ewing AveSt. Louis MO 63103, USAToll free: +1 800 493­7262 (USA and Canada)Fax: +1 314 286­6699E­Mail: [email protected]

The coulometric determination is possible by using HYDRANAL®­Coulomat AK and Coulomat CG­K reagents. The sample size for coulometric measurements should not exceed 1 mL; for very reactive ketones like cyclohexa­none, a maximum of 0.5 mL per sample is recommended.

Page 24: Analytix - Sigma-Aldrich

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Date: 06/2012Sams Code: OMC