high performance thin layer chromatography

HPTLC

INTRODUCTION Sophisticated form of thin layer chromatography. It

involves the same theoretical principle of thin layer chromatography.

It is also known as planar chromatography or Flat-bed chromatography.

Traditional Thin Layer Chromatography & its modern instrumental quantitative analysis version HPTLC are very popular for many reasons such as

visual chromatogram,

simplicity,

multiple sample handling,

low running and maintenance costs, disposable layer etc.

PRINCIPLE

HPTLC have similar approach and employ the same

physical principles of TLC (adsorption

chromatography) i.e. the principle of separation is

adsorption.

The mobile phase solvent flows through because of

capillary action. The components move according to

their affinities towards the adsorbent. The

component with more affinity towards the stationary

phase travels slower. The component with lesser

affinity towards the stationary phase travels faster.

Thus the components are separated on a

chromatographic plate.

Steps Involving in HPTLC

Sample

Preparation

Selection of

chromatography layer

Pre-washing

Pre-conditioning

Application of sample

Chromatography development

Detection of spots

Scanning & documentation4

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Sample and Standard Preparation

Solvents used are

•Methanol,

•Chloroform: Methanol (1:1),

•Ethyl acetate: Methanol (1:1),

•Chloroform: Methanol: Ammonia (90:!0:1),

•Methylene chloride : Methanol (1:1),

•1% Ammonia or 1% Acetic acid .

Selection of chromatographic layer

• Precoated plates - different support materials -

different Sorbents available

•80% of analysis takes place by silica gel GF ·

•Amino acids, dipeptides, sugars and alkaloids - cellulose

•Non-polar substances, fatty acids, carotenoids, cholesterol

- RP2, RP8 and RP18

•Preservatives, barbiturates, analgesic and phenothiazines-

Hybrid plates-RPWF254s

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Pre coated plates

• The plates with different support materials

and sorbent layers with different format and

thickness are used.

• Plates with sorbent thickness of 100-

250μm are used for qualitative and

quantitative analysis.

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Supports:

• Glass

• polyester sheets

• Aluminium sheets

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Some of the sorbents used in HPTLC:

• Silica gel 60F (Unmodified )

•Alluminium oxide

• Cellulose (microcrystalline )

• Silica gel chemically modified

Some of the binders used

Gypsum (G)

Starch (S)

Layer containing fluorescent indicator (F)

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Selection of HPTLC plates

Hand plates were available which are made up

of cellulose and other materials which are not

used much now-a –days.

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Plate size

20X20cm

10X20cm

5X10 cm

5X7.5 cm

Good cut edges of sheets is important to obtain constant Rf values.

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Pre washing of pre coated plates

The main purpose of the pre-washing is to

remove impurities which include water vapours and

other volatile substances from the atmosphere when

they get exposed in the lab environment.

Silica gel 60F is most widely used sorbent. The

major disadvantage of this sorbent is that it contain

iron as impurity. This iron is removed by using

Methanol : water in the ratio of 9:1.This is the major

advantage of the step of pre-washing.

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: Some common methods involved in

pre-washing

Ascending method

Dipping method

Continuous method

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Solvents used for pre-washing

1.Methanol

2.Chloroform: methanol ( 1:1 )

3.Choloroform: Methanol: Ammonia (90:10:1 )

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Activation of plates

Freshly opened box of HPTLC plates doesn’t need activation.

Plates exposed to high humidity or kept in hand for long time require activation.

Plates are placed in oven at 110o-120oc for 30 min prior to the sample application.

• Aluminium sheets should be kept in between two

glass plates and placing in oven at 110-120ºc for 15

minutes.

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Pre-conditioning

• Also called Chamber Saturation

•Un- saturated chamber causes high Rf values

Sample application

Usual concentration range is 0.1-1µg / µl

Above this causes poor separation

Linomat IV (automatic applicator) - nitrogen

gas sprays sample and standard from syringe

on TLC plates as bands

Band wise application - better separation.

Selection of mobile phase

•Trial and error

•one’s own experience and Literature

•Normal phase

Stationary phase is polar

Mobile phase is non polar

•3 - 4 component mobile phase should be

avoided

•Multi component mobile phase once used not

recommended for further use.

•Twin trough chambers are used only because

10 -15 ml of mobile phase is required

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Some applicators used for application of sample

a) Capillary tubes.

Samples applied in the form of spots.

Volume of 0.1-0.2μl

b) Micro bulb pipettes.

c) Micro syringes.

• Sample can apply either as

spot or band

• Volume- 1μl.

d)Automatic sample applicator.

• Sample can apply either as

spot or band.

Pre- conditioning (Chamber saturation)

•Un- saturated chamber causes high Rf values

•Saturated chamber by lining with filter paper for 30

minutes prior to development - uniform distribution of

solvent vapours - less solvent for the sample to travel -

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

H P T L C DEVELOPMENT

Vertical Development.

Vario method development.

Horizontal development.

Automatic Multiple

Development (AMD)/( Gradient ).

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Twin Trough Chambers

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Vario Chamber Development

VARIO CHROMATOGRAM

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Horizontal Development

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•HPTLC plate is developed from

both opposing sides towards the

middle.

•Plate sizes 10x10cm and 20x10cm

Post Chromatography Steps

1) Detection.

2) Photo documentation.

3) Densitometry measurements.

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Detection

UV CABINET

Detection under UV light is first choice - non

destructive

- Spots of fluorescent compounds can be seen at 254

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

length)

- Spots of non fluorescent compounds can be seen -

fluorescent stationary phase is used - silica gel GF

Detection and visualization

•Detection under UV light is first choice - non

destructive.

•Non UV absorbing compounds like ethambutol,

dicylomine etc - dipping the plates in 0.1% iodine

solution.

Quantification

•Sample and standard should be chromatographed

on same plate after development chromatogram is

scanned.

•Concentration of analyte in the sample is

calculated by considering the sample initially

taken and dilution factors.

Densitometry measurements

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Measures visible, UV absorbance or

Fluorescence.

Convert the spot/band into chromatogram

consisting of peaks

Instrumentation of HPTLC consists of following:

Lamp selector

Entrance lens slit

Monochromator entry slit

Grating

Mirror

Slit aperture disc

Mirror

Beam splitter

Reference photo multiplier

Measuring photo multiplier

Photo diode for transmission measurements.

INSTRUMENTATION

• Theory: According to the theory, the transmission of light in a translucent material can be described by:

I0 = Ia + It + Ir + Ix

Where,

I0 = Intensity of incident light. Ia = Intensity of absorbed light. It = Intensity of transmitted light. Ir = Intensity of reflected light. Ix = Intensity of light lost due to

scattering.

The Densitometer work by 2 modes:

1. Transmission mode

2. Reflectance mode

In transmission mode the ratio of It/Io is

measured and converted in to absorbance values

In reflectance mode the ratio Ir/Io is measured

and converted in to absorbance values

According to Goodman & Goodall transmission

measurements are more sensitive than

reflectance measurements

Fluorescence measurement in densitometry:

1) Measurement of direct fluorescence

2) Measurement of fluorescent quenching.

1. Direct fluorescent measurement:

• This method is followed if the spot exhibit

fluorescence when exposed to UV light.

• In this two monochromators are used for selection

of excitation & emission wavelength. The

fluorescence is measured in reflectance mode.

2) Fluorescence quenching measurement:

• As the name indicates, it utilizes the ability of analyte to

absorb & quench fluorescence light.

• In this technique fluorescent background is incorporated

into the layer. When excited by short wavelength

radiation the plate fluorescence's uniformly.

• If UV absorbing substance is present in the plate, a

portion of the fluoresced light is absorbed &

consequently quenched.

• This fluorescence diminution is measured as a function

of amount of analyte in the spot.

.

Advantages of densitometer scanner:

The purpose of scanner is to convert the

spot/band on the layer into densitogram

consisting of peaks similar in appearance

to HPLC.

The position of the scanned peaks on the

recorder chart is related to Rf values.

Quantitation is faster, reliable accurate &

reproducible

Photo-documentation With

Digital Camera

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7)DOCUMENTATION:

1. Documentation is important because labeling every single

chromatogram can avoid mistake in respect of order of

application.

2. Type of plate, chamber system, composition of mobile

phase, running time and detection method should be

recorded.

3. TO assist the analysts and researchers E .merck has

introduced HPTLC pre-coated plates with an imprinted

identification codes.

video scan software for quantitative evaluation of images capture with digistore

Documentation

Differences between TLC and HPTLC

Applications of HPTLC

Pharmaceutical industry: Quality control, content

uniformity, uniformity test, identity/purity check.

Food Analysis: Quality control , additives ,

pesticides ,stability testing ,analysis of sub-micron

levels of aflotoxins etc.

Clinical Applications: Metabolism studies , drug

screening ,stability testing etc

Industrial Applications: Process development and

optimization, In-process Q.C. check, validation etc.

Forensic : Poisoning investigations.

A) QUANTITATIVE DETERMINATION:

1) Biochemical research/Biotechnology-

Seperation of gangliosides

2) Clinical-

Inorganic & organic mercury in water & human serum. Caffeine in urine.

3) Cosmetics-

Hydrocortisone & cinchocaine in lanolin ointment

4) Environmental Analysis-

Pesticides in drinking water.

Selenium in water.

5) Food analysis-

Vitamin C in fruit juices.

Aflatoxins in food stuff

6) Pharmaceutical & chemical substance-

Content uniformity test of diclofenac sodium.

Vitamin B1 pharmaceutical products.

7) Natural products ,plant ingredients-

Glycosides in herbal drugs.

Glycyrrhizic acid in liquorice.

8) Doping analysis-

Atenalol in urine.

B) FINGER PRINT ANALYSIS-

HPTLC finger print of Valerian.

Finger print of garlic, Ashwaganda.

Finger prints for identification of liquorice, ginseng.

9) Identification and separation of phenyl

thiohydantoin -amino acid.

10) analysis of drugs in blood

EX: 1)separation of phenothiazine drugs like

chlorpromazine, acetophenazine, perphenazine,

trifluperazine and thoridazine.

11) identification of mycotoxins in admixture :

EX: detection of sterigmatocystin, zearalenone,

citrinin, ochrotoxinA, patulin, penicillic acid.

12) determination of polycyclic aromatic

hydrocarbons in particulate sample.

EX; determination of chryesene, pyrene,

fluoronthene etc.

REFERENCES

HPTLC - Quantitative Analysis of Pharmaceutical

Formulations by Dr.P.D.Sethi, Page No.3 – 72.

Pharmaceutical Analysis vol-II by Dr. A. V. Kasture,

Dr. K. R. Mahadik Nirali Publishers page no 28-30.

Textbook of pharmaceutical analysis, third edition

by S. Ravi shankar, Rx publications pages no 14.10

to 14.12

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