Some popular general use stains are Permanganate, ceric ammonium molybdate CAM , and p-anisaldehyde. These can be kept in jars which plates are dipped into, or in spray bottles.
To develop a plate with permanganate, spray or dip the plate and heat it with a heat-gun. Hold the plate face up 10 to 20 cm above the heat gun until the bulk water evaporates. Overheating will turn the entire plate brown, obscuring the spots. If glass plates are used it is often easier to see spots through the backing because it is harder to overheat. CAM and p-anisaldehyde stained plates are developed similarly. Overheating CAM stained plates turns everything blue.
There are common problems in TLC that should be avoided. Normally, these problems can be solved or avoided if taught proper techniques. Rarely, water is used as a solvent because it produces an uneven curve front which is mainly accounted for by its surface tension. Thin layer chromatography of three analgesics and caffeine under U. It is not a recommended technique in the laboratory. Due to the nature of the uv hazard polycarbonate safety spectacles which absorb short wavelength U.
The samples were dissolved in ethanol for spotting onto the plate. The TLC plate was run in an open beaker under short wavelength u. The movement of the dark purple spots samples during the running of the plate can be observed in the animation. The original movie can be viewed here.
It is easy to see which are the two active ingredients in the unknown commercial pain relief medicine by comparison of the spots with the standard reference materials running on either side caffeine and acetominophen. TLC is very simple to use and inexpensive. Undergraduates can be taught this technique and apply its similar principles to other chromatographic techniques.
There are little materials needed for TLC chamber, watch glass, capillary, plate, solvent, pencil, and UV-light. Therefore, once the best solvent is found, it can be applied to other techniques such as High performance liquid chromatography. More than 1 compound can be separated on a TLC plate as long as the mobile phase is preferred for each compound.
The solvents for the TLC plate can be changed easily and it is possible to use several different solvents depending on your desired results. As stated earlier, TLC can be used to ensure purity of a compound.
It is very easy to check the purity using a UV-light. You can modify the chromatography conditions easily to increase the optimization for resolution of a specific component. TLC plates do not have long stationary phases. Therefore, the length of separation is limited compared to other chromatographic techniques. Also, the detection limit is a lot higher. If you would need a lower detection limit, one would have to use other chromatographic techniques. TLC operates as an open system, so factors such as humidity and temperature can be consequences to the results of your chromatogram.
Retention Factor After a separation is complete, individual compounds appear as spots separated vertically. Apparatus Plates Stationary Phase As stated earlier, TLC plates also known as chromatoplates can be prepared in the lab, but are most commonly purchased.
Solvent Mobile Phase Proper solvent selection is perhaps the most important aspect of TLC, and determining the best solvent may require a degree of trial and error. How fast the compounds travel up the plate depends on two things: If the compound is soluble in the solvent, it will travel further up the TLC plate How well the compound likes the stationary phase.
If the compound likes the stationary phase, it will stick to it, which will cause it to not move very far on the chromatogram.
Useful Solvent Mixtures A solvent that can be used for separating mixtures of strongly polar compounds is ethyl acetate : butanol : acetic acid : water, Pipettes Spots are applied to the plate using very thin glass pipettes. The capillary should be thin enough to apply a neat spot, but not so thin as to prevent the uptake of an adequate quantity of analyte. Here is a popular method of producing TLC pipettes.
Heat a glass capillary in the very tip of a Bunsen burner flame just until it becomes pliable and then pull the ends apart until the center of the capillary is significantly narrower. Snap this in half and use the thin end to apply spots.
Cut the plate to the correct size and using a pencil never ever use a pen , gently draw a straight line across the plate approximately 1 cm from the bottom. Do not use excessive forces when writing on a TLC plate as this will remove the stationary phase. It is important to use a pencil rather than a pen because inks commonly travel up the plate with the solvent. An example of how black ink separates is shown in the section labeled "examples". Using TLC pipettes, apply spots of analyte to the line.
Make sure enough sample is spotted on the plate. This can be done by using the short-wave UV. A purple spot should be seen. If the spot is not visible, more sample needs to be applied to the plate. If a standard of the target compound is available, it is good practice to produce a co-spot by spotting the standard onto a spot of the unknown mixture.
This ensures the identity of the target compound. Place the plate into the chamber as evenly as possible and lean it against the side.
Never allow the bulk solvent to rise above the line you drew. Allow capillary action to draw the solvent up the plate until it is approximately 1 cm from the end. Never allow the solvent to migrate all the way to the end of the plate.
Remove the plate and immediately draw a pencil line across the solvent front. Use a short-wave UV light and circle the components shown with a pencil. The sequence involved in TLC. Visualizing If fluorescent plates are used, a number of compounds can be seen by illuminating the plate with short-wave UV.
Over-large Spot s: Spotting sizes of your sample should be not be larger than mm in diameter. The component spots will never be larger than or smaller than your sample origin spot.
If overlapping occurs, it would prove difficult to resolve the different components. Consequences would be inaccurate R f values due to the uneven advance of sample origin spots. This uneven advance can be caused by a few factors listed below. No flat bottom. When placing the TLC plate into the chamber, place the bottom of the plate on the edge of the chamber normally glass container e. Also, make sure that the TLC plate is placed in the chamber evenly.
Do not tilt the plate or sit it at an angle. Not enough solvent. There should be enough solvent depends on size of the chamber to travel up the length of the TLC plate.
Plate is not cut evenly. It is recommended that a ruler is used so that the plate is cut evenly. Streaking : If the sample spot is too concentrated, the substance will travel up the stationary phase as a streak rather than a single separated spot. In other words, the solvent can not handle the concentrated sample and in result, moves as much of the substance as it can up the stationary phase.
The area under the first peak was 36 m2 and the second was m2. The theoretical plate. Alumina was inserted into the column to act as. Background: Derived form the greek words for colour Chroma and writing graphe.
Chromatography is a method is method in which different kinds of the coloured chemical mixtures are separated. In the early s, Mikhail Tswett, a Russian botanist, was interested in the individual compounds presented in plants. He understood that removing ground up plants extracts with the dissimilar solvents will provide assorted coloured solutions. Tswett conducted an experiment which involved pouring a plant.
Chromatography is a process commonly used to separate. Mainly used in the mining industry for extraction of minerals and resources, explosives have been used as a weapon of destruction. Explosives often contain oxidising substances such as nitrates chlorates, and a binding agent such as ammonium and potassium, which can be detected and analysed to prevent such substances from being used as a weapon.
Explosives detection is critically important in many field settings e. The presence of an impurity in the molten compound reduces its vapor pressure thus lowering the melting point of the compound. Broaden the melting point range. For what tow purpose are melting points routinely used a. To determine the identity of an organic solid. To determine the purity of an organic solid.
What effects on the measured melting point would you expect in each of the following cases? The purpose of this experiment is to separate carbohydrates into its pure components such as mixtures of monosacrides by TLC. TLC is used to identify sugars in normal and pancreatic disease urine, the procedure is easy and reproducible. Thin Layer Chromatography I. Introduction Column chromatography is a technique used to separate non-volatile mixtures.
A sub-class to column chromatography is thin layer chromatography. Thin layer Chromatography is usually being used to separate compounds by the distribution between two phases. The two phases are mobile and stationary phase. It is usually performed on a sheet of glass coated with a thin layer of adsorbent silica named TLC plate.
0コメント