If you are using chromatography to analyze samples, you may wonder why should chromatography spots be small. First, you should know that chromatography spots are tiny particles surrounded by a eluting solvent. However, these particles should be spaced at least half an inch apart from the edges of the plate. This is because the eluting solvent will come in contact with the spots during separation.
Streaking chromatography spots
To get smaller spots, a slurry of silica and eluting solvent is added to the column under positive pressure. A layer of sand can be added to protect the silica gel, which may also cause streaking. The reaction mixture must be diluted to a minimum concentration in the eluting solvent before spotting. Once the reaction mixture is diluted to the required concentration, it is added gently to the top of the column.
The adsorbent plate should be removed from the developing chamber when the solvent front moves within a cm of the adsorbent surface. Streaking spots can be removed by adding ammonia or formic acid to the eluting solvent. Alternatively, streaking can be a sign of a detached adsorbant layer or scoring of the plate. Double spotting can also indicate improper application of a polar solvent.
Besides small sample size, spotting the sample with a TLC capillary should be smaller than two millimeters. The reason for this is that, if the sample spots are large, they will overlap with each other, making it difficult to separate components. Thus, small initial spots maximize separation potential. If you can avoid this, your chromatography experiments will be as accurate as they could be.
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Uneven advance of solvent
The main cause of achromatic chromatography spots being small is an uneven advance of the solvent. The solvent is usually used in the reverse phase of the chromatogram, but if you are using a polar solvent, it will tend to spread out the starting spot. The solvent used should be volatile and non-polar, since polar solvents cause chromatogram spots to be small. Aim for a small, uniform spot that is no more than 2-5 mm in diameter.
An uneven advance of the solvent can result in a spot being too small or too big. To solve this problem, the first step is to make sure the solvent is uniformly distributed over the plate. This will reduce the chances of a large spot. In addition, the solvent should be applied in one direction, and the stationary phase should be dried to 90 degrees. Using a short-wave UV light, check for a visible spot.
In order to prevent a small spot from forming, ensure the column is properly positioned. A flat bottom on a TLC plate will prevent uneven advance of the solvent. And always make sure that the glass bottles are placed in an even fashion. The bottom of the glass container will also influence the shape of the chromatographic spots. If the solvent level drops below the second mark, it is time to collect the fractions.
Using the least polar solvent
A suitable eluent has a retardation factor that is close to 0.5. If it is too high, you can adjust the eluent by reducing the polar solvent percentage or switching to a more polar solvent. It is best to use an eluent with a low Rf. The eluent should be polar enough to make chromatography spots small, but not too polar to bind the sample.
The type of solvent you choose will depend on the nature of the substance you’re trying to identify. While nonpolar solvents are fine to use for impurities, polar compounds will require a more ionic solvent in order to elute. In addition, polar solvents may dissolve the silica solid phase. The most polar solvents include pentane, hexane, and cyclohexane.
If you’re trying to identify a specific amino acid, you can try making a mixture of amino acids with a coloured product. The coloured product will make the chromatogram visible, and you can use it to identify the specific amino acid. When you use the least polar solvent, you can use the same procedure for separating a mixture into a small number of different spots.