Instrumental Analysis
Laboratory Practice
University of Pécs Faculty of Sciences
Department of Analytical and Environmental Chemistry
2014
Instrumental Analysis
Laboratory Practice
Editor: Balázs Csóka
Authors: Borbála Boros
Anita Bufa Balázs Csóka
Ágnes Dörnyei Csilla Fenyvesi-Páger
Anikó Kilár Ibolya Kiss
Lilla Makszin Tímea Pernyeszi
Reviewed: Attila Felinger Ferenc Kilár
DOI: 10.15170/TTK.2014.00001
Instrumental Analysis
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Contents
CHAPTER 1 – SYLLABUS ------------------------------------------------------------------------------------------------- 5
ELECTROANALYSIS------------------------------------ ------------------------------------------------------------------- 8
CHAPTER 2 – POTENTIOMETRY-------------------------- ------------------------------------------------------------ 8
2.1 THEORY-----------------------------------------------------------------------------------------------------------------------8 Classification of Electrochemical Methods -------------------------------------------------------------------------- 8
2.2 PRACTICE ------------------------------------------------------------------------------------------------------------------- 12 Procedure 1 – Direct potentiometry – pH measurement of buffer solutions ----------------------------------- 12 Procedure 2 – Indirect potentiometry – Acid-base titration ----------------------------------------------------- 13
2.3 QUESTIONS----------------------------------------------------------------------------------------------------------------- 15 2.4 ABBREVIATIONS, DEFINITIONS ------------------------------------------------------------------------------------------ 15
CHAPTER 3 – CONDUCTOMETRY -------------------------- -------------------------------------------------------- 16
3.1 THEORY--------------------------------------------------------------------------------------------------------------------- 16 3.2 PRACTICE ------------------------------------------------------------------------------------------------------------------- 19
Procedure 1 – Acid-base titration using conductometric end-point detection--------------------------------- 19 Procedure 2 – Titration of weak bases with strong acid: determination of the temporary hardness of the drinking water---------------------------------------------------------------------------------------------------------- 20
3.3 QUESTIONS----------------------------------------------------------------------------------------------------------------- 21
CHAPTER 4 – SPECTROPHOTOMETRY--------------------------------------------------------------------------- 22
4.1 THEORY--------------------------------------------------------------------------------------------------------------------- 22 4.2 PRACTICE ------------------------------------------------------------------------------------------------------------------- 26
Procedure 1 – Determination of the concentration of NiSO4 solution by standard addition method------- 26 Procedure 2 – Determination of methylene blue concentration ------------------------------------------------- 28
4.3 QUESTIONS----------------------------------------------------------------------------------------------------------------- 28 4.4 ABBREVIATIONS, DEFINITIONS ------------------------------------------------------------------------------------------ 29
CHAPTER 5 – OPTICAL ATOMIC SPECTROSCOPY------------ ----------------------------------------------- 30
5.1 THEORY--------------------------------------------------------------------------------------------------------------------- 30 5.1.1 Atomic emission spectroscopy--------------------------------------------------------------------------------- 30 5.1.2 Atomic absorption spectroscopy ------------------------------------------------------------------------------ 32
5.2 PRACTICE ------------------------------------------------------------------------------------------------------------------- 35 Procedure 1 – Concentration determination of potassium ion (K+) solution by atomic emission spectroscopy (calibration curve method) --------------------------------------------------------------------------- 35 Procedure 2 – Concentration determination of copper ion (Cu2+) solution with atomic absorption spectroscopy (Standard addition experiment) --------------------------------------------------------------------- 36
5.3 QUESTIONS----------------------------------------------------------------------------------------------------------------- 37
CHAPTER 6 – INTRODUCTION TO CHROMATOGRAPHIC SEPARAT ION----------------------------- 38
6.1 GENERAL DESCRIPTION OF CHROMATOGRAPHY --------------------------------------------------------------------- 38 6.2 CLASSIFICATION OF CHROMATOGRAPHIC METHODS---------------------------------------------------------------- 38 6.3 ELUTION IN COLUMN CHROMATOGRAPHY---------------------------------------------------------------------------- 40 6.4 IMPORTANT CHROMATOGRAPHIC QUANTITIES AND RELATIONSHIPS--------------------------------------------- 41 6.5. KEYWORDS, ABBREVIATIONS ------------------------------------------------------------------------------------------- 44 6.4. QUESTIONS----------------------------------------------------------------------------------------------------------------- 44
CHAPTER 7 – GAS CHROMATOGRAPHY--------------------- ---------------------------------------------------- 45
7.1 THEORY--------------------------------------------------------------------------------------------------------------------- 45 The Kováts retention index ------------------------------------------------------------------------------------------- 47 7.1.1 The main parts of GC------------------------------------------------------------------------------------------- 47
7.2 PRACTICE ------------------------------------------------------------------------------------------------------------------- 49 Procedure 1 – Calculate the Kováts retention index of unknown components.-------------------------------- 51 Procedure 2 – Qualitative analysis by standard components. --------------------------------------------------- 51 Procedure 3 – The examination of temperature as a factor affecting separation. ---------------------------- 51
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Procedure 4 – Characterize the separation of the components in the sample. -------------------------------- 51 Concepts and Abbreviations------------------------------------------------------------------------------------------ 52
7.3 QUESTIONS----------------------------------------------------------------------------------------------------------------- 52
CHAPTER 8 - HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ------------------------ 53
8.1 INTRODUCTION------------------------------------------------------------------------------------------------------------- 53 8.2 TYPES OF HPLC ----------------------------------------------------------------------------------------------------------- 53 8.3 THE HPLC INSTRUMENT ------------------------------------------------------------------------------------------------- 55
8.3.1 Flasks for the mobile phase storage -------------------------------------------------------------------------- 55 8.3.2 Pumps ------------------------------------------------------------------------------------------------------------ 55 8.3.3 Injectors ---------------------------------------------------------------------------------------------------------- 56 8.3.4 Columns ---------------------------------------------------------------------------------------------------------- 56 8.3.5 Detectors --------------------------------------------------------------------------------------------------------- 57
8.4. PRACTICE ------------------------------------------------------------------------------------------------------------------ 58 Quantitative analysis of active substances of Saridon analgetic by RP-HPLC-------------------------------- 58
8.5. KEYWORDS, ABBREVIATIONS ------------------------------------------------------------------------------------------- 61 8.6 QUESTIONS----------------------------------------------------------------------------------------------------------------- 61
CHAPTER 9 – MASS SPECTROMETRY ---------------------------------------------------------------------------- 62
9.1 THEORY--------------------------------------------------------------------------------------------------------------------- 62 9.1.1 Ion sources working under atmospheric pressure ---------------------------------------------------------- 63 9.1.2 Quadrupole mass analyzers----------------------------------------------------------------------------------- 64 9.1.3 Mass spectrum--------------------------------------------------------------------------------------------------- 66
9.2 PRACTICE ------------------------------------------------------------------------------------------------------------------- 68 Structural analysis of capsaicin and dihydrocapsaicin by electrospray – ion trap MS and MS/MS methods--------------------------------------------------------------------------------------------------------------------------- 68
9.3. KEYWORDS, ABBREVIATIONS ------------------------------------------------------------------------------------------- 70 9.4. QUESTIONS----------------------------------------------------------------------------------------------------------------- 71
CHAPTER 10 – CAPILLARY ELECTROPHORESIS ------------- ------------------------------------------------ 72
10.1 THEORY-------------------------------------------------------------------------------------------------------------------- 72 10.1.1 Introduction ---------------------------------------------------------------------------------------------------- 72 10.1.2 Instrumentation ------------------------------------------------------------------------------------------------ 73 10.1.3 Background----------------------------------------------------------------------------------------------------- 74 10.1.4 Electro-osmotic flow (EOF)---------------------------------------------------------------------------------- 74 10.1.5 Capillary zone electrophoresis ------------------------------------------------------------------------------ 76 10.1.6 Electropherogram --------------------------------------------------------------------------------------------- 77 10.1.7 Analytical parameters----------------------------------------------------------------------------------------- 77
10.2 PRACTICE------------------------------------------------------------------------------------------------------------------ 79 Measuring of preservatives and vitamin C in lime juice---------------------------------------------------------- 79
10.3 QUESTIONS---------------------------------------------------------------------------------------------------------------- 81
CHAPTER 11 – CALCULATIONS -------------------------- ----------------------------------------------------------- 82
ANSWERS TO PROBLEMS------------------------------------------------------------------------------------------------------ 87 CHEMICAL ELEMENTS LISTED BY ATOMIC MASS-------------------------------------------------------------------------- 89 STANDARD ELECTRODE POTENTIALS--------------------------------------------------------------------------------------- 89
BIBLIOGRAPHY --------------------------------------- -------------------------------------------------------------------- 90
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Chapter 1 – Syllabus
These pages give a short description of the Instrumental Analysis laboratory practice.
Instructors: the instructors of the course are the staff member of the Analytical and
Environmental Chemistry Department.
Time and place of the course: the length of each lab is 180 min. The starting time and the
location will be decided during the first week of the semester.
Goals: The laboratory course aims the use of instrumental methods for chemical and
pharmaceutical analysis. By using different instrumentations, the students are able to learn the
basic methods in the chemical laboratory.
Attendance: obligatory. According to the “Academic and Examination Regulations of the
University of Pécs” Section 2, Annex 1/A (6 a, b)1 only two absences from the practical
course (by any reason) will be accepted.
Only 10 min. tardiness of the student can be accepted, arriving later is not acceptable and it
will be marked as absence.
Each week the lesson begins with a short test. Only those students are allowed to take part in
the practice, who reach a ‘pass’ grade.
Students are not allowed to attend the practical course of another group with the same topic.
Requirements: Oral examinations are only allowed if successful written tests in the practices
and max. one ‘fail’ grade to the exercises are reached. The grade obtained for the practical
course will give a 1/3 weight into the final grade.
1 Academic and Examination Regulations of the University of Pécs (Eff. from 18. Dec. 2008) (http://aok.pte.hu/docs/th/file/COS_090618.pdf) Annex 2. Rules pertaining to attending classes - Section 1/A (6) The rules of accepting absences are as follows: a) the student who has been absent from less than 15% of the classes of the course-unit cannot be condemned for absence. b) whose absence was between 15 and 25% (for any reason), the person responsible for the course-unit shall decide on accepting the semester by examining the particular case. His/her decision shall be indicated by signing or refusing to sign the ‘end-of-semester signature’ heading in the registration book. c) he/she whose absence reaches 25% (for any reason, with or without a certified excuse) cannot be granted entry to examination.
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Homework: Every week the measured data need to be processed at home. Calculations,
graphs, theory of the measurements need to be written in the laboratory notebook. The
notebook - including all the necessary parts - should be handed over not later than 48 hours
after the lesson. Computerized methods (Excel, Origin, SPSS) are not accepted for
calculations. All the graphs should be made on millimeter squared (scale) paper.
The format of the homework should have a following layout and content.
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Date Student’s name
Instructor’s name
Title of the practice, definition of the experiments
Group number
The laboratory notebook should contain the followings:
first page:
Main goal of the experiments, used methods, number of the samples (unknowns),
calculated results – all written into the suitable place
All the other pages:
Details of the experiments
Theory of the measurements, with necessary figures
Stepwise detailed description of the measurement
o Name and identification of the samples
o The analytical method used, reagents, sample pretreatment
o Details of the instrument used (name, type), settings, working parameters
o Measured data, direct measurement results
o Calculations, including intermediate and final results
o Any problems observed during the measurements
o Other notes
In the laboratory notebook all the results, graphs, drawing, documents etc.
obtained during the experiments should be fixed (e. g. glued in)!!
Sample number(s) of the
(unknown) measured
Experimental results Evaluation, grade
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Electroanalysis
Electroanalytical methods deal with procedures where the analysis is done in an
electrochemical cell by measuring electrode potential and/or current flow. Several methods
can be distinguished depending on the parameter controlled or measured during the
electrochemical process. The three most important methods of electroanalysis are:
1 - potentiometry (measuring the electrode potential difference)
2 - coulometry (measuring the current flow through the cell as a function of time)
3 - voltammetry (the cell potential is regulated while the current is measured).
Depending of the measurements, 2-4 electrodes are immersed into the sample in the
measuring cell to do electroanalysis. Based on its functions they can be a) working (indicator)
b) reference and c) counter (auxiliary) electrodes.
Chapter 2 – Potentiometry
2.1 Theory
Classification of Electrochemical Methods
There are only three principal sources for the electroanalytical signal: potential, current,
and charge. These signals make a wide variety of experimental designs. The simplest division
of the method is between bulk methods, which measure properties of the whole solution, and
interfacial methods, in which the signal is a function of phenomena occurring at the interface
between an electrode and the solution in contact with the electrode. By measuring the
solution’s conductivity, (which is proportional to the total concentration of dissolved ions)
one is using a bulk electrochemical method. By determining the pH using a glass-electrode is
one example of an interfacial electrochemical method.
Interfacial Electrochemical Methods
Interfacial electrochemical methods can be divided into static methods and dynamic
methods. Static methods mean that no current passes between the electrodes and the
concentrations of species in the electrochemical cell does not change (static). Potentiometry is
one of the most important quantitative electrochemical methods, in which the potential of the
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electrochemical cell is measured under static conditions. Because no (or only a negligible)
current flows while measuring an electrode’s potential, the composition of the solution
remains unchanged. For this reason, potentiometry is a useful quantitative method.
As the Nernst equation was formulated in 1889, the relation between the
electrochemical cell’s potential and the concentration of electroactive species in the cell had
been clear. The development of the pH sensitive glass electrode was based on the discovery of
Cremer in 1906. Cremer discovered that a potential difference exists between the two sides of
a thin glass membrane when opposite sides of the membrane are in contact with solutions
containing different concentrations of H3O+.
Potentiometric measurements are made using simple instrumentations: a potentiometer
to determine the difference in potential between an indicator electrode and the reference
electrode which supplying a reference potential.
Potential and Concentration - The Nernst Equation
The potential of a potentiometric electrochemical cell is given as
Ecell = Ec – Ea
where Ec and Ea are potentials for the reactions occurring at the cathode and anode. These
potentials are a function of the concentrations of analyte, as defined by the Nernst equation:
alnnFRT
EE += 0
where E° is the standard-state reduction potential, R is the gas constant, T is the temperature
in Kelvins, n is the number of electrons involved in the reduction reaction, F is Faraday’s
constant, and a is the activity of the measured species, which is identical with the
concentration of the species as the conc. is lower than 10-3 mol/ dm3 .
Under typical laboratory conditions (temperature of 25 °C or 298 K) the Nernst equation
becomes
clogn
.EE
05900 +=
where E is given in volts.
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Reference Electrodes
Potentiometric electrochemical cells are constructed from two half-cells: one of the
half-cells produce a reference potential, and the potential of the other half-cell indicates the
analyte’s concentration. By convention, the reference electrode is taken to be the anode, and
the indicator electrode to the cathode.
The reference electrode’s potential must be stable so that any change in Ecell is attributed
to the indicator electrode, and, therefore, to a change in the analyte’s concentration. The most
common types of reference electrodes are: standard hydrogen electrode (SHE), saturated
calomel (Hg2Cl2) electrode (SCE) and silver/silver chloride electrode.
Silver/silver chloride electrode is based on the redox couple between
AgCl and Ag.
)aq(Cl)s(Age)s(AgCl −− +↔+
The potential of the Ag/AgCl electrode is determined by the
concentration of Cl– around the AgCl.
]Cllog[.EE AgCl/Ag−−= 05900 ( 0
AgCl/AgE = 0.222 V)
When prepared using a saturated solution of KCl, the Ag/AgCl
electrode has a potential of +0.197 V at 25 °C. As 3.5 M KCl is used
the electrode has a potential of +0.205V at 25 °C.
A typical Ag/AgCl electrode is shown in Figure 2-1. It is consists
of a silver wire, the end of which is coated with a thin film of AgCl.
The wire is immersed in a solution that contains the desired
concentration of KCl and that is saturated with AgCl. A porous plug
serves as the salt bridge.
Glass Ion-Selective Electrodes
Typical glass electrodes are manufactured of a glass with a composition of
approximately 22% Na2O, 6% CaO, and 72% SiO2. When immersed in an aqueous solution,
both the – approximately 10 nm thin – outer membrane layers become hydrated, while the
inner part is non-hydrated or dry. Hydration of the glass membrane results in the formation of
negatively charged sites (G-), formed by deprotonation of Si-OH sites of the glass
membrane’s silica framework. Sodium ions, which are able to move through the hydrated and
Figure 2 -1 Scheme of a Ag/AgCl reference
electrode
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dry layer, serve as the counterions. Hydrogen ions from solution diffuse into the membrane
and, since they bind more strongly to the glass than does Na+, displace the sodium ions
)aq(Na)s(HG)s(NaG)aq(H ++−+−+ +−↔−+
The protonation or deprotonation of G- happens as the membrane is in contact with
solution having either lower or higher pH. Since the inner side of the glass is immersed into a
pH buffer, the outer side is attacked by different amount of H+ regarding the sample’s pH,
which results in a different amount of occupied charged sites, thus charge difference occurs
between the two boundaries of the membrane. The transport of charge across the membrane is
carried by the Na+ ions.
The potential of glass electrodes obeys the equation
]Hlog[.KEcell++= 0590
over a pH range of approximately 1–12 (K is a constant).
Above pH 12, the glass membrane shows higher response
(higher selectivity) to other cations, such as Na+ and K+.
Glass membrane electrodes have been usually
produced in a combination form that includes both the
indicator and the reference electrodes, which simplifies the
measurement of pH. An example of a typical combination
electrode is shown in Figure 2 - 2.
Since the usual thickness of the glass membrane in an
ion-selective electrode is about 50-100 µm, they must be
handled carefully to prevent breakage or cracks. Glass
electrodes should not be allowed to dry out, as this destroys
the membrane’s hydrated layer. The composition of a glass
membrane changes over time, affecting the electrode’s
performance. The average lifetime for a glass electrode is
several years.
Measurement of pH
Before measuring the pH of a solution, the glass-electrode should be calibrated with
buffers of known pH. Usually the calibration is carried out with 2 buffers, in which the
electrode is immersed and the electrode potential is measured. The measured values are then
Figure 2 -2 Scheme of combined glass electrode
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used for extrapolating the pH–potential relation within the pH 1-12 working range, and by
measuring the electrode potential in an unknown solution, its pH can be calculated.
2.2 Practice
Summary
In these labs first you will get to know the instruments and method of pH
measurements. Then you will be given solutions to obtain their pH.
In the second part of the experiments, you will use a pH electrode to follow the course
of an acid-base titration. You will observe how pH changes slowly during most of the reaction
and rapidly near the equivalence point. You will compute the first and second derivatives of
the titration curve to locate the end point. From the mass of unknown acid or base and the
moles of titrant, you can calculate the molecular mass of the unknown. Sections 11-1 and 11-5
of the Harris book provide background for this experiment.
Reagents
Standard: standard 0.1 M HCl with known factor value
Methylred and phenolphthalein indicators
pH calibration buffers: pH 10 and pH 4
Unknowns of NaOH solution
Procedure 1 – Direct potentiometry – pH measurement of buffer solutions
1. Prepare the 3 buffer solutions selected by your instructor from the followings
compositions:
A (mL) : B (mL) 1. 10:40 2. 10:20 3. 20:30 4. 10:10 5. 30:20 6. 20:10 7. 40:10
Pipette the given volumes into a clean and dry 100 mL flask, and fill with water to the mark.
Take approx. 20 mL into a 50 mL beaker.
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2. Following instructions for your particular pH meter, calibrate a meter and glass
electrode, using buffers with pH values near 10 and 4. Rinse the electrodes well with distilled
water and blot them dry with a tissue before immersing in any new solution.
3. Measure, and record the pH of the selected solutions. Rinse the electrode carefully
after all measurements.
Data analysis 1.
Calculate the concentration of the free [H+] or [OH-] in mol/1000 mL units.
Use the following formulas:
pH = -log [H+] ; pH + pOH = 14
Procedure 2 – Indirect potentiometry – Acid-base titration
1. Take one of the volumetric flasks with an unknown concentration of NaOH. Fill with
water to the mark.
2. Following instructions for your particular pH meter, calibrate a meter and glass
electrode, using buffers with pH values near 10 and 4. Rinse the electrodes well with distilled
water and blot them dry with a tissue before immersing in any new solution.
3. The first titration is intended to be rough, so that you will know the approximate end
point in the next titration. Pipette 10.0 mL of unknown into a 150-mL beaker containing a
magnetic stirring bar. Place the electrode(s) in the liquid so that the stirring bar will not strike
the electrode. Add approximately 70 mL water to it in order to reach the necessary level of the
solution, regarding the pH-electrode sensible part. Add 3 drops of any of the indicators and
titrate with standard 0.1 M HCl. Add 1.0 mL of titrant at a time so that you can estimate the
equivalence volume. Write down the pH values after every addition, and continue it till
adding 20 mL of titrant.
4. Now comes the careful titration. Pipette 10.0 mL of unknown solution into a 150-mL
beaker containing a magnetic stirring bar. Position the electrode(s) in the liquid so that the
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stirring bar will not strike the electrode. If a combination electrode is used, the small hole near
the bottom on the side must be immersed in the solution. This hole is the salt bridge to the
reference electrode. Allow the electrode to equilibrate for 1 min with stirring and record the
pH.
5. Add 1 drop of indicator and begin the titration. Add 1.0 mL aliquots of titrant and
record the exact volume, the pH, and the color 30 s after each addition. When you are within 2
mL of the equivalence point, add titrant in 0.2 mL increments. The equivalence point has the
most rapid change in pH. Continue titrating beyond the equivalence point within the 2 mL
range by 0.2 mL. Then finish it by 1 mL steps and record the pH after each as you reach 20
mL aliquots of titrant added.
6. Repeat Steps 4 and 5, thus measure carefully two times. Data analysis 2.
1. Construct a graph of pH versus titrant volume.
Mark on your graph where the indicator colour change(s)
was (were) observed. Obtain the equivalence volume.
2. Fill in the data into the Table 2-1 below and
compute the first derivative (the slope, ∆pH/∆V) for each
data point. Draw the first derivative curve. From your
graph, estimate the equivalence volume as accurately as
you can, as shown in Figure 2-3.
3. Fill in the Table’s last column, compute the
second derivative (the slope of the slope, ∆(slope)/∆V).
Prepare a graph as before and locate the equivalence
volume as accurately as you can.
4. From the average of the equivalence volumes
and the molecular mass of unknown (NaOH), calculate the
mass of the unknown in mg/100 mL units.
Figure 2 -3 Titration curve and its derivatives of an acid-base titration
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2.3 Questions
1. Draw a reference electrode, and show its working principles.
2. Give a brief classification of the electroanalytical methods.
3. How is a glass electrode sensing pH?
4. Why is a reference electrode needed for potentiometric measurements?
5. Show the connection between the concentration of a solution and the electrode’s potential
immersed into it.
6. Show the place of potentiometry within the electrochemical methods.
7. Write the steps of a potentiometric titration (roughly!).
2.4 Abbreviations, definitions
indicator electrode (also known as the working electrode).
An electrode; its potential is changing as a function of the analyte’s concentration
reference electrode
An electrode; its potential remains constant. Other potentials can be measured against it.
glass electrode
An ion-selective electrode made of a thin glass membrane. Its potential develops from an H+
ion-exchange reaction on the glass membrane’s surface.
V [mL] pH ∆∆∆∆pH / ∆∆∆∆V ∆∆∆∆2pH / ∆∆∆∆V2
Table 2-1 Sample table for collecting titration data
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Chapter 3 – Conductometry
3.1 Theory
One example of bulk electrochemical methods (see classification of the electrochemical
methods) is the measurement of the solution’s conductivity. The conductance depends
directly upon the number of charged particles in the solution. All ions individually contribute
to the conduction process, but the ratio of current carried by any species is determined by its
relative concentration and its inherent mobility. The application of direct conductance
measurements to analysis is limited because of the nonselective nature of the method. It is
mostly used for the determination of total electrolyte concentration, like as a criterion of
distilled water’s purity. Indirect conductance measurement, like conductometric titrations, can
be applied for the determination of numerous substances, while it is locating the end-point of
a titration.
Important relationships Conductance – G
The conductance of a solution (in ohm -1) is the reciprocal of the electrical resistance. That is,
G=1/R
where R is the resistance in ohms.
Specific Conductance – κ
Conductance is directly proportional to the cross-sectional area A and inversely proportional
to the length I of a uniform conductor; thus,
lA
G κ=
where κ is a proportionality constant called the specific conductance. These parameters are
based upon the centimeter, thus κ is the conductance of a 1 cm3 cube of solution. The
dimensions of specific conductance are then ohm-1 cm-1.
Dividing the specific conductance by the molar concentration of the solution (c
[mol/L]) one can obtain the molar conductivity.
Λm=κ/c
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Based on Kohlrausch’ first law, the anions and cations are influencing the electric
conductivity independently, thus the molar conductivity is the sum of the molar conductivity
caused by the anions and cations separately. For strong electrolytes one can state:
Λm=Σλ++Σλ-
where
Σλ+= molar conductivity of the cations [cm2 /(Ω mol)]
Σλ-= molar conductivity of the anions [ cm2 /(Ω mol)]
Measurement of conductance A conductance measurement requires a cell to
contain the solution, the electrode and suitable
electricity to measure the resistance of the solution.
An alternating current source needs to apply in
order to eliminate the effect of electrolysis. However,
the suitable frequencies are limited to about 1000-
3000 Hz.
The conductivity electrode is build up from two
flat or cylindrical electrodes separated by a fixed
distance. The electrodes are made of platinum and to
increase their effective surface are usually platinized.
In Figure 3-1 you can find a scheme of an electrode,
and in Figure 3-2 the wiring diagram of the device.
Conductometry in practice
Conductometric measurements can be used in all type of chemical measurements, in
which the numbers of the charge transferring species are varying. These are the acid-base
reactions, reactions with precipitation or gas evaluation, complex formation etc.
In order to locate end points in titrations the conductance data are plotted as a function
of titrant volume. The two linear portions are then extrapolated, the point of intersection being
Pt plate
glass body
hole
conductomerticcell
R U
Figure 3-1 Scheme of a conductometric electrode
Figure 3-2 Schematic circuit diagram of a conductometric device
Instrumental Analysis Conductometry
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taken as the equivalence point. Sufficient number of measurements (four to six before and
after the equivalence point) is needed to define the titration curve.
Acid-Base Titration
Neutralization titrations are particularly well adapted to the conductometric end point
because of the large ionic conductances of hydrogen and hydroxide ions compared with the
conductances of the species that replace them in solution.
Titration curve of strong acid and
base is shown in Figure 3-3. The solid
line in Figure 3-3 represents a curve
obtained when sodium hydroxide is
titrated with hydrochloric-acid. Also
plotted are the calculated contributions of
the individual ions to the conductance of
the solution (broken lines). During
neutralization, hydroxide ions are
neutralized and also replaced by an
equivalent number of less mobile
chloride ions; the conductance changes to lower values as a result of this substitution. At the
equivalence point, the concentrations of hydrogen and hydroxide ions are at a minimum and
the solution exhibits its lowest conductance. A reversal of the slope occurs past the end point
as the hydrogen ion concentrations increase. With the exception of the immediate
equivalence-point region, an excellent linearity exists between conductance and the volume of
base added.
The percentage change in conductivity during the course of the titration of a strong acid
or base is the same regardless of the concentration of the solution. Thus, very dilute solutions
can be analyzed with accuracy comparable to more concentrated ones.
Figure 3-3 Titration curve of an acid-base titration with conductometric end-point detection
Instrumental Analysis Conductometry
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3.2 Practice
Procedure 1 – Acid-base titration using conductometric end-point detection
Reagents
standard 0.1 M HCl with known factor value
Materials
150-mL beaker, burette, magnetic stirrer, conductometer
Unknowns of NaOH solution
Procedure
1. Take one of the volumetric flasks with an unknown concentration of NaOH. Fill with
distilled water to the mark.
2. Pipette 10.0 mL of unknown into a 150-mL beaker containing a magnetic stirring bar.
Position the electrode(s) in the liquid so that the stirring bar will not strike the electrode. Add
approximately 70 mL water to it in order to reach the necessary level of the solution,
regarding the conductometric electrode sensible part. Titrate with standard 0.1 M HCl. Add
1.0 mL of titrant at a time so that you can estimate the equivalence volume. Write down the
conductivity values after every addition, and continue it till adding 20 mL of titrant.
3. Repeat the titration two times, using the same procedure as Step 2.
Data analysis
1. Construct a graph of conductance versus titrant volume. Determine the equivalence
points visually, calculate the average of the equivalence volume.
2. By using the “least squares method”, calculate the equations of the titration curve,
and calculate the point of intersection. (You will find some help at the end on this manual.)
3. From the average of the equivalence volumes and the molecular mass of unknown
(NaOH), calculate the mass of the dissolved unknown in mg/100 mL units.
Instrumental Analysis Conductometry
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Procedure 2 – Titration of weak bases with strong acid: determination of the temporary
hardness of the drinking water
Temporary hardness is due to the presence of calcium hydrogencarbonate Ca(HCO3)2(aq)
and magnesium hydrogencarbonate Mg(HCO3)2(aq). Both calcium hydrogencarbonate and
magnesium hydrogencarbonate decompose when heated and the water is boiled. The original
insoluble carbonate is reformed, and the precipitation of solid calcium carbonate or solid
magnesium carbonate is produced. This removes the calcium ions or magnesium ions from
the water, and so removes the hardness. Therefore, hardness due to hydrogencarbonates is
said to be temporary.
As hydrogencarbonates are titrated with HCl, the following reaction happens:
Ca(HCO3)2 + 2 H+(aq) + 2 Cl-(aq) = 2H2O + 2CO2 + Ca2+
(aq) + 2 Cl-(aq)
Mg(HCO3)2 + 2 H+(aq) + 2 Cl-(aq) = 2H2O + 2CO2 + Mg2+
(aq) + 2 Cl-(aq)
During these reactions the conductivity increases, before and after the equivalence too, but the
slopes are different. Before the equivalence the addition of Cl- and the reaction product
cations from the almost insoluble hydrogencarbonates cause it. After the equivalence the
excess of H+ and Cl- play the most important role in even more higher increase of
conductivity.
Reagents
0.1 M HCl standard with known factor value
Materials
150 mL beaker, burette, magnetic stirrer, conductometer
100 mL graduated cylinder
Procedure
1. Take the graduated cylinder and fill 100 mL tap water into it. Pour it into a 150 mL
beaker. You will titrate it without adding any more distilled water.
2. Put a magnetic stirrer bar into the water sample and begin to titrate with continuous
stirring. Position the electrode(s) in the liquid so that the stirring bar will not strike the
electrode. Titrate with standard 0.1 M HCl. Add 1.0 mL of titrant at a time so that you can
Instrumental Analysis Conductometry
21
estimate the equivalence volume. Write down the conductivity values after every addition,
and continue it till adding 20 mL of titrant. You can use a similar table to collect the data.
V(mL) 0 1 2 3 4 5 6 7 8 9 10 … …
G (µS)
V – added HCl volume in mL unit G – measured conductivity
3. Repeat the titration two times, using the same procedure as Step 2.
Data analysis
1. Construct a graph of conductance versus titrant volume. Determine the equivalence
points visually; calculate the average of the equivalence volumes.
2. By using the “least squares method”, calculate the equations of the titration curve,
and calculate the point of intersection.
3. From the average of the equivalence volumes, supposing the only Ca(HCO3)2 was
present in the sample, calculate the weight of the Ca(HCO3)2 in 100 mL water sample. From
this value calculate the temporary hardness of the water sample in German Hardness Degree
(°dH) (1 unit equals to 10 mg CaO in 1000 mL sample).
Equations to use for calculating the regression equation (least squares method)
In case of linear regression:
baxy +=
where a and b can be obtained as follow:
∑∑
−−−
=2
i
ii
)xx(
)yy)(xx(a xay
n
xayb ii −=
−= ∑ ∑
3.3 Questions
1. What is the molar conductivity?
2. Give a short description about the direct and indirect conductometric methods.
3. Which chemical reactions can be measured by conductometry? Why?
4. Why can we use conductometry for end-point detection of the acid-base titrations?
Instrumental Analysis Spectrophotometry
22
Chapter 4 – Spectrophotometry
Spectro(photo)metry is a group techniques that uses electromagnetic radiation (light) to
measure concentrations.
4.1 Theory
The wavelength (or frequency) of electromagnetic radiation varies over many orders of
magnitude, this is called electromagnetic spectrum. This wide range is divided into different
spectral regions based on the type of atomic or molecular transition that gives rise to the
absorption or emission of photons.
The energy of a photon, in joules, is related to its frequency, wavelength, or
wavenumber by the following equations where h is Planck’s constant, (h=6.626×10–34 Js), c
is the speed of light (3×108 m/s in vacuum), λ is wavelength, ν is frequency.
λ=ν= hc
hE
In absorption spectroscopy the energy carried by a photon (= particle of electromagentic
radiation) is absorbed by the analyte, promoting the analyte from a lower-energy state to a
higher-energy (=excited) state. When a sample absorbs electromagnetic radiation, its energy
level increases, because the photon is “destroyed” and its energy acquired by the sample.
Electron can be excited only when the photon’s energy matches the difference in energy (∆E)
between two energy levels of the sample molecule. One can find on the electromagnetic
spectrum that absorbing a photon of visible light causes a valence electron in the analyte to
move to a higher-energy level.
As a result of absorption, the intensity of photons energy passing through a sample
containing the analyte is attenuated. This attenuation is called as absorbance, which is the
analytical signal. A plot of absorbance as a function of the photon’s energy is called an
absorbance spectrum.
Emission of a photon occurs when a molecule in a higher-energy state returns to a
lower-energy state. The higher-energy state can be achieved in several ways, including
thermal energy, radiant energy from a photon, or by a chemical reaction.
Sources of energy
In absorption spectroscopy the energy of photons is supplied to promote the analyte to a
higher energy (but less stable) state. The absorption of the photon (and thus the energy) is
Instrumental Analysis Spectrophotometry
23
used as analytical information. The electrons in molecules can be promoted by ultraviolet or
visible range radiation. The source of this energy is often a tungsten filament (300-2500 nm)
lamp, a deuterium arc lamp, which is continuous over the ultraviolet region (190-400 nm),
xenon arc lamps (160-2000 nm), or more recently, light emitting diodes (LED) for the visible
wavelengths.
Wavelength selection
In order to get high analytical performance, only a single wavelength needs to be used
for excitation where the analyte is the only absorbing species. Unfortunately, a single
wavelength of radiation from a continuum source cannot be isolated, however, by applying a
wavelength selector (=monochromator) solves this problem, because it allows the passing of
only a narrow band of radiation to the sample.
Very simple method is to selectively absorbing (=filtering) a narrow band of radiation
using an optical filter before the radiation reaches the sample. Unfortunately filtering is
possible only at one selected wavelength. If the wavelength should be selected continuously, a
monochromator with prism or grating needs to be used.
The construction of a typical monochromator with a grating is shown in Figure 4-1.
Radiation from the source enters the monochromator through an entrance slit. The radiation is
collected by a collimating mirror, which reflects a parallel beam of radiation to a diffraction
grating. The diffraction grating is an optically reflecting surface with a large number of
parallel grooves. Diffraction by the grating disperses the radiation in space, where a second
mirror focuses the radiation onto a planar
surface containing an exit slit. In some
monochromators, a prism is used in place of the
diffraction grating.
Radiation exits the monochromator and
passes to the detector. The choice of which
wavelength exits the monochromator is
determined by rotating the diffraction grating. A
narrower exit slit provides a smaller bandwidth
and better resolution, but allows a smaller
throughput of radiation.
As the grating is rotated manually it is
Entrance slit
Exit slit
MirrorsOptical grating
Figure 4-1 Scheme of a monochromator with optical grating
Instrumental Analysis Spectrophotometry
24
called as fixed-wavelength monochromator, while in a scanning monochromator a drive
mechanism continuously rotates the grating, allowing successive wavelengths to exit.
Detectors
The visible signal can be detected by the human eye, but it has a strong limitation in
accuracy and sensitivity. In the spectrometric devices, sensitive transducers are used to
convert a signal induced by photons into electrical signal. Phototubes and photomultipliers
contain a photosensitive surface that absorbs radiation in the ultraviolet, visible, and near
infrared (IR) range, producing an electric current proportional to the number of photons
reaching the transducer. Another class of photon detectors e.g. photodiodes uses a
semiconductor as the photosensitive surface.
Transmittance and absorbance
The attenuation of electromagnetic radiation – as it passes through a sample – is
described quantitatively by two separate but related terms: transmittance and absorbance.
Transmittance (T) is defined as the ratio of the electromagnetic radiation’s intensity exiting
the sample, IT, to that incident on the sample from the source, I0.
0
T
II
T =
Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies
between 100% (no absorption) and 0% (complete absorption).
Attenuation of radiation as it passes through the sample leads to a transmittance of less
than T=1, because there are different ways in which the
attenuation occurs e.g. reflection and absorption by the
sample container, absorption by components of the
sample matrix other than the analyte, and the scattering
of radiation. To compensate for any loss of light
intensity the radiation’s intensity exiting from the blank
is taken to be I0.
The attenuation of the radiation is given as
absorbance, A, which is defined as Figure 4-2 Measuring sample and blank
Instrumental Analysis Spectrophotometry
25
T
0
I
IlogTlogA =−=
Absorbance is a linear function of the analyte’s concentration. The relationship between
absorbance and concentration is known as the Beer–Lambert law.
A = ε l c
When concentration (c) is expressed using molarity, (unit in mol L-1), effective light path
length, l (cm) and the molar absorptivity, ε (with units of cm–1 M–1) is used. Calibration
curves based on Beer–Lambert law are used routinely in quantitative analysis.
Limitations to Beer–Lambert law
deviations in absorptivity coefficients at high concentrations (>0.01M) due to
electrostatic interactions between molecules in close proximity
scattering of light due to particulates in the sample
fluorescence or phosphorescence of the sample
chemical reaction of the analyte
non-monochromatic radiation, deviations can be minimized using a relatively flat part
of the absorption spectrum such as the maximum of an absorption band
change of the solvent
Ultraviolet-Visible (UV-Vis) Spectrophotometry – Instrumentation
In absorbance spectroscopy, the instrument has a monochromator and is called
spectrophotometer. The simplest spectrophotometer is a single-beam instrument (Fig. 4-3)
equipped with a fixed wavelength monochromator, in which the light crosses through either
the sample or the blank.
Figure 4-3 Block diagram of a single-beam spectrophotometer
Instrumental Analysis Spectrophotometry
26
In a double-beam spectrometer (Fig.4-4) the chopper controls the radiation’s path,
alternating it between the sample and the blank. The signal reaching the detector is due to the
transmission of the blank (I0) and the sample (IT). A scanning monochromator allows for the
automated recording of spectra. Double-beam instruments are more versatile than single-beam
instruments, being useful for both quantitative and qualitative analyses.
4.2 Practice
Procedure 1 – Determination of the concentration of NiSO4 solution by standard
addition method
The standard addition is one of the calibration methods. The standard solution (solution
of known volume and concentration of analyte) is added to the unknown solution so any
impurities in the unknown are accounted for in the calibration. The operator does not know
how much analyte was in the solution initially but does know how much standard solution
was added, and knows how the readings changed before and after adding the standard
solution. Thus, the operator can extrapolate and determine the concentration initially in the
unknown solution.
Materials:
0.25 M NiSO4 solution
Equipments:
6 pcs. volumetric flask (100.00 mL)
pipettes
Figure 4-4 Block diagram of a double-beam spectrophotometer
Instrumental Analysis Spectrophotometry
27
Step 1. Take one of the unknown concentrations of NiSO4 solution in 100.00 mL volumetric
flask and fill to the mark with distilled water.
Step 2. Number 6 pcs 100.00 mL volumetric flasks from 1-6, and measure 10.00-10.00 mL
from solution prepared in Step 1. into them. Add different volume of NiSO4 standard
according to the table, and fill all to mark.
Step 3. Measure the absorbance at 390 nm, use water as blank. Calculate the concentration of
NiSO4 in each flask, excluding the unknown concentration.
Data analysis 1.
Step 1. Draw the concentration of the standard (in mg/100 mL units) as a function of
absorbance
Step 2. Extrapolate the regression line into the “negative concentration” region; obtain the
concentration of the unknown solution as the intercept of the regression line with the X-axis.
Step 3. Use the least-squares method to calculate linear regression. From y=ax+b you can get
the concentration, as 0=ax+b, where x will be the conc. value.
Step 4. Take care of the dilution and calculate the NiSO4 solution’s concentration in mg/100
mL units. (MNiSO4 = 155 g/mol)
Flask No.
Absorbance at
390 nm
1. 10 mL unknown sol. → fill to mark with dist. water
2. 10 mL unknown sol. + 3 mL 0.25 M NiSO4 sol. → fill to mark with dist. water
3. 10 mL unknown sol. + 6 mL 0.25 M NiSO4 sol. → fill to mark with dist. water
4. 10 mL unknown sol. + 9 mL 0.25 M NiSO4 sol. → fill to mark with dist. water
5. 10 mL unknown sol. + 12 mL 0.25 M NiSO4 sol. → fill to mark with dist. water
6. 10 mL unknown sol. + 15 mL 0.25 M NiSO4 sol. → fill to mark with dist. water
Instrumental Analysis Spectrophotometry
28
Procedure 2 – Determination of methylene blue concentration
Solution:
- 0.001 m/m% methylene blue stock solution
Equipment:
- test tubes
- pipettes (10 mL)
Step 1. At first determine the absorption spectra of the 0.001% methylene blue solution. Fill a
cuvette with the solution and measure its absorbance between 530 and 700 nm with 5 nm
steps, use water as blank.
Step 2. Prepare solutions for calibration. Take 6 test tubes and number them from 1 to 6. Fill
4 mL 0.001% methylene blue solution into the 1st, and fill to 10 mL (=add 6 mL water). Fill
5-5 mL water into all the other tubes. Add 5 mL solution from 1 to 2 and mix well. Take 5
mL solution from 2 to 3 and mix. Continue with all the tubes.
Step 3. Search for the highest absorbance value measured in Step 1. which will be the
absorption maximum. Measure the absorbance of all samples of the calibration and also the
unknown at the wavelength at the absorption maximum obtained in Step 1.
Data analysis 2.
Step 1. Draw the absorption spectrum (absorbance as a function of wavelength).
Step 2. Draw the calibration line from absorbance data obtained from calibration solutions.
Step 3. Calculate the regression line (least-squares method). Using the regression equation,
obtain the unknown methylene blue concentration (in m/m%) from its absorbance data.
4.3 Questions
1. What is a spectrum?
2. Write the steps for a calibration with standard addition method.
3. Which limitations does the Lambert-Beer law have?
4. Show the functions of the parts of a single/double beam spectrophotometer.
5. Give a rough description about the light sources/monochromators/detectors.
Instrumental Analysis Spectrophotometry
29
4.4 Abbreviations, definitions
intensity (I)
The flux of energy per unit time per area.
photon
A particle of light carrying an amount of energy equal to hν.
transmittance (T)
The ratio of the radiant power passing through a sample to that from the radiation’s
source.
absorbance (A)
The attenuation of photons as they pass through a sample
absorbance spectrum
A graph of a sample’s absorbance of electromagnetic radiation versus wavelength (or
frequency or wavenumber).
emission
The release of a photon when an analyte returns to a lower-energy state from a higher-
energy state.
continuum source
A source that emits radiation over a wide range of wavelengths.
monochromator
A wavelength selector that uses a diffraction grating or prism, and that allows for a
continuous variation of the nominal wavelength.
monochromatic
Electromagnetic radiation of a single wavelength.
spectrophotometer
An instrument for measuring absorbance that uses a monochromator to select the
wavelength.
Instrumental Analysis Atomic Spectroscopy
30
Chapter 5 – Optical Atomic Spectroscopy
5.1 Theory
Atomic spectroscopy is used for the determination of elemental composition. Analyte
can be measured at µg/g to pg/g levels, what is also called as ppm (parts per million) and ppb
(parts per billion).
The science of atomic spectroscopy includes three techniques: the atomic absorption
(need ground state atoms), the atomic emission (need excited state atoms) and the atomic
fluorescence. Either the energy absorbed, or the energy emitted is measured and used for
analytical purposes.
During atomic spectroscopy, the substances are examined in gas phase. So the first step
is the atomization, when the sample is vaporized at 2000-8000 K and decomposed into
gaseous atoms in a flame, furnace, or plasma. Concentrations of atoms in gas phase are
measured by emission or absorption of radiation at the wavelength of the element of interest.
The atomic spectroscopy is a principal tool of analytical chemistry, because it has high
sensitivity, it is able to distinguish one element from another in a complex sample.
5.1.1 Atomic emission spectroscopy
In atomic emission spectroscopy, samples are subjected to a high energy (in a flame, or
plasma) in order to produce excited state atoms, capable of emitting radiation. The emission
spectrum of an element consists of a collection of the allowable emission wavelengths. These
emission wavelengths are used as a characteristic for qualitative identification of the
examined element. For quantitative analysis, the intensity of light emitted is measured at the
characteristic wavelength of the element.
Flame emission spectrometry (FES)
The main parts of the flame atomic emission spectrometer are a nebulizer, air/acetylene
flame, and optical system (monochromator, detector) (Figure 5-1).
In flame emission spectrometry the atomiziation of the sample compounds and the
thermal excitation of the atoms happens in the flame. The sample solution goes into the
nebulizer by the flow of the oxidant. The nebulizer produces small droplets (an aerosol) from
Instrumental Analysis Atomic Spectroscopy
31
the liquid sample. The fuel (usually acetylene), oxidant (usually air), and aerosol are mixed
thoroughly before introduction into the flame. In the spray chamber, the baffles block large
droplets of liquid. The excess sample solution (about 95% of the initial sample) flows out to a
drain.
The flame
The temperature of the flame depends on the fuel and the oxidant used. The
acetylene/air combination produces a flame temperature of 2400-2700 K. The flame profile
consists of four regions (cones) with different temperatures.
In the flame the small droplets evaporate and decompose into free atoms. Depending on
the energy of flame, the free atom can be in ground state, or in excited state, or it can be
ionized. In the atomic emission experiments excited state atoms should be formed from the
sample. Many metal atoms oxidized in the outer cone. In the presence of these molecules
(oxides and hydroxides), the intensity of atomic signal decreases. A “rich” flame (rich in
fuel), with excess carbon, tends to reduce metal oxides and hydroxides and thereby increases
sensitivity. However, “rich” flames are cooler, so the amount of the excited state atoms can be
Outer cone Interconal layer Blue cone Preheating region Burner head
To drain
Fuel and Oxidant
Sample
Spray cham
Flame
Aerosol
Liquid
Monochromator
Detector
Readout device
Sample
Flame
Figure 5 -1 Flame atomic emission spectrometer Figure 5 -2 Nebulizer
Figure 5 -3 Profile of flame
Instrumental Analysis Atomic Spectroscopy
32
decreased. The choosing of the right flame condition (different flames for different elements)
is important for best analysis.
Monochromators
The monochromator selects the photons of desired energy passing through the flame
and prevents the scattered light of other wavelengths from passing from the flame towards the
detector.
Photomultiplier tube (detector)
The detector produces an electric signal when it is struck by photons exiting the
monochromator.
The photomultiplier tube is a very sensitive detector. This device consists of a cathode,
a number of dynodes and an anode. The electromagnetic radiation (photons) knocks out
electrons from the photosensitive surface of the cathode. Each photoelectron emitted from
cathode knocks out more than one electron from the first dynode. These new electrons knock
out even more electrons from the second dynode. This process is repeated several times, so
more than 106 electrons arise. The anode collects these electrons.
5.1.2 Atomic absorption spectroscopy
In atomic absorption spectrometry, ground state atoms are produced in the atom source.
If light of just the right wavelength impinges on a ground state atom, the atom may absorb the
light while it becomes excited state atom. The absorption spectrum (the absorbed radiation)
characterizes the element examined. The absorption wavelengths are used as a characteristic
Many electrons emitted from dynode
Anode Photoemissive
cathode
Photon
Photoelectrons emitted from cathode
Dynodes
Figure 5-4 Scheme of a photomultiplier tube
Instrumental Analysis Atomic Spectroscopy
33
for qualitative identification of the element. For quantitative analysis, the amount of light
absorbed is measured at the wavelength of the element of interest.
Flame atomic absorption spectrometry (FAAS)
The main parts of the flame atomic absorption spectrometer are a hollow cathode lamp
(light source), nebulizer, air/acetylene flame, and optical system (Figure 5-5).
In flame atomic absorption spectrometry, the flame will be the atom source. The sample
solution is aspirated (sucked) into the flame, where the liquid evaporates and the remaining
solid is atomized. Light of the hollow-cathode lamp is emitted from excited atoms of the same
element which is to be analyzed. Thus the radiant energy corresponds directly to the
wavelength, which is absorbable by the atomized sample. The monochromator placed after
the flame selects one analytical line from the hollow-cathode lamp and rejects as much
emission from the flame as possible. The detector measures the amount of the light (the power
of the electromagnetic radiation) that passes through the sample (in the flame).
Hollow-cathode lamp (light source)
The absorption (or emission) spectrum of the gas phase atoms consists of sharp lines
with widths of ~0.001 nm. These narrow absorption lines require the use of special light
source (hollow-cathode lamp) for atomic absorption measurements. The hollow-cathode lamp
produces such sharp lines of the correct frequency that the examined element may absorb.
A hollow-cathode lamp is filled with inert gas (Ne or Ar) under reduced pressure. The
hollow cathode is coated (or made) with the same element as that being analyzed. The inert
gas is ionized applying a high voltage (about 500 V) between the anode and the cathode. The
produced positive ions (Ar+ or Ne+ ions) are accelerated toward the hollow cathode. As these
strike the cathode, free metal atoms are ejected into the gas phase from the cathode. Gaseous
Monochromator
Detector
Readout device
Sample
Flame
Hollow-cathode lamp
Figure 5 -5 Flame atomic absorption spectrometer
Instrumental Analysis Atomic Spectroscopy
34
metal atoms are excited by collisions with high-energy electrons. The excited state atoms emit
photons. The generated atomic radiation is absorbable by the atomized sample in the flame.
Since the majority of hollow cathode lamps are single element lamps (the cathode
coated with one element), a different lamp is usually required for each element.
Atomization (Ways to form gaseous atoms)
In the atom source, gaseous atoms of the element of interest are created. These free
atoms can be produced in a flame, or in an electrically heated furnace, or in a plasma.
The electrically heated graphite furnace is used exclusively for atomic absorption
(flameless AA). A graphite furnace requires less sample amount (1-100 µL) than a flame and
is more sensitive (the residence time of the atomized sample in the optical path is several
seconds).
The inductively coupled plasma (ICP) is the hottest atom source (used for ICP-
emission, ICP-MS). This uses Ar gas to create Ar+ + e- then the hot plasma of ionized gas is
obtained by an oscillating magnetic field produced by induction coil. This atomization
technique is very expensive, both to purchase and to operate.
The Beer-Lambert law
According to the Beer-Lambert law, if the path length (l) and the molar absorption
coefficient (ε) are known and the absorbance (A) is measured, the concentration of the sample
(c) can be deduced.
A = ε l c
(-) (+)
Hollow cathode
Anode
Quartz or glass window
Figure 5 -6 Scheme of the hollow-cathode lamp
Instrumental Analysis Atomic Spectroscopy
35
5.2 Practice
Procedure 1 – Concentration determination of potassium ion (K+) solution by atomic
emission spectroscopy (calibration curve method)
Materials: 50 ppm K+ stock solution (1 ppm = 10-6 g/cm3 = 10-6 g/mL)
Equipments: 6 pcs. volumetric flask (50.00 mL); automatic pipette
Step 1. Prepare five potassium ion solutions containing K+ in 1, 2, 3, 4 and 5 ppm
concentration from a 50 ppm K+ stock solution. Dilute the appropriate volumes of the stock
solution with distilled water in 50.00 mL volumetric flasks. (Fill 1, 2, 3, 4 and 5 mL 50 ppm
K+ stock solution into the flasks, and fill all to mark.) This will be the series of the calibration
solution.
Step 2. Measure (five times!) and record the emissions of the solutions at 766.5 nm.
Use distilled water for blank. Fill in the table.
E1 E2 E3 E4 E5 EAverage
EStandard
deviation
1 ppm 2 ppm 3 ppm 4 ppm 5 ppm Unknown
Data analysis
Step 1. Calculate the average and the standard deviation of the 5 emission data obtained
for each solution.
Step 2. Plot the averaged emission intensity versus the concentration of K+ on
millimeter squared paper. Draw a linear curve graphically on the data points.
Step 3. Use the least square method to calculate the regression line.
Step 4. Calculate the unknown potassium-ion concentration in parts per million (ppm)
according to the graphically fitted calibration curve and also from the calculated regression
equation. From y=ax+b you can get the concentration, as E unknown=ax+b, where x will be the
unknown concentration.
Instrumental Analysis Atomic Spectroscopy
36
Procedure 2 – Concentration determination of copper ion (Cu2+) solution with atomic
absorption spectroscopy (Standard addition experiment)
Materials: 100 ppm Cu2+ solution; unknown Cu2+ solution
Equipments: 6 pcs. volumetric flask (25.00 mL); automatic pipette
Step 1. Prepare the following solutions in 25.00 mL volumetric flasks:
Absorbance
1. flask 0.25 mL of unknown Cu2+ solution → fill to the mark with distilled water and mix it
2. flask 0.25 mL of unknown Cu2+ solution + 0.25 mL 100 ppm Cu2+ solution → fill to the mark with distilled water and mix it
3. flask 0.25 mL of unknown Cu2+ solution + 0.50 mL 100 ppm Cu2+ solution → fill to the mark with distilled water and mix it
4. flask 0.25 mL of unknown Cu2+ solution + 0.75 mL 100 ppm Cu2+ solution → fill to the mark with distilled water and mix it
5. flask 0.25 mL of unknown Cu2+ solution + 1.00 mL 100 ppm Cu2+ solution → fill to the mark with distilled water and mix it
6. flask 0.25 mL of unknown Cu2+ solution + 1.25 mL 100 ppm Cu2+ solution → fill to the mark with distilled water and mix it
Step 2. Measure (five times!) and record the absorbance of the solutions at 324.8 nm.
The blank solution is the distilled water.
Data analysis
Step 1. Calculate the average and the standard deviation of the 5 absorbance data
obtained for each solution.
Step 2. Calculate the concentration of Cu2+ solutions in each flask, excluding the
unknown concentration of the sample solution. Prepare a table with concentration of Cu2+
solutions in the rows and 5 absorbance data; AAverage; AStandard deviation in the columns (similar
which was used in K+ concentration determination).
Step 3. Plot the averaged absorbance versus the concentration of Cu2+ on millimetre
paper. Fit a linear curve graphically on the data points.
Instrumental Analysis Atomic Spectroscopy
37
Step 4. Calculate the unknown Cu2+ concentration in the solution in parts per million
(ppm) according to the graphically fitted curve. Extrapolate the regression line into the
“negative concentration” region; obtain the concentration of the unknown solution as the
intercept of the regression line with the X-axis.
5.3 Questions
1. What is the basis of the atomic emission spectroscopy/atomic absorption
spectroscopy?
2. Describe the functions of the parts of a flame atomic emission/atomic absorption
spectrometer.
3. What is the role of the monochromator?
4. How does the photomultiplier tube work?
5. How does the hollow-cathode lamp work?
6. What are the advantages of the graphite furnace comparing to the flame?
7. What is the Beer-Lambert law?
Instrumental Analysis Chromatographic Separation
38
Chapter 6 – Introduction to Chromatographic Separation
6.1 General Description of Chromatography
Chromatography is a widely used method for the separation, qualitative identification
and quantitative determination of the closely related chemical components of complex
mixtures. All chromatographic separations use a stationary phase and a mobile phase (solvent,
eluent). The samples are dissolved in a mobile phase, which may be a gas, a liquid, or a
supercritical fluid. The mobile phase is passing through the stationary phase carrying with it
the sample mixture. The stationary phase is a phase that is fixed in place in a column or on a
solid surface.
6.2 Classification of Chromatographic Methods
Chromatographic methods have two basic types. In column chromatography, the
stationary phase is held a narrow tube through which the mobile phase is forced through by
pressure. In planar chromatography, the stationary phase is supported on a flat surface or in
the pores of a paper, and the mobile phase passes through the stationary phase by capillary
action or under the influence of gravity.
A more fundamental classification of chromatographic separations is based on the types
of mobile and stationary phases and the kinds of equilibria involved in the transfer of solutes
between the phases. Table 6-1 shows three general categories of chromatography: gas
chromatography (GC), liquid chromatography (LC), and supercritical fluid
chromatography (SFC). The names imply that the mobile phases in the three techniques are
gases, liquids, and supercritical fluids. The second column of the table reveals the various
types of liquid chromatography and gas chromatography. They differ in the nature of the
stationary phase and the types of equilibra between the phases.
Instrumental Analysis Chromatographic Separation
39
General Classification Specific Methods Stationary Phase Type of Equilibrium 1. Gas chromatography
(GC) a. Gas-liquid
(GLC) Liquid adsorbed or bonded to a solid surface
Partition between gas and liquid
b. Gas-solid Solid Adsorption 2. Liquid chromatography
(LC) a. Liquid-liquid,
or partition Liquid adsorbed or bonded to a solid surface
Partition between immiscible liquids
b. Liquid-solid, or adsorption
Solid Adsorption
c. Ion exchange Ion-exchange resin
Ion exchange
d. Size exclusion Liquid in interstices of a polymeric solid
Partition/sieving
e. Affinity Group specific liquid bonded to a solid surface
Partition between surface liquid and mobile liquid
3. Supercritical fluid chromatography (SFC), mobile phase: supercritical fluid
Organic species bonded to a solid surface
Partition between supercritical fluid and bonded surface
Chromatography is divided into categories on the basis of the mechanism of interaction
of the solute with the stationary phase:
Adsorption chromatography: a solid stationary phase and liquid or gaseous mobile
phase are used. Solute is adsorbed on the surface of the solid particles. The more strongly a
solute is adsorbed, the slower it travels through the column.
Partition chromatography: a liquid stationary phase is bonded to a solid surface, which
is typically inside the pores of porous of the silica (SiO2) chromatographic column in gas
chromatography. Solute equilibrates between the stationary liquid and the mobile phase,
which is a flowing gas in gas chromatography.
Ion-exchange chromatography: Anions such as –SO3- or cations such as –N(CH3)3
+ are
covalently attached to the stationary solid phase, usually a resin. Solute ions of the opposite
charge are attracted to the stationary phase. The mobile phase is a liquid.
Molecular exclusion chromatography: Also called size exclusion, gel filtration, or
gel permeation chromatography, this technique separates molecules by size, with no attractive
interaction between the stationary phase and solute. Rather, the liquid mobile phase passes
through a porous gel. The pores are small enough to exclude large solute molecules but not
Table 6-1 Classification of Column Chromatographic Methods
Instrumental Analysis Chromatographic Separation
40
the small ones. Large molecules stream past the particles without entering the pores. Small
molecules take longer time to pass through the column because they enter the pore and
therefore must visit a larger volume before leaving the column.
Affinity chromatography: this most selective kind of chromatography employs
specific interactions between one kind of solute molecule and a second molecule that is
covalently attached (immobilized) to the stationary phase. For example, the immobilized
molecule might be an antibody to a particular protein. When a mixture containing a thousand
proteins is passed through the column, only the one protein that reacts with the antibody binds
to the column. After all other solutes have been washed from the column the desired protein is
dislodged by changing the pH or ionic strength.
6.3 Elution in Column Chromatography
Figure 6-1 shows how two components A and B of a sample are resolved in a packed
column by elution. Elution is a process in which solutes are washed through a stationary
phase by the movement of a mobile phase. Mobile phase, which entering the column called
eluent. Fluid which emerging from the end of the column is called eluate.
The chromatogram is a graph showing the detector response as a function of elution
time. The chromatogram is useful for both qualitative and quantitative analysis. The position
Figure 6 -1 Diagram showing the separation of a mixture of components A and B by column elution chromatography
Instrumental Analysis Chromatographic Separation
41
of the peak maxima on the time axis can be used to identify the components of the sample.
The peak areas provide a quantitative measure of the amount of each species.
Figure 6-2 shows a simple chromatogram of a two-component mixture. The small peak
on the left is not retained by the stationary phase. The time tm after sample injection for this
peak to appear is sometimes called the dead or void time. The dead time (void time), tm, is the
time (min) it takes for an unretained species to pass through a chromatographic equipment
and column. The retention time, tr, for each component is the time (min) that elapses between
the injection of the sample onto the column and the arrival of the maximum concentration of
that component at the detector. Retention volume, Vr, is the volume (cm3) of mobile phase
required to elute a particular solute from the column.
6.4 Important Chromatographic Quantities and Relationships
The partition coefficient, K, is the ratio of concentrations of solute in the stationary and
mobile phases.
Partition coefficient: K = m
s
cc
where cs is the concentration of solute in the stationary phase, cm is the concentration of
solute in the mobile phase.
Figure 6-2 Chromatogram of a two-component mixtrue
Instrumental Analysis Chromatographic Separation
42
The retention volume, Vr is the volume (cm3) of mobile phase required to elute a
particular solute from the column. The volume flow rate of the mobile phase is u (volume per
unit time).
Retention volume: Vr = tr u
The adjusted retention time, tr’, for a retained solute, is the additional time required to
travel the length of the column, beyond that required by solvent.
Adjusted retention time: tr’ = tr - tm
The adjusted retention volume, Vr’, is the volume (cm3) of the eluent that passed
through the column while the component was retained on the surface. These volumes are
different for every component of the sample. It takes volume Vm to push solvent from the
beginning of the column to the end of the column.
Adjusted retention volume: Vr’ = Vr - Vm
For each peak in the chromatogram, the retention factor, k, is calculated as adjusted
retention time normalized by tm.
Retention factor: k = m
mr
ttt −
= m
'r
tt
= mm
ss
Vc
Vc
where Vs is the volume of the stationary phase, Vm is the volume of the mobile phase.
For two components A and B, the relative retention (separation factor), α, is a ratio of
their adjusted retention time.
Relative retention: α= 'rA
'rB
tt
= A
B
kk
= A
B
KK
For component B eluted after component A, the unadjusted relative retention, γ, is the
ratio of their unadjusted retention times.
Unadjusted relative retention: γ = rA
rB
tt
Instrumental Analysis Chromatographic Separation
43
In chromatography, the resolution, R, of two peaks is defined.
Resolution: R =
2BA
rArB
ww)t()t(
+−
=
2BA
rArB
ww)V()V(
+−
=
2
5890
2121 B/A/
rArB
)w()w()]t()t[(.
+−
where wA and wB are the width of A and B Gaussian peaks, and (w1/2)A and (w1/2)B are the
width at half-height of A and B Gaussian peaks.
The plate model supposes that the chromatographic column contains a large number of
separate layers, called theoretical plates. Separate equilibrations of the sample between the
stationary and mobile phases occur in these “plates”. The analyte moves down the column by
transfer of equilibrated mobile phase from one plate to the next. It is important to remember
that the plates do not really exist; they are a figment of the imagination that helps us
understand the processes at work in the column. They also serve as a way of measuring
column efficiency, either by stating the number of theoretical plates, N, in a column.
Number of theoretical plates: N=162
wt r = 5.54
2
21
/
r
wt
Plate height / Height Equivalent to a Theoretical Plate, H, is approximately the length
of column required for one equilibration of solute between mobile and stationary phases.
Figure 6-3 Resolution of some chromatographic peaks
Instrumental Analysis Chromatographic Separation
44
Plate height: H = NL
where L is length of the column.
6.5. Keywords, abbreviations
eluent, elution, eluate, mobile phase, stationary phase, retention time, retention factor,
retention volume, relative retention, chromatogram, selectivity factor, plate height, column
resolution
6.4. Questions
1. What are the major differences between liquid-liquid and liquid-solid
chromatography?
2. What are the major differences between liquid-liquid and gas-liquid
chromatography?
3. Describe how the retention factor for a solute can be calculated?
4. Describe how the number of plates in a column can be calculated?
5. How can the selectivity factor be calculated in LC?
Instrumental Analysis Gas Chromatography
45
Chapter 7 – Gas Chromatography
Gas chromatography is a widely used analytical method, which can be used for the
analysis of thermally stable, volatile organic and inorganic compounds via a separation
procedure.
Gas chromatography (GC) can be used for example in environmental science,
brewing, food industry, perfumery and flavour analysis, petrochemical industry,
microbiological analyses, pharmaceutical industry and clinical biochemistry.
The advantages include high efficiency, selectivity, requirement for small volumes of
sample, and that the separation of the components are not destroyed during the separation
process, so with related techniques (e.g, with GC-MS) the analysis can be carried out further.
7.1 Theory
In order to get a full background to this chapter it is strongly advised first to study
Chapter 6 – Introduction to Chromatographic Separation.
Chromatography encompasses a diverse and important group of separation methods
that permit the scientist to separate, isolate, and identify closely related components of
complex mixtures; many of these separations are impossible by other means.
Chromatographic methods employ a stationary phase and a mobile phase. Components of a
mixture are carried through the stationary phase by the flow of the mobile one; separations are
based on differences in migration rates among the sample components.
Gas chromatography can be used for the separation of thermally stable, volatile
organic and inorganic compounds.
Two types of gas chromatography are encountered: gas-solid chromatography and
gas-liquid chromatography. Gas-solid chromatography employs a solid stationary phase, in
gas-liquid chromatography the stationary phase is a liquid.
In gas chromatography (Figure 7-1) the most common method of sample injection
involves the use of a microsyringe to inject a liquid or gaseous sample through a self-sealing,
silicone-rubber diaphragm or septum into a flash vaporizer port located at the head of
chromatographic column (capillary column) and separated analytes flow through a detector,
whose response is displayed on a computer. Injection of the sample may be made manually or
using an autosampler. Elution is brought about by the flow of an inert gaseous mobile phase
Instrumental Analysis Gas Chromatography
46
(helium, nitrogen and hydrogen). The choice of gases is often dictated by the type of detector
used. In contrast to most other types of chromatography, the mobile phase does not interact
with molecules of the analyte; its only function is to transport the analyte through the column.
When the chromatographic conditions are properly selected, the sample components
are separated on the stationary phase and they will reach the detector in the reverse order of
their interaction strength.
The chromatographic separation factors depend on the nature and velocity of the
carrier gas, the temperature, the length and internal diameter of column, the type and
thickness of stationary phase.
The detector senses the separated components, measuring some physical or chemical
properties. The detector signal can be employed for qualitative identification and quantitative
determination of separated components.
The chromatogram is a graph showing the detector response as a function of elution
time. It has two main parameters: the retention time (tR) and the peak area. Retention time is
used for the qualitative analysis of components. Quantitative analysis is based on the area of a
peak. In the linear response concentration range, the area of a peak is proportional to the
quantity of that component.
Kolonna Gáz
palack
Szűrő Áramlás és nyomás-
szabályozó
Injektor
Detektor Jelerősítő
Vezérlés
Gas Column
Filter Amplifier
Detector Injector Flow and pressure controller
Syringe
Computer
Figure 7-1 Schematic diagram of a capillary GC system
Instrumental Analysis Gas Chromatography
47
The Kováts retention index
The Kováts retention index (Ix) can be used for the identification of components. By
definition, the retention index for a normal alkane is equal to 100 times the number of carbon
atoms in the compound. For a homologous series, the plot of the logarithm of adjusted
retention time (tR’) versus the number of carbon atoms is linear, provided that the lowest
member of the series is excluded.
Retention index:
where n is the number of carbon atoms in the smaller alkane; n+1 is the number of carbon
atoms in the larger alkane; tR,n is the adjusted retention time of the smaller alkane; tR
,n+1 is the
adjusted retention time of the larger alkane; and tR,x is the adjusted retention time of the
unknown component (tR’n+1 > tR’x > tR’n ).
7.1.1 The main parts of GC
Gas Systems
The most common mobile phases for GC are helium, nitrogen and hydrogen, which
have the advantage of being chemically inert. The choice of gases is often dictated by the type
of detector used. The gases of flame ionization detector are hydrogen and air.
Injector
Several capillary injectors are available, the most common of which is a split / splitless
injector (Figure 7-2). Injection takes place into a heated glass or quartz liner rather than
directly onto the column.
In the split mode, the sample is split into two unequal portions the smaller of which
goes onto the column. This technique is used with concentrated samples.
In the splitless mode, the entire sample is introduced onto the column.
ntt
ttI
nn
nX
RR
RRx 100
lglg
lglg*100
''
''
1
+−−
=+
Instrumental Analysis Gas Chromatography
48
Column
Two general types of columns are encountered in gas chromatography, packed and
capillary. Capillary columns (Figure 7-3) are made from fused silica, usually coated on the
outside with polyimide to give the column flexibility. The wall of the column is coated with
the liquid stationary phase. The most common type of coating is based on organo silicone
polymers, which are chemically bonded to the silanol groups on the wall of the column and
the chains of the polymers are further cross-linked.
Important factors in the separation are the length and internal diameter of column, the
type and thickness of stationary phase.
The column is ordinarily housed in a thermostated oven. The optimum column
temperature depends upon the boiling point of the sample and the degree of separation
required. The ovens can be programmed to either produce a constant temperature, isothermal
conditions or a gradual increase in temperature.
Figure 7-2 A split / splitless injector
Figure 7-3 A capillary column
Instrumental Analysis Gas Chromatography
49
Detector
Detectors used in GC vary in nature depending upon the characteristics of the analyte
and the circumstances of its determination.
The flame ionization detector (Figure 7-4) is the most widely used and generally
applicable detector for gas chromatography. The eluent is mixed with hydrogen and then with
air. When solute molecules contained in the carrier gas elute from the column and pass into
the detector they are combusted in the flame and in doing so, generate ions which move to the
collector electrode, due to the potential difference between the jet and the electrode. The
resulting ionisation current is amplified and fed to the data system.
7.2 Practice
Conditions for Gas Chromatography
(Details of the instrument used, settings, working parameters, solvents and samples)
Gas chromatograph: Agilent Technologies 6890N GC (Figure 7-5)
Injector: split-splitless, manual and autosampler, T=270°C
Column: capillary column, Hp-1ms (25 m × 0.2 mm × 0.33 µm)
Detector: flame ionization detector (T=270°C)
Temperature program:
100°C (10 min)
100°C (0.5 min)→ 35°C /min →240°C (0 min)
Anode (+)
Air
Gasket
Hydrogen-air flame
Hydrogen
Column
Cathode (-)
Figure 7-4 A typical flame ionization detector
Instrumental Analysis Gas Chromatography
50
Carrier gas: helium, flow rate 1.5 mL/min
Injection volume: 1 µL
Injection mode: splitless, manual
Solvents and Samples:
n-alkanes (octane, decane and dodecane) dissolved in n-pentane
two unknown components dissolved in n-pentane
standard components dissolved in n-pentane
Manual Injection
After cleaning the syringe several times with solvent (n-pentane), take up 1 µL of
sample and 0.5 µL air then push the needle through the rubber septum into the heated
injection port of the chromatograph. Remove the syringe then start the measurement with start
button.
Capillary Column inside
Oven
Gas
Mass Selective Detector
Autoinjection System
Autosampler
Figure 7 -5 Agilent Technologies 6890N GC
Instrumental Analysis Gas Chromatography
51
Procedure 1 – Calculate the Kováts retention index of unknown components.
Inject the sample of n-alkanes dissolved in n-pentane and record the
chromatogram.
Inject the sample of unknown components dissolved in n-pentane and record
the chromatogram.
Calculate the Kováts retention index of unknown components.
Identify the components.
Procedure 2 – Qualitative analysis by standard components.
Inject the standard components dissolved in n-pentane and record the
chromatograms.
Procedure 3 – The examination of temperature as a factor affecting separation.
Inject the sample of known components dissolved in n-pentane, measure at a
temperature program and record the chromatogram.
Compare the chromatograms of isothermal and programmed temperature
measurements.
Procedure 4 – Characterize the separation of the components in the sample.
Calculate the k, α, R, N, H, R parameters with the help of the table and the
chromatogram recorded under the practice.
Parameter Report Component 1 Component 2 tM tR tR’ k’ w α N H R
Instrumental Analysis Gas Chromatography
52
Concepts and Abbreviations
Gas chromatography, GC, injector, stationary phase, mobile phase, capillary column, flame
ionisation detector, chromatogram, retention time, Kováts retention index.
7.3 Questions
What is the gas chromatography?
What are the main parts of the gas chromatograph?
What happens to be the principle of separation of components?
How does the flame ionization detector work?
What is the chromatogram?
What is the retention time?
List some factors affecting the separation and explain their effect.
Instrumental Analysis High Performance Liquid Chromatography
53
Chapter 8 - High-performance liquid chromatography (HPLC)
8.1 Introduction
In order to get a full background to this chapter it is strongly advised first to study
Chapter 6 – Introduction to Chromatographic Separation.
HPLC is an acronym, related to a separation technique; the most widely used
analytical method to separate the components of a complex mixture. The explanation of the
term HPLC is the following:
C – Chromatography: a group of separation techniques utilizing the mass-transfer between
a stationary and a mobile phase.
LC – Liquid Chromatography : one class of chromatography, the applied mobile phase is
in liquid state.
HPLC – High Performance Liquid Chromatography: high performance is the result
of many factors:
(1) very small particles of narrow distribution range,
(2) uniform pore size and pore size distribution,
(3) high pressure column slurry packing techniques,
(4) accurate low volume sample injectors,
(5) sensitive low volume detectors,
(6) high pressure pumping systems.
8.2 Types of HPLC
Based on the chemical nature of the stationary phase, and on the retention
mechanism, HPLC can be divided into three types, which cover almost 90% of all
chromatographic applications.
I. Adsorption chromatography: the stationary phase is an adsorbent, and the retention
mechanism is based on repeated adsorption and desorption steps.
Instrumental Analysis High Performance Liquid Chromatography
54
• Normal-phase chromatography: in which the stationary phase or adsorbent is more
polar than the mobile phase or eluent. For example, the adsorbent can be silica or
alumina, and the solvent can be n-hexane or diethyl-ether.
• Reversed-phase chromatography: in which the stationary phase is nonpolar or
weakly polar and the solvent is more polar (just the opposite as in normal phase
chromatography). The stationary phases are usually chemically modified silicas,
during the modification using alkyl-chains the surface of the silica gel becomes apolar.
The mobile phases are usually polar mixtures of an organic component (methanol,
acetonitrile etc.) and water.
II . Ion-exchange chromatography: the stationary phase has a charged surface with
opposite charges on it compared to the sample ions. This technique is used almost only
for ionic or ionizable samples. The mobile phase is an aqueous buffer with controlled
pH and ionic strength.
III. Size exclusion chromatography: the stationary phase is made of a material with
precisely controlled pore size. The larger molecules rapidly pass the column, while the
smaller ones penetrate into the pores and washed out later by the mobile phase.
Figure 8-1 SEM-image of porous silica-gel particles Figure 8-2 Chemical modification of the silica using octadecyl ligands (C18)
Instrumental Analysis High Performance Liquid Chromatography
55
8.3 The HPLC instrument
8.3.1 Flasks for the mobile phase storage
Using high- pressure pumps to deliver liquid, the solvents have to be gas-free in
HPLC experiments. The excess gas in the mobile phase causes several problems during the
analysis. Because of the compressibility of the gases, the pump pressure and the flow rate
will fluctuate, and it would cause significant disturbance in the detection and in the
repeatability of the chromatographic data. Due to those reasons, the mobile phase has to be
degassed. Degassing may be accomplished by one of the following methods or their
combination:
Degassing the liquid under vacuum – heavy-walled flask is really important, in this
case.
Heating the liquid until its boiling occurs.
Placing the container of liquid in an ultrasonic bath, or inserting an ultrasonic probe
in it.
Bubbling a fine stream of helium through the liquid; helium has the unique
ability to purge other gases out of solutions.
Some instruments are equipped with built-in degasser, but its capacity is not
enough in every case.
8.3.2 Pumps
The pumps applied in the practice of HPLC have to provide a constant and almost
pulse-free flow of the mobile phase through the system. If we use one pump, the
Figure 8-3 Typical HPLC instrument with computer data acquisition station
Instrumental Analysis High Performance Liquid Chromatography
56
composition of the mobile phase is constant during the experiment, which is called isocratic
elution. With the aid of two or more pumps, using a time program, the composition of the
mobile phase is variable. When the mobile phase composition is changing during the
analysis, the technique is called gradient elution.
8.3.3 Injectors
Injectors for liquid chromatographic systems should provide the possibility of
injecting the liquid sample within a large volume range with high reproducibility and
under high pressure (up to 1200 bar). They should also produce minimum band broadening
and minimize possible flow disturbances. Generally, the most useful and widely used
sampling device for modern LC is the six-port Rheodyne valve.
In the six-port Rheodyne valve, the sample is introduced into the sample loop (in load
position) using a special syringe. A clockwise rotation of the valve rotor (inject position)
places the sample-filled loop into the mobile-phase stream, with subsequent injection of
the sample onto the top of the column through a low-volume, cleanly swept channel.
8.3.4 Columns
The column is the heart of the HPLC instrument, this is the part where the
stationary phase is immobilized, and the retentions of the compounds take place. Modern
HPLC column beds used in adsorption chromatography are small rigid porous particles with
high surface area.
Figure 8-4 Manual six-port injector with the two positions for sample introduction (E=eluent)
Instrumental Analysis High Performance Liquid Chromatography
57
Nowadays, stainless steel is chosen for the material of the column housing because it
offers the best compromise of cost, workability, and corrosion resistance. Depending on the
chromatographic procedure, the column length and diameter can change in a wide range. In
modern instruments, a column thermostat is used to ensure the constant temperature in the
column during the separation.
8.3.5 Detectors
Detectors equipped with the flow-through cell were a major breakthrough in the
development of modern liquid chromatography. Such detection was first applied by the
group of Tiselius, in Sweden in 1940, by continuously measuring the refractive index of the
column effluent. Current LC detectors have wide dynamic range, and have high
sensitivities often allowing the detection of nanograms of material. A few types of
detectors:
Refractive index detector
UV/VIS detector
Fluorescence detector
Conductivity detector
Mass-spectrometric detector (MS)
In the last decade there has been a significant progress in the development of LC/MS
interfacing systems. MS as an on-line HPLC detector is said to be the most sensitive,
selective and in the same time the most universal detector. But it is still the most expensive
one.
Figure 8-5 LC columns with different lengths and diameters
Instrumental Analysis High Performance Liquid Chromatography
58
8.4. Practice
Quantitative analysis of active substances of Saridon analgetic by RP-HPLC
Instrument
Glass flasks for the eluents
Degasser: DGU-14A VP (Shimadzu, Japan)
Pumps: LC-10AD VP (Shimadzu, Japan)
Injector: 7125 Rheodyne injector (Rheodyne, USA) with 2 µL sample loop
Diode Array Detector: SPD-M10A VP (Shimadzu, Japan)
System Controller: SCL-10A VP (Shimadzu, Japan)
PC with Labsolutions chromatographic software (Shimadzu, Japan)
Chromatographic conditions
Column: BDS Hypersil C18 (100×4.6 mm, 3µm, Thermo Scientific, USA)
Mobile phases (eluents):
o A: 0.1 v/v% trifluoroacetic acid in water
o B: 0.1 v/v% trifluoroacetic acid in methanol
Column temperature: ambient
Injection volume: 2 µL
Separation mode: for the separations different kind isocratic and gradient elution
modes will be set.
Detection wavelengths: depend on registered spectra.
Chemicals
water, methanol, thiourea, trifluoroacetic acid, paracetamol and caffeine standards, Saridon
tablet
Structural formula of paracetamol and caffeine
HO
N
H
O
N
NH3C
N
N
CH3
O
CH3
O
paracetamol caffeine
Instrumental Analysis High Performance Liquid Chromatography
59
Step 1. Column characterization
Determine the dead or void time (tm) and the dead or void volume (Vm) of the column using
thiourea as nonretained marker.
Elution steps uv (mL/min)
tm (min)
Vm (mL)
Isocratic 1
Isocratic 2
Isocratic 3
Isocratic 4
Gradient 1
Gradient 2
Step 2. Qualitative analysis of the caffeine and paracetamol standards
Determine the retention time (tr ) of paracetamol and caffeine, by injecting standard
solutions.
Calculate the following chromatographic parameters from the chromatogram of
standard solutions for paracetamol and caffeine compounds:
o adjusted (reduced) retention time, tr’
o retention volume (Vr ),
o retention factor (k) of paracetamol and caffeine, by injecting standard
solutions. Repeat the injections at least three times.
Step 3. Effect of the experimental parameters on the retention volumes of paracetamol and
caffeine
Determine the effect of the methanol concentration under isocratic conditions on the
retention volumes of the studied compounds. Try the separation using different
gradient programs.
Table 8-1 Column characterization
Instrumental Analysis High Performance Liquid Chromatography
60
Elution steps
uv (mL/min)
tp (min)
tp’
(min) Vp
(mL) Vp
’
(mL) wp
(min) Np Hp
(µm) k' p
Isocratic 1
Isocratic 2
Isocratic 3
Isocratic 4
Gradient 1
Gradient 2
Elution steps
uv (mL/min)
tc (min)
tc’
(min) Vc
(mL) Vc
’
(mL) wc
(min) Nc Hc
(µm) k' c
Isocratic 1
Isocratic 2
Isocratic 3
Isocratic 4
Gradient 1
Gradient 2
Elution steps R α
Isocratic 1
Isocratic 2
Isocratic 3
Isocratic 4
Gradient 1
Gradient 2
Step 4. Calibration curve
Dilute four times the standard solution, and measure the peak area of the standards
with five known concentrations. Construct a calibration curve of peak area versus
concentration. Calculate the peak areas from the average of the three injections.
Table 8-2 Effect of the experimental parameters on the retention volumes of paracetamol and caffeine
Instrumental Analysis High Performance Liquid Chromatography
61
Step 5. Qualitative analysis
Inject the Saridon tablet solution and determine the concentration of caffeine and
paracetamol based on the measured peak areas, and the calibration curve.
Calculate the concentration of the pharmaceutically active compounds in one tablet.
The laboratory notebook should contain the followings:
theory of high-performance liquid chromatography,
detailed parts of the HPLC equipment,
detailed description of the measurements (HPLC equipment settings, eluents,
column, working parameters),
determined and calculated chromatographic parameters.
8.5. Keywords, abbreviations
high-performance liquid chromatography, bonded stationary phase, dead volume,
derivatization, eluent strength, gradient elution, isocratic elution, guard column, porous
particle, normal-phase chromatography, reversed-phase chromatography, ultraviolet detector,
diode array detector,
LC, NP-HPLC, RP-HPLC, UV-Vis, DAD
8.6 Questions
1. What is the definition of chromatography?
2. What is liquid chromatography?
3. What is the difference between normal-phase and reversed-phase chromatography?
4. Define the retention volume, and the retention factor.
5. What is the chromatogram?
6. Define the main parts of an HPLC instrument.
7. What are the differences between isocratic and gradient elution?
8. Why is high pressure needed in HPLC?
9. How can the dead or void time be measured?
Instrumental Analysis Mass Spectrometry
62
Chapter 9 – Mass spectrometry
9.1 Theory
Mass spectrometry is a widely used technique in chemistry, biochemistry, pharmacy,
and medicine. It is also a high-performance analytical tool for structure analysis of organic
compounds. It has high sensitivity, low detection limits, high reproducibility, extremely low
sample consumption; and samples can be analyzed with different mass spectrometric
techniques even in gas, liquid or solid state. Mass spectrometry can be powerfully combined
with gas chromatography, liquid chromatography, and capillary electrophoresis.
The basic principle of mass spectrometry (MS) is to generate gas-phase ions from the
sample, to separate these ions by their mass-to-charge ratio (m/z) and to detect them
qualitatively and quantitatively by their respective m/z and abundance.
The general scheme of mass spectrometers is shown in Fig. 9-1. Basically a mass
spectrometer consists of an ion source, a mass analyzer and a detector. The mass analysis and
the detection of the ions take place under high vacuum conditions which are provided by the
vacuum system. Most of the ion sources are operated under high vacuum, although novel ion
sources have been introduced which work under atmospheric pressure. The properties of the
sample (e.g., it is in gas, liquid, or solid state, or its polarity, acidity, solubility in different
solvents, etc.) determine the sample inlet and ion source combination to be chosen for the
mass spectrometric analysis. Ion optics transfers the ions from atmospheric pressure of the ion
source to the high vacuum of mass analyzer via different vacuum stages. A data system is
used to collect and process data from the detector and, nowadays, it also controls all functions
of the instrument.
Vacuum system
Ion optics Mass analyzer Detector
Sample inlet
Data system
Ion source
Vacuum system
Ion optics Mass analyzer Detector
Sample inlet
Data system
Ion source
Figure 9-1 General scheme of a mass spectrometer
Instrumental Analysis Mass Spectrometry
63
9.1.1 Ion sources working under atmospheric pressure
Electrospray (ESI): ionization process which uses an electrical field to generate
charged droplets and subsequent gas-phase analyte ions. In many conventional ESI sources,
nebulization of the liquid-phase sample is pneumatically assisted with the addition of a flow
of nebulizer gas (e.g. nitrogen) (see Fig. 9-2).
Briefly, the electrospray ionization comprises the following steps. Potential difference
is applied between the sample inlet capillary and the counter electrode (usually between 2-5
kV). The electric field causes electrophoretic charge separation in the solution at the capillary
tip and generates a mist of highly charged droplets. The charged droplets are attracted toward
the capillary sampling orifice through a counter flow drying gas (e.g. heated nitrogen), which
shrinks the droplets and carries away uncharged material. Thus the droplets reduce in size by
evaporation of the solvent or by ‘Coulomb explosion’ (droplet subdivision resulting from the
high charge density). Finally, fully desolvated ions result from complete evaporation of the
solvent.
Drying gas
Heated capillary
Nebulizer gas
Sample inlet
2-5 kV
Drying gas
Drying gas
Heated capillary
Nebulizer gas
Sample inlet
2-5 kV
Drying gas
Drying gas
Heated capillary
Heater
Nebulizer gas
Sample Inlet
Corona discharge
Drying gas
Drying gas
Heated capillary
Heater
Nebulizer gas
Sample Inlet
Corona discharge
Drying gas
Atmospheric Pressure Chemical Ionization (APCI): a corona discharge is used to
ionize the analytes and the species of the mobile phase or solvent in the gas phase (see Fig. 9-
3). Gas phase chemical ionization process takes place at atmospheric pressure where the
ionized solvent acts as reagent gas and ionizes the sample in gas-phase reactions.
Figure 9-2 An electrospray ion source Figure 9-3 An APCI ion source
Instrumental Analysis Mass Spectrometry
64
Atmospheric Pressure Photoionization (APPI): ultraviolet light produced by an UV
lamp ionizes gas phase analytes or dopants added to the sample with subsequent gas-phase
reactions (see Fig. 9-4).
UV lamp
Dopant: e.g. toluene
Drying gas
Heated capillary
Drying gas
Heater
Nebulizer gas
Sample Inlet
UV lamp
Dopant: e.g. toluene
Drying gas
Heated capillary
Drying gas
Heater
Nebulizer gas
Sample Inlet
9.1.2 Quadrupole mass analyzers
Linear quadrupole (Q)
A linear quadrupole mass analyzer consists of four cylindrical metal rods set parallel
and mounted in a square configuration. The pairs of opposite rods are held at the same
potential which is composed of a direct voltage (U) and an alternating voltage component (V
amplitude with ω frequency); see Fig. 9-5. As an ion enters the quadrupole assembly, an
attractive force is exerted on it by the oppositely charged electrodes. Attraction and repulsion
are alternating in time, because the voltage applied to the rods and, thus, the sign of the
electric force also changes periodically. Only ions of a certain m/z value or m/z range pass the
quadrupole for a given set of voltages (U, V and ω). Overall, the analyzer acts as a mass filter.
The whole m/z range can be scanned with continuously varying the U, V voltages.
Figure 9-4 An APPI ion source
Instrumental Analysis Mass Spectrometry
65
Quadrupole Ion Trap (QIT)
A quadrupole ion trap is a mass analyzer that uses an oscillating three-dimensional
quadrupole electric field to store and then eject ions. The ion trap consists of a ring electrode
between two hyperbolic endcap electrodes (see Fig. 9-6). As a high voltage RF potential is
applied to the ring electrode and the endcap electrodes are held at ground potential, an
oscillating three-dimensional quadrupole electric field is formed in the trap. This field can
keep ions of a particular m/z range within the ion trap during the ion accumulation period.
After trapping, the mass spectrum is recorded when the ions are consecutively ejected from
the analyzer by varying the three-dimensional quadrupole field.
The main advantage of an ion trap mass analyzer is the opportunity of tandem mass
spectrometric measurements in time. Tandem mass spectrometry or MS/MS, briefly, means
multiple mass analyses. After the first mass spectrometric analysis, a precursor ion is selected
Figure 9-5 A linear quadrupole mass analyzer
Figure 9-6 A quadrupole ion trap mass analyzer
Instrumental Analysis Mass Spectrometry
66
for further analysis. It decomposes either spontaneously or as a result of additional activation
process yielding product ions or fragment ions. These ions are subjected to a second mass
spectrometric analysis, when a MS/MS spectrum is recorded.
Ion trap MS/MS is performed in one analyzer in a sequenced program of events:
1) Ion accumulation: The ion trap is loaded with ions until a maximum charge level or time
has been reached.
2) Isolation: Varying the three-dimensional quadrupole field keeps only one m/z ion, the
precursor ion, stable inside the trap.
3) Collision Induced Dissociation (CID): The precursor ion is resonantly excited to gain
energy for CID with the helium bath gas present inside the trap. The product ions are stored in
the trap. (Note step 2 and 3 can be repeated again for MS3 and so on.)
4) Scan: The product ions are mass analyzed by scanning the voltage on the ring electrode.
MSn analysis in an ion trap mass spectrometer permits multiple stages of precursor ion
isolation and fragmentation. This stepwise fragmentation permits individual fragmentation
pathways to be followed and provides extra structural information.
9.1.3 Mass spectrum
A mass spectrum is the two-dimensional representation of signal intensity or ion
abundance versus mass-to-charge ratio. The mass-to-charge ratio, m/z, is a dimensionless
quantity, because it is calculated from the dimensionless mass number, m, of a given ion, and
the number of its elementary charges, z. The most intense peak in a mass spectrum is called
base peak. In most representation the relative abundances of ions are plotted as a function of
their m/z values, i.e., the intensity of the base peak is normalized to 100 % relative intensity
and the intensities of other peaks are expressed as the percentages of the base peak intensity
(see Fig. 9-7).
Instrumental Analysis Mass Spectrometry
67
Depending on the ionization process different ions appear in the mass spectrum. In case
of electron impact ionization mass spectrometry, the peak at highest m/z results from the
intact ionized molecule, the molecular ion (represented by M+•, e.g. C4H10+• for butane in Fig.
9-7). A molecular ion is formed by the removal of one electron from a molecule to form a
positive radical ion or the addition of one electron to form a negative radical ion. In Fig. 9-7,
the molecular ion peak at m/z 58 is accompanied by several peaks (so called fragment ion
peaks) at lower m/z caused by fragmentation or dissociation of the molecular ion. In case of
electrospray ionization mass spectrometry, different ions of the molecular species can be
detected (e.g. protonated or deprotonated molecules, adduct ions): Protonated or deprotonated
molecules, represented by [M+H]+ or [M−H]−, are ions resulting from the ionization of a
molecule by the addition or removal of a proton, respectively. A cationized molecule is an
adduct ion formed by the association of a cation with the molecule, e.g. [M+Na]+, [M+K] +.
An anionized molecule is an adduct ion formed by the association of an anion with the
molecule, e.g. [M−Cl]−.
Definitions
Average mass or chemical mass of an ion or molecule is calculated using a weighted
average of the natural isotopes for the atomic mass of each element. That is the mass
calculated from the relative atomic masses of the elements.
Figure 9-7 Electron impact ionization mass spectrum of butane
Instrumental Analysis Mass Spectrometry
68
Nominal mass of an ion or molecule is calculated using the mass of the predominant
isotope of each element rounded to the nearest integer value and multiplied by the number of
atoms of each element.
Monoisotopic mass is the exact mass of an ion or molecule calculated using the exact
mass of the predominant isotope of each element.
Resolution: In a mass spectrum, the observed m/z value divided by the smallest
difference ∆(m/z) for two ions that can be separated: (m/z)/∆(m/z).
9.2 Practice
Structural analysis of capsaicin and dihydrocapsaicin by electrospray – ion trap MS and
MS/MS methods
Instrument:
Agilent 6300 LC/MSD Trap XCT Plus mass spectrometer equipped with
electrospray ion source and ion trap mass analyzer.
The sample solutions will be introduced into the ion source by direct infusion with a
syringe and a syringe pump.
Default MS parameters:
Drying gas (N2) flow rate: 4 L/min
Drying gas (N2) temperature: 325°C
Nebulizer gas (N2) pressure: 15 psi
High voltage: –3000 V or +3000 V
Mass-to-charge (m/z) range detected: 50-2200 m/z
Analysis speed (Ultra Scan mode): 26000 m/z/s
Other parameters of the ionization and mass analysis will be optimized during the
practice
Instrumental Analysis Mass Spectrometry
69
Chemicals:
water, methanol, capsaicin, dihydrocapsaicin
Structural formula of capsaicin and dihydrocapsaicin:
Step 1.
Calculate the elemental formula of capsaicin and dihydrocapsaicin. Calculate the
nominal, monoisotopic and average masses of both compounds.
Atomic Symbol
Atomic Number
Mass Number
Isotopic Abundance
Isotopic Mass
Relative Atomic Mass
H 1 1 99.985 1.007825 1.00795 2 0.015 2.014101
C 6 12 98.90 12.000000 12.0108 13 1.10 13.003355
N 7 14 99.63 14.003070 14.00675 15 0.37 15.000109
O 8 16 99.76 15.994915 15.9994 17 0.04 16.999132 18 0.20 17.999116
Step 2.
Dilute the 1mg/mL stock solutions to 10 µg/mL concentration with methanol and
water. The solvent of the diluted solutions should be water−methanol 1:1 v/v.
Step 3.
Record the mass spectra of the samples in positive and negative ion modes.
Analyze the mass spectra. Based on the m/z of the ions describe ions observed in the
mass spectra.
OHN
HO
OO
HN
HO
O
capsaicin dihydrocapsaicin
Instrumental Analysis Mass Spectrometry
70
Step 4.
Isolate the [M+H]+ and [M–H]– quasimolecular ions as precursor ions and fragment
them. Thus, record the MS/MS mass spectra for both compounds in positive and
negative ion modes.
Analyze the fragmentation mass spectra, suggest fragmentation sites of the precursor
ions and suggest empirical formula for the major fragment ions.
The laboratory notebook should contain the followings:
theory of mass spectrometry, parts of the mass spectrometer, details on electrospray
ionization, quadrupole mass analyzers, tandem mass spectrometry
detailed description of the measurements (settings, working parameters)
the elemental formula, and the calculated nominal, monoisotopic and average masses
of capsaicin and dihydrocapsaicin
(+)ESI-MS and (–)ESI-MS mass spectra with explanations of the ions
(+)ESI-MS/MS and (–)ESI-MS/MS mass spectra of the [M+H]+ and [M–H]– ions,
respectively
fragmentation sites indicated in the structural formula of the molecules
elemental formula suggested for the major fragment ions
9.3. Keywords, abbreviations
mass spectrometer, ion source, mass analyzer, electrospray ionization, atmospheric pressure
chemical ionization, atmospheric pressure photoionization, linear quadrupole mass analyzer,
quadrupole ion trap mass analyzer, mass spectrum, mass-to-charge ratio, base peak, molecular
ion, ions of molecular species (protonated or deprotonated molecules and adduct ions),
tandem mass spectrometry, precursor ion, product ion or fragment ion, average mass or
chemical mass, nominal mass, monoisotopic mass, resolution
ESI, APCI, APPI, Q, QIT, MS/MS, MSn
Instrumental Analysis Mass Spectrometry
71
9.4. Questions
1. What are the basic principles of mass spectrometry?
2. What are the main parts of a mass spectrometer?
3. What is a mass spectrum?
4. What are the tasks of ion sources? Name three ion sources.
5. What are the tasks of mass analyzers? Name two mass analyzers.
6. How does an electrospray ion source work?
7. Define tandem mass spectrometry.
8. Calculate the isotopic distribution of: (a) Br2, (b) Cl2.
9. Calculate the minimum resolution that is required from a mass analyzer to separate the
following isobaric species (i.e. species of the same nominal mass).
(a) CO (M=27.99491) and N2 (M=28.00615)
(b) 13CC6H7 (M=92.05813) and C7H8 (M=92.06260)
10. A protein (cytochrome C) electrospray – ion trap MS mass spectra is depicted on Fig. 8-
8. In the ESI-MS mass spectrum of a protein, normally a characteristic series of
multiply charged peaks present. In the positive ionization mode, the (M+zH)z+ ions are
formed in a multistep, consecutive protonation process. Thus each peak in the
following spectrum belongs to the same protein; the differences between the ions are
in their protonation and charge states.
(a) Calculate the molecular mass of the protein.
(b) Determine the charge number (z) of the ion corresponding to the base peak.
I [x10 6]
589.81 619.26
651.60
687.77
728.18
773.56
825.01
883.78
500 550 600 650 700 750 800 850 900 950 m/z 0.00
0.25
0.50
0.75
1.00
1.25
1.50
Figure 8-8 Electrospray mass spectrum of cytochrome C recorded in positive ion mode
Instrumental Analysis Capillary Electrophoresis
72
Chapter 10 – Capillary Electrophoresis
10.1 Theory
10.1.1 Introduction
Electrophoresis is the migration of electrically charged solute molecules (ions) in an
electric field. It can be performed in slab-gel format or in microfluidic capillary formats. In
capillary electrophoresis (CE), the electrophoretic separation of solutes is carried out in
narrow-bore tubes, typically 25 to 200 µm inner diameter (id), which are usually filled only
with buffer (electrolyte). Because of the high resistance of the electrolyte in the narrow-bore
capillary, very high electrical fields (100 to 500 V/cm) can be applied, which results in short
analysis times. The heat developed during electrophoresis is efficiently dissipated due to the
large surface area-to-volume ratio of the capillary.
Today, CE is a premier separation technique for the study of low molecular weight
substances (e.g., inorganic and organic ions, amino acids, purine and pyrimidine bases,
nucleosides, nucleotides, chiral drugs, vitamins etc.) as well as large structures (such as
proteins, nucleic acids, carbohydrates, oligonucleotides, DNA restriction fragments, virus
particles and even whole cells). Main advantages of CE include the extremely high separation
efficiency and resolution, minimal consumption of samples and buffers, on-capillary
detection, high speed of analysis, and the potential for quantitative analysis and automation.
In addition, the numerous operation modes offer different separation mechanisms and
selectivities (see table 10-1).
Mode Basis of separation
Capillary zone electrophoresis (CZE) Free solution mobility
Capillary isotachophoresis (CITP) Moving boundaries
Capillary isoelectric focusing (CIEF) Isoelectric point
Capillary gel electrophoresis (CGE) Size (mass)
Micellar electrokinetic chromatography (MEKC)
Hydrophobic / ionic interactions with micelle
Capillary electrochromatography (CEC) Chromatographic interactions
Table 10-1 Modes of capillary electrophoresis
Instrumental Analysis Capillary Electrophoresis
73
10.1.2 Instrumentation
The commercial instrumentation of capillary electrophoresis is illustrated in Figure 10-
1. Typical CE systems use fused silica capillaries externally coated with polyimide, like those
applied in gas chromatography. The capillary is placed in buffer reservoirs and filled with
buffer from one of the buffer vials by applying external pressure. The sample (1-10 nL) is
introduced from the sample vial, by replacing one of the vials with the sample vial and
applying either low pressure (hydrodynamic injection) or low voltage (electrokinetic
injection). After replacing the buffer vial, the electric field is applied across the capillary by a
high voltage (HV) power supply (typically 10 to 30 kV), which connects the two electrodes
immersed in the buffer vials. The resulting electrophoretic current is usually 5 to 50 µA. The
analytes start to migrate in the capillary towards the detector and they separate by reason of
their electrophoretic mobility depending on their size and charge. Optical detection can be
made directly through the capillary. UV-Visible absorption is the most widely used detection
method. An optical window in the capillary is easily created by removal of a small (1-3 mm)
section of the protective polyimide coating. Other detectors are LIF (laser induced
fluorescence), conductivity detector, mass spectrometer, etc.
Modern CE devices also include an autosampler ideal for automation of measurement
series, and a temperature regulating unit (thermostat) ensuring constant temperature during
the separation process (observe that viscosity of the electrolytes depend on temperature).
Figure 10-1 Schematic of CE instrumentation
Instrumental Analysis Capillary Electrophoresis
74
10.1.3 Background
Separation by electrophoresis is based on differences in solute velocity in an electric
field. The linear velocity of a migrating ion in an electrolyte solution is given by
v = µ E (1)
where v = ion velocity [cm/s]
µ = electrophoretic mobility [cm2/V⋅s]
E = applied electric field [V/cm]
Consequently, an increase in field strength increases the velocity. When an ion has been
accelerated to constant velocity in a constant electric field, the electric force (FE = q E) on the
ion is equal to the frictional force (FF = - 6 π η r v), which gives the relation:
qE = 6πηrv (2)
where q = ion charge
η = viscosity of electrolyte
r = solvated radius of ion
v = ion velocity
Solving for velocity and substituting equation (2) into equation (1) yields an equation
that describes the mobility in terms of physical parameters
r6q
e πη=µ
From this equation it is evident that small, highly charged ions have high mobilities
whereas large, minimally charged ions have low mobilities.
10.1.4 Electro-osmotic flow (EOF)
EOF is the bulk flow of liquid in the capillary at a constant speed induced by the electric
field. It results from the charge excess near the interior capillary wall. The inner surface of the
fused silica capillaries are covered by silanol groups (≡Si–OH), which start to dissociate to
negatively charged (≡Si–O-) groups in contact with solutions above pH 2.5. The immobilized
surface ions attract the mobile ions of opposite charge in the buffer solution (electrolyte) by
Instrumental Analysis Capillary Electrophoresis
75
electrostatic forces, which arrange themselves into two layers called electrical double layer
(Figure 10-2). The net surface charge density and the thickness of the double layer are
affected by the pH and the ionic strength of the electrolyte, respectively.
The applied electric field forces the charge excess in the diffuse double layer to move
toward the cathode. The motion of these ions will draw the bulk liquid along with them,
creating a flat (plug-like) solvent flow termed electro-osmotic flow (EOF) (Figure 10-3). The
flat profile does not contribute (except in a thin layer close to the tube wall) to a broadening of
an analyte zone, as does a parabolic profile of hydrodynamic flow generated by pumps in an
open capillary, i.e., EOF only displaces the analytes. Peak efficiency in CE, often in excess of
105 theoretical plates.
The mobility of EOF is given by the Smoluchowski equation:
πηζεε
=µ4
0EOF
where µEOF = electro-osmotic mobility
ζ = zeta potential
ε = relative permittivity (dielectric constant of electrolyte)
εo = relative permittivity of the vacuum
η = viscosity of electrolyte
Figure 10-3 Plug-like flow profile of EOF
Figure 10-2 Representation of the double layer at the capillary wall
Instrumental Analysis Capillary Electrophoresis
76
The zeta potential is the potential difference created close to the wall (Figure 10-4),
and is strongly dependent on the ionic strength and pH of the buffer.
10.1.5 Capillary zone electrophoresis
CZE is the simplest form of CE, because the capillary is only filled with buffer.
Separation occurs because analytes migrate in discrete zones and at different velocities, based
on their charge to size ratio. Separation of both anionic and cationic solutes is possible due to
the superposition of electro-osmotic flow on to analyte mobility (Figure 10-5).
As depicted in Figure 10-5, EOF causes movement of all species, regardless of charge,
in the same direction (from the anode to the cathode). The ions migrate with a resultant
migration velocity owing to their own electrophoretic mobility and the mobility of the EOF,
whereas neutral analytes are transported by the EOF. Cations migrate fastest as the
electrophoretic attraction towards the cathode and the EOF are in the same direction. Neutral
Figure 10-4 The electrostatic potential in the double layer
Figure 10-5 Migration order of ions in capillary zone electrophoresis in the presence of EOF
Instrumental Analysis Capillary Electrophoresis
77
molecules are not separated from each other, and anions migrate slowest as they are attracted
to the anode but are still carried by the EOF toward the cathode (the magnitude of EOF can be
more than an order of magnitude greater than the electrophoretic mobilities of the analytes).
If the capillary wall is pre-treated (coated) with a neutral surfactant (either a viscous
polymer or a covalently attached polymer), the walls will be uncharged and the EOF will be
eliminated. In these circumstances, anions and cations can migrate in opposite directions.
When analyzing proteins, stable neutral coatings are needed to reduce effectively protein
adsorption onto the capillary wall.
10.1.6 Electropherogram
The result of an electrophoretic run is an electropherogram, where migration time is
plotted on the X axis and absorbance data are plotted on the Y axis (Figure 10-6). Qualitative
information of compounds is provided by the position of peaks (migration time), while
quantitative information (concentration of compounds) can be obtained from the peak height
or peak area.
10.1.7 Analytical parameters
Electrophoretic mobility
The mobility is characteristic constant for an ion in a given electrolyte, and it can be
determined from an electrophoresis experiment. Upon replacing v with l/t and E with U/L in
equation (1), the mobilities of the analytes can be determined by the following formula:
Figure 10-6 Electropherogram of benzyl derivatives
Instrumental Analysis Capillary Electrophoresis
78
UtLl
a ⋅⋅=µ
where µa = apparent mobility
U = applied voltage
l = effective capillary length (from injection end to the detector)
L = total capillary length
t = migration time
In the presence of electro-osmotic flow, the measured mobility is called the apparent
mobility, µa = µe + µEOF. The effective mobility, µe, can be extracted from µa by
independently measuring the EOF using a neutral marker (e.g. acetone) that moves at a
velocity equal to the EOF.
Efficiency
In separation science, two related concepts for measuring the efficiency of separation
are widely used, the plate height (H) and the plate number (N). The theoretical plate number
for a Gaussian peak can be determined directly from an electropherogram, using the following
formula:
2
21
545
ω=
/
t.N
or
2
16
ω= t
N
where t = migration time
ω1/2 = temporal peak width at half peak height
ω = temporal peak width at the baseline
Theoretical plate number can be related to the HETP (height equivalent to a theoretical plate),
H, by
Nl
H =
where l = effective capillary length
Instrumental Analysis Capillary Electrophoresis
79
Low values of H are favorable and are in the µm range for high-efficiency separation.
Resolution (R)
The resolution is a quantitative measure of the degree of separation of two sample
components and is defined as:
21
t2R
ω+ω∆=
where ∆t = difference between the migration times of the analytes
ω1 and ω2 = widths of the peaks at the baseline
10.2 Practice
Measuring of preservatives and vitamin C in lime juice
Running conditions:
Instrument:
Buffer:
Capillary length (total and effective):
Injection mode:
Temperature:
Voltage:
Polarity:
Detection wavelength:
Instrumental Analysis Capillary Electrophoresis
80
Samples:
Benzoic acid, sorbic acid and ascorbic acid (vitamin C) of known concentration
(standard solutions). Lime juice.
CH3 OH
O
O
OH
O
OHOH
O
OH
OH
Sorbic acid (E200) Benzoic acid (E210) Vitamin C (E300)
MW = 112.12 g/mol MW = 122.12 g/mol MW = 174.14 g/mol
λmax = 255 nm λmax = 225 nm λmax = 265 nm
Tasks
1. Make the electrophoretic run of a mixture of standards.
2. Identify the peaks (compounds) according to their charge/mass ratio.
3. Determine t (migration time) and ω1/2 (peak width at half height) of the peaks.
4. Calculate µa (apparent mobility) of each compound.
5. Determine separation efficiency by the calculation of parameters N, H and R.
6. Make the electrophoretic run of lime juice.
7. Identify the peaks (qualitative analysis) and calculate the concentration of compounds
(quantitative analysis) by comparing the electropherogram to that of standard
mixture.
The laboratory notebook should contain the followings:
theory of the analytical method
parts of the CE
settings, working parameters
detailed description of the measurement
electropherograms
calculations
results
Instrumental Analysis Capillary Electrophoresis
81
10.3 Questions
1. What is the principle of separation in capillary zone electrophoresis?
2. Define ion velocity and electrophoretic mobility.
3. What is electro-osmotic flow (EOF)? Describe its benefits and flow-profile.
4. What will be the order of migration of components in an uncoated capillary at pH 9?
5. What are the main parts of a CE instrument?
6. Name two injection modes in CE.
7. What kind of detectors can be applied in CE?
8. What is an electropherogram?
Instrumental Analysis Calculations
82
Chapter 11 – Calculations
1. Calculate the molarity of a potassium dichromate solution prepared by placing 10.3000 g
of K2Cr2O7 in a 50.00 mL volumetric flask, dissolving, and diluting to the calibration
mark.
2. Calculate the molar concentration of NaCl, to the correct number of significant figures,
if 2.1915 g of NaCl is placed in a beaker and dissolved in 50.00 mL of water measured
with a graduated cylinder. This solution is quantitatively transferred to a 300.00 mL
volumetric flask and diluted to volume. Calculate the concentration of this second solution
to the correct number of significant figures.
3. 50.00 mL of 0.200 mol/L NaOH solution is neutralized with 20.00 mL of sulfuric acid.
Determine the concentration of the acid in mol/L .
4. 20.00 mL HCl solution was titrated with 0.175 mol/L (f = 0.995) KOH solution. 17.23 mL
basic solution was added to reach the equivalence point. What was the concentration of
the sample in mol/L and mg/100 mL?
5. A sample of liquid containing phosphoric acid, it was completely neutralised by 21.60 mL
of 0.500 mol/L sodium hydroxide solution.
What mass (in milligrams) of phosphoric acid was in the sample?
OH3PONaNaOH3POH 24343 +⇔+
6. 60.00 mL of an acidified dichromate(VI) solution with a concentration 0.050 mol/L was
titrated against a 0.600 mol/L Fe2+ solution. What volume of Fe2+ solution would be
required to reach the end point of this titration?
OH7Cr2e6H14OCr 232
72 +⇔++ +−+−
7. A 0.1784 g sample of a monoprotic acid neutralizes 16.40 mL of 0.085 mol/L KOH
solution. Calculate the molar mass of the acid.
−++ +⇔ eFeFe 32
Instrumental Analysis Calculations
83
8. 0.3000 g of aspirin (an acid) was titrated with sodium hydroxide solution of concentration
0.100 mol/L. If the aspirin required 16.45 mL of the NaOH solution to neutralise it,
calculate the percent purity of the aspirin .
9. 150.00 mL of an aqueous sodium chloride solution contains 0.0045g NaCl.
Calculate the concentration of NaCl in parts per million (ppm).
(1 ppm =1µg/g or 1 µg/mL)
10. Using the systematic approach, calculate the pH of the following solutions
1. 0.050 mol/L HClO4;
2. 10–7 mol/L HCl;
3. 0.025 mol/L HI.
11. Consider the titration of 20.00 mL of 0.050 mol/L strong acid with 0.100 mol/L strong
base. Find the pH at the following volumes of base added and make a titration graph of
pH versus Vb.
Vb = 0.00, 1.00, 3.00, 5.00, 9.00, 9.50, 10.00, 10.50, 11.00, 12.00 and 15.00 mL.
12. What is the pH at the equivalence point when 0.100 mol/L acetic acid (pKs = 4.76) was
titrated with 0.050 mol/L NaOH solution?
13. What will be pH of the original and the final solutions as you mix 13.00 mL 0.200 mol/L
(f = 0.980) HCl solution and 20.00 mL 0.160 M (f = 0.948) KOH solution?
14. How the originally 12.50 pH of the 100.00 mL NaOH solution change after every
addition, as we add 5 times 2.00 – 2.00 mL of 0.100 mol/L HCl solution?
15. Calculate the potential of a copper electrode immersed in:
a. 0.035 mol/L Cu(NO3)2
b. 0.055 mol/L in NaCl and saturated with CuCl
c. 0.025 mol/L NaOH and saturated with Cu(OH)2.
Instrumental Analysis Calculations
84
16. Calculate the potential of a platinum electrode immersed in a solution that is
a. 0.0508 mol/L in K4Fe(CN)6 and 0.00548 mol/L K3Fe(CN)6;
b. 0.0360 mol/L in FeSO4 and 0.00725 mol/L Fe2(SO4)3;
c. Prepared by mixing 50.00 mL 0.075 mol/L Ce(SO4)2 with an equal volume of
0.125 mol/L FeCl2 (assume solutions were 1.000 mol/L in H2SO4 and use
formal potentials).
17. Calculate the theoretical cell potential of the following cells. If the cell is short-circuited,
indicate the direction of the spontaneous cell reaction.
a. Zn Zn2+ (0.200 mol/L) Co2+ (5.04·10-4 mol/L) Co
b. Pt Fe3+ (0.250 mol/L, Fe2+ (0.085 mol/L) Hg2+ (0.0412 mol/L) Hg.
18. Calculate the energy in joules of one photon of radiation with a wavelength of 15.00 µm.
19. Calculate the wavelength and the energy in joules associated with a signal at 280 MHz.
20. Prepare the solutions by diluting 4×10-2 w/w % FeSO4 solution. Take 2.50 mL of the
stock solution, and dilute to 15.00 mL with water. Then take 5.00 mL to the next and
dilute to 15.00 mL. Follow this 2nd sequence 3 times. What will be the concentration (in
% ) of the last (5th) solution?
Instrumental Analysis Calculations
85
21. You should make a calibration with standard addition. Calculate the standard
concentrations in each flasks in mol/1000 mL units.
You have a 0.150 mol/L standard ZnCl2 solution. The solutions were prepared according
this table:
22. A 6.25·10-5 mol/L solution of potassium permanganate has a transmittance of 37.5% when
measured in a 1.8 cm cell at a wavelength of 525 nm. Calculate the absorbance of this
solution and the molar absorptivity of KMnO4.
23. The logarithm of the molar absorptivity of phenol in aqueous solution is 3.812 at 211 nm.
Calculate the range of phenol concentration that can be used if the absorbance is to be
greater than 0.100 and less than 1.000 with 0.75 cm cell.
24. A typical simple infrared spectrophotometer covers a wavelength range from 5 µm to 35
µm. Express its range in wavenumbers and in hertz (Hz).
25. Consider a chromatography column in which Vs = Vm/3. Find the retention factor if K =
3 and K = 30.
26. The retention volume of a solute is 57.30 mL for a column with Vm = 15.70 mL and Vs =
11.40 mL. Calculate the retention factor and the partition coefficient for this solute.
Flask No.
mol/L
1. 10.00 mL unknown solution → fill to 100.00 mL with dist. Water
2. 10.00 mL unknown solution+ 4.00 mL standard solution → fill to 100.00 mL with dist. Water
3. 10.00 mL unknown solution+ 8.00 mL standard solution → fill to 100.00 mL with dist. Water
4. 10.00 mL unknown solution+ 12.00 mL standard solution → fill to 100.00 mL with dist. Water
5. 10.00 mL unknown solution+ 16.00 mL standard solution → fill to 100.00 mL with dist. Water
6. 10.00 mL unknown solution+ 20.00 mL standard solution → fill to 100.00 mL with dist. Water
Instrumental Analysis Calculations
86
27. Retention times in a gas chromatogram are 1.15 min for CH4, 7.35 min for pentane, 7.57
min for unknown, and 8.02 min for hexane. Find the Kováts retention index for the
unknown component.
28. The packed column in gas chromatography had an inside diameter 4.6 mm. The
measurement volumetric flow rate at the column outlet was 35.0 mL/min. If the column
porosity was 0.38, what was the linear flow velocity in cm/s?
29. Substances A and B have retention time of 16.25 min and 18.20 min, respectively, on a
25.0 cm column. An unretained species passes though the column in 1.35 min.
The peak widths (at base) for A and B are 1.21 and 1.33 min, respectively.
Calculate
a. The column resolution;
b. The average number of plates in the column;
c. The plate height.
30. A protein required 5.3 min to travel 80.0 cm to the detector in a 95.0 cm long capillary
tube with 31.2 kV between the ends. Find the apparent electrophoretic mobility.
Instrumental Analysis Calculations
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Answers to problems
1. c = 0.700 mol/L;
2. c = 0.125 mol/L;
3. c = 0.250 mol/L;
4. c = 0.150 mol/L, m = 547.5 mg in 100.00 mL;
5. m = 0.3528 g;
6. V = 30.00 mL;
7. M = 127,9 g/mol, HI;
8. m = 296.1 mg, 98.7%;
9. 30 ppm;
10.
a. pH = 1.3;
b. pH = 7;
c. pH = 1.6;
11. pH = 1.30; pH = 1.37; pH = 1.52; pH = 1.70; pH = 2.46; pH = 2.77; pH = 7.00;
pH = 11.21; pH = 11.51; pH = 11.79; pH = 12.16
12. pH = 8.78;
13. pH = 12.17;
14. pH = 12.46; pH = 12.42; pH = 12.38; pH = 12.34; pH = 12.29;
15.
a. E = 0.294 V;
b. E = 0.199 V;
c. E = - 0.138 V;
16.
a. E = 0.303 V;
b. E = 0.723 V;
c. E = 0.690 V;
17.
a. E = - 0.409 V;
b. E = 0.0145 V;
18. E = 1.326·10-20 J;
19. E = 1.856·10-25 J; λ = 1.071 m;
Instrumental Analysis Calculations
88
20. c1 = 6.67·10-3 %; c2 = 2.22·10-3 %; c3 = 7.41·10-4 %; c4 = 2.47·10-4 %; c5 = 8.23·10-5 %;
21. c1 = 6.0·10-3; c2 = 1.2·10-2; c3 = 1.8·10-2; c4 = 2.4·10-2; c5 = 3.0·10-2;
22. A = 0.426, ε = 37.864 L/mol·cm;
23. c1 = 2.056·10-5 mol/L, c2 = 2.056·10-4 mol/L;
24. υ1 = 6·1013 s-1, υ2 = 8.57·1012 s-1;
25. k1 = 1.0, k2 = 10.0;
26. k = 2.65, K = 3.65;
27. I = 534;
28. uo = 9.237 cm/s
29. R = 1.535, NA = 2886, NB = 2996, N = 2941, H = 0.085 mm;
30. µo = 7.6·10-8 m2/·Vs;
Instrumental Analysis Calculations
89
Chemical elements listed by atomic mass
Name Symbol Atomic Mass
Hydrogen H 1.0079 Carbon C 12.0107 Nitrogen N 14.0067 Oxygen O 15.9994 Sodium Na 22.9897 Magnesium Mg 24.3050 Phosphorus P 30.9738 Sulfur S 32.0650 Chlorine Cl 35.4530 Potassium K 39.0983 Chromium Cr 51.9961 Manganese Mn 54.9380 Iron Fe 55.8450 Nickel Ni 58.6934 Cobalt Co 58.9332 Copper Cu 63.5460 Zinc Zn 65.3900 Tin Sn 118.7100 Iodine I 126.9045 Mercury Hg 200.5900 Lead Pb 207.2000
Standard Electrode Potentials
Half reaction Ered (V) Ce4+(aq) + e– → Ce3+(aq) in 1.000 M H2SO4 +0,680 Hg2+(aq) + 2e– → Hg(l) +0.854 Fe3+(aq) + e– → Fe2+(aq) +0.771 Cu+(aq) + e– → Cu(s) +0.521 Fe(CN)6
3–(aq) + e– → Fe(CN)64–(aq) +0.360
Cu2+(aq) + 2e– → Cu(s) +0.337 Co2+(aq) + 2e– → Co(s) –0.277 Zn2+(aq) + 2e– → Zn(s) –0.763
Instrumental Analysis
90
Bibliography
Daniel C. Harris – Quantitative Chemical Analysis
F. James Holler, Douglas A. Skoog and Stanley R. Crouch – Principles of
Instrumental Analysis
D. A. Skoog, D. M. West, F. J. Holler and S. R. Crouch – Fundamentals of
Analytical Chemistry
David Harvey – Modern Analytical Chemistry
David G. Watson – Pharmaceutical Analysis
Jürgen H. Gross – Mass Spectrometry – A Textbook
E. de Hoffmann and V. Stroobant – Mass Spectrometry Principle and Applications
David Heiger – High performance capillary electrophoresis
DOI: 10.15170/TTK.2014.00001