Chromatographic methods of analysis : educational aid

The Ministry of Science and Higher Education of the Russian Federation 
Kazan National Research Technological University 
R. Bakeeva, S. Garmonov
CHROMATOGRAPHIC  
METHODS OF ANALYSIS 
Educational aid 
Kazan 
KNRTU Press 
2022 
1 

UDC 543.544(075) 
Published by the decision of the Editorial Review Board  
of the Kazan National Research Technological University 
Reviewers: 
PhD., Professor G. Ziyatdinova 
PhD., Professor L. Nikitina 
Bakeeva R. 
Chromatographic methods of analysis : educational aid / 
R. Bakeeva, S. Garmonov; The Ministry of Science and Higher Education of the Russian Federation, Kazan National Research Technological University. – Kazan : KNRTU Press, 2022. – 92 p.
ISBN 978-5-7882-3221-8 
Basic principles of chromatographic methods of analysis are described. 
Theoretical questions like fundamentals of chromatography and classification of chromatographic methods, gas chromatography, high performance 
liquid chromatograph, main characteristics of the chromatographic process, 
chromatogram processing methods, planar chromatography, gel chromatography are considered. Examples of control questions and tests are given. 
Methods for conducting laboratory work are given. A glossary of chromatographic terms is provided. 
Designed for students majoring in Chemical Technology programmes 
(18.03.01) and studying Analytical Chemistry in English. 
Prepared at the Department of Analytical Chemistry, Certification and 
Quality Management. 
UDC 543.544(075) 
ISBN 978-5-7882-3221-8 
© R. Bakeeva, S. Garmonov, 2022 
© Kazan National Research Technological 
University, 2022 
2 

CONTENTS 
 
1. CHROMATOGRAPHIC METHODS OF ANALYSIS ................................................................................................................. 
5 
1.1. Fundamentals of Chromatography and Classification of Chromatographic Methods 
......................... 
5 
1.2. Gas Chromatography ................................................................................................................................................................ 
7 
1.3. High Performance Liquid Chromatography .............................................................................................................. 
15 
1.4. Main Characteristics of the Chromatographic Process 
....................................................................................... 
22 
1.5. Chromatogram Processing Methods 
............................................................................................................................. 
26 
1.6. Planar Chromatography ....................................................................................................................................................... 
29 
1.7. Gel Chromatography .............................................................................................................................................................. 
37 
2. LABORATORY WORKS ................................................................................................................................................................... 
42 
2.1. Laboratory works on gas-liquid chromatography ................................................................................................. 
42 
Laboratory work 1. Qualitative Analysis of a Hydrocarbon Mixture 
............................................................... 
42 
Laboratory work 2. Determination of Quantitative Hexane Content in a Mixture  
of Hydrocarbons ........................................................................................................................................................................... 
45 
Laboratory work 3. Evaluation of the Qualitative and Quantitative Composition  
of Turpentine ................................................................................................................................................................................. 
48 
Control Questions and Tasks ................................................................................................................................................ 
50 
Test ...................................................................................................................................................................................................... 
51 
2.2. Laboratory Works on High-Performance Liquid Chromatography .............................................................. 
54 
Laboratory work 4. Liquid Chromatograph Device and Testing of its Operation ................................... 
54 
Laboratory work 5. The Obtaining a Chromatogram of the Test Mixture  in Reverse-Phase 
Chromatography. Determination of Chromatographic Column Efficiency 
.................................................. 
57 
Laboratory work 6. Identification of Naphthalene in a Test Mixture of Aromatic  
Hydrocarbons by Retention Time and Spectral Ratio. Determination of the Error  
in Determining the Spectral Ratio of Naphthalene ................................................................................................. 
60 
Laboratory work 7. Definition of Adulteration of Gasoline 
.................................................................................. 
64 
Control Questions and Tasks ................................................................................................................................................ 
67 
Test ...................................................................................................................................................................................................... 
69 
3 

3.3. Laboratory Works on Planar Chromatography 
........................................................................................................ 
72 
Laboratory work 8. Separation and Detection of Halides 
..................................................................................... 
72 
Laboratory work 9. Separation of Amino Acids by Thin-Layer Chromatography .................................... 
75 
Control Questions 
........................................................................................................................................................................ 
76 
Test ...................................................................................................................................................................................................... 
77 
Bibliography 
.............................................................................................................................................................................................. 
81 
Glossary ....................................................................................................................................................................................................... 
82 
Definitions of chromatographic terms 
............................................................................................................................. 
82 
Terms and Definitions of GLC from the Current GOST 17567-81. Types 
of Chromatography ..................................................................................................................................................................... 
85 
Gas chromatograph and its structural elements. In accordance with state 
standard 17567-81 
..................................................................................................................................................................... 
86 
General concepts used in gas chromatography 
.......................................................................................................... 
88 
Main parameters of the gas chromatographic process .......................................................................................... 
89 
4 

.  C H R O M A T O G R A P H I C  M E T H O D S  O F  A N A L Y S I S  
Chromatography is a separation process in which the compound to be determined is partitioned between the mobile and stationary phases. The mobile 
phase can be liquid or gas, and the stationary phase can be solid or liquid. 
During separation, the substances to be separated are concentrated. Chromatographic methods are universal, have high selectivity and sensitivity of analytical determinations, and therefore are widely used in analysis. Among 
the various chromatography options, the most widely used are high performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), 
and planar chromatography.  
The basic concepts and normative terminology adopted in chromatography are systematized and unified by the IUPAC Commission (International 
Union of Pure and Applied Chemistry). 
Chromatography is the science of intermolecular interactions and 
the transfer of molecules or particles in a system of phases that are immiscible and move relative to each other. Chromatography is a process of differentiated multiple redistribution of substances or particles between immiscible and moving relative to each other phases. This leads to the isolation of 
the concentration zones of the individual components of the initial mixtures 
of substances or particles. Chromatography combines the separation and determination of mixtures of substances or particles, based on the difference in 
the speed of their movement in a system of immiscible and moving relative 
to each other phases. 
1.1. Fundamentals of Chromatography and 
Classification of Chromatographic Methods 
Chromatographic processes are based on various hydrodynamic, sorption 
and diffusion phenomena. Sorption processes go through two stages: 
the stage of approaching equilibrium and the equilibrium stage. The first 
stage is characterized by a certain speed. The last step plays a critical role in 
achieving chromatographic separation. Sorption is the absorption of gases, 
vapors or dissolved substances by solid or liquid absorbents. If the sorbate is 
absorbed by the entire volume of the sorbent, then the process is called 
5 

absorption, and if it is concentrated on the surface of the sorbent, then the 
process is called adsorption. Usually, adsorbents are solid bodies with a developed surface, Silica gels, alumogels, active carbons, molecular sieves, and 
porous polymer sorbents are widely used in chromatography for this purpose. 
Liquid absorbents are not used by themselves in chromatography; they are 
usually applied to the surface of solid materials with a relatively small surface, which are called solid carriers. 
There are principles of chromatographic separation such as: 
• any chromatographic separation involves the movement of the analyzed sample through the layer of the stationary phase; 
• the movement of the components of the mixture is carried out by 
gas or liquid (of mobile phase); 
• the components of the analyzed mixture move at different effective 
velocities because the stationary phase contributes to the selective 
retardation. 
The speed of movement of various substances at different speeds is 
due to two factors: the strength of binding to the adsorbent and the affinity 
for the solvent (eluent). 
The variety of modifications and variants of the chromatographic 
method necessitates their systematization or classification. When classifying 
according to the state of aggregation of phases, chromatography methods can 
be divided into four groups (Table 1.1). 
 
Table 1.1. Classification of chromatographic methods according 
to the state of aggregation of phases 
Stationary 
Mobile 
Varieties of the method 
Possible options 
phase 
phase 
Redox, adsorption-complexing, thin-layer chromatography 
Solid 
Liquid 
Adsorption chromatography; ion exchange 
chromatography; sediment chromatography 
Solid 
Gaseous 
Gas adsorption chromatography 
Chromathermography, thermal dynamic (warm dynamic) 
Liquid 
Liquid 
Liquid Partition Chromatography 
Columnar; one-dimensional; 
two-dimensional; circular; 
electrophoretic; thin layer.  
Reversed phases method 
Liquid 
Gaseous 
Gas-liquid partition chromatography 
Chromatography of gases, liquids, stepwise, capillary chromatography 
6 

Classification of chromatographic methods is also possible according 
to the mechanism of the process of separating compounds, according to 
the type of adsorbent deposition on the substrate, and the action of physical 
fields (Tables 1.2, 1.3). 
Table 1.2. Classification of chromatographic processes according 
to the mechanism of the separation process 
Mechanism of the separation 
Scheme 
Variant's name 
process 
By molecular size 
Sieve  
chromatography 
Physical adsorption or dissolution 
Molecular  
chromatography 
Ion exchange 
Ion exchange  
chromatography 
Due to hydrogen bonding, chemical 
affinity, etc. 
Chemisorption 
chromatography 
Table 1.3. Classification of chromatographic methods according to 
the type of application of the sorbent onto the substrate and the action 
of physical fields 
The nature of the adsorption proScheme 
Variant’s name 
cess 
In a cylindrical layer of sorbent 
Column  
chromatography 
In a layer of sorbent on a flat surface 
Planar  
chromatography 
In a liquid film or sorbent layer 
placed on the inner wall of the tube 
Capillary  
chromatography 
In the fields of electric, magnetic, 
centrifugal and other forces 
Chromatography 
under the action 
of physical fields 
1.2. Gas Chromatography 
Gas chromatography (GC) is a method for the separation and determination 
of volatile compounds. The components of the analyzed mixture are 
7 

distributed between the stationary phase (solid or liquid) and the mobile 
phase (carrier gas). In this case, the mobile phase is in the state of gas or 
vapor and is an inert gas. The stationary phase is in the column. It can be 
a solid or liquid that is applied to a solid inert carrier or uniformly coats 
the inner walls of the column. 
Gas chromatography is a universal method for separating mixtures of 
substances that evaporate without decomposition. In this case, the components of the mixture to be separated move along the chromatographic column 
with the carrier gas flow. The principle of separation is the unequal affinity 
of substances for the volatile mobile phase and the stationary phase in 
the column. The components of the mixture are selectively retained by 
the stationary phase, since their solubility in this phase is different. This is 
how separation occurs. Components with greater solubility take longer to exit 
the liquid phase than components with less solubility. Then the substances 
leave the column and are registered by the detector. The detector signal in 
the form of a chromatogram is recorded by a computer.  
Gas liquid chromatography (GLC) uses a liquid stationary phase that 
is distributed as a thin film on the surface of an inert solid support. GLC is 
the most selective chromatographic separation method, since there is a large 
selection of liquid phases, allowing operation in a wide temperature range 
(20–400 °C). Low limits of detection, rapidity, accuracy, and simplicity of 
operations provided the GLC method with wide application for the analysis 
of complex objects.  
As a result of movement along the stationary phase, the components 
in the vapor state come into contact with the adsorbent or liquid. There is 
a multiple transition of a substance from one phase to another, that is, 
the sorption-desorption cycle. This process is due to the physicochemical 
properties of the mobile and stationary phases, as well as the kinetic parameters of the establishment of interfacial equilibrium. 
The separation of the mixture takes place in the column, which is 
the main component of the chromatograph. Typically, columns are filled 
with a solid inert carrier, on which a non-volatile liquid is applied as a thin 
film. Columns are made of steel, glass or polymer material. They are twisted 
into a spiral in accordance with the dimensions of the thermostat of the chromatograph. Capillary columns are also used. These are tubes of small diameter, on the walls of which a thin film of liquid or adsorbent is applied. 
The solid support, the nature and amount of the liquid phase, the length 
and temperature of the column are important factors that determine the separation of mixtures. 
8 

The solid carrier is designed to hold a thin uniform film of the liquid 
phase. Materials based on silica, fluorocarbon polymers, polystyrene, and 
copolymers of styrene and divinylbenzene are used as solid carriers. Examples of silica-based carriers are diatonite and kieselguhr (eg spherochromes, 
chromatoin, hesosorbs). Theoflon, polychrome are examples of fluorocarbon 
polymers. 
The requirements for solid media are as follows: 
• large specific surface area (from 1 to 20 m2/g); 
• small and uniform pore diameter; 
• inertness (minimal chemical and adsorption interactions with 
the sample); 
• mechanical strength (carrier should not be crushed during filling 
and operation of the column). 
The content of the stationary liquid phase is usually 5–15 % of 
the amount of the solid support. 
The choice of the liquid phase in the GLC method depends on 
the composition of the analyzed mixture and is carried out taking into account the following requirements: 
• good dissolving ability of the components of the mixture, since at 
low solubility the components leave the column quickly and 
the separation is unsatisfactory; 
• selective dissolution of the mixture components; 
• low volatility and chemical inertness at a given column temperature. 
The selection of the column's stationary liquid phase and chromatography conditions will be easier if complete sample information is available. 
These are the expected components and their structure, the boiling point 
range.  
In order to achieve effective separation, the liquid phase must be similar in chemical structure to the components of the analyzed mixture. So, polar compounds (for example, alcohols) are better separated in the polar liquid 
phase. In this case, the components of the same nature leave the column in 
the sequence corresponding to the increase in their boiling points.  
The choice of carrier gas depends on the efficiency of the column, 
the sensitivity and the principle of operation of the detector. Typically, helium and nitrogen are used as carrier gases. The carrier gas must meet the 
following requirements: 
9 

• inertness, excluding the possibility of interaction with the sample 
or the stationary phase; 
• providing the necessary diffusion characteristics that determine 
the efficiency of the column; 
• no influence on the detector readings; 
• purity; 
• accessibility. 
The carrier gas, as a rule, enters the chromatograph column from 
a compressed gas cylinder. In isothermal chromatography, the column resistance does not change during analysis. A pressure regulator is used to 
maintain a constant pressure at the inlet to the column and, therefore, to maintain a constant carrier gas velocity. At a certain temperature, the constant 
velocity of the carrier gas ensures that the retention time of the analytes remains constant. The optimum carrier gas velocity is usually determined experimentally. It increases with increasing length and inner diameter of 
the column.  
The intensity of separation of the mixture depends on the temperature. 
Typically, a temperature increase of 30 °C doubles the rate of movement of 
the components of the mixture. The result of increasing the temperature is 
a reduction in the duration of the analysis. 
As a rule, as the temperature is lowered, a more complete separation 
is achieved. The optimum temperature should not be too high, as this will 
impair separation. It should not be too low as this tends to increase the retention time. The temperature is set at approximately the average boiling point 
of the mixture.  
When choosing the column temperature, the physicochemical properties of the liquid phase (boiling, melting, and destruction points) are also 
taken into account. Chromatography at a constant temperature along the entire length of the column is an isothermal process. 
In isothermal mode, the separation of complex mixtures is difficult. 
This is especially true for mixtures of homologues, the boiling points of 
which differ significantly. To improve the separation of such mixtures, 
the temperature of the column is changed over time according to a specific 
program (gas chromatography with temperature programming). 
Separation improves with increasing column length. However, 
the chromatographic peaks are blurred. The length of packed columns is usually in the range of 1 to 4 m; the length of capillary columns is from 15 to 
100 m. Packed columns are called columns of large diameter, which can be 
10