H.P.T.L.C.

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

PRESENTED BY :- PRAMOD H.DALAL

GUIDED BY : Mr. S.P. WATE

CONTENT :
Introduction Principle of HPTLC Difference between HPTLC & TLC Features of HPTLC Instrumentation Steps involved in HPTLC Factor affecting HPTLC References

1)INTRODUCTION:
a) In HPTLC several sample can simultaneously handle even sample present in divergent nature. b) It is a sophisticated & automated form of TLC. c) It can be considered a time machine & allow you to do many things at a time usually not possible with other analytical technique. d) This technique widely used for both Quantitative & Qualitative i.e.(identification & estimation) of constituent mixture.

2)PRINCIPLE :
Same theoretical principle of TLC. Separation may result due to adsorption or partition or by both phenomenon depending upon the nature of adsorbents used on plate and solvent system used for development..

Parameter Layer of Sorbent Efficiency Separations Analysis Time Solid support

HPTLC 100m High due to smaller particle size generated 3 - 5 cm Shorter migration distance and the analysis time is greatly reduced Less

TLC 250m

10 - 15 cm Slower

Wide choice of stationary phases like silica gel for Silica gel , normal phase and C8 , C18 for reversed phase modes Alumina & Kiesulguhr New type that require less amount of mobile phase Auto sampler

Development chamber Sample spotting Scanning

More amount Manual spotting

Use of UV/ Visible/ Fluorescence scanner scans Not possible the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer

Features of HPTLC

Simple

& rapid Several analysts work simultaneously Lower separation time Less cost Low maintenance cost Low mobile phase consumption per sample No interference from previous analysis Visual detection

possible

3) VARIOUS STEPS INVOVED IN HPTLC :

a) Sample Preparation b) Selection of chromatographic layer c) Plates d) Pre washing e) Conditioning f) Sample application g) Pre conditioning h) Mobile phase i) Chromatographic development j) Detection of spots k) Scanning & documentation.

Sample & Standard preparation

Selection of chromatographic layer

Layer pre - washing Layer pre - conditioning Application of sample and standard Chromatographic development Detection of spots Scanning and documentation of chromatoplate Figure 1. Schematic procedure for HPTLC

a)Sample Preparation

: Not demanding as for other chromatographic development. Several steps for sample pre treatment may be necessary such as Sampling, Mechanical crushing, Extraction, Filtration & Enrichment of minor compounds. For normal phase chromatography using silica gel / Alumina pre-coated plate , solvent generally should be nonpolar and volatile type. Since polar solvent tends to give circular shape at origin. For reversed phase chromatography polar solvent used for dissolving the sample.

b)Selection of chromatographic layer :

Depends on nature of material to seperated Commonly used material are Silica gel 60 F, Alumina, Cellulose etc. These material can be coated on hand made plates with or without binders Pre coated plate are commercially available & are commonly used. Pre coated plate costly than hand made plate.

c) Plates :
Size : 2020cm, 1020cm, 510 cm, 57.5 cm

(1) Types of plate :

A)Hand made plates : B) Pre coated plates : (2) Plate size :

Basic difference in TLC & HPTLC plates

TLC : 5-20 m particle size. HPTLC : 4-8m particle size.

A)Hand made plates :

Size : 20 20 cm. Material used : cellulose, cellulose with starch as binder, silica gel, silica gel G , silica gel with starch, acelytated cellulose with calcium sulfate . B) Pre coated plates : The pre coated plates with different support material (Glass, Aluminium, plastic ) & with different sorbent layer are available. Sorbent thickness 100 250 m used for qualitative & quantitative analysis .

2) Plate size :
Pre-coated HPTLC plate size 2020 cm with alumium or polyester support. Before handling the pre-coated plates, note the direction of the application of sorbent as chromatographic development have to must be performed in that direction only. Good cut edges of sheets is important to obtain constant Rf value.

Supports Materials
Materials Advantage 1.Ressistant to heat and chemicals 2.Easy to handle and offers superior flat surface for work even in roll forms 2.Unbreakable 3.Less packing material 4.Spots can be cut and eluted thus eliminates dust from scrapping 1.Increasesed resistance Disadvantage 1. Fragility 2.Relatively High wt 3.Costs more additional packaging

Glass

for

Polyester

sheets 1.More economical as produced 1.Charring reactions if

temperature exceeds 120oc as the plates are dimensionally unstable beyond this temperature

(0.2 mm thick)

Aluminum
Sheets(0.1mm)

temperature 1.Eluents containing high concentration of mineral acids or ammonia can attack chemically on aluminum

d) Pre-washingsg
Plates need to be pre-washed to remove water vapour or other volatile impurities. Mainly methanol is used. To avoid any possible interference due to impurities with the chromarographic seperation particularly in case of quantitative work, it is always recommended to clear the plate before actual chromatography, this process k/n as Pre-Washing.
S

Solvents used for pre-washing

1.Methanol 2.Chloroform: methanol ( 1:1 ) 3.Choloroform: Methanol: Ammonia (90:10:1 ) 4.Methylene chloride: Methanol ( 1:1 ) 5.Ammonia solution (1%)

e) Conditioning :

The pre-washed plates or Plates exposed to

humidity & surrounding are need to be activated by placing them in oven at 110o-120oc for 30 min, this process k/n as conditioning. Plates are placed in oven at 110o-120oc for 30 min prior to the sample application.
.

f)_ Sample application :

It is important step for obtaining good resolution & results. Application of 1-5 l is most satisfactory for HPTLC. Sample application carried by Linomat & Nanomat type applicator on the plate which gives uniform, safe and standard results.

Diag.: Linomat

2) CAMAG

Linomat

Automated sample application device. Sample is loaded in micro syringe (Hamilton Syringe) 1l capacity. Sample can apply either as spot or band by programming the instrument with parameters like spotting volume ,band length etc.

1) CAMAG Nanomat: Samples applied in the form of spots. The volume is controlled by disposable platinum iridium of glass capillary which has volume of 0.1-0.2l.

Diag. :CAMAG NANOMAT

g) Pre conditioning (chamber saturation) :

Suppose the plate is introduced into an unsaturated chamber, the solvent evaporates from the plate mainly at the solvent front, therefore large quantity of the solvent evaporates & large quantity of solvent required for a given distance. Suppose the plate is introduced into an saturated chamber, solvent vapour distributed throughout the chamber, then plate is placed on the chamber, it soon gets pre-loaded with solvent vapour, since less amt. of solvent required to travel a particular distance, resulting lower Rf values. Low polarity mob. Phase is no need for saturation. Saturation is desirable in case of highly polar mobile phase. For reverse phase saturate chamber with polar solvent

h ) Mobile phase :
Mobile phase should be of high graded.

Avoided the use of mobile phase containing more than three or four components. Chemical properties ,analytes and sorbent layer factors should be considered while selection of mobile phase. Mobile phase optimization is necessary while performing HPTLC. Chambers containing multi component MP are not generally used.

Ascending development, Descending development Horizontal development After development, plates are removed from the chamber & dried to remove traces of mobile phase.

Types of chamber :
1.Twin trough chamber 2.Rectangular chambers 3. V-shaped chambers 4.Sandwitch chamber 5.Horizontal development chamber 6.Automatic development chamber

1.Twin trough chamber

2.Rectangular chambers

Problem encountered during chromatographic development :

Tailing Diffusion
Tailing :
This is occur due to the presence of traces of impurities or presence of more than one ionic species of substance being chromatographed. This is reduced by buffering the mobile phase system with acidic (1-2% acetic acid) or basic (ammonia) solution.

Diffusion :
This may aries due to non-uniformity of mobile phase, longitudinal diffusion between mobile phase & stationary phase .

j) Detection of Spot (Evaluation) Generally, detection by iodine vapour Alternatively by Visual detection at 254nm UV-Light The detection sensitivity depends on the specifying for the reagent employed. Iodine is the universal detection reagent. Both spraying & dipping technique are used for applying detection reagents.

k) Scanning and 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. 4. Suppliers name, item number, batch no. , individual plate no. are imprinted near upper edge of precoated plates. This will not only help in traceability of analytical data, but will also avoid manipulation of data at any stage as coding will automatically get recorded using photo-documentation.

 The purpose of the scanner is to convert spot /band on the layer into chromatogram . The position of the scanned peak on the recorder chart are related to Rf values of the spot on the layer & peak height . The signal which are measured represent the adsorption of transmitted or reflected light that passes through the spot compaired to blank portion of the sorbent layer. To carry out HPTLC densitometric analysis, three or four standard and purified samples are applied on the same plate. After development , chromatogram is scanned. The calibration curve consisting of scan area standard VS amount of analyte is construced & amount of analyte in the sample represented by scan area is interpolated from standard curve. Depending upon the analytical problem two types of calibration: Single level calibration and double level calibration . Single level calibration utilized in stability testing, dissolution profile and multi-level calibration used in linear regression, non-linear regression etc.

Factor affecting HPTLC

Type of stationary phase Mobile phase Layer thickness Temperature Mode of development Amount of sample Dipping zone others

Applications of HPTLC:
Pharmaceutical industry: Quality control, content uniformity, uniformity test, identity/purity check. Food Analysis: Quality control , additives , pesticides ,stability testing ,analysis of submicron levels of aflotoxins etc Clinical Applications: Metabolism studies , drug screening ,stability testing etc Industrial Applications; Process development and optimization, In-process check ,validation etc. Forensic : Poisoning investigations

 HPTLC-

Quantitative analysis of pharmaceutical Formulations by P.D. Sethi www.pharmainfo.net http://images.google.co.in/images?q=hpt lc+plates&ie=ISO-8859-1&hl=en http://images.google.co.in/images?svnum =10&hl=en&lr=&ie=ISO-8859-1&q=linomat www.camag.com http://www.infoexpo.ch/abstract

QUESTIONS :
Q) Explain tech. of HPTLC. What are merit over column

chromatography ? write its application.(20) Ans : HPTLC merit over Column Chromatography:
1) Apparatus is smaller 2) Quantity of material required is lesser 3) Faster procedure 4) Above points make the over all procedure cheaper 5) The combinations of solvents that can be used is more 6) The Rf value can be measured easily, once a suitable visualizing technique is used . 7) All these makes it better suited for analytical purposes (but not for separation purposes which is actually one of its disadvantages

Q) Described the essential component of HPLC & HPTLC ? (20)

Thank

You