HPTLC- Piyush Seminar (Final)

- Piyush L.

Agrawal M.pharm

– (Q.A.)

HPTLC- High Performance Thin Layer Chromatography

INDEX

Monday, April 10, 2023P.L.Agrawal2

• Chromatography?• Why HPTLC over TLC • Why HPTLC over HPLC• Theory & Principle of HPTLC Instrumentation• Comparative specifications of HPTLC• Steps involved in HPTLC• Application and advancement in HPTLC

Introduction:

Monday, April 10, 20233

Chromatography:- Chromatography is a non-destructive procedure for resolving a multi-component mixture of trace minor or major constituents into its individual fractions.

chromatography can be applied both qualitative & quantitatively but it is primarily a “Separation technique”

Chromatography may be defined as a method of separation in which separating a mixture of component through equilibrium distribution between two phases.

P.L.Agrawal

Chromatography technique is based on-


Difference in the rate at which the components of a mixture moves through a porous medium (stationary phase) under the influence of some solvent or gas(mobile or moving phase) It involves following steps-

Adsorption or retention of a substances on a stationary phase

Separation of the adsorbed substances by a mobile phase

Recovery of the separated substances by a continuous flow of mobile phase (Elution)

Qualitative & quantitative analysis by eluted substances

Chromatography

P.L.Agrawal5

GLC- is that the sample to be analyzed or its derivative must be volatile & stable within possible temperature range

HPLC-require solubility in appropriate solvent & sample must be free from all insoluble substances

TLC-TLC is subjected to least limitations. TLC is practiced in two forms, first by using it as a qualitative tool for separation of simple mixtures with speed & low cost and second by using it as a powerful separation tool for quantitative analysis with high sample through out ,which is now referred as HPTLC. Monday, April 10, 2023

• A sophisticated and automated form of TLC.

Principle of Chromatography:

The principle underlying C-techniqueThe basis of all forms of chromatographic is the ”partition or distribution co-efficient (Kd) which describe the way in which compound distribution itself between two immiscible phase i.eKd = Concentration in solvent-A of compd.

Concn. Of compound in solvent BThe term effective distribution coefficient is defined as the total amount of substance present in one phase divided by the total amount present in other phase.


Main Difference of HPTLC


TLC HPTLC Layer of Sorbent

•250µm •100µm

Efficiency •Less•High due to smaller particle size generated

Separations •10 - 15 cm •3 - 5 cm

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

Solid support•Silica gel , Alumina & Kiesulguhr

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

Development chamber

•More amount •New type that require less amount of mobile phase

Sample spotting

•Manual spotting

•Auto sampler

Scanning •Not possible

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

Why HPTLC over HPLC?


Parameters HPLC HPTLCSimultaneous Processing

Simultaneous processing of sample & standard is not possible, I.s. is frequently required for precision & accuracy of analysis

Simultaneous processing of sample & standard under similar conditions leads to better analytical precision & accuracy of analysis ,Less need for Internal std.

Flexibility Limited Flexibility Extreme flexibility for various steps

Operating skills Skilled & well trained personnel are essential

Less skill is needed for operating HPTLCTechnically, it is simple to learn & operate

High sample Throughput

Even with total automation, HPLC cannot compete with HPTLC in terms of sample output in given time

High sample Throughput of similar or different nature of samples, hence lower analysis time and less cost per analysis

Cost HPLC columns are very expensive Low cost pre-coated HPTLC plates/rolls are available

Sample preparation

Sample preparation is the most critical, is laborious & time consuming and may require membrane filtration in some cases

Sample preparation is very simple

Solvent system Degassing/filtration of solvent is absolutely essential

Solvents need no prior treatment like filtration and degassing

Sample application

Normally fix volume is selected Injected/Introduced Manual as well as automatic.

Variable volume can be applied Spot or band Manual or automatic

Solvent grade Solvents of only HPLC grade are recommended to be used.

Solvents of analytical grades are suitable.


Method to be used for analysis depends upon several parameters like –

• Solubility or Volatility of sample• Concentration of analyte• Detection limit• Cost of ANALYSIS• No. of samples under analysis• Sample prepn. & Separation technique

Features of HPTLC


Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard

Several analysts work simultaneously Lower analysis time and less cost per analysis Low maintenance cost Simple sample preparation - handle samples of

divergent nature No prior treatment for solvents like filtration and

degassing Low mobile phase consumption per sample No interference from previous analysis - fresh stationary

and mobile phases for each analysis - no contamination Visual detection possible - open system

SOP for HPTLC


Plate material & labeling Pre-coated HPTLC plates, 20x10 / 10x10 cm Project number_year/month/day_plate number

Parameters for sample application 8 mm bands, spray-on

Detailed description of development 6 cm, chamber saturation, humidity control

Derivatization Dipping whenever possible

Densitometry MWL scan Scan at the max. WL

Digital documentation UV 254 nm / 366 nm / white light

Advantages of HPTLCFairly simple ,InexpensiveRapid , Extremely flexible and Visual

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

Handling of Plates


• Plate material Handle plates with extreme caution to avoid any damage to the layer.

• Store plates in the original package with the lid closed.

• Remove plate from storage only immediately prior to use.

• Plates are generally handled only at the upper edge to avoid contamination.

• Unless otherwise necessary Merck HPTLC plates silica gel 60 F 254 in the format 10x10 cm or 20x10 cm are used.

• For reproducibility studies and quantitative analyses plates are pre-washed.

Preparation and storage of developing solvents


• Developing solvents consisting of more than 1 component are prepared by measuring the required volume (respectively weight) of each component separately and transferring them into a solvent bottle of appropriate size. The bottle is closed with a lid and shaken to ensure proper mixing of the content.

• Volumes smaller than 1 mL are measured with a suitable micropipette.

• Volumes up to 20 mL are measured with a graduated volumetric pipette of suitable size.

• Volumes larger than 20 mL are measured with a graduated cylinder of appropriate size.

• To minimize volume errors developing solvents are prepared in a volume that is sufficient for one working day.

Sample and Standard Preparation


• To avoid interference from impurities and water vapors

• Low signal to noise ratio - Straight base line- Improvement of LOD

• 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 • Dry the plates and store in dust free atmosphere

MobilePhase


The mobile phase is the solvent or solvent mixture moving through the stationary phase on the TLC/HPTLC plate during development.

Mobile phase should be chosen taking into consideration chemical properties of analytes& sorbentlayers.

Use of mobile phase containing more than three or four components should be avoided as it is often difficult to get reproducible ratios of different components.

Advantages:Mobile phase evaporates before derivatizationDoes not interfere with determination of the position of solute

spots/ bandsSmaller volume of mobile phase required

Selection of mobile phase


Trial and error - one’s own experience and Literature Normal phase : Stationary phase is polar, Mobile phase is non

polar Polar compounds retained because of higher affinity with the

stationary phase while Non-polar compounds eluted first as low affinity

-Reversed phase: Stationary phase is non polar, Mobile phase is polar

- Polar compounds eluted first because of lower affinity with stationary phase while Non-Polar compounds retained

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 10 -15 ml of mobile phase is

required Components of mobile phase should be mixed introduced into the

twin - trough chamber

Dipping is done by specially designed chambers designated as A, B, C each containing 10ml of cleaning solvent. The plates to be washed are dipped into each one after the other keeping for 1 min and eventually discarding the earlier solvent.

Continuos mode-Excellent results are obtained with continuos mode technique in which the plates are washed in a chamber closed by a lid having a slit.


Activation of pre-coated plates


• Freshly open box of plates do not require activation

• Plates exposed to high humidity or kept on hand for long time to be activated by placing 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.

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.

Development


• Plates are developed in a saturated Twin Trough Chamber according to the following procedure:1. Prepare the appropriate volume (10 mL for 10x10 cm,

20 mL for 20x10 cm TTC) of developing solvent.2. Open chamber and place a correctly sized (10x10 cm;

20x10 cm) piece of filter paper in the rear trough.3. Pour solvent into chamber so that the filter paper is

thoroughly wetted and adheres to rear wall of TTC.4. Tilt chamber to the side (about 45[degrees]) so that

the solvent volume in both troughs equalizes.5. Set chamber on bench, replace the lid and let

chamber equilibrate for 20 minutes.6. Mark the desired developing distance (70 mm from

lower edge of plate) with a pencil on the right edge of the plate.

Application of sample and standard


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 HP TLC plates as bands · Band wise application - better separation - high response to densitometer

Linomat IV auto sampler

Development


1. Slide off the lid to the side.2. Insert the plate into the front trough. The layer

should face the filter paper and the back of the plate is resting against front wall of CHAMBER.

3. Replace lid.4. Develop plate to the mark.5. Open lid, remove plate.6. Dry the plate (vertically in direction of

chromatography) 5 minutes in a stream of cold air.

7. After each development remaining mobile phase and filter paper are discarded. Prior to being prepared for the next run the chamber is dried and, if necessary, also cleaned.

Chromatographic development Chromatographic development and dryingand drying


After development, remove the plate and mobile phase is removed from the plate

To avoid contamination of lab atmosphereDry in vacuum desiccator - avoid hair drier

- essential oil components may evaporate

Detection and visualization


• 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

• 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

Quantification


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

Scanning speed is selectable up to 100 mm/s - spectra recording is fast - 36 tracks with up to 100 peak windows can be evaluated

Calibration of single and multiple levels with linear or non-linear regressions are possible · When target values are to be verified such as stability testing and dissolution profile single level calibration is suitable

Statistics such as RSD report automatically Concentration of analyte in the sample is calculated by

considering the sample initially taken and dilution factors

Documentation


• E - Merck introduced plates with imprinted identification code - supplier name. Item number, batch number and individual plate number - Avoid manipulation of data at any stage - coding automatically get recorded during photo documentation

• Validation of analytical method All validation parameters such as precision, accuracy, LOD, LOQ, Ruggedness, Robustness can be performed

Factors influencing the HPTLC separation of spots

It is depend on separating efficiency and selectivity of the separating system.

Type of stationary phase (sorbent).Type of precoated plates. Layer thickness. Binder in layer.Mobile phase (solvent system).Solvent purity.Size of developing chamber.



Saturation of chamber. Sample volume to be spoted. Size (diameter) of initial spot. Solvent level in the chamber. Gradiant,RH,Temperature Flow rate of solvent. Separation distance. Mode of development. Proper documentation

Applications of HPTLC :


Separation of Analgesics like, Ascorbic acid, caffeine, paracetamol in methanol (each 1mg/ml).◦ Mobile phase : Dichloromethane-ethyl acetate-

ethanol (50:50:10)◦ Stationary Phase : Plates of silica gel 60F 254

(Merck), Sample volume : 1µl as spot.

In identification of antibiotics, e.g. Isoniazid◦ Mobile Phase : Chloroform- Methanol-Glacial acetic

acid (GAA) (9:1:0.1v/v)◦ Stationary Phase : HPTLC pre-coated plate, silica gel

60F254-aluminium (Marck), Sample volume : 4µl


• It is use in bioequivalence studies , e.g. Azithromycin

Mobile Phase : Dichloromethane-methanol(3:7v/v)─n-hexane-ethylacetate-dietylamine (15:5:2) Stationary Phase : HPTLC pre-coated plate, Kiesel gel 60F254-aluminium (Marck), Sample volume : 20µl

• To calculate percentage recovery of compound, e.g. Propranolol, hydrochlorthiazide

Mobile Phase : toluene-methanol-ethylacetate-ammonia(8:2:1v/v) Stationary Phase : HPTLC pre-coated plate 60F254-glass (Marck), Sample volume : : 10µl


• Multidimensional and Multimodal Separations by HPTLC in Photochemistry in Bulk Drug and Pharmaceutical Dosage

• HPTLC Fingerprint Analysis: A Quality Control for Authentication of Herbal Phytochemicals

• HPTLC in Herbal Drug Quantification

• HPTLC Determination of Artemisinin and Its Derivatives in Bulk and Pharmaceutical Dosage

• Stability-Indicating HPTLC Determination of Imatinib Mesylate

TLC/HPTLC in Biomedical Applications Analytical Aspects of High Performance

Thin LayerChromatography Quantitative Analysis and Validation of

MethodUsing HPTLC Quantification of Low Molecular Mass

CompoundsUsing Thermostated Planar

Chromatography


Advances in analytical methods and novel detection

systems are reviewed from publications from 1995 onwards. This covers aflatoxins B[1], B[2], G[1], G[2], and their metabolites in food, animal feed and biological matrices such as blood and urine.

Improved extraction techniques, new clean-up methods and optimized methods for specific matrices are summarized. It also highlights methods such as thin layer chromatography (TLC) and high performance-TLC (HPTLC), which are particularly suited to developing countries and advances that have been made in TLC quantification through low cost detection and scanning systems.

Novel developments in detection of aflatoxins are assessed such as the application of surface plasmon resonance biosensors, flow injection monitoring, fiber optic sensors, capillary electrokinetics,electrochemical transduction, and immunological-based rapid test kits.



• High performance thin-layer chromatography analysis was used to analyze neutral lipids in the digestive gland-gonad (DGG) complex of the marine snails Ilyanassa obsoletus and Littorina littorea infected with larval trematodes

• For rapid and sensitive screening of lipid biochemical abnormalities of scaling skin disorders a sequential, one-dimensional high-performance thin-layer chromatographic method (HPTLC) has been developed


• High-performance thin layer chromatography (HPTLC) was used to determine neutral lipids in the cecal contents and mucosa of domestic chicks and ICR mice, and in the cecal mucosa of chicks infected with Zygocotyle lunata (Trematoda). • These findings showed that this parasite can alter the neutral lipid composition of the host cecal mucosa• Reproducible results have been obtained by degradative

charring as well as fluorescence detection. By fluorescence detection the method is particularly suitable for the determination of minor amounts of cholesterol sulfate and other sterols.


•All major human stratum corneum lipid classes, i.e., cholesterol sulfate, glucosylceramides, six major ceramide fractions, free sterols, free fatty acids, triglycerides, sterol esters, squalene, and n-alknes, are separated and quantitated after a stepwise development of a single silica gel 60 HPTLC-plate using three consecutive solvent systems

Cyclophosphamide is an alkylating agent widely used from cancer chemotherapy to immunotherapy purposes. In paediatrics oncology, oral cyclophosphamide prescribed at low dosages for a long time treatment is currently investigated. This treatment is a putative well tolerated regimen for children treated for a wide variety of recurrent solid tumours. For these purposes, new oral formulations more convenient for children than cyclophosphamide 50mg tablets are needed. Thus, we present a rapid method for the assay of cyclophosphamide in various pharmaceutical preparations using high-performance thin-layer chromatography (HPTLC)


HPTLC- Piyush Seminar (Final)

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