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The entire process can be visualized with the scheme in Fig. It starts from a clearly defined analytical goal, method selection, optimization, and development, which is called prevalidation considerations before arriving at the elaboration of a validation protocol and is the starting point of the actual validation.

After performing all the experiments described in the validation protocol, obtained data are evaluated and compared with the acceptance criteria. If all criteria are met, the method can be regarded as valid.

In a less-formal approach, some validation data may be incorporated from experiments, which were conducted previously as part of the method development.

The above approach is widely accepted for validation of qualitative HPTLC methods for identification during routine use. It is possible that the validation method in different situations may require some changes in the standard validation protocol. Such changes may include restrictions with respect to relative humidity, waiting times, precision, etc. The validation protocol is a key instrument for structuring, regulating and documenting the validation processes, depending on the quality management system.

The following elements must be included: Selectivity Ability of the developed analytical method is to detect analyte quantitatively in the presence of other components which are expected to be present in the sample matrix.

Results are expressed as Resolution. If the expected impurities or related substances are available, they should be chromatographed along with the analyte to check the system suitability, retention factor, tailing factor, and resolution. Precision Precision provides an indication of random error. Accuracy Accuracy of an analysis is determined by systematic error involved.

It is defined as closeness of agreement between the actual value and mean analytical value obtained by applying the test method a number of times. The accuracy is acceptable if the difference between the true value and mean measured value does not exceed the RSD values obtained for repeatability of the method. This parameter is very important for formulated pharmaceutical dosage forms as it provides information about the recovery of the analyte from sample preparation and effect of matrix.

A blank matrix and known impurities must be available to test the accuracy of the method. Experiments are usually recommended to evaluate ruggedness of a HPTLC method like sample preparation: composition, quantity of solvent, pH, shaking time, temperature and number of extractions; sample application: volume applied, spot shape and size, band and spot stability; separation: at least on three different plates; chromatographic conditions: chamber saturation, eluent composition, eluent volume, temperature, humidity and development distance; spot visualization: postchromatographic derivatization, spraying, dipping, reaction temperature and time; quantitative evaluation: drying of plates, detection and wavelength.

Srivastava Once the analytical method is developed, it should be performed independently by three analysts well conversant with practical aspects of the technique, analyzing the same sample under same experimental conditions to check reproducibility of the method.

If this is not possible, then amount of analyte to be applied has to be increased. Limit of detection LOD is determined on the basis of signal to noise ratio. Mean of 15 noise peak areas and their absolute SD values are determined. LOD is the amount of applied sample producing a peak area which is equal to the sum of mean blank area and three times standard deviation. Stability Analyte should not decompose during development of the chromatogram and should be stable in solution and on the sorbent for at least 30 and 15 min, respectively.

The complete removal of organic solvent should be avoided. Sample preparation, in most cases, a 5-min sonication with methanol, followed by centrifugation and using the supernatant as test solution, yields satisfactory results. Derivatization is optimized with the goal of convenience, safety, and reproducibility. Botanical Reference Materials BRM of known adulterants are used to ensure sufficient specificity of the method.

Small modifications of the mobile phase composition are applied to fine-tune separation. Each step of the optimization process is documented for complete traceability.

The optimization of the chromatographic mobile phase proved to be possible when the number of experimental determinations of separation parameters for each compound is obtained for more than one distinct compositions of mobile phase, at least equal with the number of variable use in the mathematical model.

A mobile phase optimization program based on an original mathematical approach is to be developed for its performances by applying on three sets of compounds. The original optimization procedure starts from the idea that 1 An Overview of HPTLC: A Modern Analytical Technique with Excellent Potential 15 into a mixture of three solvents the quantitative measure of the choused chromatographic parameter is dependent on composition of mobile phase through an equation of dependency with six or seven parameters, taking into consideration the molar fraction of the solvents.

The optimization procedure is included in a program and applied on three sets of previously studied compounds through high-performance thin-layer chromatography with three solvents. The mobile-phase optimization process proved to be able to provide accurate, precise, and reproducible method of characterization and analysis of chromatographic parameters.

HPTLC: Automation For the past 50 years, both automatic and automated instruments have been used to monitor and control process stream, such as density, viscosity, and conductivity. It is necessary to distinguish between the characteristics of automatic and automated devices. According to the current definitions of the International Union of Pure and Applied Chemistry IUPAC , both devices are designed to replace, refine, extend or supplement human effort and facilities in the performance of a given process.

The unique feature of automated devices is the feedback mechanism, which allows at least one operation associated with the device to be controlled without human intervention. An automatic photometer might continuously monitor the absorbance of a given component in a process stream, generating some type of alarm if the absorbance exceeds a preset value. By contrast, an automated system could transmit absorbance values to a control unit that adjusts process parameters temperature and amount of additional reagent to maintain the concentration of the measured component within preset limits.

In spite, of this fundamental difference, the terms automatic and automated are often interchanged. The use of automated sample processing, analytics and screening technology for profiling absorption, distribution, metabolism, excretion, and physicochemical properties is becoming more widespread. The use and application of these technologies is both diverse and innovative.

High throughput screening technologies have been utilized enabling the profiling of an increased number of compounds. Although the drivers for using these technologies are common, different approaches can be taken.

Control Systems, Safe, efficient, and economical operations of chemical processes are ever more dependent in the use of online analyzers. The use of analytical measurements of component properties in near real time for process control during manufacturing is becoming more common. The combination of online analyzers and advanced control technologies holds an enormous economic potential. Advances in science and technology have raised an increasing demand for control analyses and posed various challenges to analytical chemists such as the need to develop new methods exhibiting as much selectivity, sensitivity, sample and reagent economy, throughput, cost-effectiveness, simplicity, and environmental 16 MM.

Srivastava friendliness as possible. The large of number of samples, with which analysts can be confronted, imposes the use of expeditious automatic methods. Despite the major conceptual and operational differences between partly and fully automated methods, the two are frequently confused. Thus, a fully automated method allows the whole analytical process to be completed with no intervention from the analyst; also, it can by itself make the decision as to whether the operating conditions should be altered in response to the analytical results.

All methods are deemed automated simply because one or several steps of the analytical process are performed in an automated manner. However, an automated method should be capable of completing all steps including sampling, sample preparation and dissolution, interference removal, aliquot withdrawal, analyte measurement, data processing, result evaluation, and decision making, and also of restarting the whole process in order to adapt it to the particular needs of a new sample if needed.

A fully automatic method is very difficult to develop especially for solid samples, the first steps in the analysis of which can rarely be performed in an inexpensive manner. Usually, the operations posing the greatest difficulties among those involved in such steps are those requiring some mechanical handling, automation of which is only possible in most cases by using robot arm adapted to the particular chemical operations to be performed.

Because this equipment is too expensive for most analytical applications, fully automated methods for the analysis of solid samples are very scant and largely restricted to the control of manufacturing processes. The automation of analyses involving fluid samples is facilitated by their usually adequate homogeneity and easy mechanical handling by the use of peristaltic or piston pumps, or some other liquid-management devices.

This is not the case with solid samples, analysis of which frequently involves their prior conversion into liquids by dissolution. The dissolution step is the bottleneck of analytical processes involving solid samples as it is frequently slow and must be performed manually.

The earliest automatic methods used dedicated devices suited to the particular application. This restricted their scope to very specific uses such as the control of manufacturing processes or in those cases where the number of samples to be analyzed was large enough to justify the initial effort and investment required. The computer-controlled techniques have introduced a great number of advantages to HPTLC systems mainly a dramatic decrease of the needed sample and reagents volumes, and have allowed the introduction of the concept of unit laboratory operations.

HPTLC: Hyphenation Over the past several years, the concept of hyphenation has gained rapid growth in the pharmaceutical industry because of its ability to produce a large number of compounds with a wide range of structural diversity in a short time. The combinational approach hyphenation has received a significant recognition compared to a traditional one-compound-at-a-time approach. Today, thin-layer chromatography has been successfully hyphenated with high-performance liquid chromatography HPLC , mass spectroscopy MS , Fourier transform infra-red FTIR , and Raman spectroscopy to give far more detailed analytical data on separated compounds.

Flow rates typical for mobile phase can be applied to the layer. A splitter in the transfer line to the spray-jet applicator is required to accommodate higher flow rates from wider-bore columns. The column eluent is nebulized by mixing with nitrogen gas and sprayed as an aerosol onto the layer. The spray head is moved horizontally on one line within a defined bandwidth. The main problems are more on the detection and data handling side than separations. It is simpler to obtain mass spectral information from the solution phase using liquid chromatography—mass spectrometry LC—MS than to either quantify or identify separated bands by thin layer chromatography— mass spectrometry TLC—MS.

High-Performance Thin-Layer Chromatography—Mass Spectrometry HPTLC—MS The combination of chromatographic separations with mass spectrometric detection is considered an indispensable tool for problem solving in analytical chemistry and increasingly for routine analytical methods. Mass spectrometric detection brings an added level of information, complementary to the chromatographic process that improves the certainty of identification and the specificity of detection.

Mass spectral information can generally be obtained from sample sizes typical of common analytical methods. The challenge was to develop an automated system for in 18 MM. Srivastava situ acquisition of mass spectral data directly from layers with retention of the spatial integrity of the chromatographic separation.

This is certainly not a simple problem but is a problem of some importance, since it restricts the range of applications that HPTLC is considered suitable. Morlock, assistant professor at the Institute of Food Chemistry of the University of Hohelnheim in Stuttgart, Germany, modified ChromeXtractor and demonstrated the performance of this versatile interface in comparison to other technical solutions for hyphenation.

The substance of interest is eluted directly form the HPTLC plate and is transferred online into the mass spectrometer. If the target zone is not visible, it can be marked either under UV nm or UV nm, by extrapolation of the adjacent zone made visible by derivatization.

The HPTLC—MS interface is operated in semiautomatic mode which means that after manual positioning of the zone the piston is lowered at the push of a button. Moving a lever starts the solvent flow through the layer and extracts the zone.

Hyphenating HPTLC with MS appears to hold considerable promise for those analysts who previously have had reservations towards the use of planar chromatography. The hyphenation opens a new dimension for the technique and makes it more prestigious from the scientific view.

Recently, HPTLC—MS has been successfully used for the identification and quantification of amino acid in peptides, fast identification of unknown impurities, problem-solving technique in pharmaceutical analysis, identification of botanicals, screening for bioactive natural products in sponges, determination of ginkgolides A, B, and C and bilobalide in Ginkgo bilodes and identification of Hoodia gordonii a popular ingredient of botanical slimming products.

It is because of the robustness and simplicity of use of HPTLC and the need for detection techniques that provide identification and determination of sample. Almost all chemical compounds yield good IR spectra that are more useful for the identification of unknown substances and discrimination between closely related substances. The information content of UV—VIS spectra is rather poor and rarely enables unambiguous identification of a substance; furthermore, a chromophore is needed for UV detection.

These reasons make this hyphenated technique more universally applicable. The potential of this method is demonstrated by its application in various fields of analysis such as drug, forensic, food, environmental, and biological analysis, etc. Such a system benefits from the high spatial resolution of the laser, simple transfer of analyte molecules, compatibility with modern mass spectrometric systems and less fragmentation under atmospheric pressure.

One drawback of such a system is that the cost for a traditional pulsed laser system is relatively high which somehow counteracts the advantage of TLC in the low costs. The size of the laser system is also not ideal for a miniaturization of the whole analytical system. The initial efforts were carried out on a graphite plate photon-absorbing material.

A continuous wave diode laser replacing traditional pulsed lasers was employed for this purpose.


High-Performance Thin-Layer Chromatography


DIN 66230 PDF

High-performance thin-layer chromatography (HPTLC)



High-Performance Thin-Layer Chromatography (HPTLC)


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