1. Satish Pradhan Dnyanasadhana College, Thane. Department of Chemistry T
Satish Pradhan Dnyanasadhana College, Thane. Department of Chemistry T.Y.B.Sc. Analytical Chemistry Sem-V UNIT- 2.4 HPTLC

2. 2.4 High Performance Thin Layer Chromatography (HPTLC)
2.4.1 Introduction to HPTLC
2.4.2 Techniques in HPTLC
A) Stationary Phase
B) Sample Application
C) Mobile phase
2.4.3 Determination by Detectors in HPTLC.
Detectors: Single beam densitometer, Double beam densitometer,
Flourimetric Detector
2.4.4 Comparison between TLC and HPTLC
2.4.5 Advantages and Limitations

3. Subjected leads to based on
Sample having various components
Subjected
Interactions Mobile phases, component, stationary phase
leads to
Differential migration of components
based on
Difference in physical and chemical properties of components governs
Relative affinity of components towards stationary and mobile phase thus
Component having less affinity towards stationary phase move fast or via versa
Formation of different bands or zones after traveling different distances

4. Introduction
Recent developments in the practice of thin-layer chromatography have resulted in a breakthrough in performance which has led to the expression 'high performance thin-layer chromatography'.
These developments have not been the result of any specific advance in instrumentation (as with HPLC), but rather the culmination of improvements in the various operations involved in TLC.
The three chief features of HPTLC are summarized below.

5. Stationary Phase
HPTLC can be regarded as the most advanced form of modern TLC. In HPTLC plates featuring small particles with a narrow size distribution. As a result, homogenous layers with a smooth surface can be obtained. HPTLC uses smaller plates (10 _ 10 or 10 _ 20 cm) with significantly decreased development distance (typically 6 cm) and analysis time (7–20 min). HPTLC plates provide improved resolution, higher detection sensitivity, and improved in situ quantification and are used for industrial pharmaceutical densitiometry quantitative analysis. Normal phase adsorption TLC on silica gel with a less polar mobile phase, such as chloroform–
methanol, has been used for more than 90% of reported analysis of pharmaceuticals and drugs

6. Mobile Phase
The selection of mobile phase is based on adsorbent material used as stationary phase and physical and chemical properties of analyte. General mobile-phase systems that are used based on their diverse selectivity properties are diethyl ether, methylene chloride, and chloroform combined individually or together with hexane as the strength-adjusting solvent for normal-phase TLC and methanol, acetonitrile, and tetrahydrofuran mixed with water for strength adjustment in reversed-phase TLC. Separations by ion pairing on C-18 layers are done with a
mobile phase such as methanol–0.1 M acetate buffer (pH 3.5) containing 25 mM sodium pentanesulfonate (15.5:4.5). Accurate volumetric measurements of the components of the mobile phase must be performed separately and precisely in adequate volumetric glassware 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.

7. Methods of sample application :
Due to the lower sample capacity of the HPTLC layer, the amount of sample applied to the layer is reduced. Typical sample volumes are nL which give starting spots of only mm diameter; after developing the plate for a distance of 3-6cm, compact separated spots are obtained giving detection limits about ten times better than in conventional TLC. A further advantage is that the compact starting spots allow an increase in the number of samples which may be applied to the HPTLC plate. The introduction of the sample into the adsorbent layer is a critical process in HPTLC. For most quantitative work a platinum-iridium capillary of fixed volume (100 or 200 nL), sealed into a glass support capillary of larger bore, provides a convenient spotting device. The capillary tip is polished to provide a smooth, planar surface of small area (CU 0.05 mm2), which when used with a mechanical applicator minimizes damage to the surface of the plate; spotting by manual procedures invariably damages the surface.

8. Development of Chromatogram
Separations obtained in HPTLC are affected by the vapor phase, which depends on the type, size, and saturation condition of the chamber during development. The interactions of these three phases as well as other factors, such as temperature and relative humidity, must be controlled to obtain reproducible TLC separations.
HPTLC plates are developed in flat-bottom chambers, twin-trough chambers, or horizontal-development chambers. In general, saturated twin-trough chambers fitted with filter paper offer
the best reproducibility. For development of plates in a saturated TTC, initially prepare the appropriate volume of mobile phase. Place a correctly sized piece of filter paper in the rear trough of TTC and carefully pour prepared mobile phase into chamber so that the filter paper is thoroughly wetted and adheres to rear wall of TTC. Tilt TTC to the side (about 45_) so that the solvent volume in both troughs equalizes. Set chamber on bench, replace the lid and let chamber equilibrate for 20 min. Mark the desired developing distance (70 mm from lower edge of plate) with a pencil on the right edge of the plate. Slide off the lid to the side and place the plate into the front trough in such a way that the layer and filter paper should face each other and the
back of the plate is resting against front wall of TTC. Replace the lid and develop plate to the mark. Remove plate from chamber and dry it (vertically in the direction of chromatography) for 5 min in a stream of cold air. 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.

9. The availability of scanning densitometers
Commercial instruments for in-situ quantitative analysis based on direct photometric measurement have played an important role in modern thin-layer chromatography. Although double beam instruments are available, single beam single wavelength operation is mainly used in HPTLC since the quality and surface homogeneity of the plates are generally very good.
High performance thin-layer chromatography has found its greatest application in the areas of clinical (e.g. analysis of drugs in blood) and environmental analysis.

10. Most modern HPTLC quantitative analysis are performed in situ by measuring the zones of samples and standards using a chromatogram spectrophotometer usually called a densitometer or scanner with a fixed sample light beam in the form of a rectangular slit. Generally, quantitative evaluation is performed with the TLC Scanner 3 using winCATS software. It can 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. Calibration of single and multiple levels with linear or nonlinear regressions are possible. When target values are to be verified, such as stability testing and dissolution profile, single level calibration is suitable. Concentration of analyte in the sample is calculated by considering the sample initially taken and dilution factors.
Quantification

11. Advantages: HPTLC Technically, it is simple to learn and operate.
Several analysts work simultaneously on the system.
Lower analysis time and less cost per analysis and Low maintenance cost.
Visual detection possible – as it is an open system.
Availability of a great range of stationary phases with unique selectivity for
mixture components.
Chromatographic layer (sorbent) requires no regeneration as TLC/HPTLC plates are disposable.
Ability to choose solvents for the mobile phase is not restricted by low UV transparency or the need for ultra-high purity. Corrosive and UV-absorbing mobile phases can be employed.
No prior treatment for solvents like filtration and degassing.
There is no possibility of interference from previous analysis as fresh stationary and mobile phases are used for each analysis.

12. Advantages: HPTLC No carry over, hence no contamination.
Repetition of densitometric evaluation of the same sample can be achieved under different conditions without repeating the chromatography to optimize quantification, since all sample fractions are stored on the TLC/HPTLC plate.
Samples rarely require cleanup. High sample throughput since several samples can be chromatographed simultaneously.
Lower expenditure of solvent purchase and disposal since the required amount of mobile phase per sample is small.
In addition, it minimizes exposure risks of toxic organic effluents and reduces possibilities of environment pollution.
Accuracy and precision of quantification is high because samples and standards are chromatographed and measured under the identical experimental conditions on a single TLC/HPTLC plate.
Sensitivity limits of analysis are typically at nanogram(ng) to picogram(pg) levels.
Use of different universal and selective detection methods.
HPTLC is a modern adaptation of TLC with better and advanced separation efficiency and detection limits.

13. Application
HPTLC is one of the most widely applied methods in phytochemical analysis. It is due to its numerous advantages, e.g., it is the only chromatographic method offering the option of presenting the results as an image.
HPTLC provides identification as well as quantitative results. It also enables the identification of adulterants.
In case of complex samples, the resolving power of traditional one-dimensional chromatography is usually inadequate; hence special modes of development are required. Multidimensional and multimodal HPTLC techniques include those realized in one direction (UMD, IMD, GMD, BMD, AMD) as well as typical two-dimensional methods realized on mono- or bi-layers.
HPTLC Fingerprint Analysis: A Quality Control for Authentication of Herbal Phytochemicals.