1. -Sailee Gurav
MSc Part 1- Biochemistry

2.  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

3.  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.

4. ◦ 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

5. 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.

6.  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

7.  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

9. 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….

10. 1. One dimensional development
2. Two dimensional development
3. Horizontal development
4. 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

11.  Purity of sample
 Examination of reaction
 Identification of compounds
 Biochemical analysis
 In pharmaceutical industry
 Separation of multicomponent
pharmaceutical formulations
 In food and cosmetic industry

12. • 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

13.  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

14. a.manual plate coater
b.Automatic plate
coater
Drying Rack
Platecutter
Immersion Devise
Plate heater

15.  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

16.  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
Application of sample and standard

17.  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

18.  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

19.  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

20. 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.

21. 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

22. Success of chromatography → depends on the
following.
1. Removal of impurities
2. No. of components to be separated
3. Length of the column used
4. Affinity differences b/w components
5. Quality of adsorbent used

23. They act as
▫ Solvent
▫ Developer
▫ Eluent
COLUMN CHARACTERISTICS
Column - Neutral glass
Column dimensions - length & diameter ratio
(10:1,30:1 or 100:1)

24.  Bottom portion of the column – packed with glass
wool/cotton wool
 Above which adsorbent is packed
 After packing a paper disc kept on the top
Two types of packing techniques are there.
1.Dry packing
2.Wet packing

25.  Adsorbent is packed in the column in dry form
 Fill the solvent, till equilibrium is reached
Wet Packing Technique
 ideal & common technique
Adsorbent + M.P in a beaker & poured in to column
S.P settles uniformly & no crack in the column of
adsorbent

26. Samples dissolved in M.P & introduced into the
column at once → eluted
ELUTION PROCEDURES
Two techniques
1)Isocratic elution techniques
2)Gradient elution techniques
Isocratic elution techniques
(Iso means – same)
Same solvent composition or solvent of same polarity
used throughout the process of separation

27. ( 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-Visually
Colorless components- Different properties which
can be used are – uv / visible detector,
flourescence detector, Flame ionization detector

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

29. High 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.

30.  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