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REVIEW ARTICLE Vishal Modi et.al / IJIPSR / 4 (3), 2016, 384-407

Department of Pharmaceutical Chemistry ISSN (online) 2347-2154

Available online : www.ijipsr.com April Issue 384

A REVIEW ON VARIOUS APPLICATIONS OF

HIGH PERFORMENCE THIN LAYER CHROMATOGRAPHY

(HPTLC) IN PHARMACEUTICALS

1Vishal Modi*, 2SanchitaBhatkar, 3Parixit Prajapati, 4TarashankarBasuri

1,4Head of Department, Department of Pharmaceutical Chemistry, SSR College of Pharmacy, sayli road, silvassa-396230, INDIA

2Department of Quality Assurance Techniques, SSR College of Pharmacy, sayli road, silvassa-396230, INDIA

3Department of Pharmaceutical Chemistry, SSR College of Pharmacy, sayli road,

silvassa-396230, INDIA

Corresponding Author:

Vishal Modi

Head of Department, Department of Pharmaceutical Chemistry,

SSR College of Pharmacy,

sayli road, silvassa-396230, INDIA

Email: [email protected]

Phone: +91 9824931330

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

High performance thin layer chromatography (HPTLC) is automated and sophisticated form of thin layer

chromatography (TLC). For the past few decades, due to the vast chemical diversity compounds natural

sources have been gaining importance. This led to phenomenally increase in the demand for herbal drug

medicines and need has been felt for ensuring the quality, safety, and efficacy of herbal drugs. Using

modern analytical techniques, it need small amount of sample for detection. This review has an attempt to

focus on the application of HPTLC with some examples of drugs, herbal drugs and

formulations analyzed by HPTLC.

Keywords: High Performance Thin Layer Chromatography (HPTLC), Drugs, Herbal formulation.




INTRODUCTION

Chromatography means the group techniques used for the separation of a complex mixture of

compounds by their distribution between two phases, was invented by Russian botanist Mikhail

Semyonovich Tswetin 1901, during his research on plant pigments. Other separation method is

not as powerful and applicable as in chromatography. It is the most simple, versatile technique

which is employed in analytical chemistry and pharmaceutical analysis. The fact that has genuine

reasons is,

1) To all types of chromatography and material, sensitive methods of detection are available that

can be separated in small quantities, in which it identified and assayed from the complex

mixtures.

2) relatively, chromatographic separations are fast and completion of an analysis can be done in a

short interval of time.

Finally, to achieve good accuracy and precision the procedure is well established to controlled

and the apparatus is well maintained[1].

High performance thin layer chromatography (HPTLC) basically depends upon the full

capabilities of thin layer chromatography (TLC). As it is useful in analysis of qualitative method

and it combines the art with quickness at a moderate cost of chromatography[2].

The parameters that affect the separation of individual constituents present in a complex mixture

in which it includes: partition coefficient, retention factor and capacity factor of the individual

constitutes on the plate, selectivity of the mobile phase and stationary phase to the solutes, and

height of the plate that decide the separation efficiency and also the resolution of the individual

constituents within a complex mixture.

The partition coefficient is the analytes molar concentration in the stationary phase to that in

the mobile phase.

Retention factor Rf , a fundamental qualitative value and is expressed as the ratio of migration

distances of a mix relative to the mobile phase.

Capacity factor k, a fundamental characteristic that determines its qualitative chromatographic

behaviour and it can be shown as the ratio of the retention time in stationary phase to that in

mobile phase and is influenced by chemical nature of two phases[3].




The efficacy of separating two components from mixture in a chromatogram is termed as

resolution and is influenced by the selectivity of the individual components between the stationary

and the mobile phase [1,4].

PRINCIPLE

HPTLC take place with high speed capillary flow range of the mobile phase. There are three main

steps that includes,

Sample to analysed to chromatogram layer volume precision and suitable position are

achieved by use of suitable instrument

Solvent migrates the planned distance in layer by capillary action in this process sample

separated in its components.

Densitometer is used for scanning separation tracks with light beam in visible or UV region.

FEATURES OF HPTLC [4,5]

Several analysts works simultaneously.

Lower analysis time and less cost per analysis.

Cost effective.

No prior treatment for solvents (filtration and degassing).

Per sample low mobile-phase consumption.

Visual detection possible.

Compounds unabsorbed by UV are detected by post chromatographic derivatization.

Several analysts work simultaneously on the system.

Availability of a good range of the stationary phases with unique selectivity. Chromatographic

layer requires no regeneration as TLC/HPTLC plates are disposable.

Repetition of densitometric evaluation of the same sample under different conditions can be

achieved by not repeating the chromatography for optimize quantification.

Samples rarely require clean up.

Minimum use of solvent because the required amount of mobile phase on each sample is

small.

Accuracy and precision of quantification is high.

Use of different universal and selective detection methods [4].




Advantages

samples can be detected in quantities such as nano-gram range,

due to automation minimum handling and human errors,

better accuracy and sensitivity.

Disadvantages

bulky instrumentation, require large space, has many folds expensive,

requires stringent condition of operation like dust free environment, and

technically skilled person with the knowledge [1,5].

STATIONARY PHASE:

HPTLC uses small particles along with a distribution of narrow size in HPTLC plates and as

a result with a homogenous layers smooth surface can be obtained.

Smaller plates dimensions having 10 × 10 or 10 × 20 cm are uses in HPTLC with significantly

decreased development distance (6 cm) and analysis time of 7–20 min.

HPTLC plates provide improved resolution and in situ quantification, higher detection

sensitivity and are used for industrial pharmaceutical analysis.

Normal phase adsorption TLC on silica gel along with a less polar mobile phase, such as

chloroform– methanol, has been mostly used. Hydrocarbon- impregnated silica gel plates,

lipophilic C-18, C-8, C-2; phenyl chemically-modified silica gel phases; developed with a

more polar aqueous mobile phase.

Other pre-coated layers are used that includes cellulose, aluminum oxide, magnesium oxide,

magnesium silicate, polyamide, kieselguhr, ion exchangers, and polar modified silica gel

layers containing bonded amino, diols, cyano, and thiol groups. Optical isomer separations are

carried out on a chiral layer which can be produced from C-18 modified silica gel

impregnated with a Cu (II) salt and an pure hydroxyproline derivative on a silica layer with a

chiral selector such as brucine, on molecularly imprinted a-agonists polymer, or on cellulose

with mobile phases having added chiral selectors.

To prepare layers, mixtures of sorbents have been used with special selectivity properties.

HPTLC plates need to be stored under appropriate conditions[6].




MOBILE PHASE:

Basically, the selection of mobile phase is depends upon adsorbent material used as stationary

phase and physical as well as chemical properties of analyte. General mobile-phase systems

are used based on their diverse selectivity properties are methylene chloride, diethyl ether, and

chloroform combined individually or together with hexane being a strength-adjusting solvent

for normal-phase TLC. For strength adjustment methanol, acetonitrile, and tetra hydro furan

mixed with water in reversed-phase TLC.

Ion pairing seperations are done on C-18 layers with a mobile phase that is methanol–0.1 M

acetate buffer (pH 3.5) containing 25 mM sodium pentane sulfonate (15:4.5). Accurate

volumetric measurements of components of the mobile phase must be performed precisely

and separately in adequate volumetric glassware and shaken to ensure proper mixing of the

content. With the help of suitable precised micropipette volumes are measured smaller than 1

ml. Volumes larger than 20 ml are measured with appropriate size of a graduated cylinder. To

minimize volume errors, developing solvents are prepared that is sufficient for one working

day[6].

CLASSIFICATION OF HPTLC:

Figure no.1: Classification of HPTLC

HPTLC techniques are classified into four classes i.e. Classical, High performance, Ultra thin-

layer and preparative chromatography. They differ in the particle size distribution and thickness

of the sorbent layers with classical TLC. For classical, high-performance and preparative thin-




layer chromatography, the mean particle sizes are 5, 12, and 25 μm respectively, whereas Ultra-

thin layer chromatography have a monolithic layer with 1–2 um macro-pores[7]. The thickness of

the sorbent layers which is 0.5–2 mm, 10 um, 200 um, 250 um for preparative, ultra-thin, high-

performance, and classical sorbent layers, respectively[8].

Common Methodology for HPTLC Analysis: For a qualitative and quantitative analysis

method development in thin-layer chromatography is the most significant steps. During a

new analytical procedure, always starts with wide literature survey[9] i.e. primary

information about the physicochemical characteristics and nature of the sample that

includes structure, polarity, volatility and solubility[10].

Selection of the stationary phase-

Stationary phase selection depends upon the type of compounds to be separated[11]. HPTLC

plates provide advanced and improved resolution and in situ quantification and detection of

higher sensitivity[12,13].

Mobile phase selection and optimization-

The selection of mobile phase is depend upon an stationary phase adsorbent material and

physical and chemical properties of analyte[14,15].

The Table no. 1 gives the details of mobile phase that is used in detection of some chemical

compounds.

Table no. 1: Mobile phase generally used in detection of some chemical compounds

Sr.

No.

Chemical Compounds Mobile Phase

1. Polar compounds:

Anthraglycosides, Arbutin,

Alkaloids, Cardiac Glycosides,

Bitter Principles, Flavonoids,

Saponin.

Ethyl Acetate: Methanol: Water [100:13.5:10]

2. Lipophilic Compounds:

Essential oils, Terpenes,

Coumarin, Napthoquinons,

Toluene: Ethyl Acetate [93:7]




Velpotriate

3. Alkaloids Toluene: Ethyl Acetate: Diethyl Amine [70:20:10]

4. Flavonoids Ethyl Acetate: Formic Acid: Glacial Acetic Acid: Water

[100:11:11:26]

5. Saponins Chloroform: Glacial Acetic Acid: Methanol: Water

[64:32:12:8]

6. Coumarins Diethyl Ether: Toluene [1:1] Saturated with 10% Acetic

Acid

7. Cardiac Gylcosides Ethyl Acetate: Methanol: Water [1000:13.5:10] Or

[81:11:8]

8. Bitter Drugs Ethyl Acetate: Methanol: Water [ 77:15:8]

9. Essential Oils Toluene: Ethyl Acetate [93:7]

10. Lignans Chloroform: Methanol: Water [70:30:4]

Chloroform: Methanol [90:10]

Toluene: Ethyl Acetate [70:30]

11. Pigments Ethyl Acetate: Formic Acid: Glacial Acetic Acid: Water

[100:11:11:26]

12. Pungent Testing Toluene: Ethyl Acetate [70:30]

13. Terpenes Chloroform: Methanol: Water [65:25:4]

14. Triterpens Ethyl Acetate: Formic Acid: Formic Acid [50:50:15]

Toluene: Chloroform: Ethanol [40:40:10]

PROTOCOL USED FOR DRUG ANALYSIS[1]:

1. Selection of Chromatographic Layer

2. Sample and Standard Preparation

3. Activation of Precoated Plates

4. Application of Sample and Standard




5. Selection of Mobile Phase

6. Preconditioning (Chamber Saturation)

Figure no. 2: Steps involved in HPTLC

1. Selection of Chromatographic Layer

With different supporting materials i.e. plastic, aluminium, sorbent layers and glass, the precoated

plates are available in different thickness and formats. Usually plates with solvent thickness of

100–250 mm are usually available for qualitative and quantitative analysis.

Commonly available precoated plates are:

More than 80% of analysis is done on silica gel 60F plates.

Aluminum oxide: Basic substances, alkaloids and steroids are available.

Microcrystalline cellulose: Basically Amino acids, sugars, antibiotics are available

RP-2, RP-8and RP-18: These are the chemically modified silica gel plates commonly used for

analysis of steroids, carotenoids, fatty acids, and cholesterol.

2. Sample and Standard Preparation

From impurities, low signal-to-noise ratio, straight base line and improvement of limit of

detection (LOD) are employed to avoid interference. If noise contribution from the electrical and

optical noise can be lowered then an increase in signal-to-noise (S/N) ratio can be resulted. Below




250 nm, the main cause of signal variation. Reduction of layer thickness from 0.25 to 0.15 mm is

an alternative and preferred method of maintaining the light flux for a favourable S/N ratio.

Solvents used for sample preparations are ammonia, methanol, chloroform, ethyl acetate,

methylene chloride or acetic acid.

A good solvent system is one that does not put anything on the solvent front, but moves all

components of the mixture off the baseline. Between Rf 0.15 and 0.85 range, the peaks of interest

should be resolved. The elution power of the mobile phase depends on a property called eluent

strength[16]. The more non-polar the compound, the faster it will elute than that to the polar.

3. Activation of Precoated Plates

Freshly open box of plates does not require any activation. Plates which are kept on hand for long

time requires activation. The plates is activated by placing the plates in an oven at 110–120°C for

300 prior to spotting. Aluminium sheets should be placed in between two glass plates and kept in

oven at 110–120°C for 15 min[17].

4. Application of Sample and Standard

The concentration range is 0.1–1 mg/ml; above this ranges it lead to poor separation. Sample and

standard can be sprayed or applied with nitrogen gas sprays on TLC plates as bands or spot by

using automatic sample applicator[18].

With sufficiently high concentration of analyte, Pharmaceutical preparation is simply dissolved in

a suitable solvent that will solubilize the analyte[17,20]. It is a critical step of application of the

sample and to obtain good resolution for quantification in HPTLC[18]. Sample application

techniques depend on factors like the type of workload, sample matrix and time

constraints[19,20].

5. Selection of Mobile Phase

Poor grade of solvent used in mobile phases preparation was found to decrease resolution, Rf

reproducibility and spot definition. It also based on one’s own experience and literature. Taking

into consideration, sorbent layer mobile phase and the chemical properties of the analytes should

be chosen. Using three or four components in mobile phase should be avoided as it is often

difficult to get the reproducible ratios of different components[21].




6. Preconditioning (Chamber Saturation)

On the separation profile, chamber saturation has pronounced influence. High Rf values are cause

by unsaturated chamber. Filter paper lining for 30 min prior to development in saturated chamber

leads to uniform distribution of solvent vapours and less solvent requires for the sample to

travel[22].

7. Chromatographic Development and Drying

Although chromatogram development is the most crucial step in the HPTLC procedure14. In twin-

trough or horizontal-development chambers, HPTLC plates are developed. For the best

reproducibility, saturated twin-trough chambers are fitted with filter paper. Twin-through

chamber avoids solvent vapour preloading and humidity[23,24]. It is, therefore, for completely

automatic development the AMD system (Automated multiple developments) is now available. In

classical sense with fixed plate positioning the development is always made ascending, and on

each run only 8 ml mobile phase have been used. The distance of development is determined by

the time control. Until all chromatographic steps have been completed the chamber is not opened.

8. Detection and Visualization

Under detection of UV light is first step and is non-destructive. Spots of fluorescent compounds

can be seen at 254 nm i.e. short wave length or at 366 nm i.e. long wave length. Fluorescent

stationary phase such as silica gel GF, spots of non-fluorescent compounds can be used. Non-UV-

absorbing compounds being visualized by using 0.1% iodine solution. If individual component

does not respond to UV, then derivatization is needed with visualizing agent.

By quenching of fluorescence due to UV light (200-400 nm) detection of separated compounds

on the sorbent layers is enhanced. This process is commonly known as Fluorescence quenching.

Visualization at UV 254 nm: F254 should be described as phosphorescence quenching. In this

instance, after the source of excitation is removed the fluorescence stays for a short period. It is

longer than 10 seconds but, very short lived. With UV wavelength at 254 nm, F254 fluorescent

indicator is excited and emits green fluorescence. Compounds that absorb radiation at 254 nm

reduce this emission on the layer, and the compound zones are located where a dark violet spot on

a green background is observed. This quenching is caused with conjugated double bonds by all

compounds. Anthraglycosides, coumarins, flavonoids, propyl phenols in essential oils, some




alkaloid type such as indole, isoquinoline and quinoline alkaloids etc. should be detected under

254 nm[15,25].

Visualization at UV 366 nm: F 366 should be described as Fluorescence quenching. In this after

the source of excitation is removed, instance the fluorescence does not remains21. This quenching

is shown by all anthraglycosides, coumarins, flavonoids, phenolcarboxylic acids, some alkaloid

types (Rauwolfia, Ipecacuanha alkaloids)[15].

Visualization at white light: By viewing their natural color in daylight (White light) zone

containing separated compounds can be detected[26].

Derivatization:

Derivatization is a procedural technique that modifies functionality of an analyte’s to enable

chromatographic separations[27]. Derivatization can be performed either by spraying the plates

with a suitable reagent[28,29]. For better reproducibility, immersion is the preferred

derivatization technique.

Quantification:

Sample and standard sample should be chromatographed on same plate and after development,

chromatogram is scanned. Densitometry is a simplest way of quantifying the desired sample

components directly applied on the plate. The resolution of compounds to be separated on the

chromo-plate is followed by measuring the optical density of the separated spots directly on the

plate. Under the same condition the sample amounts are determined by comparing them to a

standard curve from reference materials. The evaluation of original data using the conventional

methods of scanning by measuring the transmitted light by the optical density as a function of the

concentration of the sample or standard that is delivered on stationary phase. The scanning

densitometer is an advanced workstation for evaluation of TLC/HPTLC and by measuring the

objects by absorbance of fluorescence[30].

VARIOUS APPLICATIONS OF HPLTC:

HPTLC is widely used and applied methods for the analysis in clinical chemistry, biochemistry,

pharmaceutical industries, forensic chemistry, food and drug analysis, environmental analysis,

cosmetology and also in other areas. Due to its various and number of advantages. Other than this




include simplicity, low costs, high sample capacity, parallel analysis of samples, rapid results and

multiple detection. Le Roux et al[31] evaluated that in clinical trials, HPTLC technique is used for

determination of salbutamol serum levels and established as a proper method for determining

samples from the serum. In HPTLC, many lipids have also been analyzed and studied; 20

different lipid subclasses were separated with the reproducible and promising results. Related to

clinical medicine many reports on studies have already been published in many journals. In the

analysis of drugs in serum and other tissues HPTLC is now strongly recommended[32].

Applications of HPTLC include quantification of active ingredient and herbal drugs, phyto-

chemical and biomedical analysis, fingerprinting of formulations, and check of presence of

adulterants in the formulations.

HPTLC IN PHARMACEUTICAL PRODUCTS:

HPTLC is also used in many pharmaceutical preparations and dosage forms for analyzing the

purity and efficacy. Puranik et al developed and in solid dosage form a simultaneous

determination of ofloxacin and ornidazole has been done which is a simple, rapid and accurate

chromatographic methods (HPLC and HPTLC). The amount of ofloxacin and ornidazole

determined as percentage of label claimed that was found to be 100.23% and 99.61% with mean

percent recoveries that is 100.47 and 99.32%, respectively. Both these methods are simple,

precise, accurate, selective, and rapid and also successfully applied for the determination of

prepared mixtures and tablets and capsules[33]. In pharmaceutical preparations method was

established for analysis of celecoxib, etoricoxib, and valdecoxib. Małgorzata Starek et al reported

that the procedure can be used for selective analysis of drugs, and from auxiliary substances

repeatable results are obtained without interference[34]. HPTLC method was used to analyze

samples of fixed-dose tablets of lamivudine, stavudine, and nevirapine[35]. Accurate and precise

two simple HPTLC methods were established in Mexicord capsules for the determination of

mexiletine hydrochloride[36]. Determination of olanzapine on silica gel 60F252 layers uses

mobile phase as a methanol-ethyl acetate (0.8+0.2, v/v) developed and validated for quantitative

analysis[37]. Further, both as a bulk drug and in tablet formulation analysis paracetamol,

diclofenac potassium, and famotidine was developed and validated[38].




According to the ICH guidelines the method was validated for linearity, accuracy, precision,

specificity, and robustness. This method is readily used for QC and stability testing of different

dosage forms like tablets and capsules, and also for bulk drug analysis of omeprazole[39,40].

HPTLC APPLICATIONS IN DRUG ANALYSIS

The details regarding HPTLC determination of pharmaceutical products in various formulations

are given in Table 2.

Table no. 2: HPTLC determination of pharmaceutical products in various formulations

Drug Dosage form Technique used Reference

Moxonidine Tablet Stationary phase:Silica gel, 60F 254

Mobile phase:

methanol:toluene:triethylamine(4:6:0.1(v/v/v).

Densitometricquantification at 266 nm.

Kakde et

al.,

2012

Cefixime

Trihydrate and

Ambroxol

Hydrochloride

Pharmaceutical

dosage form

Stationary phase: silica gel 60 F254.

Mobile phase: acetonitrile:methanol:triethylamine

(8.2:1:0.8, v/v/v).

Densitometricmeasurements of spots at 254 nm

Deshpande

et al.,

2010

Ofloxacin and

Ornidazole

Solid dosage

Form

Stationary phase: silica gel60F254.

Mobile phase: dichloromethane:

methanol: 25% ammonia solution (9.5:1:3 drops v/v)

Detection was carried out at 318 nm. The mean R f value

of ofloxacin and ornidazole

was found to be 0.16 and 0.56, 0.78

Puranik et

al.,

2011

Mexiletine

Hydrochloride

Mexicord

Capsules

Stationary phase: horizontal chambers on RP C18F254s

and normal phase amino (NH2) HPTLC.

Mobile phase: chloroform:tetrahydrofuran: hexane:

ethylamine (3:2:5:0.1 v/v/v/v).

Densitometricmeasurements were achieved in the UV

Pietra et

al.,

2011




mode at 217 nm.

Olanzapine Formulations Stationary phase: silica gel 60F254.

Mobile phase: methanol:ethylacetate (8:2, v/v).

Olanzapine was quantified by densitometric analysis at

285 nm.

Patel et al.,

2010

Omeprazole Capsule dosage

Form

Stationary phase: silica gel 60F254.

Mobile phase: chloroform:methanol (9:1v/v).

Densitometric analysis at 302 nm .Rf

value of 0.39 +/- 0.12.

Jha et al.,

2010

Lamotrigine Tablets Stationary phase: silica gel 60F254.

Mobile phase: toluene:acetone:ammonia (3:7:0.5, v/v/v).

Densitometric detection at

312 nm.

Koba et al.

2013

Exemestane Bulk and

pharmaceutical

dosage form

Stationary phase:silica gel 60 F254.

Mobile phase: chloroform:methanol(9.2:0.8 (v/v).

Densitometric scanning at 247 nm.

Mane et al.,

2010

Itraconazole Bulk drug and

in

pharmaceutical

dosage form.

Stationary phase: aluminium plate precoated with silica

gel 60F254.

Mobile phase: Toluene:Chloroform:Methanol [5:5:1.5

(v/v)].

Densitometric analysis at 260 nm. Rf 0.52 ± 0.02.

Parikh et

al.,

2011

Aceclofenac Tablet

formulation

Stationary phase: Aluminium backed silica gel 60 F254

plate.

Mobile phase: toluene: ethyl acetate: glacial acetic acid,

(6:4:0.02v/v.

Densitometry analysis at 282 nm.

Suganthi et

al.,

2013




HPTLC IN HERBAL PRODUCTS:

HPTLC is an ideal screening tool for adulterations and for evaluation and monitoring of

cultivation, extraction processes and testing of stability. Diterpenoids 1β,3α,8β-trihydroxy-

pimara-15-ene(A),6α,11,12,16-tetrahydroxy-7-oxo-abieta-8,11,13-triene (B) and 2α,19-

dihydroxy-pimara-7,15-diene (C) are used as chemical markers for the standardization of

Photiniaintegrifolia plant extracts[41].

A simple HPTLC method has been developed for the simultaneous determination of isovitexin,

orientin, vitexin and isoorientin both pure and in commercial samples of bamboo-leaf flavonoids

and is also used for manufacturing QC of bamboo-leaf flavonoids[42]. Many reports present the

evidence of utilization of fingerprinting analysis in HPLTC of natural origin of drugs, thus the

increasing acceptance of natural products is well suited and provide the core scaffolds for future

drugs[43-49].

Table no. 3 HPTLC determination of herbal plants

Active

constituent

Herbal plant Technique used Uses

Androgphlide

and

Wedalolactone

Andrographispaniculata

and Eclipta alba

Stationary phase: Precoated s ilica gel 60

F254 plates.

Mobile phase: Toluene: Acetone:

Formic Acid (9:6:1)

Detection wavelength: 254nm

Hepato-protective

formulations

Phyllanthin

and

Hypophyllanth

in

Phyllanthus Stationary phase: Per activated silica gel

60 F254 plates.

Mobile phase: Hexane: Acetone: Ethyl

Acetate (24:12:8)

Detection wavelength: 580 nm

Rf value: 0.24 and 0.29

Viral infections, liver

disorders, bacterial

infections.

Eugenol Ocimum sanctum Stationary phase: Aluminium-blacked Cardiopathy, Blood

disorders, Asthma,




silica gel 60 F254 plates.

Mobile phase: Toluene: Ethyl Acetate:

Formic Acid

(90:10:1v/v)

Densitometric measurement: 280nm. Rf

value is 0.77

Bronchitis, Skin

diseases.

Hyperforin Hypericumperforatum Stationary phase: silica gel 60 F254

plates.

Mobile phase: Petroleum Ether: Ethyl

Acetate (90:10)

Anti-inflammatory,

Anti-depressant,

Healing agent

Corosolic acid Lagerstroemia speciosa Mobile phase: Chloroform: Methanol

(9:1)

Plates scanned at 20nm

Antidiabetic activity

Harding grass Phalarisaquatical Stationary phase: silica gel 60 F254

precoated zone HPTLC glass

Mobile phase: Acetate: Chloroform: 7N

NH4OH in Methanol (8:2:2,v/v/v)

Bladder diseases

Valerenic Acid Valerianajatamansi

and Valerianaofficinalis

Stationary phase: Precoated silica gel

60F254Aluminum plates Mobile phase:

Hexane: Ethyl Acetate: Acetic Acid

(80:20:0.5 v/v)

Anddensitometric determination was

carried out after derivatization with

anisaldehyde–sulphuric acid reagent at

700 nm, in absorption–reflectance mode.

Sedative and

spasmolytic activity

Rutin Amaranthusspinosus

Linn

Stationary phase: silica gel 60

F254 HPTLC plates

Mobile phase: Ethyl Acetate:

Formicacid: Methanol: Distilled water in

Antidiabetic,

antithrombotic,

antiinflammatory,

and anticarcinogenic




the proportion 10:0.9:1.1:1.7 (v/v). activity.

HPTLC IN OTHER FIELDS:

In recent years, the developing world shows HPTLC as a globally accepted practical solution for

characterization of small molecules in quality assessment. It is used for steroids, pesticides and

purity control of chemicals[50] and also used for analysis of vitamins, water-soluble food dyes,

pesticides in fruits and vegetables and also in other stuff[51,52] the analysis of stem cell lipids by

offline HPTLC-MALDI-TOF MS. Other than this Chemicals of forensic concern[53,54]

including abuse drugs, adulterations, poisons, chemical weapons, and illicit drugs are also

detected.

HPTLC AS BIOMARKER IN PHARMACOGNOSTICAL RESEARCH:

Indian Systems of Medicine has been performed for various pharmacological activities such as

hepatoproctative[55] in many plants HPTLC analysis is carried out. It used as a rapid method to

control raw plant material quality and formulations based on the Lawsoniainermis plant[56].

Micheliachampaca L. is commonly known as champa that is a reservoir of numerous bio-

markers[57].

HPTLC method for the estimation of curcumin can be used routinely with good reliability and

reproducibility in marketed turmeric powder[58]. HPTLC method is also a very powerful tool for

identification of adulterants in herbal products which is based on the characteristic image

produced and for determining the presence and the quantification of both inadvertent substitution

as well as intentional adulteration of prescription drugs.

HPTLC IN THE FORENSIC SCIENCES

Thiopental is used as an induction agent as general anesthesia and also manage the intracranial

pressure in traumatic brain injuries. The potential for accidental or intentional abuse of thiopental

is high because of rapid onset of action. A HPTLC method has been reported for the thiopental

evaluation in the post-mortem blood by simple and rapid HPTLC. High amount of thiopental in

post-mortem blood which can help to conclude that fatal doses of thiopental caused the death is




revealed analysis of toxicological. Exstesy tablets, seized from around Jakarta by narcotic police,

were physically characterized and chemical profiled by HPTLC-densitometry[59].




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