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Key words:
Separation techniques, compounds and their physicochemical properties (molecular volume/size, polarity, molecular interactions), mobile phase, stationary phase, liquid chromatography, thin layer chromatography, column chromatography, retardation factor, elution, chromatogram development, qualitative and quantitative analysis with chromatography techniques, eluotropic series, elution strength.
Literature:
D.A. Skoog, F.J. Holler, T.A. Nieman: Principles of Instrumental Analysis; 637 - 718
Search on www pages “Thin-layer chromatography principles” For example: MIT Digital Lab Techniques Manual you find on http://www.youtube.com/watch?v=e99nsCAsJrw&feature=player_detailpage Basic equipment for modern thin layer chromatography: www.camag.com/downloads/free/brochures/CAMAG-basic-equipment-08.pdf other examples: en.wikipedia.org/wiki/Thin_layer_chromatography www.chemguide.co.uk/analysis/chromatography/thinlayer.html www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/th in_layer_chrom.html
Theoretical background
Chromatography is the separation technique in which separated solutes are distributed between two phases: stationary and mobile. The first phase can pose a layer of sorbent/adsorbent (0.1 to 0.25 mm in thickness) fixed to a carrier plate made of glass, plastic or aluminum (used in technique named as thin-layer chromatography, TLC) or placed inside of a steel tube as a column bed (used in a technique named as high-performance liquid chromatography, HPLC, or generally in column liquid chromatography, LC). The second phase, mentioned above, constitute liquid or gas phase. Various organic (e.g. methanol, hexane, acetone) and inorganic (e.g. water) solvents or their mixtures (e.g. acetone and
hexane, methanol and water) can be used as the mobile phases. So each chromatographic system consists of: a) stationary phase, b) mobile phase, c) mixture of components to be separated. A solution of the component mixture is usually introduced into the chromatographic system by injection (in HPLC or classical column chromatography in entrance to the column) or by spotting/application onto start line (in TLC). In column chromatography the mobile phase is pumped through the adsorbent bed or its flow is caused by gravitation as it is demonstrated in Fig 1A. In thin layer chromatography mobile phase is driven into movement by capillary forces (solvent wets adsorbent layer on the chromatographic plate by capillary forces) as it is demonstrated in Fig 1B. Under such circumstances mixture components migrate along the stationary phase (adsorbent) according to the direction of flow of the mobile phase.
Fig. 1. (A) Classical column chromatography, (B) chromatogram development in conventional chamber (in cuboid vessel)
Migration velocities of mixture components are slower from that o the mobile phase. It is because of time, which separated molecules spend in the stationary phase. Arrangement of solute zones on the chromatographic plate after chromatogram development is demonstrated in Fig. 2.
Mobile phase Station ary phase
Valve
Mobile phase
Chromatographic plate Chromatographic chamber
A B
Fig.3. A simplified model of silica gel surface
There are also silica based adsorbents, which are non-polar, i.e. chemically modified silica. Modified silica gel is formed by chemical reaction of its surface with e.g. trichlorooctadecylsilane or other reagents. Thus the surface polarity decreases and then its hydrophobicity increases.
2. Aluminum oxide Aluminum oxide (Al 2 O 3 ) is another adsorbent, which is often used as stationary phase in laboratory practice. TLC aluminum oxide plates usually comprise neutral or basic aluminum oxide. These kinds of plates provide distinct separation features with regard to a pH range of the mobile phase used. Under aqueous conditions basic compounds can be well separated with basic aluminum oxide plates, while neutral compounds can be successfully separated with neutral aluminum oxide ones. 3. Cellulose Cellulose is the next adsorbent used as a stationary phase in chromatography systems, especially in TLC. Macromolecules consisting of D-glucose units coupled -glycosidically at positions 1 and 4 by oxygen atoms stand for this adsorbent. A section of a cellulose chain is shown in Fig. 4.
Fig. 4. Fragment of cellulose macromolecule
There are two kinds of cellulose layers used in TLC, native cellulose (400 -500 units per chain) and micro-crystalline cellulose that is prepared by the partial hydrolysis of regenerated cellulose and comprises between 40 and 200 units per chain. Similarly to the silica gel, cellulose surface can be modified by esterification (e.g. acetylation).
Table.1. TLC stationary phases (adsorbents), mechanism of separation and examples of compounds separated with TLC
Stationary Phase
Chromatographic Mechanism Typical Application
Silica Gel Adsorption (^) lipids, aflaxtoxin, bile acids, vitamins, alkaloidssteroids, amino acids, alcohols, hydrocarbons,
Silica Gel RP reversed phase fatty acids, vitamins, steroids, hormones,carotenoids
Cellulose, kieselguhr partition^
carbohydrates, sugars, alcohols, amino acids, carboxylic acids, fatty acids Aluminum oxide
adsorption amines, alcohols, steroids, lipids, aflatoxins, bile acids, vitamins, alkaloids
Solvents
As it has been mentioned above, the choice of the mobile phase for chromatographic separation is dependent on interactions between mixture components in question with stationary phase. If polar interactions are involved in this process then solvents of dispersive character to molecular interaction (like hexane) in mixture with polar ones (e.g. ethyl acetate) are chosen as mobile phase solution. Analogously, if dispersive interactions predominate between adsorbent surface and solutes then solvents of polar properties (methanol or acetonitrile) in mixture with water are preferred.
Eluotropic series of solvents Solvents are arranged in a series according to increase of their elution strength in a chromatographic system with given stationary phase. Each adsorbent (stationary phase) possess its own eluotropic series of solvents.
Mechanisms of chromatographic separation Several mechanisms are involved in solute separation in chromatographic system. The most often applied mechanisms of chromatographic separation are presented in Fig. 5.
Fig. 5. The mechanisms of chromatographic solute separation often applied in laboratory practice
Adsorption mechanism of chromatographic separation is very often used for solute separation. Migration of solute in chromatographic system in which adsorption mechanism is involved depends on:
- molecular interactions of solute with stationary phase,
- molecular interactions of solute with solvent (eluent, mobile phase components).
If polar solutes are strongly bonded (adsorbed) to polar stationary phase then relatively polar (strong) solvent as the mobile phase has to be applied to elution of such solutes. If the solvent is too “weak” then migration of the solutes is small, the solutes show short migration distances. It can be said their retention is strong. Usually under such circumstances mixture components are not well resolved.
If the mixture components are nonpolar their molecular interactions (e.g. dipole – induced dipole or/and London dispersion forces) with polar adsorbent are weak. The solutes are then weakly attracted by polar stationary phase (show weak affinity with the stationary phase), and can be easily eluted from the chromatographic system. It can be said their retention is small. Generally speaking, if stationary phase is more polar than mobile phase then chromatographic system is named as normal phase system. Analogously, if mobile phase is more polar than stationary phase then chromatographic system is named as reversed phase system.
Possible interactions of various solute molecules with silica gel stationary phase are presented in Fig. 6.
Fig.6. Influence of various functional groups in solute molecule on its migration distance and retardation factor. The coloured, dashed lines indicate hydrogen bonds between solute molecule and silica stationary phase
increase of solute migration distances
Inrease oenhance of solute retardation factor, RF
distances of the solute zones 1, 2, 3, 4, respectively; b - the mobile phase migration distance (distance of solvent front migration)
Substance showing RF value of 0.4 spends 2/5 of the experiment (chromatogram development) time in the mobile phase and 3/5 of the experiment time in the stationary phase. The solute with RF values of 0.6 spends 3/5 of the chromatogram development time in the mobile phase and 2/5 of the chromatogram development time in the stationary phase. It means the first solute migrated shorter distance in comparison with the second one. The difference of the RF values is equal to 0.2. The solute zones on chromatographic plate migrated different distances, and then their separations is possible..
The retardation factor can be converted into retention factor, k, with the following equation
(3) This factor is a measure of retention of solutes in column chromatography systems. It expresses how many times longer a solute spends in the stationary phase in comparison to that in the mobile phase. The separation factor, α, is another chromatographic parameter. It determines separation selectivity of two solutes in a given chromatographic system. Its value can be equal to or higher than 1.0. It is calculated with the following equation:
(4) If is equal to 1.0 then two solutes cannot be separated. Then one should search another chromatographic system, which enables to obtain higher separation factor than 1.0.
Application of chromatography The main application of chromatographic processes involves:
- resolution of mixtures into their components,
- purification of substances (including technical products) from their contamination,
- determination of homogeneity of chemical substances,
- comparison of substances suspected of being identical,
- quantitative separation of one or more constituents from complex mixture
- concentration of materials from dilute solutions (plant extracts).
EXPERIMENTAL PART
HORIZONTAL DS CHAMBERS (www.chromdes.com)
In standard version (DS-II) the Horizontal DS Chamber for TLC consists of a flat PTFE plate (4) with five rectangular depressions: two containers/reservoirs (2) of eluent and a central tray with three troughs (7) and the chromatographic plate (3). The chamber is covered with a large cover plate (1).
Principle of action
Development of chromatogram is started by shifting the plates (1) to the chromatographic plate (3) which brings a narrow zone of the absorbent layer on the plate (3) into contact with the eluent from one or two sides. Fig. 8 shows the situation before chromatogram development and Fig. 9 during chromatogram development. The eluent in containers/reservoirs (2) is covered with the glass plates (1) so that a vertical meniscus of the eluent is formed. Because the bottom of the containers/reservoirs (2) is slightly slanted, the meniscus moves in the direction of the chromatographic plate (3) during the development process, to the complete absorption of the eluent by the adsorbent layer.
1 3 6 5 1
Fig. 8
4 2 8 7 8 2
1 3 6 5 1
Fig. 9
4 2 8 7 8 2
1 – cover plate of eluent reservoirs, 2 – eluent reservoirs, 3 – chromatographic plate, 4 – PTFE plate, 5
- large cover plate, 6 – cover plates of troughs, 7 – troughs for vapour saturation, 8 – eluent (blue area)
Step 5 Calculate retardation factor, RF, of investigated solutes and use them to fill Table 1. Table.1. The values of migration distance (mm) and retardation factor, RF, of solutes in systems with silica gel and different solvents, is the elution strength
Solute
Hexane = 0.
Toluene = 0.
Acetone = 0. The solvent front migration distance, start
- finish (b) Migration distance (a)
RF Migration distance (a)
RF Migration distance (a)
RF
Dye 1 Dye 2 Dye 3 Dye 4
Mixture
Formula to use: RF = a/b, RF – retardation factor Answer the questions:
Which solvent is characterized by the highest elution strength?
Arrange the eluotropic series for solvents/eluents used.
What components comprise the investigated sample mixture?
Step 6
Apply the data from Table 1 for calculation of the data in Table 2.
Table 2. The values of separation factor, α, of solutes chromatographed in systems with silica gel and different solvents, is the elution strength
Solute
Hexane = 0.
Toluene = 0.
Acetone = 0. Separation factor Separation factor
Separation factor Dye 1/ Dye 2 Dye 2/ Dye 3 Dye 3/ Dye 4
Formulas to use:
Place for calculations:
Answer the question:
Indicate chromatographic system, which is characterized by the highest values of separation factor?
Indicate the chromatographic system, which facilitates good separation of all investigated mixture components.
Step 7
Apply the data from Table 3 for calculation of the data in Table 4.
Table 4. Separation factor, α, values of solutes in systems with aluminum oxide and solvents/eluents specified
Formulas to use:
Solute
Hexane = 0.
Toluene = 0.
Acetone = 0. Separation factor (α)
Separation factor (α)
Separation factor (α) Dye 1/ Dye 2 Dye 2/ Dye 3 Dye 3/ Dye 4
Answer the question:
For which solvent the separation factor shows the highest values?
PART III
COMPARISON OF ELUTION STRENGTH OF SOLVENTS IN SYSTEMS WITH SILICA
AND ALUMINA
Step 8 Comparison of the results obtained for the systems with silica gel and aluminum oxide. Fill in Table 5 with appropriate data.
Table.5. The values of retardation factor, RF, obtained for the systems with silica gel and aluminum oxide
Solute
Hexane Toluene Acetone Silica gel
Aluminum oxide
Silica gel
Aluminum oxide
Silica gel
Aluminum oxide Dye 1 Dye 2 Dye 3 Dye 4
Mixture
Answer the question: Have you obtained the same results for the systems with silica gel and aluminum oxide? If not then try to explain the difference/s?
PART IV
ELUTION STRENGTH OF MIXED SOLVENTS IN SYSTEMS WITH
SILICA GEL
Step 9 Pour 2 mL portion of the eluent solution (5%, 10%, 40% v/v, acetone in hexane) into the shallow reservoir of single chromatographic chamber (one solution into one chromatographic chamber). Step 10 Put a piece of blotting paper on the chamber bottom. Step 11 Pour the solvent on the blotting paper (approximately 0,5 mL of solvent).
Answer the questions: Does composition of the mobile phase influence on migration distance of solute zone/s?
Arrange the solvent mixtures/solutions in respect of their elution strength in silica gel system
