Should I Change The Adsorbent To Differentiate Between Two Rf Values

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ISSN (Online): 0975-8232,
ISSN (Impress): 2320-5148

AN OVERVIEW ON Sparse LAYER CHROMATOGRAPHY

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AN OVERVIEW ON THIN LAYER CHROMATOGRAPHY

Archana A. Bele* and Anubha Khale

H. G. Higher of Pharmacy, Jogeshwari (West), Mumbai, Maharashtra, Bharat

Abstruse:In the present article attempt has been made to explicate the basic ideas and the significance of Thin layer Chromatography (TLC) in dissimilar belittling methods. As TLC is less time consuming, low cost, and can be performed with less complicated technique it has a broad application in pharmaceutical analysis. If performed precisely 32 amino acids can exist separated by TLC. Likewise it has a wide application in identifying impurities in a compound. It tin can exist used as a preliminary analytical method prior to HPLC. The concept of TLC is simple and samples usually require simply minimal pretreatment. TLC can be used to monitor the progress of a reaction, identify compounds present in a given substance. TLC is also used to separate the identical compounds in a mixture. Many standard methods in industrial chemistry, environmental toxicology, nutrient chemistry, water, inorganic and pesticide assay, dye purity, cosmetics, plant materials, and herbal analysis rely upon TLC as the preferred arroyo.

Keywords:Thin layer chromatography, capillary action,Mobile phase,

Rƒ value

INTRODUCTION: Sparse layer chromatography (TLC) is a chromatography technique used to separate mixtures. Chromatography was discovered by M. Tswett in 1906.Sparse layer chromatography is performed on a sail of glass, plastic, or aluminum foil, which is coated with a sparse layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary stage. After the sample has been applied on the plate, a solvent or solvent mixture (known every bit the mobile phase) is drawn up the plate via capillary activity. Considering different analytes ascend the TLC plate at different rates, separation is achieved.

Thin layer chromatography can be used to: Monitor the progress of a reaction, identify compounds present in a given substance, decide the purity of a substance. Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For example, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the more polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the bounden places.

Consequently, the less polar compound moves higher upward the plate (resulting in a higher Rf value). If the mobile stage is changed to a more polar solvent or mixture of solvents, information technology 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 lot use a mixture of ethyl acetate and heptane as the mobile phase, adding more than ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will ordinarily not outcome in reversed order of running of the compounds on the TLC plate.

Principle of TLC ¹ : Sparse layer chromatography uses a thin glass plate coated with either aluminum oxide or silica gel as the solid phase. The mobile phase is a solvent called co-ordinate to the properties of the components in the mixture. The principle of TLC is the distribution of a compound between a solid stock-still stage (the sparse layer) applied to a drinking glass or plastic plate and a liquid mobile phase (eluting solvent) that is moving over the solid phase. A pocket-size corporeality of a compound or mixture is applied to a starting point merely above the bottom of TLC plate.

The plate is then developed in the developing chamber that has a shallow puddle of solvent just below the level at which the sample was applied. The solvent is fatigued up through the particles on the plate through the capillary action, and as the solvent moves over the mixture each compound will either remain with the solid phase or dissolve in the solvent and move up the plate. Whether the chemical compound moves up the plate or stays behind depend on the physical properties of that individual compound and thus depend on its molecular structure, particularly functional groups. The solubility rule " Similar Dissolves Like" is followed. The more than like the physical properties of the compound to the mobile phase, the longer it volition stay in the mobile phase. The mobile stage will carry the most soluble compounds the furthest up the TLC plate. The compounds that are less soluble in the mobile phase and take a higher analogousness to the particles on the TLC plate will stay behind ¹.

Rƒ values : The behavior of an private chemical compound in TLC is characterized by a quantity Known as Rƒ and is expressed as a decimal fraction. The Rƒ is calculated by dividing the altitude the compound traveled from the original position by the distance the solvent travelled from the original position (the solvent front).

Rƒ = Distance of center of spot from starting signal

Distance of solvent forepart from starting signal

The Rƒ value is a abiding for each component only under identical experimental condition. Information technology depends upon number of factors as;

Nature of adsorbent: Different adsorbents will give different Rƒ value for same solvent. Reproducibility is only possible for given adsorbent of abiding particle size and binder. Plates should exist stored over silica gel in desiccators before utilise and the sample should be applied quickly and so that the water vapor in the atmosphere is not adsorbed by the plate. Because of the difficulties associated with activation procedures, information technology is far better to use plates stored at room temperature and non to activate them.
The mobile phase: The purity of solvents and quantity of solvent mixed should be strictly controlled. Information technology should be fabricated freshly for each run if ane of the solvents is very volatile or hygroscopic. Example- acetone.
Temperature: Although precise control of temperature is not necessary, the tank should exist kept away from sources of heat, direct sunlight etc. As the temperature is increased, Volatile solvents evaporate more than quickly, solvents run faster, and Rƒ values generally decrease slightly.
Thickness of layer: Standard plates approximately 250 micrometer is the preferable thickness of layer. Below 200, the Rƒ values vary considerably. The layers may be of college or lower thickness in individual compounds.
Developing tank: Information technology is important that saturated conditions are attained for running TLC plates. This is best achieved by using small tanks with filter newspaper liners and sufficient solvent, and by leaving the tank to equilibrate for at to the lowest degree 30 minutes earlier running the plates. A well plumbing equipment chapeau is essential.
Mass of sample: Increasing the mass of sample on the plate will often increment the Rƒ of drug, especially if it normally tails in the system. Yet, if a plate is grossly overloaded, this too will give a tailing spot and will have the effect of evidently decreasing the Rƒ value. The two situations are commonly easy to distinguish by the intensity of the spot.
Chromatographic Technique: Depending upon the development technique used i.e. ascending, descending, horizontal etc, the Rƒ value change for the same solvent system.

Plate preparation ¹: TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a pocket-sized amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as thick slurry on an unreactive carrier sheet, ordinarily glass, thick aluminum foil, or plastic. The resultant plate is stale and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.one- 0.25 mm for analytical purposes and around 0.5- 2.0 mm for preparative TLC.

Capillary spotters: Identify a melting point capillary and in the dark bluish part of the Bunsen burner flame. Hold it at that place until it softens and starts to sag. Quickly remove the capillary from the flame and pull on both ends to nearly 2-three times its original length. If you pull the capillary inside the flame, you will have a "piece of fine art", simply not a good spotter. Allow the capillary to cool down, and then break it in the middle. Make sure to intermission off the airtight end on one of them.

Spotting the plate: The sparse terminate of the spotter is placed in the dilute solution; the solution volition rise upwardly in the capillary (capillary forces). Touch the plate briefly at the start line. Allow the solvent to evaporate and spot at the aforementioned identify once more. This way you lot will go a full-bodied and small spot. Endeavor to avoid spotting as well much textile, because this will deteriorate the quality of the separation considerably ('tailing'). The spots should be far enough away from the edges and from each other as well. If possible, yous should spot the compound or mixture together with the starting materials and possible intermediates on the plate.

Location of spots: The position of various solutes separated by TLC can be located by various methods. Colored substances can be seen straight when viewed confronting stationary phase, while colorless substances can exist detected just by making them visible by making utilise of some spraying agent, which produces colored areas in the region which they occupy.

Specifically in TLC following can be used for spraying the invisible spots:

Being purely inorganic in nature, corrosive agents may besides exist used for spraying on the invisible spots.
Dilute solution of Potassium dichromate in concentrated sulfuric acrid. In the procedure, potassium dichromate (yellowish) is reduced to chromic sulfate (green) by near of the organic compounds, specially used for sugars.
Vapors of sulfur trioxide, produced on warming fuming sulfuric acid, chars organic compound and makes them visible every bit dark spots.
Solution of potassium permanganate.
Iodine vapors.

Other mutual reagents include saturated solution of hydrogen sulphide, 0.2N aqueous ammonium sulphide, 0.ane% alcoholic quercetin, 0.2% methanolic 1-(2-pyridylazo)- two- napthol, 1% methanolic oxine , and 0.5% aqueous sodium rhodizonate. If the adsorbent used for the TLC plate contains a fluorescing cloth, the solutes can exist viewed under ultraviolet light.

Evolution solvents ²: The choice of a suitable solvent depends upon: Nature of substance, and adsorbent used on the plate. A evolution solvent should be such that, does non react chemically with the substances in the mixture under examination. Carcinogenic solvents (benzene etc) or environmentally dangerous solvents (dichloromethane etc) should always be avoided. Solvent systems range from non-polar to polar solvents. Non-polar solvents are generally used, as highly polar solvents cause the adsorption of any component of the solvent mixture. Ordinarily used evolution solvents are petroleum ether, carbon tetrachloride, pyridine, glycol, glycerol, diethyl ether, formamide, methanol, ethanol, acetone, and n-propanol.

Mobile Phase: For silica gel chromatography, the mobile stage is an organic solvent or mixture of organic solvents. As the mobile phase moves pass the surface of the silica gel information technology transports the analyte pass the particles of the stationary phase. However, the analyte molecules are only free to move with the solvent if they are not leap to the surface of the silica gel. Thus, the fraction of the time that the analyte is bound to the surface of the silica gel relative to the time it spends in solution determines the retentivity gene of the analyte. The power of an analyte to demark to the surface of the silica gel in the presence of a particular solvent or mixture of solvents can exist viewed equally a the sum of two competitive interactions. First, polar groups in the solvent can compete with the analyte for binding sites on the surface of the silica gel. Therefore, if a highly polar solvent is used, it will interact strongly with the surface of the silica gel and will leave few sites on the stationary phase costless to bind with the analyte. The analyte volition, therefore, move speedily pass the stationary phase. Similarly, polar groups in the solvent tin interact strongly with polar functionality in the analyte and prevent interaction of the analyte with the surface of the silica gel.

This outcome also leads to rapid movement of the analyte pass the stationary phase. The polarity of a solvent to exist used for chromatography tin can be evaluated by examining the dielectric constant (å) and dipole moment (ä) of the solvent. The larger these 2 numbers, the more than polar is the solvent. In addition, the hydrogen bonding ability of the solvent must also be considered. For case methanol is a potent hydrogen bond donor and will severely inhibit the ability of all merely the most polar analytes to bind the surface of the silica gel.

Developing a Plate ⁱⁱ: A TLC plate can be developed in a chalice or closed jar. Place a small corporeality of solvent ( mobile stage) in the container. A small spot of solution containing the sample is practical to a plate, nigh one centimeter from the base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a sealed container. The solvent moves upwards the plate by capillary activity and meets the sample mixture, which is dissolved and is carried up the plate by the solvent.

Dissimilar compounds in the sample mixture travel at unlike rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By irresolute the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) tin be adapted. The solvent level has to be below the starting line of the TLC, otherwise the spots volition dissolve away. The lower border of the plate is then dipped in a solvent. The solvent (eluent) travels up the matrix by capillarity, moving the components of the samples at various rates because of their unlike degrees of interaction with the matrix (stationary phase) and solubility in the developing solvent. Non-polar solvents will strength not-polar compounds to the top of the plate, because the compounds dissolve well and do not interact with the polar stationary phase. Let the solvent to travel upward the plate until ~1 cm from the top. Take the plate out and mark the solvent forepart immediately. Practise not permit the solvent to run over the edge of the plate. Next, let the solvent evaporate completely.

Precautions during sample application;

Sample should be dissolved in a nonpolar solvent equally polar solvent has a tendency to spread out the starting spot.
Solvent used for dissolving sample should be volatile.
While applying sample, the surface of the adsorbent should not be disturbed equally this distorts the shapes of the spots on subsequent developed chromatogram, hindering the accuracy of quantitative measurements.
The sample spot should be inside 2-v mm in diameter.

The TLC Experiment:

LC CHAMBER FOR DEVELOPMENT
WITH A LID OR A CLOSED JAR

After ~5 Min After ~10 Min After Drying

Visualization: When the solvent front has moved to within about i cm of the top end of the adsorbent (later on 15 to 45 minutes), the plate should be removed from the developing bedchamber, the position of the solvent forepart marked, and the solvent immune to evaporate. If the components of the sample are colored, they can be observed directly. If not, they tin sometimes be visualized past shining ultraviolet low-cal on the plate or past allowing the plate to correspond a few minutes in a closed container in which the atmosphere is saturated with iodine vapor. Sometimes the spots tin can be visualized past spraying the plate with a reagent that will react with one or more of the components of the sample.

Analysis: The components, visible as separated spots, are identified by comparison the distances they have traveled with those of the known reference materials. Measure the distance of the first line to the solvent forepart. Then mensurate the distance of eye of the spot to the commencement line. Divide the distance the solvent moved by the distance the individual spot moved. The resulting ratio is called Rf-value. As the chemicals being separated may be colorless, several methods exist to visualize the spots.Often a small amount of a fluorescent chemical compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV₂₅₄). The adsorbent layer volition thus fluoresce light green by itself, but spots of analyte quench this fluorescence, Iodine vapors are a general unspecific color reagent, Specific colour reagents exist into which the TLC plate is dipped or which are sprayed onto the plate. Once visible, the Rf value, or retentivity gene, of each spot can be adamant by dividing the altitude traveled past the production by the total altitude traveled by the solvent (the solvent forepart). These values depend on the solvent used, and the type of TLC plate, and are not physical constants.

Preparative TLC ⁸: TLC can also exist used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. The mixture is not "spotted" on the TLC plate as dots, but rather is applied to the plate every bit a thin even layer horizontally to and just above the solvent level. When developed with solvent the compounds split in horizontal bands rather than horizontally separated spots. Each band (or a desired band) is scraped off the backing material.

The backing fabric is and then extracted with a suitable solvent (e.g. DCM) and filtered to give the isolated material upon removal of the solvent. For pocket-size-scale reactions with easily separated products, preparative TLC can be a far more than efficient in terms of time and cost than doing chromatography. Obviously, the whole plate cannot be chemically developed or the product will be chemically destroyed. Thus this technique is all-time used with compounds that are colored, or visible under UV light. Alternatively, a small department of the plate can be chemically developed e.g. cut a section out and chemically developing it, or masking most of the plate and exposing a small section to a chemical developer like iodine.

Applications ^{one-6, nine, 10}: Sparse layer chromatography has been a useful tool in numerous applications of pharmaceutical importance.

TLC of amino acids : TLC of amino acids is more difficult than TLC of inks, because amino acids are colorless. Therefore, one cannot come across the spots with the naked eye one time the plate is fully developed and stale. To see the spots, it is necessary to use either the ninhydrin or the black-light visualization techniques.

E.thou., Amino acids, proteins and peptides ^viii: A mixture of 34 amino acids, proteins and peptides has been successfully separated and isolated from urine using silica gel plates. All these substances were found to be ninhydrin positive. The development were carried out first with chloroform-methanol-20%ammonium hydroxide (2:two:i) and then with phenol-h2o.

Pharmaceuticals and drugs: TLC is used in the identification, purity testing and decision of the concentration of agile ingredients, auxiliary substances and preservatives in drugs and drug preparations, procedure command in constructed manufacturing processes. Various pharmacopoeias have accustomed TLC technique for the detection of impurity in a drug or chemical

E.m., Antibiotics: Penicillin's have been separated on silica gel 'Yard' by using the ii solvents, acetone- methanol (1:1) and iso-propanol-methanol (three:7). As the detecting agent, the iodine-azide reaction was employed by spraying the stale plates with a 0.1 %
iodine solution containing iii.5% of sodium azide.

Separation of multicomponent pharmaceutical formulations: Information technology is also used in separation of multicomponent pharmaceutical formulations.
Qualitative assay of alkaloids: It is used in qualitative analysis of alkaloids in control stage of both pharmaceutical formulations and vegetable drugs. TLC has been used for the isolation and determination of alkaloids in toxicology where the 30-60 minute runs give a bully advantage in comparison to the 12-24 hours required for newspaper chromatography. Purine alkaloids take been separated past TLC on silicic acid, silica gel and aluminum oxide. The spots are visualized by spraying outset with an alcoholic iodine-potassium iodine solution followed by 25% HCl- 96% ethanol (i:1).
Clinical chemistry and Biochemistry: For the determination of active substances and their metabolites in biological matrices, diagnosis of metabolic disorders such as phenylketonuria, cystinuria and maple syrup illness in babies. It serves every bit an useful tool in analysis of urinary constituent derived from lipids in analysis of many urinary constituents such as steroids, amino acids, porphyrins and bile acids. Urinary analysis by TLC is most effective when done in conjunction with other chromatographic processes, so that minor metabolites can be detected and resolved completely gratis of other components.
Cosmetology: In the identification of dye raw materials and end products, preservatives, surfactants, fat acids, constituents of perfumes.
Food Analysis: For the decision of pesticides and fungicides in drinking water, residues in vegetables, salads and meat, vitamins in soft drinks, banned additives in Germany (e.one thousand. sandalwood extract in fish and meat products), compliance with limit values (e.k. polycyclic compounds in drinking water, aflatoxins in milk and milk products). A typical separation of dyes in spinach looks like this:
Analysis of Heavy Petroleum Product ⁸: Thin-layer chromatography (TLC), which is usually used in the analysis of circuitous mixtures, is seldom used in the investigation of petroleum products, possibly the most circuitous objects. In item, with respect to heavy petroleum products, no such data has been constitute in the literature. At the same time, the simplicity, economic system, and efficiency of this technique in comparing with column chromatography are advantages that are widely known. TLC technique used (in the preparataive variant) for a rapid determination of the grouping composition of heavy petroleum products (asphalts, pitches, resids), and in connection with
spectroscopic studies of the chemical limerick of the fractions obtained.
Separation of aromatic amines: Cationic and non-ionic surfactant-mediated systems have been used as mobile phases in sparse-layer chromatographic separation of aromatic amines on silica gel layers. The result of surfactant concentration below and higher up its critical micellar concentration on mobility of amines was examined. The influence of organic and inorganic additives such as alcohols, urea, NaCl and NaBr in micellar solutions on the mobility and separation efficiency of amines is also assessed.

Applications related to Organic Chemistry ^1-half-dozen:

It has been widely used for checking number of other separation processes. TLC
has likewise been practical successfully in various purification processes, checking
of distillation fractions and for checking the progress of purification past molecular
distillation.
TLC has been used as an analytical tool in organic chemistry due to its high speed of separation and its applicability in a large number of chemical compounds. It'due south important use is in the separation and isolation of individual components of a mixture, simply in organic chemistry it has also been used for: Checking the purity of samples, as purification procedure, for identification of organic compounds, for studying diverse organic reactions, in characterizing and isolating a number of compounds such as acids, alcohols, glycols, amides, alkaloids, vitamins, amino acids, antibiotics, food stuffs and exam of reaction. The reaction mixture is examined past TLC to assess whether the reaction is complete or otherwise.The method is also used in checking other separational processes and purification processes like distillation, molecular distillation etc.
High sensitivity of TLC is used to check purity of sample, considering loftier sensitivity enables impurities to exist observed in so called pure samples. With the help of TLC it is possible to know whether a reaction is complete and had followed the expected course. The nature of byproducts can too be ascertained by using TLC. If the reaction does not proceed as desired or expected, then an test of the behaviour of the spots with standard reagents may sometimes give information for the rapid identification of the products.

Problems in TL C:

Over-large Spots: Sample spots made using TLC capillaries should be no larger than 1-2 mm in diameter, because component spots in the adult plate will be no smaller than, and volition commonly be larger than, the size of the initial spot. If the initial spot is larger than two mm in bore, then components with similar Rf values may non be resolved considering their spots volition be so large that they volition overlap considerably and may appear to be 1 large spot. Modest initial spots, on the other hand, maximize the potential of complete separation of components.

Uneven Accelerate of Solvent Front: A common problem in TLC is uneven advance of solvent forth the plate. Instead of a straight line, the solvent front end may appear to bow either upward or down in the center. Uneven accelerate of solvent leads to uneven advance of substance spots, and inaccurate Rf values result. A frequent cause of uneven solvent advance is the use of a developing chamber that does not have a flat bottom. Drinking glass bottles usually have bottoms that bend upward from the edges to the center. If the bottom of the TLC plate is placed on this curved surface, the shape of the solvent advance line may mirror the shape of the container bottom. It is therefore of import to apply flat-bottomed developing tanks in TLC. A bowed solvent front may also issue if too little developing solvent is placed in the sleeping room; if the plate is cut improperly, so that the sides are not exactly perpendicular to the lesser edge; and if the slide is excessively tilted in the bedroom. Care in choosing and using a developing sleeping accommodation is the best defense against curved solvent fronts. Water is seldom used as a developing solvent because it has a trend to produce a dramatically curved front end. This may exist due to its unusually high surface tension.

Streaking: Sometimes a substance will movement forth a TLC plate as a long streak, rather than every bit a single detached spot. This is the result of spotting the plate with too much substance, more than the moving solvent can handle. The solvent moves as much substance as it tin, merely a substantial amount of substance is left behind. The substance is dragged along by the solvent leaving a trail of substance that may sometimes bridge the entire distance between the starting line and the solvent front. Streaking can be eliminated by systematically diluting the spotting solution until development and visualization prove the substances moving every bit single spots, rather than elongated streaks.

Specific TLC Procedures:

Separartion for alanine, glycine, threonine, and proline: TLC of amino acids is more hard one cannot see the spots with the naked center once the plate is fully developed and dried. To see the spots, it is necessary to use either the ninhydrin or the black-calorie-free visualization techniques. Notice the spots, and make up one's mind whether or not a chosen solvent system has been effective in moving an amino acid or in separating a mixture. Therefore the procedure of finding an effective solvent organisation can be long and painstaking. As points of general information, amino acids are quite polar and tend to move on silica gel plates with polar solvents. They have Rf values close to 1 when h2o or full-bodied ammonia is used as the developing solvent, probably considering of their high solubility in h2o. Diluting a polar solvent with a less polar one results in smaller Rfvalues, roughly in proportion to the amount of less polar solvent used, Thus, alanine, glycine, threonine, and proline all have Rf values of around 0.60 when adult with a 50/50 mixture of water and n-propanol, and around 0.40 when developed with a 30/70 mixture of concentrated NH_iii and northward-propanol. The following procedure assumes the employ of 50/50 h2o/n-propanol every bit the developing solvent, but one can try other polar/not-polar combinations.

Experimental Procedure In the hood, set 10 mL of a mixture consisting of fifty% 1-propanol and fifty% water by volume, and pour almost half of this into a make clean developing tank. Make sure that the level of liquid in the tank is no higher than v mm, and shut the chapeau. Prepare a solution of about 0.001 g of amino acid in 0.ii mL of water. Dissolve the acid, and then draw some solution up in a spotting capillary and double-spot a properly marked and activated TLC plate. Permit the plate to dry for five minutes, and so lower the plate into the developing tank then that its bottom is submerged in the developing solvent. Close the lid, and let the plate to develop until solvent has risen to the pencil line at the top of the plate.

Remove the plate from the tank and place information technology in an oven at l ^oC to dry out. When the plate is dry, visualize it using ninhydrin spray or iodination. Circle the amino acrid spots with pencil, and summate Rf values. Compare the measured Rf values with the values for the amino acids. On this basis, identity of amino acid is done. In combination with other data , obtain, data which will assist unambiguously to identify amino acid. Suppose that amino acid has Rf fivealue similar to that of alanine, ane should and so prepare a small corporeality of alanine solution and spot information technology alongside amino acrid on a new TLC plate. Develop, dry, and visualize the plate to confirm that amino acid indeed has exactly the same value as alanine, and that the spot is the aforementioned shape and color. Finally, information technology is very of import to be observant of item in doing TLC. In improver to the Rf value for a substance, the shape of the spot produced by a detail developing solvent and the shade of color produced by iodine or ninhydrin can be characteristic of the substance. For example, when alanine, glycine, threonine, and proline are spotted side-by-side on a plate and developed with 70% n-propanol/30% conc NH₃ following observations tin can be made:

Amino Acrid	Solvent	Spot Colour subsequently Iodination	Spot Color with Ninhydrin	R_f Value	Spot Shape
alanine	50/50 water/n-propanol	white on brown bkgrnd	regal	0.65	circle
glycine	30/70 conc NH₃/due north-propanol	white on brown bkgrnd	pinkish	0.25	elongated oval
glycine	50/50 h2o/due north-propanol	white on brown bkgrnd	pinkish	0.55	circle
threonine	50/50 water/n-propanol	white on brown bkgrnd	purple	0.57	circle
proline	50/l water/n-propanol	white on brown bkgrnd	yellow with pink border	0.65	circle

Rf values of amino acids

Separation of dyes in spinach: On a rest weigh out 0.5 grams of fresh spinach and combine with 0.5 grams of anhydrous magnesium sulfate and one.0 grams of sand. Transfer these materials to a mortar and using a pestle grind the mixture until a fine dry powder is obtained (grind the mixture really well). The anhydrous magnesium sulfate will remove the water from the leaves. Transfer the powder (two.0 grams full) to a small test tube and combine with 2.0 mL of acetone. Stopper the exam tube and shake vigorously for approximately ane minute. You need to brand sure that the solid and solvent are well mixed.

Allow this mixture to stand for ten minutes, and then using a pipette carefully transfer the solvent above the solid into a small micro centrifuge tube. Employ care non to transfer whatsoever of the solid textile. The solvent extract should be green. Cap the micro centrifuge tube to minimize solvent evaporation. Obtain a TLC bedroom (a 400 mL glass beaker covered with parafilm or aluminum foil) and add developing solvent (a mixture of petroleum ether, acetone, cyclohexane, ethyl acetate and methanol). The solvent should completely cover the bottom of the chamber to a depth of approximately 0.5 cm. Keep the sleeping accommodation covered so that evaporation doesn't alter the limerick of the solvent. Allow the TLC plate to develop (separation of pigments) for approximately x minutes. As the solvent moves upwards the TLC plate you should see the unlike colored pigments separating.

Remove the TLC plate from the sleeping room when the solvent front is approximately ane.0cm from the top of the TLC plate. With a pencil, mark the level of the solvent front (highest level the solvent moves upwards the TLC plate) as shortly as you remove the strip from the sleeping accommodation (the solvent evaporates and disappears chop-chop). Also measure the pigment distances quickly as some pigments (especially the beta-carotene) may fade over time.

Beta-carotene is the about non-polar pigment (highest Rf) and its band volition exist xanthous. Chlorophyll a has a larger Rf than does chlorophyll b. For the post-obit calculations mark the center of the initial pigment dot; this will be the starting point for all the following measurements. Also mark the middle point of each pigment band and the solvent front. The literature gives Rf values of 0.61 and 0.49 for pheophytin a and pheophytin b. Utilize these values to help identify which spots are due to those compounds. Use the pure extract of ß-carotene to place the location of that ring.

Identification of naphthodianthrones: Hypericum perforatum extracts containing naphthodianthrones take been identified past TLC using silica gel plates with fluorescence indicator and ethyl acetate-formic acrid-water(xxx:ii:iii five/v/5) or toluene- ethyl acetate-formic acid-water(fifty:xl:5:5 v/v/five/v) as the mobile phase. TLC can also exist used for the identification of indolic alkaloids isolated from various Rauwolfia Species past using silica gel plates as stationary species; acetone-calorie-free petroleum-diethyl amine(2:vii:i v/five/v) and ane% solution of ammonium cerium (IV) sulfate for the visualization of spots.
Identification of drugs: Aspirin, acetaminophen, ibuprofen, and caffeine: Describe a lite pencil line about 1 cm from the end of a chromatographic plate, and on this line spot aspirin, acetaminophen, ibuprofen, and caffeine, which are available as reference standards. Use a separate capillary for each standard. Make each spot equally modest as possible, preferably less than 0.v mm in bore. Use the blower to facilitate the evaporation of the solvent between applications. Examine the plate under the ultraviolet (UV) lite to see that plenty of each compound has been applied; if non, add more than. On a dissever plate run three of the unknowns and one of the aspirin standard. The unknown sample is prepared by crushing a function of a tablet, adding this pulverization to a test tube or small vial along with an appropriate corporeality of ethanol, and so mixing the suspension. Not all of the tablet will dissolve, just enough will go into solution to spot the plate. The binder starch or silica will non dissolve. The 1% solutions should exist prepared and ready to apply. Later on the solvent has risen to nearly 2/3 of the length of the plate, remove the plate from the developing chamber. Rapidly mark the solvent front with a pencil and allow the solvent to dry. Examine the plate under UV light to see the components as nighttime spots confronting a bright green-blue groundwork. Outline the spots with a pencil. The spots can also be visualized by putting the plate in an iodine sleeping accommodation made by placing a few crystals of iodine in the bottom of a capped jar. Calculate the Rf values for the spots.

Substances:	Aspirin	Caffeine	Acetaminophen	Ibuprofen
Rf Values:	0.45	0.08	0.24	0.lx

Separation of Inorganic Ions: TLC has been used for separating cationic, anionic, purely covalent species and also organic derivatives of the metals. In order to carry out TLC of groups of cations, silica gel is first washed with acid and water to remove impurities of sodium, magnesium, calcium and iron. Merely this treatment removes the calcium sulphate folder. Therefore, calcium sulphate must be replaced past starch or another suitable binder. After washing and drying of TLC plate, the spots of cations or anions to exist separated are applied on this plate. The plate is then kept in a close chamber and the lower office of the plate is then dipped into a solvent. Information technology is than removed from sleeping accommodation and dried, visualized for spots by suitable visualizing reagents.

REFERENCES:

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Sr No: 6

Page No: 256-267

Size: 493KB

Download: 25685

Cited By: 86

Language: English

Licence: IJPSR

Authors: Archana A. Bele* and Anubha Khale

Authors Address: Lecturer, H.K. College of Pharmacy, Near MHADA Complex, Jogeshwari(W), Bombay, Maharashtra, India

Email:

Received: 28 July, 2010

Revised: 16 November, 2010

Accustomed: 18 January, 2010

DOI: http://dx.doi.org/10.13040/IJPSR.0975-8232.2(2).256-67

Published: one-February-2011

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