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INTRODUCTION OF CHROMATOGRAPHYè
Technique for separating or analysing a mixture of gases, liquids, or dissolved substances. This is brought about by means of two immiscible substances, one of which (the mobile phase) transports the sample mixture through the other (the stationary phase). The mobile phase may be a gas or a liquid; the stationary phase may be a liquid or a solid, and may be in a column, on paper, or in a thin layer on a glass or plastic support. The components of the mixture are adsorbed or impeded by the stationary phase to different extents and therefore become separated.
is the substance that is to be separated during 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.
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. 
The technique is used for both qualitative and quantitive analyses in biology and chemistry.
IN paper chromatography,
the mixture separates because the components have differing solubilities in the solvent flowing through the paper and in the chemically bound water of the paper.
IN thin-layer chromatography,
a wafer-thin layer of adsorbent medium on a glass plate replaces the filter paper. The mixture separates because of the differing solubilities of the components in the solvent flowing up the solid layer, and their differing tendencies to stick to the solid (adsorption). The same principles apply in column chromatography
In gas-liquid chromatography
, a gaseous mixture is passed into a long, coiled tube (enclosed in an oven) filled with an inert powder coated in a liquid. A carrier gas flows through the tube. As the mixture proceeds along the tube it separates as the components dissolve in the liquid to differing extents or stay as a gas. A detector locates the different components as they emerge from the tube. The technique is very powerful, allowing tiny quantities of substances (fractions of parts per million) to be separated and analysed.
is carried out on a large scale for the purification and collection of one or more of a mixture's constituents; for example, in the recovery of protein from abattoir wastes.
is carried out on far smaller quantities, often as little as one microgram (one-millionth of a gram), in order to identify and quantify the component parts of a mixture. It is used to determine the identities and amounts of amino acids in a protein, and the alcohol content of blood and urine samples. The technique was first used in the separation of coloured mixtures into their component pigments.
Thin layer chromatography
(TLC) is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase.
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC can be used.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
Thin layer chromatography finds many applications, including:
- assaying the radiochemical purity of radiopharmaceuticals
- 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
- Monitoring organic reactions.
A number of enhancements can be made to the original method to automate some steps, to increase the resolution achieved with TLC and to allow more accurate quantitation. This method is referred to as HPTLC, or "high performance TLC".
PREPARATION OF TLCè
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 small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110°C. The thickness of the adsorbent layer is typically around 0.1 - 0.25mm for analytical purposes and around 0.5 - 2.0mm for preparative TLC.
Development of a TLC plate, a purple spot separates into a red and blue spot
.Chromatogram of 10 essential oils coloured with vanillin reagent.
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 reactions and for the qualitative analysis of reaction products.
A small spot of solution containing the sample is applied to a plate, about 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 up the 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 the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column.
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 silica gel 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, an apolar stationary phase should be used, such as C18-functionalized silica.
TLC can also be 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 as a thin even layer horizontally to and just above the solvent level. When developed with solvent the compounds separate in horizontal bands rather than horizontally separated spots. Each band (or a desired band) is scraped off the backing material. The backing material is then extracted with a suitable solvent (e.g. DCM) and filtered to give the isolated material upon removal of the solvent. For small-scale reactions with easily separated products, preparative TLC can be a far more efficient in terms of time and cost than doing column chromatography. Obviously, the whole plate can not be chemically developed or the product will be chemically destroyed. Thus this technique is best used with compounds that are coloured, or visible under UV light. Alternatively, a small section of the plate can be chemically developed e.g. cutting a section out and chemically developing it, or masking most of the plate and exposing a small section to a chemical developer like iodine.
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
- Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.
- Iodine vapors are a general unspecific color reagent
- Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate
- In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for example mass spectrometry, a technique known as Far-Eastern blotting.
Once visible, the Rf value , or Retention factor, 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. Eluent on the thin layer is put on top of the plate
Applications of thin-layer chromatography and high-performance thin-layer chromatography for the separation, detection, and qualitative and quantitative determination of pesticides, other agrochemicals, and related compounds are reviewed for the period mid-2000 to mid-2004. Analyses are covered for a variety of samples, such as food, crops, biological, environ-mental, pharmaceuticals, and formulations, and for residues of pesticides of various types, including insecticides, herbicides, and fungicides, be-longing to different chemical classes. In addition to references on residue analysis, studies of pesticide-structure relationships, metabolism, degrada-tion, adsorption, uptake, dissipation, mobility, and lipophilicity are covered, many of which make use of thin-layer radio chromatography.
Reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a "co-spot" with both. A small (3 by 7cm) TLC plate takes a couple of minutes to run. The analysis is qualitative, and it will show if the starting material has disappeared, i.e. the reaction is complete, if any product has appeared, and how many products are generated (although this might be under-estimated due to co-elution). Unfortunately, TLCs from low-temperature reactions may give misleading results, because the sample is warmed to room temperature in the capillary, which can alter the reaction—the warmed sample analyzed by TLC is not the same as what is in the low-temperature flask. One such reaction is the DIBALH reduction of ester to aldehyde.
As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.
In one study TLC has been applied in the screening of organic reactions for example in the fine-tuning of BINAP synthesis from 2-naphthol. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are place separately on the base line, then reacted and then instantly analyzed
TLC as a Pilot Method for Column LC
Experimental data for 50 pesticides, presented as log k (HPLC) vs RM (TLC) correlations, showed that RP-18W F254 layers can be applied successfully as a pilot technique to choose mobile phases for C18 HPLC. Pesticides were spotted as 0.5% solutions and the HPTLC plates developed for 9 cm in the horizontal mode in a Teflon DS chamber. Zones were detected under 254 nm UV light. Mobile phases used to determine corre-lation coefficients were acetonitrile-water (70 + 30 and 60 + 40), methanol-water (80 + 20 and 70 + 30), and THF-water (50 + 50 and 40 + 60) .
Application of thin-layer chromatography with fluorescence scanning densitometry for analysing saturates in heavy liquids derived from Co-pyrolysis of biomass and plastics
-The herbal drug Pyrethrum flows (from the flowers of Pyrethrum ci-nerariaefolium, Asteraceae, a Dalmatian plant) has insecticidal properties related to the esters of pyrethrums' and cindering that are its main chemical constituents. Petroleum ether, chloroform, and ethanol extracts of the herbal drug were analyzed on silica gel 100 F254 plates with n-heptanes -acetone (4 + 1) mobile phase. Visualization of analysts was first done under 254 nm UV light and then by spraying with a solution of anisaldehyde in sulfuric acid. The herbal drug was also analyzed by the TAS technique for thermo-micro separation and application of volatile samples to the TLC plate
A mixed microbial culture capable of degrading end sulfate, the toxic metabolite of the organ chlorine insecticide, endosulfan. The products of degradation were characterized by TLC on neutral alumina F254 plates using petroleum ether-acetone (85 + 15) or chloroform-ethyl acetate (3 + 1) as the mobile phase and detection by spraying with silver nitrate-saturated 5% aqueous methanol and exposure to UV light. Zones were scraped and eluted for further analysis by GC/MS
Seven OP pesticides were determined in water at 0.1 μg L−1 by SPE enrichment on SBD-1 (RP-polymer phase) and C18 cartridges followed by separation on silica gel 60 F254 plates by development with n-hexane-acetone (75 + 30) for 10 cm in a saturated chamber . Zones were quantified by scanning fluorescence quenched zones at 220 nm with a dual wavelength, flying-spot densitometer in the reflectance mode; a densitogram is shown in Fig. 5. Calibration plots were linear be-twin 100 and 2000 ng for all pesticides, and correlation coefficients, r, were >0.999. Recovery rates were between 94 and 102% for both cartridge types except for chlorpyriphos on C18; relative standard deviation (RSD) values were 0.7-4.9%. In addition, an enzymatic inhibition screening method was described using 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcou-Marin (maleimide-CPM) as fluorogenic reagent. It reacts with thiocholine released after hydrolysis of acetylcholine with acetyl cholinesterase at pH 8 to form a strongly blue fluorescent background against which the OP pesticides can be detected as dark zones with a limit of detection (LOD) of 1-10 g spot−1 
A new TLC method for determination of 2,4-D (2,4-dichlorophe-noxyacetic acid); 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); and 2-naphtho-xyacetic acid (NAA) was reported based on TLC separation and spectro-metric quantification. The compounds were separated on silica gel G plates with the mobile phase dioxins-acetic acid (4 + 1; RF values 0.63, 0.68, and 0.52, respectively) and detected as blue, blue, and green zones, respectively, by hydrolysis with aqueous NaOH followed by spraying with p-anisidine (0.2% in methanol), N-chlorosuccinimide (0.1% in water), and sodium nitroprusside-MnO2 (10 mg mL−1, 1 + 1). Zones of standards and samples were scraped from the plate, eluted with 2 M NaOH, and measured by visible-mode spectrometry for analysis of the pesticides in water, wheat, rice, blood, and urine samples
TLC-densitometry was successfully applied as a stability indicating method to separate and quantify clotrimazole (CZ), alone or in presence of byproducts, impurities, and its acid degradation products, in pharmaceutical formulations. TLC was performed on silica gel 60 F254 plates by development with a mobile phase consisting of chloroform-acetone. TLC was used for isolation and purification of antifungal metabolites produced by Bacillus cereus.
A conventional TLC system, which includes a vertical developing tank, and a High Performance TLC (HPTLC) system, with a horizontal developing chamber and the use of HPTLC plates, has been used. The analytical method involves in both cases the measurement of two chromatograms per sample: the first, on a silica gel berberine-impregnated plate, for detection of saturates using the phenomenon of berberine-induced fluorescence; and the second, on a silica gel plate, for detection of aromatic-polars and polars, by native fluorescence.
Although the HPTLC system is more sensitive and faster, both techniques represent an improvement with regard to current methods for analyzing these kinds of products. However their application depends on the particular solubility of each sample and on its slope of the fluorescent response-sample load regression.
Thin layer chromatography, which is typically abbreviated as TLC, is a type of liquid chromatography that can separate chemical compounds of differing structure based on the rate at which they move through a support under defined conditions.
TLC is useful in detecting chemicals of security concern, including chemical weapons, explosives, stabilizing chemicals for rocket propellants, and illicit drugs. For example, the Forensic Service Center of Lawrence Livermore National Laboratory has designed a computerized and portable TLC machine that can be taken to the field, and which has the ability to analyze 20 samples at a time. Analysis can be completed within 30 minutes.
TLC as it is still practiced today was introduced by Justus Kirchner in 1951. From its beginning, the technique was an inexpensive, reliable, fast, and easy to perform means of distinguishing different compounds from each other. The method was qualitative—it showed the presence of a compound but not how much of the compound was present. In the late 1960s, TLC was refined so that it could reliably measure the amounts of compounds. In other words, the technique became quantitative. Further refinement reduced the thickness of the support material and increased the amount of the separating material that could be packed into the support. In High Performance TLC (HPTLC) the resolution of chemically similar compounds is better than with conventional TLC, and less sample is required. HPTLC requires specialized analysis equipment, and so is still not as popular or widespread as conventional TLC.
In TLC a solution of the sample is added to a layer of support material (i.e., grains of silica or alumina) that has been spread out and dried on a sheet of material such as glass. The support is known as the plate. The sample is added as a spot at one end of the plate. The plate is then put into a sealed chamber that contains a shallow pool of chemicals (the solvent), which is just enough to wet the bottom of the plate. As the solvent moves up through the plate support layer by capillary action, the sample is dragged along. The different chemical constituents of the sample do not move at the same speed, however, and will become physically separated from one another. The positions of the various sample constituents and their chemical identities are determined by physical methods (i.e., ultraviolet light) or by the addition of other chemical sprays that react with the sample constituents