Thin layer chromatography.

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Thin Layer chromatography Prepared by: Dhara Patel (10BPH052) Guided by: Ms Komal Dave

Topics Included:

Topics Included 2

Introduction:

Introduction In 1938, Izmailov and Shraiber described the basic principle underlying the process and used it for the separation of plant extracts. In 1944, Consden,Gorden & Martin used filter papers for separating the Amino acids. In 1950, Kirchner identified terpenes on filter paper. In 1958, STAHL develop standard equipment for analysing by THIN LAYER CHROMATOGRAPHY . 3

Principle :

Principle The Principle is based on ADSORPTION Chromatography and PARTATION Chromatography The component with more affinity towards the stationary phase travels slower. The component with lesser affinity towards the stationary phase travels faster. 4

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5 Advantages of TLC:

Reverse Phase Chromatography:

Reverse Phase Chromatography In this the stationary phase is Non-polar & mobile phase is polar & it is widely used in pharmaceutical analysis. 1) Polar compound get eluted first. 2) Non-polar compounds are retained for long time. Comparison of Normal phase & Reverse phase : Normal phase Reverse phase Stationary phase Polar Non-polar Mobile phase Non-polar Polar Compound eluted first Non-polar Polar Compound eluted last Polar Non-polar Example of stationary phase Silica gel C 4 ,c 8 –bonded phase 6

Stationary phase:

Stationary phase NAME COMPOSITION Silica gel H Silica gel without binder Silica gel G Silica gel + CaSO4 Silica gel GF Silica gel + Binder + fluorescent indicator Alumina Al 2 O 3 Without Binder Al 2 O 3 G Al 2 O 3 + Binder Cellulose powder Cellulose Without Binder Cellulose powder Cellulose With Binder Polyamide powder Polyamide Fuller’s earth Hydrous magnesium alumina Magnesium Silicate magnesol 7

Mobile phase:

Mobile phase Nature of the substance to be separated i e weather it is polar or non-polar according to “eluotrophic series” Mode of Chromatography. Nature of Stationary phase. Separation i e Analytical or Preparative. Examples: 1) Petroleum ether 6) Acetone 2) benzene 7) Alcohols 3) Chloroform 8) Water 4) Ethyl Acetate 9) glycol 5) Pyridine 10) Glycerine 8

Glass plates:

Glass plates Three types : they are , 1) Full plate : 20cm × 20 cm. 2) Half plate : 20cm × 10 cm. Quarter plate : 20cm × 5 cm. Microscopic slides can also be used for Monitoring the progress of a chemical reaction. 9

Preparation & Activation of plates:

Preparation & Activation of plates The T L C plates can be prepared by following techniques : 1) Pouring. 2) Dipping. 3) Spraying. 4) Spreading. Activation: It is nothing but removing of water/ moisture & other adsorbed substance from the surface of any adsorbent by heating. 10

Application of sample :

Application of sample 11

Development tank:

Development tank “ EDGE EFFECT ” 12

Development technique:

Development technique Different development techniques are : Ascending development. Descending development. Multiple/repeated development. Stepwise development. Two Dimension development. Gradient elution. 13

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14

Detection:

Detection The R f value is calculated for identification "Rf value is the ratio of distance travelled by The solute to the distance travelled by the solvent front.” Distance travelled by solute Distance travelled by solvent front 15 R f =

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16 Factors affecting R f :

Developed TLC:

Developed TLC 17

Applications:

Applications 18

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High performance thin layer chromatography (HPTLC) is the sophisticated form of TLC. It is as simple to use as standard TLC, but has many advantages: It is more sensitive. It is possible to run more samples in a shorter period of time. The amount of solvent used is much smaller. The technique is widely used in many fields both for qualitative and quantative analysis. The main difference between TLC and HPTLC is particle size of coated material which is 5-20ℳm for TLC and 4-8ℳm for HPTLC. 19 HPTLC

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20 Steps involved in HPTLC

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TLC HPTLC Particle size and shape is less uniform. Particle sixe and shape is more uniform. Required thickness of the layer is 250ℳm. Required thickness of the layer is 100ℳm. Solvent front is usually 10 to 15cm. Solvent front is usually 3 to 5cm. Time required for development is more. Time required for development is 30 to 40% less then TLC. Sample capacity for 20×20cm plate is 2-4 in number. Sample capacity for 20×20cm plate is 20 -30 in number. Sample required is 2-10ℳ l. Sample required is 0.1-0.5ℳ l. Maximum modification possible is two dimensional development. Maximum modification possible is circular , anticircular & opposite development. 21 TLC & HPTLC

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TLC CHROMATOGRAPHY PAPER CHROMATOGRAPHY It requires less amount of the substance. It requires more amount of the substance. Less time consuming More time consuming Sharpness of separation is good Sharpness of separation is less than TLC Capacity of thin layers of adsorbent is higher. Less in paper chromatography. Strong acids can be safely identified. It is not possible in paper Corrosive reagents may be coated on glass plate. Also not possible in paper Physical strength is more Physical strength is less It can be heated in oven Not possible Sensitivity is more Sensitivity is less 22

Reference :

Reference Pharmaceutical Analysis by Dr. A. V. Kasture, Dr. K.R.Mahadik, Dr. S.Wadodkar, Dr. H. N. More, Nirali Prakashan, volume 2, 13th edition, 2009. Text book of pharmaceutical analysis by Dr. S. Ravi Shankar. Rx Publication, Tiruneveli: Edition 1997, 1999, 2001. 23

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24 Thank you.

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