TLC analysis

International Islamic University Malaysia,
Kuantan campus

DEPARTMENT OF BIOTECHNOLOGY
KULLIYYAH OF SCIENCE

SBT 4534
ANALYTICAL METHODS FOR FUNCTIONAL FOODS & NUTRACEUTICALS

LABORATORY REPORT

LAB 2:
FAT & OIL FROM OIL-SEEDS: PART II THIN LAYER CHROMATOGRAPHY ANALYSIS (TLC)

COORDINATORS:
ASSOC. PROF. DR. JAMALUDDIN BIN MOHD DAUD

ABDUL AZIM ADNAN
1016741
INTRODUCTION TLC chromatography or thin layer chromatography is a type of planar chromatography. TLC is routinely used by researcher in the field of phyto-chemicals, biochemistry etc. to identify the components in a compound mixture like alkaloids, phospholipids, amino acids etc. It is a semi quantitative method of analysis and its sophisticated version or highly precise quantitative version is High performance thin layer chromatography (HPTLC). Similar to other chromatographic methods TLC is also based on the principle of separation. The separation depends on the relative affinity of compounds towards stationary and mobile phase. The compounds that under the influence of mobile phase (driven by capillary action) travel over the surface of stationary phase. During this movement the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus separation of components in the mixture is achieved. Once separation occurs individual components are visualized as spots at respective level of travel on the plate. Their nature or characters are identified by means of suitable detection techniques.

TLC System consists of
a) TLC plates preferably ready made with stationary phase: These are stable and chemically inert plates on to whose surface a thin layer of stationary phase is applied. The stationary phase on the plates is of uniform thickness and consists of fine particle size.
b) TLC chamber: This is used for the development of TLC plate. The chamber maintains uniform environment inside for proper development of spots. It also prevents the evaporation of solvents and keep the process dust free.
c) Mobile phase: This comprises of a solvent or solvent mixture recommended for the purpose. The mobile phase used should be particulate free and of highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, stationary phase.
d) A filter paper moistened in the mobile phase, to be placed inside the chamber. This helps uniform rise in mobile phase over the length stationary phase.

OBJECTIVE
1. To separate the lipid using thin layer chromatography (TLC) method.
2. To analyze triacylglycerol and detect their spots using UV and spraying agents.
3. To develop skills including use of solvent system for TLC separation method.

MATERIAL
Oil extracts from lab 1
Solvents; chloroform, methanol, diethyl ether, distilled water and acetic acid
Aluminum TLC plate
Developing tanks
Spraying agent: 10% sulphuric acids
Heating oven
UV lamp detector

PROCEDURE
1. The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize.
2. A thin mark is made at the bottom of the plate with a pencil to apply the sample spots.
3. Then samples solutions are applied on the spots marked on the line at equal distances.
4. The mobile phase is poured into the TLC tanks to a level few centimeters above the tanks bottom.
5. Then the plate prepared with sample spotting is placed in TLC tanks such that the side of the plate with sample line is towards the mobile phase. Then the chamber is closed with a lid.
6. The plate is immersed such that sample spots are well above the level of mobile phase but not immersed in the solvent as shown in the picture for development.
7. Sufficient time is allowed for development of spots. Then the plates are removed and allowed to dry. The sample spots are visualized in suitable UV light chamber.

RESULT
A. Hexane: Diethyl ether: Acetic acid by a ratio 90: 30: 1

Spot
Spot distance (cm)
Rf
1
2.0
0.27
2
2.5
0.33
3
2.8
0.37
4
3.4
0.45
5
4.0
0.53
6
5.1
0.68
7
5.7
0.76
8
7.2
0.96

B. Hexane: Dichloromethane: Acetic acid by a ratio 90: 10: 1

C. Chloroform: Methanol: Acetic acid by a ratio 80: 10:1
Spot
Spot distance (cm)
Rf
1
1.1
0.15
2
5.8
0.77
3
7.3
0.97

DISCUSSION The chromatographic tanks are developed in a 150-mL beaker or jar containing the eluent. The tank need to be closed to keep the atmosphere in the beaker saturated with solvent vapor. When the TLC plates are removed from the eluent, the solvent is allowed to evaporate. Oilseed is colorless. The oilseed separating spots can be visualized by spraying the plate with a reagent that will react with one or more of the components of the sample. The position of spots than can be determined by placing the plates under a short wavelength ultraviolet lamp. There are three type of solvent mixture (eluent) with their respective ratio are used in this experiment. These three solvents are not always necessary. Sometimes one solvent will be appropriate. However, more than one solvent allow us to adjust the eluent polarity easily when a developed plate does not give an appropriate result. A suitable eluent is one that gives a retardation factor (Rf, also called retention factor) close to 0.5 upon developing the plate. Thus, from the result, the most suitable solvent mixture (eluent) with respective ratio to extract the oilseed compound is hexane: diethyl ester: acetic acid in the ratio of 90: 30:1 (v/v/v). It managed to reveal eight spots on the plate compare to other eluents. Besides the Rf value quite good since the Rf value lied between 0.27 – 0.96. During the chromatography, the eluent distributes the compounds present in the sample over the alumina. The compound sample which spotted onto the stationary phase on the plate has two choices either adsorbs to the solid phase or being eluted by the mobile phase. The eluent has good polarity if compare to other eluents. Overall, the eluent is competed with the sample for a space on the stationary phase) coated on the plate. The more polar compound will win this competition and adhere to the stationary phase, while the rest of the mixture will move with the mobile phase (upwards). The Rf value is the ratio of the distance travelled by the center of the spot to the distance simultaneously travelled by the mobile phase.

By definition the Rf values are always less than unity. They are usually given to two decimal places. From the result in this particular eluent, the Rf value is varied between all those spots. The Rf value in an ascending order are 0.27, 0.33, 0.37, 0.45, 0.53, 0.68, 0.76 and 0.96. The lowest Rf value is 0.27. This means that the eluent is quite non-polar for that particular spot. Thus the spot is observed with a smaller Rf and this is proven by its position on the plate which closer to the baseline. On the other hand, the highest Rf value is calculated as 0.96 and it is simply means that eluent is very polar to that particular spot as its position is closer to the solvent front.

The eluent need to be adjusted because it is too polar since there is a spot which very close to the solvent front. The eluent need to be adjusted since the optimum Rf value is lied between 0.3 - 0.7. How to improve the Rf value? We can reduce the eluent polarity so that the spot will be lower down on the plate and the Rf value therefore will become smaller as well. We can either choose a different eluent or adjust the solvent ratio by increasing the percentage of the nonpolar solvent relative to the polar solvent in the eluent.

There are many advantages of calculating the Rf value for each spot on the plate. Rf value can be used to identify a particular chemical substance. It is like a 'fingerprint' to identify an unknown substance based on known Rf value of a list of substance. Besides, the factor that affects the Rf value of TLC is the polarity of the solvent. If the solvent is too polar all of the compounds can run right up the plate resulting in poor separation. The Rf value is also used to established the identity of the spots of TLC plate. In fact the Rf value is ratio of the distance the solvent travels to the distance compound travels. Here are some other important factors that can affect Rf values; the absorbent uniformity on the thin layer plate, same concentration (spotting is too weak or strong), room temperature during the mobile phase and development distance of the solvent during the mobile phase can all affect the results.

CONCLUSION We had done the experiment successfully since we managed to separate the lipid compound. The lipid compound is separated into triglyceride and fatty acid respectively. The spot is visualized very clear under the UV light. However the eluent need to be slightly been adjusted since it is too polar for the compound. Overall, we had learnt the fundamental concept and principle of the TLC method for separation of oil and fat from the oilseed.

REFERRENCE

Retrieved from http://www.chem.ualberta.ca/~orglabs/Techniques%20Extra%20Info/TLC.html#top

Retrieved from http://bheem.hubpages.com/hub/tlc-thin-layer-chromatography-Principle-Procedure

Retrieved from http://en.wikipedia.org/wiki/Thin_layer_chromatography