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Department: Biotechnology Course Title: Analytical Chemistry & CourseTeacher: Dr. Saba Rauf Instrumentation Ch.hr. 2+
LECTURE 8
THIN LAYER
CHROMATOGRAPHY - TLC
8.1 Introduction
8.2 Principle
8.3 Procedure
8.4 Visualizing technique
8.5 Interpretation of data
8.6 Application
8.7 Limitations
8.1 Introduction
Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. a) The stationary phase - is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. b) The mobile phase - is a developing liquid which travels up the stationary phase, carrying the samples with it. Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.
Department: Biotechnology Course Title: Analytical Chemistry & CourseTeacher: Dr. Saba Rauf Instrumentation Ch.hr. 2+ Thin Layer Chromatography is a technique used to isolate non-volatile mixtures, identifying substances and testing the purity of compounds. TLC is a useful technique because it is relatively quick and requires small quantities of material. Stationary phase Description Application Solvent Silica gel G Silica gel with average particle size 15μm containing ca 13% calcium sulfate binding agent Used in wide range pharma test Hexane Butanol Chloroform Methanol Ethanol Acetonitrile Silica gel G254 Silica gel G with fluorescence added Same application with Silica gel G where visualization is to be carried out under UV light.
Department: Biotechnology Course Title: Analytical Chemistry & CourseTeacher: Dr. Saba Rauf Instrumentation Ch.hr. 2+ inside for proper development of spots. It also prevents the evaporation of solvents, and keeps the process dust free. Mobile phase- This comprises of a solvent or solvent mixture. The mobile phase used should be particulate- free and of the highest purity for proper development of TLC spots. A filter paper- It has to be placed inside the chamber. It is moistened in the mobile phase. It ensures that the mobile phase rises uniformly throughout the TLC plate’s length.
8.4 How to PERFORM TLC
With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots. Then, samples solutions are applied on the spots marked on the line in equal distances. The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom. A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect).
8.5 VISUALIZING Technique
Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the plate with the sample
Department: Biotechnology Course Title: Analytical Chemistry & CourseTeacher: Dr. Saba Rauf Instrumentation Ch.hr. 2+ line is facing the mobile phase. Then the chamber is closed with a lid. Sufficient time is given for the development of spots. The plates are then removed and allowed to dry. The sample spots are then seen in a suitable UV light chamber, or any other methods as recommended for the given sample.
8.6 INTERPRETING the Data
Some common techniques for visualizing the results of a TLC plate include: UV light Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with Iodine KMnO4 stain (organic molecules) Ninhydrin Reagent: often used to detect amino acids and proteins
Department: Biotechnology Course Title: Analytical Chemistry & CourseTeacher: Dr. Saba Rauf Instrumentation Ch.hr. 2+ Thin layer chromatography can be used to identify natural products like essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc. It is used in the food industry, to separate and identify colours, sweetening agent, and preservatives
8.8 LIMITATIONS of TLC
Thin Layer Chromatography plates do not have longer stationary phase. The results generated from TLC are difficult to reproduce. TLC operates as an open system, some factors such as humidity and temperature can be affecting the final outcome of the chromatogram. It is only a qualitative analysis technique and not quantitative.
