Adsorption Chromatography

-Sailee GuravMSc Part 1- Biochemistry

It utilizes mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase.

Equilibriation between mobile & stationary phase accounts for the separation of different solutes.

Types : TLC,HPTLC,COLUMN CHROMATOGRAPHY

Adsorption chromatography

Separation depends on affinity of compounds towards stationary & mobile phase.

Compounds under influence of mobile phase travel over surface of stationary phase.

Component -more affinity -Stationary phase travels - slower

Component -lesser affinity -Stationary phase-travels faster.

Separation of components in mixture is achieved.

TLC Principle

◦ Normal or Reversed phase chromatography

◦ Prepare a plate with proper sorbent material

◦ Prepare mobile phase

◦ Mark the plate

◦ Apply the sample

◦ Develop the plate

◦ Detect the analytes

General Procedure

Stationary phase Adsorbents mixed with water or other solvents→

slurry Silica gel H ( Silica gel without binder ) Silica gel G ( Silica gel + CaSO4 ) Silica GF (Silica gel + binder + fluorescent

indicator) Alumina, Cellulose powder etc.

Practical Requirements

Specific dimensions- 20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm Microscopic slides can also be used

Preparation & activation of TLC plates Pouring Dipping Spraying Spreading

Glass plate

After spreading → Air dry Activated by heating at about 100˚C for 30 min. Then plates may be kept in desiccators

Application of sample Using capillary tube or micropipette Spotting of sample

Development tank Better to develop in glass beakers, jars to avoid more

wastage of solvents When standard method is used, use twin trough tanks

Activation of Plates

Depends upon various factors:

Nature of the substance

Nature of the Stationary Phase

Mode of Chromatography

Separation to be achieved, Analytical/Preparative

e.g. → pyridine, carbon tetrachloride, acetone, water, glycerol, ethanol, benzene….

Mobile phase

1. One dimensional development2. Two dimensional development3. Horizontal development4. Multiple development

Detecting or visualizing agents

Iodine chamber method Sulphuric acid spray reagent UV chamber for fluorescent compounds

Specific methods Qualitative analysis Rf value –distance travelled by solute/ distance travelled by solvent

Quantitative analysis Direct & indirect method

Development technique

Purity of sample Examination of reaction Identification of compounds Biochemical analysis In pharmaceutical industry Separation of multicomponent

pharmaceutical formulations In food and cosmetic industry

Applications of TLC

• HPTLC is well known & versatile separation method • Lot of advantages

• Fast & inexpensive

• It does not require time consuming pretreatments

• It is very useful in quantitative and qualitative analysis of pharmaceuticals

Principle same as TLC

HPTLC

Steps involved in HPTLC

Selection of chromatographic layer Sample and standard preparation Layer pre-washing Layer pre-conditioning Application of sample and standard Chromatographic development Detection of spots Scanning Documentation of chromatic plate

a.manual plate coater

b.Automatic plate coater

Plate coater Drying Rack

Platecutter

Immersion DevisePlate heater

 80% of analysis - silica gel GF Basic substances, alkaloids & steroids-

Aluminum oxide Amino acids, dipeptides, sugars & alkaloids

- cellulose Sample and Standard Preparation Solvents used are Methanol, Chloroform,

Ethyl acetate Dry the plates and store in dust free

atmosphere

Selection of chromatographic layer

Plates exposed to high humidity in an oven at 110-120oc for 30' prior to spotting 

Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120oc for 15 minutes.

Usual concentration range is 0.1-1ug

Activation of pre-coated plates

Application of sample and standard

Un- saturated chamber causes high Rf values 

Saturated chamber - lower Rf values.

Chromatographic development and drying

After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere

Dry in vacuum desiccator

Pre- conditioning (Chamber saturation)

Detection under UV light Spots of fluorescent compounds can be

seen at 254 nm (short wave length) or at 366 nm (long wave length) 

Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution

When individual component does not respond to UV - derivatisation required for detection

Detection and visualization

Sample and standard should be chromatographed on same plate - after development chromatogram is scanned 

Camag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode

Concentration of analyte in the sample is calculated by considering the sample initially taken and dilution factors

Quantification

Column of stationary phase is used Solid – S.P Liquid – M.P

Principle Mixture of components dissolved in the mobile

phase is introduced in to column. Components moves depending upon their relative

affinities.

COLUMN CHROMATOGRAPHY

Stationary phase Adsorbent should be: Spherical in shape Mechanical stability must be high They shouldn’t react chemically It should be useful for separating for wide

variety of compounds It should be freely available & inexpensive

Practical requirements

Success of chromatography → depends on the following.

1. Removal of impurities2. No. of components to be separated3. Length of the column used4. Affinity differences b/w components5. Quality of adsorbent used

They act as ▫ Solvent ▫ Developer ▫ Eluent

COLUMN CHARACTERISTICSColumn - Neutral glassColumn dimensions - length & diameter ratio

(10:1,30:1 or 100:1)

Mobile phase

Bottom portion of the column – packed with glass wool/cotton wool

Above which adsorbent is packed After packing a paper disc kept on the topTwo types of packing techniques are there.1.Dry packing2.Wet packing

Preparation of the column

Adsorbent is packed in the column in dry form Fill the solvent, till equilibrium is reached

Wet Packing Technique ideal & common techniqueAdsorbent + M.P in a beaker & poured in to column S.P settles uniformly & no crack in the column of

adsorbent

Dry Packing Technique

Samples dissolved in M.P & introduced into the column at once → eluted

ELUTION PROCEDURESTwo techniques1)Isocratic elution techniques2)Gradient elution techniques Isocratic elution techniques(Iso means – same)Same solvent composition or solvent of same

polarity used throughout the process of separation

Introduction of the sample

( gradient – gradually) Solvents of gradually ↑ polarity or ↑ elution

strength are used during the process of seperation.

E.g. benzene, chloroform, ethyl acetate DETECTION OF COMPONENTS

Colored components-VisuallyColorless components- Different properties

which can be used are – uv / visible detector, flourescence detector, Flame ionization detector

Gradient elution techniques

►Seperation of mixture of components►Purification process►Isolation of metabolites►Estimation of drugs in formulations

APPLICATIONS

Current ResearchHigh performance thin layer chromatographic method for the simultaneous

estimation of camylofin dihydrochloride and mefenemic acid in pharmaceutical tablet

Objective: The objective of the present aims to develop new, simple, precise and accurate High Performance Thin Layer Chromatographic method for the estimation of Camylofin dihydrochloride and Mefenemic acid in pharmaceutical dosage forms.

Method: Chromatographic separation of the drugs were achieved employing merck precoated silica gel 60 F254 (0.2 mm thickness) on aluminium sheets as stationary phase with a solvent system of chloroform: methanol: ammonia in the ratio of 6:4:0.1 v/v/v, densitometric quantification of the separated bands was done at 270nm.The saturation time of the chamber and the developing distance was set at 30 minutes and 8cm respectively. The method was validated as per ICH guidelines.

Results: The Rf values were found to 0.46 for Camylofin dihydrochloride and 0.35 for Mefenemic acid .The proposed method was found to be linear in the concentration range of 320-480 ng / band for Camylofin dihydrochloride and 1600-2400ng/ band for Mefenemic acid. The average recovery was found to be 100.62% w/w and 100.02% w/w for Camylofin dihydrochloride and Mefenemic acid respectively.

Conclusion: The novel HPTLC method developed is precise, specific and accurate. Satisfactory results were obtained from validation of the method. Hence the proposed method is suitable in the quality control of estimation Camylofin dihydrochloride and Mefenemic acid in pharmaceutical dosage forms.

Current Research:http://www.ijppsjournal.com/Vol6Issue1/8263.pdf HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD FOR

THE SIMULTANEOUS ESTIMATION OF CAMYLOFIN DIHYDROCHLORIDE AND MEFENEMIC ACID IN PHARMACEUTICAL TABLET

http://bheem.hubpages.com/hub/HPTLC-High-performance-thin-layer-chromatography-Principle-Instrumentation

http://www.pharmainfo.net/reviews/basic-principles-hptlc College Analytical Chemistry,Himalaya Publishing House

Edition : 19th (2011),K.B.Baliga. Biophysical Chemistry Principles & Techniques,Himalaya

Publishing House ,Edition : 6th (2012),By Avinash Upadhyay, Kakoli Upadhyay, Nirmalendu Nath,

Practical Biochemistry Principles & Techniques, Cambridge low-price editions,Edition:5th,Edited By Keith Wilson & John Walker

Reference