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Experiment 9 Thin-Layer Chromatography

By Yi Jun-Lau Feb 04, 2015 3875 Words
Title: Thin-Layer Chromatography
Objectives:
Part1: 1. To learn the technique of TLC and the visualization of colourless components. 2. To identify an unknown drug by a TLC comparison with standard compounds. Part 2: To learn the separation technique by using Thin Layer Chromatography plate in separating a mixture of compounds into individual pure compound by using Spinach Leaf. Introduction:

(i) General Concepts
Chromatography is a common and powerful method used to separate and analyze complex mixtures. Using this technique, the course of a reaction can be followed, and the products can be separated and isolated. In this method, the components within the mixture are distributed between two phases: a stationary phase and a mobile phase which moves through the stationary. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partioned. The material to be separated is place onto the stationary phase and is then carried along by the mobile phase. The components of the mixture are absorbed by the stationary phase to different degrees and it is thus the various rates of migration for each component on the adsorption the slower the material passes through the system. The types of chromatography is divided into few types which include gas chromatography(GC), high performance liquid chromatography(HPLC), thin layer chromatography(TLC), and column chromatography(CC). However, only TLC and CC are applied in this experiment. (ii) Principles of Chromatography

The separation of the components of a mixture depends on the phase each component remains in and the rate at which each travels. The stationary phase does not move, while the other mobile phase travels past the fixed phase. Due to various functional groups, each compounds travel past the stationary phase at a different speed. The strength of the following types of interactions: ion-dipole, and van der Waals forces. Different types of chromatography use various types of stationary and mobile phases. In this experiment, the solid phase is silica gel while the mobile phase is an organic solvent (may be a single type or a mixture of solvents). This means that the compounds to be separated must choose between being absorbed to the solid silica gel or moving along in the organic solvent. The silica gel is either pack into a column or adhered to a sheet of glass, plastic or aluminium, depending on the type of chromatography.

When a column is used, the compound mixture is placed on top and the solvents are run down the column separating the mixture along the way. With the silica gel on a plate, the compounds are placed close to the bottom and the mixture is separated as the solvent travels up by means of capillary action. Since silica gel is a porous form of SiO2, the surface of gel contains Si-OH and Si-O-Si functional groups. With silica gel, the dominant interactive force between the adsorbent and the materials to be separated are of the dipole-dipole type. Highly polar molecules interact fairly strongly with the polar Si-O bonds of these adsorbent and will tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules thus generally tend to move through the adsorbent more rapidly than the polar species. Figure 1 illustrates the general column chromatography setup and Figure 2 illustrates the movement of a compound mixture in a column and their separation and isolation.

Another factor that establishes the rate at which a compound travels past silica gel is the polarity of the solvent. A polar solvent will compete for silica absorption sites, disallowing the compounds to do so. This promotes a faster rate at which all compounds travel. The order in which the compounds to do so. This promotes a faster rate at which all compounds travel. The order in which the compounds move remains the same, while moving faster as the polarity of the eluent (solvent system) increases.

(iii) Thin Layer Chromatography (TLC)

TLC involves the same principles of separation as column chromatography but the apparatus and technique for development is different. Instead of a column, the silica (or alumina) is adhered to a plate of glass, plastic or aluminum. A capillary spotter I used to apply the dissolved sample onto the plate about 1cm from the bottom (aline with pencil is drawn). Once the solvent has evaporated, only the sample remains. The plate is carefully placed into a closed developing chamber, which as a shallow layer of solvent that does not submerse the spot. The chamber is lined with a folded piece of filter paper to ensure a uniform and saturated atmosphere of solvent vapour.

The plate is removed when the solvent front has reached about 0.5cm from the top, and is quickly marked with pencil. The capillary action of the solvent causes the initial spot to be separated into individual components that may be visualized by colour identification or with the following techniques for colourless compounds: (a) Irradiation with ultraviolet light

(b) Reversible staining with iodine vapour (formation of brown spot which fade) (c) Spraying with a reagent that irreversibly colors the spots, e.g H2SO4, KMnO4

Figure 1 illustrates the general principles showing the separation of the same mixture of 3 components, A, B and C.

The rate at which a compound moves in respect to the solvent front, R f , is characteristic of that compound under standard conditions. The R f value is calculated by dividing the distance each spot has travelled (measures from the pencil line to the middle of the spot) by the distance the solvent front travelled from the pencil line. This is illustrated in Figure 2. The advantages of TLC are the very small quantities of sample required and the great ease and rapidly with which it is resolved. For these reasons, it is often used to monitor the progress of a reaction by running the crude sample beside the reaction sample on the same plate. The following are some common uses of TLC: (a) To determine the number of components in a mixture.

(b) To determine the identity of two substances
(c) To monitor the progress of a reaction.
(d) To determine the effectiveness of a purification
(e) To determine the appropriate conditions for a column chromatographic separations. (f) To monitor column chromatography

Figure 2: Illustration of a developed TLC plate and Rf calculation

(iv) Factors affecting resolution of separations
(a) Adsorbents: Weight ratio of adsorbent to sample is important to obtain accurate separation. If too much sample is applied, the active adsorbing sites will be saturated and the column will be flooded, resulting in poor separations. In most cases, a ratio of 20 to 1 is satisfactory but sometimes up to 100 to 1 is necessary. Even the ratio of column height to diameter is important; about 8 to 1 is considered optimal. (b) Solvents: The solvent (or solvent mixture) is important to the compound separation, keeping in mind that the more polar the solvents, the faster the compound move. In some cases, a solvent system may increase in polarity by gradually changing the composition of the solvent mixture.

(c) Functional group: Compounds with highly polar groups are strongly adsorbed and eluted less readily than less polar (or polarizable) compounds. The strengths of adsorption for compounds having the following types of polar functional groups decreases in the order listed below. However, variations may occur depending on the overall structure of each specific compound.

RH, R-X, alkenes, R-OCH3, R-CO2R’, RCOR’, RNH2, R-OH, R-CO2H

Increasing absorption on polar stationary phases

Experimental Procedure:
Part 1: TLC Analysis of Analgesic Drugs
In this experiment, TLC will be used to examine the composition of various analgesic (pain relieving) drugs. Among these are the aspirin and acetaminophen. Caffeine is sometimes added to these formulations to overcome drowsiness. A: Spotting the TLC plate

1. A TLC plate was obtained from instructor. The edges of the plate was held carefully. 2. The plate was set down on a clean and dry surface, then a line was drawn across the plate about 1.0 cm from the bottom of the plate by using a 2B pencil. 3. Five lines of 2-3 mm were drawn, spaced about 0.6 cm apart and running perpendicularly through the lines across the bottom of the TLC plate. 0.5 cm must be spaced from each side of the edges. 4. 5 different analgesics were spotted on each 2-3 mm lines. Firstly, acetaminophen was spotted on the plate, followed by caffeine, unknown A, aspirin and lastly the unknown B. The plate was examined under the UV light to check whether enough each solution has been applied. B: Developing the TLC plate

1. A developing chamber was prepared by using a 250 ml beaker, a half-piece of filter paper inside and aluminium foil to cover. 2. Mixture of 1:3 of ethyl acetate : hexane was poured into the beaker to the depth of about 1cm. The TLC plate was placed in the developing chamber. 3. After the solvent has risen to near the top of the plate, the plate was removed. The solvent front was marked with a pencil. The solvent was allowed to evaporate from the plate in the fumehood. Part C: Visualization

1. The colourless compounds were visualized by illumination of the plate with UV lamp. 2. The spots were outlined by using a 2B pencil. The spots may be visualized by putting the plate in an iodine chamber for a couples of minutes. Part D: Comparison of the unknown with reference standards

1. The plate was sketched in notebook and the Rf value was calculated for each spot. 2. The unknown drug was determined based on Rf value.
PART 2: Separation of Spinach Leaf Pigments by T.L.C.
A. Prepare the Developing Tank
1. 10 cm3 of the mobile phase- the mixture of 9.5 cm3 CH2Cl2 and 0.5 cm3 CH3OH was obtained from the fume hood. It was poured into a 150 cm3 Developing Tank. A 70 mm x 70 mm square of filter paper was inserted and the mobile phase was swirled to wet the paper. The beaker was covered with a watch glass. B. Make a Sample Spotter

Only one is required.
C. Extraction Procedure
1. 10 g of spinach was grinded using a mortar and pestle to which a portion of sand (2 spatulas has been added as an extra abrading agent). 20 cm3 of acetone was added and it was continued grinding until the extract is green and the spinach was completely pulped. 10 cm3 of petroleum spirit was added and it was mixed quickly using the pestle. A Pasteur pipette was used to make the transfer, about 5 cm3 of the liquid part of the mixture was transferred into the 10 cm3 glass vial. The vial was capped immediately. Two layer was formed- lower pale yellow layer and an upper intense green layer. D. Spot the Plate

1. A Merck T.L.C. plate (15 mm x 66 mm) was obtained and a dot was made with a pencil half way along the short edge, 10 mm up from the bottom. The upper green layer of spinach extract was spotted on the plate several times. 2. The stationery phase was solid called silica-gel.

E. Develop the Chromatogram
1. About 5 minutes was taken for the plate to develop. The solvent front was marked and observed under UV light. The observations under both UV and visible light conditions were recorded. It was done immediately as the spots fade as they exposed to light and air after the chromatography was completed. F. Record result

1. An accurate drawing of T.L.C. plate was made and the identity of the sample, the type of stationery phase, the mobile phase, the colour of each discernible spot, the method of visualization and the Rf value for each compound were recorded.

Results:
Distance travelled by solvent front: 8.5 cm
Type of stationary phase used: silica gel
Type of mobile phase used: eluent (mixture of 2:1 ethyl acetate : hexane) Part 1:
TLC plate

Spot
Compound
Observations
Rf value
Conclusion

Visible light
UV light (254 nm)
UV light (365 nm)
Iodine Chamber

1
Acetaminophen
No visible spot was observed.
A big tailing black spot was observed.
No visible spot was observed.
Yellowish-brown spot was observed.

-
2
Caffeine
No visible spot was observed.
A medium black spot was observed.
No visible spot was observed.
No visible spot was observed.

-
3
Unknown A
No visible spot was observed.
A medium black spot was observed.
No visible spot was observed.
Yellowish-brown spot was observed.

Unknown A is Acetaminophen.
4
Aspirin
No visible spot was observed.
A small grey spot was observed.
No visible spot was observed.
No visible spot was observed.

-
5
Unknown B
No visible spot was observed.
A small grey spot was observed.
No visible spot was observed.
No visible spot was observed.

Unknown B is Aspirin.
* Big spot: About 0.5 cm diameter
* Medium spot: About 0.3 cm diameter
* Small spot: About 0.2 cm diameter
Part 2:
Sample used: extract of spinach leaf

Spot
Observations
Rf value
Conclusion

Visible light
UV light (254 nm)
UV light (365 nm)

A
A yellow spot was observed.
A yellow spot was observed.
No visible spot was observed.

Spot A is Xanthophylls.
B
An orange spot was observed.
An orange spot was observed.
An orange-yellow spot was observed.

Spot B is Carotenes.
C
An green spot was observed.
An grey spot was observed.
An green spot was observed.

Spot C is Chlorophylls and Phaeophytins.
Discussion:
In this experiment, the eluent (solvent) is prepared by using a mixture of ratio of 3:1 hexane and ethyl acetate. The polarity of this solvent cannot be too low because the polar compounds may not be able to carry by the eluent and it will not be separate, hence it will cause the separation not observable. If the solvent of too high polarity is used, the polar compound will travel very fast that the separation between non-polar compound and polar compound became small and poor separation will be observed. The solvent mixture was believed that it has optimized solubility for these organic compounds such as acetaminophen was spotted on the plate, followed by caffeine, unknown A, aspirin and lastly the unknown B to dissolve in the solvent. In another words, the compounds can be easily to be carried by the solvent in the TLC plate. Before the plate is placed into the solvent, a half folded filter paper was dipped inside the solvent in a beaker which is covered by a piece of Para film. This is to create a system that full with the vapours of organic solvent and the solvent was allowed to travel up along the plate quickly. After the plate was introduced into the solvent, the solvent start to migrate itself and the compounds move along with solvent until the solvent front has been reached. Besides that, the “tailing” effect need to prevent excess sample is dropped in each single spotting because it will cause “tailing” effect which may cause no colour spot was observed under the visualization. To prevent this ‘tailing’ effect, we need to quickly remove the capillary tube after the first small droplet is applied to the TLC plate and wait it evaporate then only further start to applied the sample again. The UV light (365nm) is used to examine whether the sample is applied enough to the TLC plate or not. As those 5 solutions are UV active, they will show purplish-grey under the 365nm UV light. Once we observed the dark purplish-grey colour spot on the TLC plate for 5 of the spots, it was claimed that enough samples had been applied to the TLC plate. More samples should be applied to the TLC plate if the spot does not appear in dark purplish-grey. There are three components in the TLC plate which are the adsorbent, the development solvent and the organic compounds that analyzed. Silica-gel consists of a three dimensional network of thousands of alternating silicon and oxygen bonds. It is a very polar and is capable of hydrogen bond due to its partial positive charge in silicon and partial negative in oxygen. The silica gel tends to bind the compounds (on stationary phase) as the development solvent tried to dissolve the compounds (on mobile phase) in order to carry the compounds along the plate as the solvent travels up. All the compounds are possible to be adsorbed into the stationary phase however the adsorption time of these organic compounds in the particular phase is depends on the polarity of each compound. The more polar the compound is, the longer the time taken that the compound adsorbed into the stationary phase so it eluting speed is slower (more time on stationary phase). Less polar compounds are weakly adsorbed, so the time taken for less polar compounds to be adsorbed on stationary phase is shorter. As a result, the less polar compounds can travel further along the plate compared to the more polar compounds. As in the iodine chamber, the colourless spot of acetaminophen solution and unknown A were turned into fade brown colour spot. After the TLC plate was dried, it was place under a UV light in order to see how the mixtures separated and to also reveal their polarity. When under the UV light the spots were marked using a pencil and were later measured to find the Rf values. By understanding the relative polarities of these organic compounds, silica-gel is more polar caused the more polar compound to be more attracted to it. Therefore, the more polar compound will moved slowly while the less polar compound will moved faster on the TLC plate. Hence, in this experiment, caffeine was the most polar compound because it contained an amine and amide groups which its Rf value was 0.1294 cm. Acetaminophen and unknown A were the second most polar compound due to alcohol and amide group which had 0.2588 cm Rf value while Aspirin and unknown B were 0.4353 cm and 0.4588 cm of Rf value respectively due to carboxylic acid and ester groups. Their relative polarity from the highest to the lowest was shown: Caffeine, Acetaminophen, Unknown A, Aspirin and Unknown B. Based on the experiment, unknown A was determined as Acetaminophen while unknown B was determined as Aspirin. This is because the Rf value of unknown A and unknown B are the same with the Rf value of acetaminophen and aspirin respectively. The results for aspirin and unknown B were slightly different may due to the sample size applied are not the same.

In the second part of the experiment, the spinach was being applied on a TLC plate. The TLC plate showed there are total four components in spinach. The Rf value of the first spot, second spot, and third spot are 0.5882, 0.9059, and 0.9059 respectively. The colours of each spot in the TLC plate have been recorded, which the compounds with Rf value of 0.5882 showed yellow colour for both visible light and UV light (254 nm) but no visible spot observed under UV light (365 nm) while the second spot showed orange colour for both visible light and UV light (254 nm) but orange-yellow spot observed under UV light (365 nm). For spot C, there was an green spot was observed under visible light and UV light (365 nm) but it showed grey spot under the short wavelength UV light. The yellow spot was predicted as the xanthophylls while the green spot is predicted as chlorophyll. Xanthophylls and chlorophylls are the pigments that usually can be found in the plant. The orange spot was predicted as the carotones and the grey spot was phaeophytins. Here is the polarity strength of compound that determine from experiment: (non-polar) Xanthophylls < Chlorophyll < Phaeophytins < Carotenes (polar) Carotenes is the most non-polar compound among those 4 compounds from the extract of spinach leaf. So, it had the highest Rf value as it abstracts weakly to silica-gel and it had the furthest distance travelled. Rf values that obtained for carotenes, it is the lowest which was 7.7 cm away from the bottom line. It was followed by the phaeophytins which position at TLC was 7.7 cm. Chlorophylls contained several polar C-O and C-N bonds and Mg2+ chelated to the N atoms. In this experiment, the distance travelled of chlorophylls was 7.7 cm. Xanthophylls contain their oxygen either as hydroxyl groups or as pairs of hydrogen atoms that are substituted by oxygen atoms acting as a bridge (epoxide). Therefore, its distance travelled was 5.0 cm. TLC plate was allowed to separate these pigments in the extract of spinach leaf. The spots of carotenes including β-carotenes and α-carotenes. For endocyclic double bond which shifted one position out of its conjugation relative to β isomer and several oxygen-containing carotene derivatives are called xanthophylls. It was occurred as yellow or orange spots on the TLC plate. Besides, the green chlorophylls a and b was seen while the grey spots for phaeophytins a and b also spotted. Phaeophytins was the chlorophylls which the Mg2+ ions was replaced by two H+ ions.

Precaution steps:
1. Students should wear gloves, lab coats and goggles all the time while conducting experiment. 2. The beaker should not be moved after the TLC plate had introduced into the beaker. 3. Do not look directly to the ultraviolet lamp.

4. Avoid to use pen on the TLC plate as its ink is a chemical that may react with the solvent. 5. Do not touch on the surface of silica gel of TLC plate by using finger. 6. The mobile phase should be disposed into the waste chemical bottle. 7. When spot the sample, it is adviced to drop by drop and allow the drop in small total surface because the big total surface will cause the “tailing” effect. Conclusions:

1. In conclusion, thin layer chromatography is a useful technique when trying to identify compounds and see how they separate a mixture of compounds into individual pure compound. 2. It is also a useful tool to see how polar or nonpolar a compound is. 3. The unknown drugs by a TLC comparison with standard compounds were determined, that is, unknown A as Acetaminophen while unknown B is Aspirin. 4. Spot A in part 2 of experiment is Xanthophylls. Spot B is Carotenes. Spot C is Chlorophylls and Phaeophytins. References:

1. Column Chromatography, 21 August 2009. Available from:
<http://www.chemguide.co.uk/analysis/chromatography/column.html> [Accessed 11 June 2013] 2. Experiment 6 — Thin-Layer Chromatography, 2009. Available from: < https://www.amherst.edu/system/files/media/1058/Exp6.pdf> [Accessed 14 June 2013] 3. Fedessenden, R.J., Fedessenden, J.S., & Feist, P. 2001, Organic Laboratory Techniques, Brooks/Cole, Canada, pp 119-140. [Accessed 11 June 2013] 4. Gilbert, J.C., Martin, S.F., Experimental Organic Chemistry: A Miniscale and Microscale Approach: A Miniscale and Microscale Approach 5TH edition, Brooks/Cole, Canada, pp 180-191. [Accessed 11 June 2013] 5. Thin Layer Chromatography (TLC), 2013, Thin Layer Chromatography (TLC). Available from: < http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html> [Accessed 8 June 2013].

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