1. Chromatographic Methods of Analysis
Lecture 15
Chromatographic Methods of Analysis
Associate prof . L.V. Vronska
Associate prof . M.M. Mykhalkiv

2. Outline The basic concepts of chromatography.
Classification of chromatographic methods.
Theoretical bases of chromatography.
The main parts of chromatographic equipment.
Gas chromatography.
Theoretical base of liquid chromatography
HPLC – high-performance liquid chromatography
TLC – thin-layer chromatography
Paper chromatography
Ion-exchange chromatography

3. 1. The basic concepts of chromatography
Chromatography is a process which is based on multiple repetition of sorption and desorption of substances. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase.
Chromatography (from Greek χρώμα:chroma, color and γραφειν:graphein to write) is the collective term for a family of laboratory techniques for the separation of mixtures.

5. Thin layer chromatography is used to separate components of chlorophyll

6. Sorbtion is divided on:
Sorbtion is absorption process of the gaseous or dissolved substance (sorbate) by firm substance or liquid (sorbent), reverce process is called desorbtion.
Sorbtion is divided on:
Adsorption is a process that occurs when a gas or liquid solute accumulates on the surface of a solid or a liquid (adsorbent), forming a film of molecules or atoms (the adsorbate).
Absorption is a physical or chemical phenomenon or a process in which atoms, molecules, or ions enter some bulk phase - gas, liquid or solid material.
Chemisorption is a classification of adsorption characterized by a strong interaction between an adsorbate and a substrate surface, as opposed to physisorption which is characterized by a weak Van der Waals force.

7. At constant temperature adsorption increases:
- with increase of solution concentration;
- with increase in pressure of gas.
Adsorption is usually described through isotherms, that is, the amount of adsorbate on the adsorbent as a function of its pressure (if gas) or concentration (if liquid) at constant temperature. The quantity adsorbed is nearly always normalized by the mass of the adsorbent to allow comparison of different materials.

8. Langmuir isotherm (red) and BET isotherm (green)
Often molecules do form multilayers, that is, some are adsorbed on already adsorbed molecules and the Langmuir isotherm is not valid. In 1938 Stephan Brunauer, Paul Emmett, and Edward Teller developed a model isotherm that takes that possibility into account. Their theory is called BET theory, after the initials in their last names

9. The Langmuir equation or Langmuir isotherm or Langmuir adsorption equation relates the coverage or adsorption of molecules on a solid surface to gas pressure or concentration of a medium above the solid surface at a fixed temperature. The equation was developed by Irving Langmuir in The equation is stated as:
where: n - quantity (mol) of the adsorbed substance at equilibrium;
n∞ - a maximum quantity of substance which can be adsorbed on sorbent;
b - constant;
C - concentration.

10. The quantity of the adsorbed substance will be defined:
In the range of small concentration an isotherm of adsorption is straight line. It is really if bc <<1 so (1 + bc) → 1, then
This equation of straight line adsorption (Henry's equation).
The quantity of the adsorbed substance will be defined:
- Concentration of substance;
- Sorbent affinity.

11. 2. Classification of chromatographic methods.
1. On physical nature of mobile and stationary phase:
Mobile phase
The gaseous
The liquid
stationary phase
The firm
gas-solid chromatography
Liquid adsorption chromatography, thin layer, ion-exchange, ionic, precipitation (sedimentation) chromatography
Partition gas-liquid chromatography
Partition liquid chromatography, HPLC, gel-chromatography

12. 2. On depending of sorbtion mechanism:
Molecular (interaction between a stationary phase (sorbent) and divided mix components at the expense of intermolecular interaction (Van der Waals forces);
chemisorption (ion-exchange, sedimentation, chelation chromatography, oxidation-reduction).

13. 3. Type separation: frontal chromatography;
elution - more often a used chromatography;
displacement chromatography.

15. Advantage - concentration of solution does not decrease.
Lack – often bridging of components zone because they are not divided by solvent.

17. 4. Techniques by chromatographic bed shape
Column chromatography (it is a separation technique in which the stationary bed is within a tube)
Planar chromatography:
The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography)

18. 3. Theoretical bases of chromatography
A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

19. !!! adjusted time is proportional to chromatographic resolution of the given component of investigated mix.

20. adjusted time depends from:
The nature chromatographic compounds;
The nature of a mobile phase;
The nature and weight of stationary phase;
Speeds of mobile phase movement;
Column temperatures (in a gas chromatography);
Lengths of a column;
Partition coefficient (than it is more for substance, the more its retention time).

21. retention volume - the volume of mobile phase needed to move a solute from its point of injection to the detector (Vr).
baseline width - the width of a solute’s chromatographic band measured at the baseline (w).

22. r=/s r= ( - 0) / (s - 0)
Peak height h or h’ (from a point of crossing of tangents with a zero line).
Peak width µ0,5 - distance between peak points on half of height (or on any other mark on height).
Relative retention time r and relative adjusted time tr is calculated on equation:
r=/s r= ( - 0) / (s - 0)
!!! Relative retention times:
Less depend on external conditions, than retention time;
Resolve the serial analysis without standard samples of defined substances.

23. Often retention time isn’t measured, but measure retention distance l - distance on chromatogram from a point which corresponds of introduction sample moment to an absciss which correspond of peak maximum.
The retention volume it depends on speed  of mobile phase movements
V =   
The retention factor (delay) R is a relation of moving speed w of the given component and the speed u of mobile phase movements:
R =  / or R = 0 /
The capacity factor k - is equal to ratio of relative retention time =  - 0 of given components to 0:
k = ( - 0) / 0
The more the capacity factor k, than more time of investigated component in stationary phase.

24. Martin and Synge theory (theoretical plate)
At the beginning of a chromatographic separation the solute occupies a narrow band of finite width. As the solute passes through the column, the width of its band continually increases in a process called band broadening. Column efficiency provides a quantitative measure of the extent of band broadening.
In their original theoretical model of chromatography, Martin and Synge treated the chromatographic column as though it consists of discrete sections at which partitioning of the solute between the stationary and mobile phases occurs. They called each section a theoretical plate and defined column efficiency in terms of the number of theoretical plates, N, or the height of a theoretical plate, H

25. !!! A column’s efficiency improves with an increase in the number of theoretical plates or a decrease in the height of a theoretical plate.
The number of theoretical plates in a chromatographic column is obtained by combining equations:
Alternatively, the number of theoretical plates can be approximated as
where w1/2 is the width of the chromatographic peak at half its height.

26. Rate theory (kinetic theory)

28. Van Deemter equation is an equation showing the effect of the mobile phase’s flow rate on the height of a theoretical plate.
where:
A = Eddy-diffusion
B = Longitudinal diffusion
C = mass transfer kinetics of the analyte between mobile and stationary phase
u = Linear Velocity.

29. The constant A depends on the size of particles, their density or density of column filling. The constant B is connected with diffusion factor of molecules in a mobile phase. The constant C characterises kinetics of sorption - desorption process, material transfers and other effects.
There is some disagreement on the correct equation for describing the relationship between plate height and mobile-phase velocity.

30. At small mobile-phase velocity the height equivalent to a theoretical plate (HETP) decreases, and then starts to increase.
The optimum mobile-phase velocity corresponds to a minimum in a plot of H as a function of u.
Optimum velocity of division which gives us a considerable quantity of theoretical plates, and accordingly small HETP, is calculate:
So, the kinetic theory gives a basis for optimisation of chromatographic process.

31. The goal of chromatography is to separate a sample into a series of chromatographic peaks, each representing a single component of the sample. Resolution is a quantitative measure of the degree of separation between two chromatographic peaks, 1 and 2, and is defined as or
For two peaks of equal size, a resolution of 1.5 corresponds to an overlap in area of only 0.13%. Because resolution is a quantitative measure of a separation’s success, it provides a useful way to determine if a change in experimental conditions leads to a better separation.

32. Three examples of chromatographic resolution.
If peaks are mutually bridging, definition of peak width of each substance is impossible on separation degree  :

33. In chromatography: the qualitative analysis is based on definition of retention time and quantitative - on definition of peak height or area.

34. 4. The main parts of chromatographic equipment.
A gas chromatograph with a headspace sampler

47. Requirements to adsorbent (Al2O3, silicagels, the activated coal, porous capillaries on the basis of styrene, divinyl benzene, synthetic zeolites):
Necessary selectivity;
Chemical inertness to mix components;
Availability.

51. To group of differential detectors belong:
Differential (concentration change appear instant); are often applied
Detectors
Integrated (fix concentration change for whole time interval), are applied not often
To group of differential detectors belong:
Thermal Conductivity Detector (TCD)
flame ionization detector
electron capture detector (ECD)
Others depending on properties of system, a modular condition of phases.

59. 5. Gas chromatography.

60. Gas-liquid chromatography (GLC), or simply gas chromatography (GC), is a common type of chromatography used in organic chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound.

61. The moving phase (or "mobile phase") is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen.
The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column.
The gaseous compounds being analyzed interact with the walls of the column, which is coated with different stationary phases. This causes each compound to elute at a different time, known as the retention time of the compound. The comparison of retention times is what gives GC its analytical usefulness.

62. Qualitative analysis:
Generally chromatographic data is presented as a graph of detector response (y-axis) against retention time (x-axis), which is called a chromatogram. This provides a spectrum of peaks for a sample representing the analytes present in a sample eluting from the column at different times. Retention time can be used to identify analytes if the method conditions are constant. Also, the pattern of peaks will be constant for a sample under constant conditions and can identify complex mixtures of analytes. In most modern applications however the GC is connected to a mass spectrometer or similar detector that is capable of identifying the analytes represented by the peaks.

63. Pharmacopoeia offers three methods for identification:
1) comparison of retention times of analyzed substance in the investigated sample and in a comparison solution (a standard solution of investigated substance);
2) comparison of relative retention times of analyzed substance in the investigated sample and a comparison solution (if precision of condition chromotographic analyses isn’t possible);
3) comparison of chromatogram of investigated sample with chromatogram of comparison solution or with chromatogram, resulted in separate article (for preparations of herbal and animal origin).

64. Separation process is based on differences in fugitiveness and solubilities separated components.
The quantitative analysis can be made only in the event that the substance is heat-resistant, that it evaporates reproducibility and eluateed from column without decomposition. At substance decomposition on chromatogram are artificial peaks which belong to decomposition products.

65. Quantitive analysis:
The area under a peak is proportional to the amount of analyte present in the chromatogram. By calculating the area of the peak using the mathematical function of integration, the concentration of an analyte in the original sample can be determined. Concentration can be calculated using a calibration curve created by finding the response for a series of concentrations of analyte, or by determining the relative response factor of an analyte. The relative response factor is the expected ratio of an analyte to an internal standard (or external standard) and is calculated by finding the response of a known amount of analyte and a constant amount of internal standard (a chemical added to the sample at a constant concentration, with a distinct retention time to the analyte).

66. Symmetry factor of peak: 0.05/2А,
Pharmacopoeia demands to make definition of the quantitative contents through the areas of peaks, if symmetry factor of peak from 0.8 to 1.20 it is possible to apply in place of the areas by peak heights.
In case of usage of temperature programming the quantitative analysis is made only through the peak areas.
Symmetry factor of peak: 0.05/2А,
where:  width to peak on 1/20 of peak height;
A - distance between a perpendicular lowered from peak maximum and forward border of peak on 1/20 peak height.

67. Methods of quantitive analysis
1. Normalization method.
Accept the sum of all peak parametres (h, or S, or  width of all peaks) for 100 %. Then the ration of height of individual peak to sum of heights or the ration of one peak area to sum of the areas and multiply on 100 will characterise W(component) (%) in a mix.
This method is used when it is identical dependence of value of the measured parametre from concentration of all components of a mix.

68. 2. Normalization method with calibration factors
In this method accept the sum of all peak parametres for 100 % with due regard for sensitivity of the detector. Differences in sensitivity of the detector are considered with the aid of correction factors for each component. One of dominating components of a mix consider comparative and correction factor for it accept equal to one. Calibration factor Кі is calculated:
Сi - concentration of i-component in a modelling mix with standard substance;
Сstan - concentration of standard substance;
Пstan and Пi - parametres of substance peaks - standard and i-component.

69. For 100 % accept the sum of corrected parametres КіПі and result of the analysis is calculated such as in normalization method:
Advantage: 1) metered flow of sample isn’t necessary;
2) not absolute identity of analysis conditions at repeated definitions.

70. 3. A method of absolute calibration (the most exact).
Experimentally define dependence of peak height or area from substance concentration and plot calibration chart. On calibration chart calculate concentration of analyzed substance.
It is the basic method of definition of impurity.
Advantage: does not demand separation of all components on sample, but only that substance are necessary for defining.

71. 4. A method of the internal standard.
Is based on introduction in an analyzed mix of precisely known quantity of standard substance. As standard substance choose the substance similar on physical and chemical properties to components in mix, but not necessarily it is component of a mix.
To an investigated solution and a standard solution of investigated substance add strictly identical quantity of the internal standard.
After chromatography measure peak parametres of an analyzed component and the internal standard on chromatogram of investigated solution and the same parametres on chromatogram of standard solution of defined substance.

72. Advantage: 1) the account of chromatograph duty (t°,  mobile phase).
2) increase of analysis accuracy because of independence of reproducibility parallel chromatographic experiment series and chromatography of standard and investigated solutions.

73. Requirements to the internal standard:
Good solubility in sample and chemical inertness to components of an analyzed mix, stationary phase and the firm carrier.
The internal standard choose from compounds which similar to objects of the analysis on structure and fugitiveness.
Quantity of the internal standard in sample select so that the ration of peak areas of the standard and defined substance was close to 1.
The peak of the internal standard on chromatogram should take places affinity to peaks of compounds - objects of the analysis, not being imposed neither on them, nor on peaks of other substances.
The internal standard should not contain impurity which are imposed with peaks of defined substances-components of sample.
If define in sample two and more substances (which considerably differ retention times) so it is expedient to use 2 and more internal standards.

74. Application in the pharmaceutical analysis, technology and the toxicological analysis:
1. Quality assurance of substances and medicinal forms - identification and quantitative definition of the flying residual solvents, which may be in drugs after their reception.
2. Definition of ethanol content and other organic solvents in ready drugs.
3. Definition of preservatives (Nipagin, sorbic acid) in children's syrups.
4. Definition of some preservatives in injection solutions
5. Quality assurance galena preparations.

75. 6. Theoretical base of liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane.
Precondition of occurrence and application:
Substances with big molar weight
Nonvolatile substances
thermally unstable substances
Ionic substanses

76. LC is based on adsorption from a solution.
Adsorptive balance between solution and adsorbent will be co-ordinated with the equation of Langmuir isotherm, in the range of the diluted solutions the isotherm is linear.
Selectivity of adsorption depends by nature forces of interaction between adsorbed substances and adsorbent.
H = 2Rr(1 – Rr)Uts,

78. !!! The choice of mobile phase is always more important, than a choice of stationary phase.
The stationary phase should retention divided substances.
The mobile phase (solvent or more often mix of solvents) should provide different capacity of a column and effective separation in optimum time.
The stationary phase is porous finely dispersed materials with a specific surface is more 50 m2/g

79. Stationary phase
Polar: SiO2, Al2O3, MexOy, and also with graft polar groups (–NH2, - ОН, diols)
Non-polar: dag, kieselguhr, diatomite.

80. Polar stationary phases is used for separation:
non-polar, low-polar and mean polar substance
Non-polar stationary phases is used for separation:
they don’t have selectivity to polar molecules
These sorbents are put on superficial-porous carriers.

81. Mobile phase From it depends: Selectivity of separation;
Efficiency of column;
velocity of chromatographic zone movement.

82. Requirements to a mobile phase:
Should dissolve investigated sample;
Small viscosity (diffusion factors D of sample components are high enough);
Possible excretion from it divided components;
It must be inertness in relation to materials of all parts of chromatograph;
Meets the requirements of the chosen detector;
The safe;
The cheap.
!!! As it has been told, eluting force of a mobile phase – solvent influences on seperation.

83. Solvents (eluents) divide on weak and strong.
Eluting force of solvent shows, in how many time energy of eluent sorption more than energy of eluent sorption, chosen as the standard, for example, n-heptane.
Solvents (eluents) divide on weak and strong.
Weak eluents are a little adsorbed by stationary phase therefore distribution factors D of sorbate is high.
Strong eluents are strongly adsorbed by stationary phase, therefore distribution factors D of sorbed substances (sorbate) is low.

84. Force of solvents increases in range:
For example: SiO2 – stationary phase.
Force of solvents increases in range:
pentane (0)  CCl4 (0,11)  C6H6 (0,25)  CHCl3 (0,26)  CH2Cl2 (0,32)  acetone (0,47)  dioxane (0,49)  acetonitrile (0,5) ethanol  methanol.

85. In liquid adsorptive chromatography eluotropic Snyder's series:
Sequence of solvents according to their increase eluting forces is named eluotropic series.
In liquid adsorptive chromatography eluotropic Snyder's series:
pentane (0)  n-hexane (0,01)  cyclohexane (0,04)  CCl4 (0,18)  benzene (0,32)  CHCl3 (0,38)  acetone (0,51), ethanol (0,88)  water, СН3СООН (very big).

86. Eluotropic series for reverse phase chromatography on С18:
methanol (1,0)  acetonitrile (3,1),
ethanol (3,1)  isopropanole (8,3) 
n-propanole (10,1)  dioxane (11,7).

87. Methods of elution
When a separation uses a single mobile phase of fixed composition it is called an isocratic elution. It is often difficult, however, to find a single mobile-phase composition that is suitable for all solutes.
gradient elution is the process of changing the mobile phase’s solvent strength to enhance the separation of both early and late eluting solutes.

90. Rules of thumb help us at choice of eluent
Rules of thumb help us at choice of eluent. Sorption increases at increase number of olefinic linkage and ОН-groups in compounds.
Sorption decrease (for organic compound): acids  alcohols  aldehydes  ketones  esters  unsaturated hydrocarbon  saturated hydrocarbon.

91. 7. HPLC – high-performance liquid chromatography

93. Liquid chromatograph of firm
Manufacturers:
Agilent Technologies

94. In HPLC both contacting phases – stationary (SP) and mobile (MP) phase are liquids.
The method distributive or a liquid-liquid chromatography is based on substance distribution between two phases which do not mix.
Separation of components is based on differences of their distribution factors between SP and MP.

95. Normal phase partition chromatography
stationary phase: always polar solvent (water, alcohol) is fixed on firm carrier – silica gel, diatomite, cellulose, Al2O3.
mobile phase: non-polar solvent – isooctane, benzene , etc.

96. Reverse phase partition chromatography
Stationary phase: non-polar solvent is fixed on firm carrier
Mobile phase: polar solvent (water, alcohol, buffer solutions, strong acids)

99. Stationary phase in normal phase chromatography
In liquid–liquid chromatography the stationary phase is a liquid film coated on a packing material consisting of 3–10 µm porous silica particles. The stationary phase may be partially soluble in the mobile phase, causing it to “bleed” from the column over time. To prevent this loss of stationary phase, it is covalently bound to the silica particles. Bonded stationary phases are attached by reacting the silica particles with an organochlorosilane of the general form Si(CH3)2RCl, where R is an alkyl or substituted alkyl group.

100. To prevent unwanted interactions between the solutes and any unreacted –SiOH groups, the silica frequently is “capped” by reacting it with Si(CH3)3Cl; such columns are designated as end-capped.
The properties of a stationary phase are determined by the nature of the organosilane’s alkyl group. If R is a polar functional group, then the stationary phase will be polar. Examples of polar stationary phases include those for which R contains a cyano (–C2H4CN), diol (–C3H6OCH2CHOHCH2OH), or amino (–C3H6NH2) functional group. Since the stationary phase is polar, the mobile phase is a nonpolar or moderately polar solvent.

101. In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chain. Most reversephase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less

103. display status of computer

104. status of pump, injector on display of chromatograph

105. Status of photodiode array detector on display of chromatograph
(UV - lamp)

106. Location of samples in autosampler

107. autosampler

109. Instead, the sample is introduced using a loop injector
Instead, the sample is introduced using a loop injector. Sampling loops are interchangeable and available with volumes ranging from 0.5 µL to 2 µ L.
Sample introduction by microsyringe

114. Types of detector:
Refractive index – generalpurpose detector (sensitivity ~ 10–6 g);
UV/Vis-detector (sensitivity 10–9 g) (254 nm);
photometric і spectrophotometric;
Photodiode array;
Fluorescent detector for definition toxines, microbiological objects, vitamins;
Conductometric (0,01 µg/mL – 100 mg/mL).

122. Chromatogram
(three component)

123. The qualitative analysis (pharmacopoeia):
Comparison of retention times of analyzed substance in the investigated sample and a comparison solution (a standard solution of the same substance) – is used more often;
Comparison of relative retention times of analyzed substance the investigated sample and a comparison solution (a standard solution of the same substance) – is applied, when is possible non-reproducible conditions of chromatography;
Comparison of chromatogram of the investigated sample with chromatogram of comparison solution or with chromatogram, resulted in separate article (for preparations of plant and animal material).

124. The quantitative analysis (pharmacopoeia) for main component:
For quantitative definition to define the peak areas or peak height (pharmacopoeia):
Heights are considered only at isocratic elution and at factor of asymmetry
Peak areas are considered only at gradient elution.
The quantitative analysis (pharmacopoeia) for main component:
Absolute calibration
Method of the internal standard.

125. The quantitative analysis (pharmacopoeia) of impurities:
Quantitative definition of impurity with usage of comparison solution (known concentration of impurity);
Method of internal normalisation;
Comparison with the diluted solution of the main substance;
Method of standard additives (for accuracy increase use of a method of the internal standard is possible).

126. At a technique necessarily mark:
Column parametres (the sizes, a sorbent, commercial mark, the size of particles of stationary phase);
Column temperature;
Speed and structure of mobile phase;
Detector type.

127. 8. TLC – thin-layer chromatography
Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with the a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.

128. A small spot of solution containing the sample is applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a sealed container. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent.

129. the Rf value , or Retention factor, of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front).
or relative Retention factor:

130. Factors which influence on value Rf:
Quality and activity of sorbent;
Sorbent moisture;
Thickness of a layer of a sorbent;
Quality of solvent

131. The number of theoretical plates n
lb – distance from start line to a stain bottom edge;
ll – distance which shows length of a stain in direction of movement of solvent front.
the height of a theoretical plate
separation factor

132. Sorbents in ТLС:
Silica gel;
Aluminium oxide;
Starch;
Cellulose and some other substances with high adsorptive ability
TLC:
Ascending Chromatography
Descending Chromatography
Circular Chromatography

133. Substrates for a sorbent (plate):
Glass;
Aluminium foil;
Polyester film.
The choice of solvents depends from:
The sorbent nature;
Properties of investigated substances.

134. Zones on chromatogram are displayed physical or chemical methods after termination of carrying out chromatography.
Physical method – UV-light or radio autographies (photographic materials – a paper or films).
Chemical method – chromatogram are displayed by reactant solution spraying

135. Equipment for ТLС:
A sealed container with suitable solvents;
Chromatographic plates (Merck, Silufol, Sorbfil and others);
Micro syringes, TLC Manual Sampler or Micropipettes;
TLC Viewing Box 254/366 nm;
TLC SPRAYER (ATOMIZER) and TLC Spray Cabinet;
TLC Plate Heater;

136. A sealed container with suitable solvents
The chamber purpose is to carry out development of the chromatogram. The chambers are made of an aggressive reagent resistant glass of two sizes: for 10 x 10 cm plates and for 15 x 15 cm plates. Smaller chambers are provided with dividing ledge, located on the bottom of the chamber.
The chambers are equipped with glass lids.

137. Chromatographic plates (Merck, Silufol, Sorbfil and others)

138. Micro syringes, TLC Manual Sampler or Micropipettes
The Micro Syringes are intended for application of samples on a plate.
Capacity - 10 µl.
For the sake of qualitative sample application a needle tip is cut under 90°.
The micropipettes are intended for liquid dozing during the process of preparing different working solutions. Volume of dozed liquid is up to 0,1 ml.

139. The MANUAL SAMPLER is used for dosage sampling onto a TLC plate in form of spots and without damage to the layer. Manual sampler can be used together with the Plate Heater.

140. TLC Viewing Box 254/366 nm
The TLC VIEWING BOX is intended for detecting analyzed colorless but UV active substances on TLC plates (for example, medicines, pesticides and some forms of toxins) by either their fluorescence induced by UV light at the wavelength of 366 nm, or their absorbing at the wavelength of 254 nm, provided the TLC layer contains a fluorescent indicator F254.
The Viewing Box is suitable for inspecting thin-layer chromatograms in an undarkened room.

141. TLC Viewing Box

142. TLC SPRAYER (ATOMIZER) and TLC Spray Cabinet
The sprayer is intended for transfer a liquid reagents required for derivatization onto a TLC plate. This all glass sprayer combined in one casing both injection system and the reagent bottle. The spray is fixed on the PVC pump.
The SPRAY CABINET ensures safe spraying on TLC Plate. While spraying the fixed on a stand plate should be placed inside the spray cabinet. The cabinet is connected by the pipe with exhaust ventilation. The cabinet is made of chemical-proof material which is unaffected by aggressive reagents.

143. TLC Plate Heater
The PLATE HEATER is intended for heating to a given temperature a TLC plate at different analysis stages. Heating at the stage of application samples and standards provides compact spots and correspondingly, increases plate effectiveness. The device is provided with a removable ruler with 5 mm divisions for accurate application of samples.
The temperature of 200x100 mm heating area is selectable between 35 and 125°Ñ and is uniformly maintained over the entire heating surface. Programmed and actual temperature are digitally displayed.

144. Separation of black ink on a TLC plate
Chromatogram of 10 essential oils coloured with vanillin reagent.

145. The qualitative analysis in ТСХ at identification (pharmacopoeia):
Comparison stain of investigated substance and standard substance. They must be identical on colouring (colour of fluorescence), retention factor and size on chromatogram.
!!! Check of operability of TLC-PLATES: the standard mix of indicators – sudan red G, bromcresol green, methyl red and methyl orange.
suitable solvents: methanol – toluene (20:80).
When the front of solvents will pass ⅔ of plate length, it is considered suitable if on chromatogram 4 divided stains are accurate:
Stain of bromcresol green with Rf is no more 0,15;
Stain of methyl orange with Rf from 0,1 to 0.25;
Stain of methyl red with Rf from 0,35 to 0,55;
Stain of sudan red G with Rf from 0,75 to 0.98.

146. Quantitative analysis:
Direct definition on a plate by means of measurement transmission of light or reflexions of light, fluorescence.
Directly on a plate or on the separate cut strips of plates by means of radio-activity counters.
After removal of stationary phase, its dissolution in corresponding solvent and measurements of a suitable physical indicator or a radio-activity of the received solution.

147. Thin layer chromatography finds many applications, including:
assaying the radiochemical purity of radiopharmaceuticals
determination of the pigments a plant contains
detection of pesticides or insecticides in food
analysing the dye composition of fibers in forensics, or
identifying compounds present in a given substance
monitoring organic reactions.

148. 9. Paper chromatography
Paper chromatography is Chemestrial technique for separating and identifying mixtures that is or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.

149. The paper is then dipped into a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper, also this can be explained as differential adsorption of the solute components into the solvent. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper.

150. Technique of paper Chromatography:
Ascending Chromatography
Descending Chromatography
Circular Chromatography
The qualitative analysis – as in TLC.
The quantitative analysis:
Visual estimation – comparison of colouring intensity of stains;
Estimation of stain areas;
Cutting of stains and their weighing of investigated sample and a standard solution;
Eluating of substances from stains and the next definition by a physical and chemical method.

151. 10. Ion-exchange chromatography
Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes.
It is often used in protein purification, water analysis, and quality control.

152. Ion exchange chromatography retains analyte molecules based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase.
Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group:

153. Anion exchange chromatography retains anions using positively charged functional group:
Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position and thus retention time.
The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.

154. Thanks for your attention!