A drug may be defined as a substance meant for diagnosis, cure, mitigation, prevention, or treatment of diseases in human beings or animals or for alternating any structure or function of the body of human being or animals. Pharmaceutical chemistry is a science that makes use of general laws of chemistry to study drugs i.e., their preparation, chemical natures, composition, structure, influence on an organism and studies the physical and chemical properties of drugs, the methods of quality control and the conditions of their storage etc. The family of drugs may be broadly classified as
1. Pharmacodynamic agents
2. Chemotherapeutic agents
Aims and scope of drug analysis:
The aims and scope of drug analysis can be summarized in one sentence as follows:
"The aim of drug analysis (with emphasis on industrial drug analysis) is the analytical
Bulk drug materials,
The intermediates of their syntheses,
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Products of drug research (potential pharmacons),
The impurities and degradation products of drugs,
Biological samples containing the drugs and their metabolites
With the aim of obtaining data which can contribute to
The high quality,
The maximum efficacy and
Maximum safety of drug therapy and
The maximum economy of the production of drugs.
Chromatographic methods are commonly used for the quantitative and qualitative analysis of raw materials, drug substances, drug products and compounds in biological fluids. The components monitored include chiral or achiral drug, process impurities, residual solvents, excipients such as preservatives, degradation products, extractables and leachables from container and closure or manufacturing process, pesticide in drug product from plant origin, and metabolites.
The objective of a test method is to generate reliable and accurate data regardless of whether it is for acceptance, release, stability or pharmacokinetic study. Data are generated for the qualitative and quantitative testing during development and post approval of the drug products. The testing includes the acceptance of raw materials, release of the drug substances and products, in process testing for quality assurance, and establishment of the expiration-dating period.
For the above purposes there is a need to develop simple, accurate and reliable methods for the determination of drugs in pharmaceutical dosage forms.
A survey of literature reveals that many analytical methods are available for the estimation of drugs like, Anastozole, Gatifloxacin and Ambroxol, Ranitidine oral solution many of them suffer from one disadvantage or the other, such as low sensitivity, lack of selectivity and simplicity etc. However, no TLC or HPTLC method for the quantitative determination of these drugs is available and so the present work involves the use of HPTLC method for the quantification of these drugs as TLC/HPTLC, a well recognized routine analytical technique proves to be more economical for analysis of pharmaceuticals than other chromatographic methods because of its advantages like a disposable stationary phase, static detection free of time constraints, storage device for chromatographic information wider range of detection possibilities, utilization of smaller volumes of solvents, minimum sample clean up and simultaneous estimation of several components in a short time is also possible.
2. INTRODUCTION TO CHROMATOGRAPHY
Chromatography (from Greek chroma: colour and "grafein" to write) is the collective term for a family of laboratory/analytical techniques for the separation of components in a mixture for qualitative identification, quantitative estimation, isolation and purification. The most appropriate definition of a chromatography seems to be given by keulemans according to whom "chromatography is a physical method of separation in which components to be separated are distributed between two phases, one of these phases constituting a stationary bed of large surface area, the other being a fluid which percolates through or along with the bed. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated. The basic principles involved in chromatography, viz., adsorption, partition, selective permeation, ion-exchange, etc.
Chromatography may be preparative or analytical. Preparative chromatography seeks to separate the components of a mixture for further use and thus facilitates a form of purification. Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. The two are not mutually exclusive.
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It was the Russian botanist Mikhail Semyonovich Tswett who invented the first chromatography technique in 1900 during his research on chlorophyll. He used a liquid-adsorption column containing calcium carbonate to separate plant pigments. The method was described on December 30, 1901 at the 11th Congress of Naturalists and Doctors in St. Petersburg. The first printed description was in 1903, in the Proceedings of the Warsaw Society of Naturalists, section of biology. He first used the term chromatography in print in 1906 in his two papers about chlorophyll in the German botanical journal, Berichte der Deutschen Botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for the German Botanical Society.
In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the Chemistry Nobel Prize for their invention of partition chromatography. Since then, the technology has advanced rapidly. Researchers found that the principles underlying Tswett's chromatography could be applied in many different ways, giving rise to different varieties of chromatography described below. Simultaneously, advances continually improved the technical performance of chromatography, allowing the separation of increasingly similar molecules.
The analyte is the substance which is to be separated during chromatography.
Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.
A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture. Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.
A chromatograph is the equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
The effluent is the mobile phase leaving the column.
An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A better definition: The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In one case of HPLC the solvent consist of a carbonate/bicarbonate solution and the sample is the anions being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.
The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography.
The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
Again the solute also refers to the sample components in partition chromatography.
The solvent refers to any substance capable of solubilizing other substance, especially the liquid mobile phase in LC.
The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
Preparative chromatography is used to nondestructively purify sufficient quantities of a substance for further use, rather than analysis.
Chromatography is a method of separating mixtures and identifying their components i.e. it's a separation method that exploits the differences in partitioning behavior of analytes between a mobile phase and a stationary phase to separate components in a mixture. The interaction of the components of a mixture with the two phases is influenced by several different intermolecular forces, including ionic, dipolar, non-polar, and specific affinity and solubility effects. There are two theories of chromatography, the plate and rate theories.
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Types of Chromatography:
Chromatography characterized as a separation method based on the differential migration of solute through a system of two phases, one is mobile phase another one is stationary phase. Chromatography is a technique by which the components in a sample, carried by the liquid or gaseous phase, are resolved by sorption, desorption steps on the stationary phase.
Chromatography is mainly divided into two categories:
Separation is mainly due to the interaction between solute and surface on the adsorbent. In this, stationary phase is solid and mobile phase is liquid.
Eg: TLC, HPTLC, GC
Separation based on partition between two phases. In this mode, both stationary phase and mobile phase are liquids.
Eg: HPLC, GLC, Paper Chromatography.
Thin Layer Chromatography - TLC:
Thin layer chromatography (TLC) is a separation technique, which came into general use following the pioneering work of Stahl. TLC also known as Planar chromatography or flat bed chromatography is a simple, reliable, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are present in a mixture. It is a multi-stage distribution process. TLC gained popularity from 1956 when Stahl described equipment and procedure for the preparation of chromatoplates and demonstrated the usefulness in the fractionation of substances.
As identification is prerequisite before proceeding with quantitation, TLC is used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound (preferably both run on the same TLC plate). Also, it is one of the most widely used techniques for rapid identification of drugs and its formulations. It is equally applicable to the drugs in their pure state, to those extracted from pharmaceutical formulations and to biological samples.
Of the three chromatographic techniques, TLC, GLC, HPLC which is applicable for an analysis depends on several parameters such as solubility or volatility of the sample, required separation efficiency, concentration of the analyte, detection limit, cost of analysis, number of samples under analysis, sample preparation and other requirements of the sample to be separated.
TLC in Pharmacy:
The most valuable use of thin layer chromatography in pharmaceutical work is to provide means of assessing low levels of impurities in medicinal substances. For this purpose, the substance is applied to the chromatographic surface and, after chromatography, any secondary spots to be seen in the chromatogram after appropriate visualization are compared to size and intensity with those of low loadings of expected impurities that have simultaneously been subjected to chromatography on the same plate.
As an adjunct to identification, thin layer chromatography may be used by comparing the behavior of the material to be identified with that of a standard substance, usually an authentic specimen of the substance being examined. If the two substances move identical distances during chromatographic process and if the two substances when mixed together and then subjected to chromatography, move as a single substance, it may be presumed that the two substances are identical. This presumption may be strengthened by repeating the procedure using a different system of chromatography; in general, if two substances behave identically in as many as three fundamentally different systems, the presumption of identity becomes very strong.
Its wide range of uses include assaying radiochemical purity of radio pharmaceuticals, determination of the pigments a plant contains detection of pesticides or insecticides in food, analyzing the dye composition of fibers in forensics, or identifying compounds present in a given substance.It is a quick, generic method for organic reaction monitoring.
The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed, TLC is often used for monitoring chemical reaction and for the qualitative analysis of reaction products.
A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluant, is the mobile phase, and it slowly rises up the TLC plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the stationary phase.
As the solvent moves past the spot that was applied, equilibrium is established for each component of the mixture between the molecules of that component, which are adsorbed on the solid, and the molecules, which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others and adsorbed according to their adsorption capabilities. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates.
Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silicagel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the most polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places.
Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. Practically this means that if you use a mixture of ethyl acetate and heptane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will not result in reversed order of running of the compounds on the TLC plate. If a reversed order of running of the compounds is desired, a polar stationary phase should be used, such as C18-functionalized silica.
The appropriate solvent in context of thin layer chromatography will be one which differs from the stationary phase material in polarity. If polar solvent is used to dissolve the sample and spot is applied over polar stationary phase TLC, the sample spot will grow radially due to capillary action, which is not advisable as one spot may mix with the other. Hence, to restrict the radial growth of sample-spot, the solvent used for dissolving samples in order to apply them on plates should be as non-polar or semi-polar as possible when the stationary phase is polar, and vice-versa.
Criteria for identification of an analyte by TLC:
Rf value of an analyte should agree within ï‚±3% compared to standard material used under similar conditions.
Visual appearance of the analyte should be indistinguishable from that of standard material.
For confirming the identity, co-chromatography is mandatory. As a result, only the spot supposed to be due to analyte should be visible and no additional spot should appear.
All of the above might sound like TLC is quite an easy procedure. Complications arise because of the spots are everywhere and sometime they are blurred and streaked. As with any technique, with practice better understanding and results are achieved. However the common problems that are generally encountered in TLC:
The compound runs as a streak rather than a spot. - The sample was overloaded. The TLC was run again after diluting your sample. Or, the sample might contain many components, which creates many spots, which run together and appear as a streak.
The sample runs as a smear or as an upward crescent Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Addition of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent resolves the problem and separations are accomplished.
The sample runs as a downward crescent. -Likely, the adsorbent is disturbed during the spotting, causing the crescent shape.
The plate solvent front runs crookedly. -Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crookedly run plates make it harder to measure Rf values accurately.
Many, random spots are seen on the plate. - One should make sure that no organic compound was accidentally dropped on the plate. When a TLC plate was left lying on the workbench, organic compound may be splashed or dropped on the plate.
No spots are seen on the plate. -The compound spotted may not be enough, perhaps because the solution of the compound is too dilute. So the solution should be concentrated or it should be spotted several times in one place, allowing the solvent to dry between applications. For the compounds, which do not show up under UV light; another method of visualizing the plate must be tried. If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead of allowing them to move up the plate by capillary action. Thus, no spots will be seen after the plate is developed.
Various steps involved in TLC -HPTLC:
Steps involves in the development of HPTLC method
Plates used for TLC may be either Hand-made or precoated.
These are generally not used now a day as pre-coated plates are easily available.
Silica gel or Silica gel G: Suspend 30g of silica gel or silica gel G in 60-65 ml of water, mix in an electric stirrer to get homogeneous slurry. The plates so coated may be dried in air or by heating for 45 min at 110Â°C. In case of silica gel G, the slurry must be used within 2 min. Activation of plates can be carried out by heating at 110Â°C for 30 min. Layer thickness is usually 250ïm.
The pre-coated plates with different support material (glass, aluminium, plastic) and with different sorbent layers are available in different format and thickness by various manufacturers. Usually plates with sorbent thickness of 100-250ïm are used for qualitative and quantitative analysis, however for preparative TLC work, plates with sorbent thickness of 1.0-2.0 mm are available in addition to chemically modified layers.
Glass support: It is resistant to heat and chemicals, easy to handle and always offers superior flat and smooth surface for chromatographic work; disadvantage being fragility, relatively high weight, additional packing material, higher production cost. The precoated plates with glass support are therefore most expensive ready to use layers.
Polyester (plastic) sheets (0.2 mm thick): More economical as they are produced even in roll forms, unbreakable, less packing material, less shelf space for storage, can be cut to any required format. Spots can be cut and eluted, thus eliminating dust of scrapping. Charring reactions are possible but temperature should not exceed 120Â°C as the plates are dimensionally unstable beyond this temperature.
Aluminium sheet (0.1 mm thick): Aluminium sheets as support offer the same advantage as polyester support but with increased temperature resistance. However with eluents containing high concentration of mineral acids or concentrated ammonia, one may find problem, as they will chemically attack aluminum. Aluminium sheets are otherwise compatible with organic solvents and organic acids such as formic acid and acetic acid.
Commonly available precoated plates with their applications:
Silica gel 60F (unmodified): More than 80% analyses are done on this layer. This layer available with suitable indicator.
Aluminium oxide: Basic substances, alkaloids and steroids.
High purity silica gel 60: Aflotoxins
Cellulose: amino acid, dipeptides, sugars, antibiotics and other labile compounds which cannot be chromatographed on active layers of silica gel.
Preparatory plates: Layer thickness of 1-2 mm, large sample volumes can be applied as streak. These are usually available as pre-coated soft layers, to be used when separated substances are to be recovered, usually gives less sample resolution than analytical plates.
Precoated TLC/HPTLC plates in size of 20x20 cm with aluminium or polyester support are usually procured mainly for economic reasons. These plates can be cut to size and shape to suit particular analysis by using general-purpose scissors.
Prewashing of precoated plates:
Sorbents with large surface area not only absorb water vapours and other impurities from atmosphere but other volatile substances often condense particularly after the packing has been opened and exposed to laboratory atmosphere for a long time. Such impurities including elutable components of the binder usually give dirty zones and fail to give reproducible results. It is only for these reasons that precoated plates are always packed with glass or foil side upward (coated layer downward). To avoid any problem due to impurities with the chromatographic, particularly in case of quantitative work, it is always recommended to clear the plates before actual chromatography.
This process is called Prewashing of Plates. Ascending, dipping, continuous mode are the common methods of cleaning the plates. Ascending technique takes somewhat longer time but cleaning effect is superior, however, active dirt gets accumulated at the solvent front. Therefore compounds of interest that migrate at the rear end of the solvent front are partially obscured by overlapping fluorescence of the surface contaminants. Thus compounds with high Rf value 75 and above will be difficult to scan particularly under fluorescent mode. This difficulty is often overcome by cutting 10 to 20 % of the upper portion of pre washed plate before proceeding with chromatographic separations.
The other most commonly employed method for prewashing is Dipping. Quicker dipping process yields rather uniform clean layer but cleaning effect is often not as good as Ascending technique. Excellent results are obtained if the plates subjected for prewashing by continuous mode for some time, i.e., in a chamber closed by a lid having a slit.
After washing, the plates must be dried for a sufficient time to ensure complete removal of the washing liquid (usually for methanol 30-60 min at 105 ° are required).use of hot or cold air (hair drier) should be avoided as laboratory air which is usually contaminated is blown over the layer and purpose of cleaning the layer is defeated. The washed plates should always be stored in a dust free atmosphere at ambient conditions. Preferably desiccators of suitable dimensions should be used for storing both cleaned and uncleaned plates. No grease should be used for sealing the dessicator. Use of drying agents is also not necessary.
Activation of precoated plates:
Freshly opened box of TLC/HPTLC plates usually does not require activation. However, plates exposed to high humidity or kept on hand for long time may have to be activated by placing in oven at 110-120Â°C for 30 minutes prior to sample spotting (aluminium backed plates should be activated by keeping between two glass plates). This step removes water that has been physically absorbed on the surface of the sorbent. After the plates are removed from prewash chambers, they should always be dried in vertical position as in horizontal position drops of solvent may fall on the plate as a result of condensation. Activation at higher temperature and for longer time should be avoided as it may lead to very active layers and there will be risk of samples being decomposed or artifact being formed. In such cases, it is advisable to resort to use of RPTLC plates.
The sample preparation is not as demanding as for other chromatographic techniques, however, several steps for sample pretreatment may be necessary such as sampling, mechanical crushing, extraction, filtration and enrichment of minor compounds. Proper sample preparation is an important prerequisite for success of thin layer chromatographic separation. The sample preparation procedure is to dissolve the dosage form with complete recovery of intact compound(s) of interest and minimum matrix with a suitable concentration of analyte(s) for direct application on the HPTLC plate. Besides maximizing the yield of analytes in the selected solvent, stability of analytes during extraction and analysis must be considered and ensured. Therefore, the choice of a suitable solvent for a given analysis is very important. For normal phase chromatography using silica gel precoated plates, solvent for dissolving the sample should be non-polar and volatile as far as possible, since polar solvents are likely to induce circular chromatography at origin, particularly when sample is applied in increment on top of each other, leading to spreading of spot, thus loss of separation efficiency. For reversed phase chromatography, usually polar solvents are used for dissolving the sample, however, such polar solvents must wet the sorbent so that sample penetrates the layer uniformly. Clean-up steps in the sample preparation, if necessary must be optimized.
It is preferable to keep the solvent as simple as possible and quantity employed be limited to ensure complete extraction of analytes and minimum of extraneous component. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones.
Choice of solvent for the sample:
It should dissolve the analytes.
Should be reasonably volatile.
Should have low viscosity.
Wets the sorbent layer.
Should be a weak chromatographic solvent for the analyte.
For TLC on silica gel, the use of weakest (least polar) solvent which allows quantitative dissolving and spotting of sample and there is no preliminary development and separation within the initial spot at the origin, is recommended. Rf values of the components of the interest in the selected solvent for preparation of the sample should be less than 0.1%.
Evaporation of solvent:
Whenever sample requires concentration, use of rotary evaporator with attached round bottom flask is recommended. The solution should be first evaporated completely to dryness and then the residue is dissolved in same or different solvent for application to the TLC layer. Use of non-polar solvent to dissolve the residue will help to remove polar impurities, which will remain undissolved. Solvents with high boiling point or polarity are difficult to remove solvent layers during application. If a small amount of solvent is left after application, it can cause serious effects on the separation by causing zone spreading or deformation. Use of hot air to dry solvent at origin should normally be discouraged as it can cause decomposition of heat labile substances on the surface of an active sorbent.
Application of sample:
Sample application is the most critical step for obtaining good resolution. The sample should be completely transferred to the layer, however, under no circumstances, the application process should damage the layer, as damaged layer results in unevenly shaped spots. Wherever possible, use of automatic application devices is recommended for quantitative analysis. While using graduated capillaries, one must ensure that they fill and empty completely.
Usually application of 1-10ïl volume for TLC and 0.5-5ïl for HPTLC is recommended keeping the size of starting zone down to minimum; 2-4mm (TLC) and 0.5-1mm (HPTLC) in the concentration range of 0.1-1ïg/ïl for TLC/HPTLC. Substance zones, which are too large from the beginning, cause poor separation, as during development, spots tend to become large and more diffused. This difficulty is more pronounced in case of substances with high Rf values. It is therefore recommended that solution should be applied in small increments with intermediate drying particularly when the sample solution is predominantly aqueous.
However, volume and concentration primarily depend on the component under analysis and their sensitivity to various detection techniques. If too much sample is applied, it may not be absorbed uniformly throughout the layer leading to overloading, as a result trailing zones and poor resolution is observed. Problem arising out of such overloading when unavoidable can best be overcome by applying the sample as band, the only apparent disadvantage being that only fewer samples can be accommodated on a given plate.
Advantages of application of sample as a band are:
Better separation because of rectangular area in which the compounds are present on the plate.
Equal Rf values of the compounds from sample and reference solution.
Matrix effect of extracted and applied excipients are significantly reduced as solution is distributed over a larger area.
Response of densitometer is higher than observed from an equal amount/equal volume of the same solution applied as a spot. It appears that light may not gain access to all the sample material applied as a spot. This is supported by the observation that range of linearity is small for point wise application than for band wise application.
Application of different volumes as bands from one solution gives same concentration response curve as obtained by application of equal volumes of solution with different concentration. This correlation is absent when sample solution is applied as a spot. This is significant, as, while preparing the concentration response curve, one need not prepare solutions with different concentration while applying the sample as a band.
Generally speaking, spot broadening in the direction of development is smaller in the case of band wise application.
Larger quantities of the sample can be handled for application, thus reducing the need for concentration step, which may be quite damaging in case of labile substances.
Position of plate for densitometric scanning is less critical as composition of the compounds is uniform in the entire area of band.
Poor grade of solvent used in preparing mobile phases have been found to decrease resolution, spot definition more signal to noise ratio and Rf reproducibility. Mobile phase commonly called solvent system is traditionally selected by controlled process of trial and error and also based on one's own experience in the field. It is often possible that few layer-solvent combinations already reported in the literature for compounds of interest or similar compounds may be suitable in a given analytical problem with minor modifications. Nevertheless, it should not be forgotten that such conditions may have been chosen due to availability rather than suitability and often improvements are required. However, mobile phase should be chosen taking into consideration chemical properties of analytes and the sorbent layer. Use of mobile phase containing more than three or four components should normally be avoided, as it is often difficult to get reproducible ratios of different components.
Solvent composition is expressed by volumes (v/v) and usually sum of total volume is 100.
Various components of Mobile phase should be measured separately and then placed in the mixing vessel. This will not only prevent the contamination of solvent stock by evaporation from already partially filled mixing vessel but also any possible volumetric error arising due to volumes expansion or contraction on mixing.
Laboratories equipped with complete HPTLC system usually use smaller development chamber such as twin trough chambers (10x10 cm) where comparatively smaller volumes of mobile phase, usually 10-15 ml is required. It is advisable that different components of the mobile phase should be measured with volumetric pipettes.
Different components of mobile phase should be first mixed in mixing vessel and then introduced into the developing chamber.
Chambers usually containing multi-component mobile phase once used is not recommended for re-use for any future development work as composition of mobile phase is likely to change during chromatographic development, due to differential evaporation and adsorption by the layer and also once the chamber is opened, each solvent component will evaporate disproportionately depending on their volatilities.
Mobile phase should be as simple as possible and permissible by analytes and sample matrix.
Some form of mobile phase optimization is generally necessary when performing HPTLC.
Optimization of mobile phase:
First level: Neat solvents from different selectivity area were tested. Within selectivity area solvents may give similar separation. Usually diethylether, ethanol, methanol, tetrahydrofuran, dimethyl formamide, dichloromethane, ethylacetate, Acetonitrile, methylethylketone, toluene and chloroform are used as neat solvents.
If acceptable resolution and medium Rf range is achieved, the analyst can directly try level 3, i.e., exploring the suitability of solvent mixture. However, if level 1 doesn't yield satisfactory results, then proceed with level 2.
Second level: From level 1, solvents which leave the main fraction/component of the analyte near the starting point or close to the solvent front, are required to be adjusted in strength. If Rf values are too high, solvent strength should be decreased by adding non-polar solvents such as n-hexane, toluene. If Rf values are too low, the solvent strength is required to be increased by addition of methanol, ethanol or water. In such case, usual ratio of 9:1, 8:2, 7:3, 6:4, 5:5 are tried.
Third level: Mixtures of solvents from different selectivity group are investigated; the strength is adjusted, if necessary. These solvent mixtures can be binary, ternary or even quaternary. Usually, one should start with the centre and corner position of selectivity such as in the case of binary mixtures, the ratios are 1:1, 9:1, 1:9, and for tertiary mixtures 1:1:1, 8:1:1 and 1:1:8 ratios should be first tried. At this level, addition of small amount of acidic (acetic acid) or basic (triethylamine) modifiers significantly enhance the separation efficiency of mobile phase.
Fourth level: At this level, final optimization of the mobile phase to be used for a particular separation is made. To get the best separation, small variations in the proportions of different solvents may have to be made.
Preconditioning (Chamber saturation):
Chamber saturation has pronounced influence on the separation profile. When the plate is introduced into an unsaturated chamber, during the course of development, the solvent evaporates from the plate mainly at the solvent front. Therefore, larger quantity of the solvent shall be required for a given distance, hence resulting in increase in Rf values. If the tank is saturated (by lining with filter paper) prior to development, solvent vapours soon get uniformly distributed throughout the chamber. As soon as the plate is placed in such a saturated chamber it soon gets preloaded with solvent vapours, hence less solvent shall be required to travel a particular distance resulting in lower Rf values.
Development and drying:
Ascending, descending, two-dimensional, horizontal, multiple overrun (continuous), gradient, radial (circular), anti-radial (anti-circular), multimodal (multi-dimensional), forced flow planar chromatography are the most common modes of chromatographic development. Rectangular glass chambers, twin-trough chambers, V-shaped chambers, sandwich chambers, horizontal development chambers, vario-KS chambers, circular and anti-circular, U chambers and automated multiple development chambers are commonly used for carrying out different types of TLC development.
It is important that once the chromatogram is developed, it should be handled with utmost care. Application of reagents if required has to be homogeneous ensuring uniform reaction and finally stabilizing of end reaction product.
After development the plate is removed from the chamber and mobile phase is removed as completely and as quickly as possible. This step should preferably be performed in fume cupboard laid horizontally so that while mobile phase evaporates, the separated substances will migrate evenly to the surface where they can be easily detected. Usually analysts may employ hand dryer to effect faster removal of the mobile phase. It is precisely for these reasons that drying of chromatogram should preferably be done in vacuum desiccator with protection from heat and light.
Factors/parameters influencing the TLC separation and resolution of spots:
Type of stationary phase (sorbent), its particle size and activity.
Type of plates (precoated or hand-made).
Layer thickness (any deviation in layer thickness).
pH of the layer.
Binder in the layer.
Mobile phase (solvent system).
Type and size of developing chamber.
Degree of chamber saturation.
Solvent for the sample preparation.
Solvent/mobile phase level in the chamber.
Sample volume spotted.
Size of the initial spot.
Temperature (Rf values usually increase with the rise in temperature).
Greater the distance between different spots and smaller the initial spot diameter of the sample, better the resolution. While describing the result of any TLC/HPTLC procedure, various parameters and conditions under which results for a specific analysis have been obtained must be documented. This is absolutely essential for possible reproducible results.
Evaluation of a thin layer chromatogram:
The evaluation depends on the purpose of a chromatographic analysis. For qualitative determination, often localization of substances is sufficient. This can be easily achieved by parallel runs with reference substances.
The Rf value:
A parameter often used for qualitative evaluation is the Rf value (retention factor) or the 100-fold value hRf.
Once visible, the Rf value of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants.
Distance traveled by the solute
Rf = --------------------------------------
Distance traveled by the solvent front
Rf values are between 0 and 1, best between 0.1 and 0.8, ideal between 0.4-0.6.
The distance traveled by a substance should be measured from the centre of the spots which are round but for the spots showing tailing, the measurement is done from the middle of most dense areas. As the Rf values are likely to be affected by number of factors, it is desirable that the authentic sample and the drug under analysis should be run simultaneously on the same plate. This is the procedure usually used for pharmacopoeial purposes.
If reproducible Rf values are to be obtained, it is, however, essential that several parameters such as chamber saturation, constant composition of solvent mixtures, constant temperature etc. are strictly controlled.
It is conventional to develop plates for a distance of 10cm, which may require up to 2 hours for a viscous mobile phase particularly in winter season. However, running of the plate for shorter distance has advantage i.e. increase in sensitivity and a saving of material. The major disadvantage being incomplete resolution, about 20% decrease in resolution by decreasing the development distance from 10cm-5cm. It is always preferable to develop the plate for a predetermined distance. Whenever the plate is removed from the chamber the position of the solvent front should be quickly marked before the solvent evaporates from the plates.
Detection and visualization:
One of the most characteristic feature of TLC/HPTLC is the possibility to utilize post chromatographic off-line derivatization. With availability of many visualization reagents, findings can be confirmed which the HPLC is lacking. These visualization reactions are possible for identification even if the separation is not optimal.
The zones can be located by various physical, chemical, biological-physiological methods. There is apparently no difficulty in detecting colored substances or colorless substances absorbing in short -wave UV region or with intrinsic fluorescence. The substances which do not have above properties have to be transferred into detectable substances by means of chromogenic or fluorogenic reagents which are more expensive, time-consuming and complicated.
Detection sensitivity depends on the specificity for the reagent employed. Iodine is the universal detection reagent, the detection is usually non-destructive and reversible.
Detection under UV light is the first choice and is non-destructive in most of the cases and is commonly employed for densitometric scanning.
Derivatization reactions are essentially required for detection when individual compound does not respond to UV or does not have intrinsic fluorescence. It is not significant whether derivatization is pre- or post-chromatographic.