1. Basics of liquid chromatography:
Thin-layer chromatography (TLC)
Column liquid chromatography (CLC)
Teresa Kowalska
Institute of Chemistry
University of Silesia
9, Szkolna street
Katowice, Poland

2. Outline of presentation
Separation sciences; the notion
Chromatography; the IUPAC definition
Classification of chromatographic techniques
Classification criteria
Different classifications
Analytical and preparative chromatography mode
Analytical chromatography mode
Preparative chromatography mode
Planar chromatography
Column chromatography
Basic mechanisms of separation in liquid chromatography
Adsorption
Partition
Parameters of retention
Parameters of resolution
Applications of liquid chromatography – general view
Applications of TLC
Applications of CLC
Conclusions

3. Separation sciences
Separation sciences are the sciences focusing on the theory of the techniques targeting the separation of the mixtures of different chemical species.
Examples of the separation techniques:
Distillation
Cristallization
Extraction
Separation with use of membranes
Electrochemical techniques
Chromatography
etc.

4. Chromatography – the IUPAC definition
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary, while the other moves in a definite direction.
Chromatography – the IUPAC definition

5. Hence, the first message to remember from the IUPAC definition
From this definition it simply comes out that each chromatographic system is composed of the following two elements:
stationary = immobile phase,
mobile phase,
and of course, of a mixture of substances to be separated.

6. Classification of chromatographic techniques Classification criteria
Geometrical
Physical
Chemical

7. Geometrical criterion
In this case, the criterion is the geometrical shape of stationary phase.
If (solid) stationary phase is thin and flat (i.e., it looks like a sheet of paper), then the chromatographic mode is planar chromatography.
Planar chromatography additionally sub-divides into:
paper chromatography
thin-layer chromatography

8. Geometrical criterion (continued)
If stationary phase assumes a cylindrical shape and is encapsulated in a tubular container, then the chromatographic mode is column chromatography.

9. Physical criterion liquid gas solid Chromatography mode Mobile phase
liquid-liquid chromatogr.
(partition chromatography)
liquid-gaseous chromatogr.
liquid
gas
solid-liquid chromatogr.
(adsorption chromatography)
solid-gaseous chromatogr.
solid
Chromatography mode
Mobile phase
Stationary phase
This classification is based on physical parameter, i.e., on the condensation degree of stationary phase and mobile phase.

10. Physico-chemical mechanism
Chemical criterion
This classification is based on physico-chemical mechanism of distribution of the analyte between the stationary and mobile phase. This classification is still open, as novel chromatographic modes are still invented and added to the list.
Electrophoresis
Isoelectric focusing
Physical adsorption + electrophoresis
Electrochromatography
Mixed mechanisms
Different mixed modes
Isotachophoresis
(Bio)receptor coupling mechanism
(Bio)affinity chromatography
Molecular sieves
Size-exclusion chromatogr.
Coulomb forces + molecular sieves + physical adsorption
Ion-exclusion chromatography
Coulomb forces + physical adsorption
Ion-pair chromatography
Chemisorption
Ion-exchange chromatography
The Nernst liquid-liquid partition
Partition chromatography
Physical adsorption
Adsorption chromatography
Physico-chemical mechanism
Technique

11. Stationary phases in liquid chromatography
Paper chromatography
In paper chromatography, stationary phase is chromatographic paper (cellulose).

12. Stationary phases in liquid chromatography
Thin-layer chromatography (TLC)
Octadecyl silica (ODS; C18; RP-18)
Octyl silica (C8; RP-8)
Dimethyl silica (C2; RP-2)
3-Cyanopropyl silica (CN)
3-Aminopropyl silica (NH2)
Diol silica
Phenyl silica
etc.
Silica gel (silica; SiO2)
Aluminium oxide (alumina; Al2O3)
Florisil (MgO·SiO2)
Microcrystalline cellulose powder
Polyamide 6 (Nylon 6; ε-aminopolycaprolactame)
Second generation stationary phases
First generation stationary phases

13. Stationary phases in liquid chromatography (continued)
High-performance liquid chromatography (HPLC)
Octadecyl silica (ODS; C18; RP-18)
Octyl silica (C8; RP-8)
Dimethyl silica (C2; RP-2)
3-Cyanopropyl silica (CN)
3-Aminopropyl silica (NH2)
Diol silica
Phenyl silica
etc.
Second generation stationary phases
Non-pressurized column liquid chromatography (CLC)
Silica gel (silica; SiO2)
Aluminium oxide (alumina; Al2O3)
First generation stationary phases

14. water + over 80 organic solvents
Mobile phases in liquid chromatography
(both planar and column chromatography mode)
water + over 80 organic solvents
Basic demands posed on solvents used as mobile phase components:
They should not be toxic;
They should not be explosive;
They should not be volatile;
They should not be viscous;
They should be easily miscible with other solvents used as mobile phase components;
They should be chemically inactive (they should not react with components of a separated mixture;
They should be the low-molecular-weight compounds;
etc.

15. Analytical and preparative chromatography mode
Each isotherm of distribution of an analyte between the stationary and mobile phase of the chromatographic system can be divided into the linear and non-linear part.
linear part
favorable: convex
unfavorable: concave
non-linear part
Linear part of isotherm: analytical chromatography mode
Non-linear part of isotherm: preparative chromatography mode

16. Simple isotherms of analyte distribution between stationary and mobile phase
Isotherms are mathematical models which illustrate the course of the distribution.
The Langmuir isotherm
(the simplest model of the convex isotherm)
The anti-Langmuir isotherm
(the simplest model of the concave isotherm)
adsorbed monolayer
adsorbed multilayer _
where qi and qs - analyte concentration on stationary phase and maximum analyte concentration on stationary phase, respectively, ci – analyte concentration in mobile phase, and K – the isotherm constant.
qi = qs
1 + K ci
K ci
1 – K ci
where qi and qs - analyte concentration on stationary phase and maximum analyte concentration on stationary phase, respectively, ci – analyte concentration in mobile phase, and K – the isotherm constant.

17. Analytical chromatography mode Main targets:
Identification of each separated chemical species (qualitative analysis);
Separation of a mixture of substances to individual chemical species;
Quantification of each separated chemical species (quantitative analysis).
Planar chromatography
Column liquid chromatography
Calibration curve
In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration.

18. Preparative (and process) chromatography mode
Main targets:
The laboratory isolation of selected chemical species from reaction mixture on preparative scale;
The industrial isolation of selected chemical species from reaction mixture on technological scale.
E.g., chemical laboratory of organic synthesis
Technological establishment (e.g., pharmaceutical orbiotechnological industry

19. Planar chromatography
Basic notions
chromatographic chamber
mobile phase
chromatographic plate
Most often, the analysis is carried out in vertical chromatographic chambers.
Horizontal chromatographic chambers (known as sandwich chambers) are also used.
Mobile phase migrates upward, owing to capillary action.
This upward migration mode is called the ascending mode.
In the course of mobile phase migration, a mixture of compounds undergoes separation to individual chemical species. This process is called the development of the chromatogram.
The plate with separated chemical species is called the chromatogram.
Planar chromatogram
Vertical chromatographic chamber
Horizontal (sandwich) chromatographic chambers

20. Visualization and acquisition of planar chromatograms
(a) Destructive
Spraying with conc. H2SO4 and heating
Organic compounds become fully dehydrated and carbonized. As a result, black or dark brown spots of the separated zones are visualized on the white background.
(b) Non-destructive
(i) Physical visualization in iodine vapours
Iodine easily evaporates. If we place the developed chromatogram in a tightly closed chamber with a vessel filled with iodine crystals, iodine vapours will better adsorb on separated chromatographic zones than on chromatographic plate.
Hence, the brown spots will appear of the pale yellow background.
The process is reversible.
Chromatogram visualized in iodine vapours

21. Visualization and acquisition of planar chromatograms
(continued)
(ii) Physical visualization by staining separated zones with dyeing reagents
Video imaging and acquisition of chromatograms in a digital form

22. Visualization and acquisition of planar chromatograms
(continued)
Densitometric scanning of the chromatograms in UV-Vis light
Densitometer
Densitogram
Chromatogram
Transformation of planar chromatograms into a digital signal, which enables quantitative analysis

23. Hyphenated technique: TLC-MS
(thin-layer chromatography with mass spectrometric detection)
The separated chromatographic bands can be eluted directly to mass spectrometer. This can be obtained with use of the TLC-MS interface (which is a specially designed device to elute separated chromatographic bands and to introduce them directly to mass spectromter).
TLC-MS interface

24. Hyphenated technique: TLC-MS
(thin-layer chromatography with mass spectrometric detection)
Practical example:
„Fingerprinting” of botanical material
(a)
(b) 1 2 3 4 5
(a) The densitogram, (b) the videoscan, and (1)-(5) the mass spectra of the five separated chromatographic bands derived from the essential oil sample of Salvia hians

25. Column liquid chromatography (CLC)
Basic notions
Non-pressurized column liquid chromatography
(an omnipresent tool in laboratories of organic synthesis, etc.)
Preparative lab-scale column
Mobile phase moves down the chromatographic column under the inluence of gravitation.
Separated fractions can be automatically collected with use of an automatic fraction collector.
Small-scale sample preparative isolation / purification applications
Automatic fraction collector

26. Column liquid chromatography (CLC) (continued)
Basic notions
Slightly pressurized column liquid chromatography, known as
FLASH CHROMATOGRAPHY (FC)
(an advanced lab scale tool for rapid isolation of compounds)
FC preparative lab-scale column
Illustration of preparative separation
Automatic fraction collector
Mobile phase moves down the chromatographic column under he influence of mechanical force (the pump)

27. Column liquid chromatography (CLC) (continued)
Basic notions
Macroscale preparative chromatography
Chromatographic column systems used in biotechnology
(e.g., for macro-scale isolation and purification of proteins)

28. Column liquid chromatography (CLC) (continued)
Basic notions
High-performance liquid chromatography (HPLC)
Chromatographic systems used for micro-scale separation, identification, and quantification of the compound mixture
(micro-scale analytical mode)
A flow scheme for HPLC system
The HPLC system
The HPLC column

29. Detection systems applied in CLC
All detection systems applied in CLC are based on advanced physical principles. Below, a list is given of the most widely employed physical principles used in the CLC detection systems:
light refraction;
UV/Vis absorption spectroscopy;
IR absorption spectroscopy;
1H NMR spectroscopy;
mass spectrometry;
fluorometry;
Rayleigh light scattering.
Coupling of an advanced HPLC separation technique with the physically advanced (mostly spectroscopic) detection techniques results in the so-called hyphenated (i.e., hybrid) analytical techniques.

30. Main application areas for chromatography
Analytical mode
Classical analytical chemistry
Life sciences analysis (in the first instance, pharmaceutical and biomedical analysis)
Environmental analysis
Geochemical analysis
Extraterrestrial analysis
Preparative and technological mode
Pharmaceutical industry
Biotechnology