Lab.2. Thin layer chromatography

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1 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; Search on www pages Thin-layer chromatography principles For example: MIT Digital Lab Techniques Manual you find on Basic equipment for modern thin layer chromatography: other examples: en.wikipedia.org/wiki/thin_layer_chromatography 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.

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.

2 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. Mobile phase Station ary phase A B Valve Chromatographic plate Chromatographic chamber 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.

3 Solvent front Start line Fig. 2. Thin layer chromatogram of dyes, 1 and 10 dye mixture, 2 9 single dyes The time the separated molecules spend in the stationary phase depends on their interactions with stationary and mobile phases. It means the mixture components can be separated in the chromatographic system if they demonstrate different migration distances, i.e. if they show different energy of molecular interactions with components of the chromatographic system. Following molecular interactions of solutes with elements of stationary and mobile phases can take place in any chromatographic system: hydrogen bond, dipole dipole, dipole induced dipole, ion dipole, instantaneous dipole induced dipole (London dispersion forces), ion ion. The stationary phase 1. Silica gel Silica gel is composed of silicon dioxide (silica). The silicon atoms are bonded via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel -OH groups are attached to the silicon atoms. So, on the surface of silica gel Si-O-H groups are present instead of Si-O-Si ones. This makes silica surface very polar. Fig. 3 shows the model of a small part of the silica surface.

Fig.3. A simplified model of silica gel surface There are also silica based adsorbents, which are non-polar, i.e. chemically modified silica.

Thus the surface polarity decreases and then its hydrophobicity increases. 2.

TLC aluminum oxide plates usually comprise neutral or basic aluminum oxide.

Under aqueous conditions basic compounds can be well separated with basic aluminum oxide plates, while neutral compounds can be successfully

4 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

5 There are two kinds of cellulose layers used in TLC, native cellulose ( 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 Silica Gel Silica Gel RP Cellulose, kieselguhr Aluminum oxide Chromatographic Mechanism Adsorption reversed phase partition adsorption Typical Application steroids, amino acids, alcohols, hydrocarbons, lipids, aflaxtoxin, bile acids, vitamins, alkaloids fatty acids, vitamins, steroids, hormones, carotenoids carbohydrates, sugars, alcohols, amino acids, carboxylic acids, fatty acids 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.

6 The strength of solvent to elute solute molecules from the adsorbent surface (stationary phase) is characterized by polarity index (P ), which ranges from 0 (for non-polar solvent, e.g. pentane) to 10.2 (very polar one, water). When the mobile phase is a mixture of two solvents A and B then its polarity index, P AB, is calculated according the following formula: P AB = φ A P A + φ B P B (1) Where P A and P B are the polarity indexes of pure solvents A and B, respectively, and φ A and φ B are the molar fractions of A or B in the mobile phase, respectively. The polarity of a solvent can be evaluated by examining its dielectric constant (ε), dipole moment (δ) and ability to hydrogen bond formation. Table.2. Properties of solvents applied in liquid chromatography Solvent Dielectric constant Dipole moment Ability to hydrogen Polarity (P ) Elution strength [D] bond Alumina Silica formation hexene not form toluene not form chloroform H-donor dichloromethane H-donor tetrahydrofuran H-acceptor ethyl acetate H-acceptor acetone H-acceptor acetonitrile H-acceptor propanol H-acceptor/ H-donor ethanol H-acceptor/ H-donor methanol H-acceptor/ H-donor Source Wikipedia

7 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: 1. molecular interactions of solute with stationary phase, 2. molecular interactions of solute with solvent (eluent, mobile phase components).

8 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. increase of solute migration distances Inrease enhance o of solute retardation factor, R F 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

9 A shape of the separated molecule also influences on its bonding with the stationary phase surface. Flat molecules can be more strongly retained by the adsorbent surface than branched ones. The solute molecules with multiple polar groups are in position to more strongly interact with the surface of polar stationary phase than the solute molecule with lower number of polar groups. However, due to steric hindrance all polar groups of the solute molecule cannot take part in molecular interactions with the adsorbent surface. In such case prediction of solute retention is more complicated. In adsorption chromatography solute elution is based on displacement of its molecules from stationary phase surface by solvent molecules. It is because solvent molecules show ability to interacts with the stationary phase. It means in any chromatographic system adsorption of the solute molecules is not permanent state. Affinity of solute with the mobile phase components (such as solubility) also influences on its retention. Stronger molecular interactions of the solutes and mobile phase components lead to decrease of solute retention, the solutes are then easily eluted from any chromatographic system (TLC and HPLC). Retention and separation parameters Retardation factor, R F, is a characteristic parameter of investigated solute/s in a given chromatographic system. It corresponds to relative migration of solute/s in comparison to solvent migration. R F values range from 0.0 to 1.0. Definition of R F is presented by the equation 2 and in Fig. 7. (2) A Solvent front B b 1 2 M Start line a1 a2 a3 a4 Fig. 7. (A) Solutes applied on the start line of the chromatographic plate and (B) chromatographic plate after chromatogram development; a1, a2, a3 and a4 the migration

10 distances of the solute zones 1, 2, 3, 4, respectively; b - the mobile phase migration distance (distance of solvent front migration) Substance showing R F 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 R F 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 R F 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: 1. resolution of mixtures into their components, 2. purification of substances (including technical products) from their contamination, 3. determination of homogeneity of chemical substances, 4. comparison of substances suspected of being identical, 5. quantitative separation of one or more constituents from complex mixture 6. concentration of materials from dilute solutions (plant extracts).

11 EXPERIMENTAL PART HORIZONTAL DS CHAMBERS ( 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 Fig Fig 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)

12 PROCEDURE Draw slightly marked start lines (use a soft pencil!) on a 5 x 10 cm chromatographic plates (glass carrier plates with thin layer of adsorbent, ca. 0.2 mm in thickness, eg. silica gel, aluminium oxide) about 1 cm from its bottom edge (5 cm in length, see Figs. 7A and 7B). PART I ELUOTROPIC SERIES OF SOLVENTS IN SYSTEM WITH SILICA GEL Brief description: Step 1 Apply side by side about 5 μl of sample solutions [mixture + several single dye solutions] onto the star line of the chromatographic plate using spotting capillary tubes. Fill the capillary by dipping it in the dye solution then gently touch the tip of the capillary tube to the adsorbent layer and make the spot (the smaller the spot the better results). Clean the capillary tube with acetone. Repeat the application procedure with the remaining solutes investigated. Remember that each solute requires clean capillary tube for sample application. NOTE: The spots applied should be placed on start line, which is 1.0 cm apart from lower edge of the chromatographic plate (see Figures 5 A, B), and the neighbouring spots on the start line should be approximately 1 cm apart. Step 2 Add 2 ml of solvent (hexane, acetone, ethyl acetate or toluene) to the reservoirs of the chromatographic horizontal DS chambers (one solvent to one chamber). Then insert the chromatographic plate with spots applied on it into the chromatographic chamber. Start to develop chromatograms in each chamber. Step 3 When the solvent front approaches to the end (finish line) of the chromatographic plate, then remove the wet plate from the chamber. Place the plate in a laboratory hood to complete evaporation of solvent. Step 4 Measure distances travelled by the solute zones from the start line (origin) to the middle of the spot for all compounds. Record the obtained data in Table 1.

13 Step 5 Calculate retardation factor, R F, of investigated solutes and use them to fill Table 1. Table.1. The values of migration distance (mm) and retardation factor, R F, of solutes in systems with silica gel and different solvents, is the elution strength Solute Hexane = 0.00 Toluene = 0.22 Acetone = 0.56 The solvent front migration distance, start finish (b) Migration R F Migration R F Migration R F distance distance distance (a) (a) (a) Dye 1 Dye 2 Dye 3 Dye 4 Mixture Formula to use: R F = a/b, R F retardation factor Answer the questions: Which solvent is characterized by the highest elution strength? Arrange the eluotropic series for solvents/eluents used.

14 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 = 0.00 Toluene = 0.22 Acetone = 0.53 Separation factor Separation Separation factor 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.

15 PART II. ELUOTROPIC SERIES OF SOLVENTS IN TLC SYSTEMS WITH ALUMINUM OXIDE Use the procedure from PART I, steps 1-6, for aluminum oxide plates. Table.3. Migration distance (mm) and retardation factor, R F, values of solutes in systems with aluminum oxide and different solvents; is the elution strength Solute Hexane = 0.00 Toluene = 0.29 Acetone = 0.56 The distance of solvent front migration, start finish (b) Migration R F Migration R F Migration R F distance distance distance (a) (a) (a) Dye 1 Dye 2 Dye 3 Dye 4 Mixture Answer the questions: Which solvent has the highest elution strength? Arrange the eluotropic series of solvents for chromatographic systems with aluminum oxide.

16 Step 7 Apply the data from Table 3 for calculation of the data in Table 4. Table 4. Separation factor, α, values solvents/eluents specified of solutes in systems with aluminum oxide and Formulas to use: Solute Hexane = 0.00 Toluene = 0.29 Acetone = 0.56 Separation factor Separation factor Separation (α) (α) 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.

17 Table.5. The values of retardation factor, R F, obtained for the systems with silica gel and aluminum oxide Hexane Toluene Acetone Solute Silica Aluminum Silica Aluminum Silica Aluminum gel oxide gel oxide gel 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).

18 Step 12 Insert the chromatographic plate with applied samples (spots) into the chromatographic chamber. The adsorbent layer should be placed face down in the chromatographic chamber. Cover the chromatographic chamber with the glass cover plate. Step 13 Equilibrate chamber atmosphere with solvent vapours for 15 min. Step 14 Start chromatogram development. When the solvent front reaches the finish line, remove the wet chromatographic plate from the chamber. Place the plate in a laboratory hood, to dry the adsorbent layer of the chromatographic plate. Step 15 Measure the migration distances of solute zones (distance from the start/origin to the middle of solute zone for all compounds) and record the obtained values in Table 6. Step 16 Calculate retardation factor, R F, values of the solutes. Table 6. Migration distance (mm) and retardation factor values of investigated solutes in the systems with silica gel and acetone + hexane Eluent 5% acetone in hexane 10 % acetone in hexane 40% acetone in hexane The migration distance of solvent front (start finish) (b) Migration R F Migration R F Migration R F distance (a) distance distance (a) (a) Dye 1 Dye 2 Dye 3 Dye 4 Mixture

19 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

Experiment 1: Thin Layer Chromatography

Experiment 1: Thin Layer Chromatography Part A: understanding R f values Part B: R f values & solvent polarity Part C: R f values & compound functionality Part D: identification of commercial food dye