Development of Chromatograms

Standard solutions of amino acids are prepared in a suitable solvent such as 70% EtOH or 0.1 molL"1 HCl in 95% ethanol. These solutions are generally applied as tight spots, 1-2 cm from the bottom of each layer, using a glass capillary or Hamilton syringe. In the beginning a higher concentration, e.g. 500 ng or more, is applied; however, the detection limits are determined for the system developed by repeating the experiment with lower concentrations.

The chromatograms are generally developed in rectangular glass chambers, which should be paper-lined for good chamber saturation and pre-equilibrated for 20-30 min with solvent before use. The time taken depends on several factors such as the nature of the adsorbent, the solvent system and the temperature. The developed chromatograms are dried in an oven between 60 and 100°C, and the cooled plates are usually sprayed with ninhydrin reagent. Heating at 90-100°C for 5-10 min produces blue to purple zones of all amino acids except proline (yellow spot).

The same method is adopted for both one- and two-dimensional modes. The locating reagent is used after the second run, and a more polar solvent is generally used to develop the chromatogram in the second dimension.

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Solar Panel Basics

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