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 HPTLC TLC

Layer of Sorbent

100µm 250µm

Efficiency High due to smaller particle size generated Less

Separations 3 - 5 cm 10 - 15 cm

Analysis Time Shorter migration distance and the analysis time is greatly reduced

Slower

Solid support Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes

Silica gel , Alumina & Kiesulguhr

Development chamber

New type that require less amount of mobile phase More amount

Sample spotting Auto sampler Manual spotting

Scanning Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer

Not possible

Features of HPTLCFeatures 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) Platesd) Pre – washinge) Conditioningf) Sample applicationg) Pre – conditioningh) Mobile phase i) Chromatographic development j) Detection of spotsk) 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 non-polar 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 seperatedCommonly used material are Silica gel 60 F,

Alumina, Cellulose etc.These material can be coated on hand made

plates with or without bindersPre – coated plate are commercially available &

are commonly used.Pre – coated plate costly than hand made plate.

c) Plates :Size : 20×20cm, 10×20cm, 5×10 cm, 5×7.5 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 µm particle size. HPTLC : 4-8µm 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 µm used for qualitative &

quantitative analysis . 2) Plate size : Pre-coated HPTLC plate size 20×20 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.

Materials Advantage Disadvantage

Glass 1.Ressistant to heat and chemicals2.Easy to handle and offers superior flat surface for work

1. Fragility2.Relatively High wt3.Costs more for additional packaging

Polyester sheets (0.2 mm thick)

1.More economical as produced even in roll forms2.Unbreakable3.Less packing material4.Spots can be cut and eluted thus eliminates dust from scrapping

1.Charring reactions if temperature exceeds 120oc as the plates are dimensionally unstable beyond this temperature

Aluminum Sheets(0.1mm)

1.Increasesed temperature resistance

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

Supports Materials

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

d) Pre-washingsg

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

Solvents used for pre-washing

e) Conditioning : The pre-washed plates or Plates exposed to humidity exposed to humidity & surrounding are need to be activated by placing them in oven at 110110oo-120-120ooc for 30 min, c 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 µl 1-5 µl is most satisfactory for HPTLC.• Sample application carried by Linomat & Linomat & Nanomat Nanomat type applicator on the plate which gives uniform, safe and standard results.

Diag.: Linomat

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

2) CAMAG Linomat

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 0.1-0.2μl.

Diag. :CAMAG NANOMAT

g) Pre –conditioning (chamber saturation) :

•Suppose the plate is introduced into an unsaturated chamberplate 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 chamberthe 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 Low polarity mob. Phase is no need for saturation.• Saturation is desirable in case of highly polar mobile phase.• For reverse phase 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 chamber2.Rectangular chambers3. V-shaped chambers4.Sandwitch chamber5.Horizontal development chamber6.Automatic development chamber

1.Twin trough chamber

2.Rectangular chambers

Problem encountered during chromatographic Problem encountered during chromatographic development development :

• Tailing• Diffusion

•Tailing :•This is occur due to the presence of traces of impurities 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 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 pre-coated 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 calibrationtwo types of calibration: Single Single level calibration level calibration and double level calibration double level calibration .Single level calibration utilized in stability testing, dissolution profile and multi-level calibration used in linear regression, non-linear regression etc.

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

Factor affecting 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 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=hptlc+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)

You”You” “Thank