In this experiment, image analysis techniques are applied to quantify the amino acids present in a given mixture.
- To quantify amino acids present in a given mixture by analyzing the intensity of the spots detected in the Thin Layer Chromatography plate.
Image quantification studies are one of the most important aspects of image analysis. The accuracy of the information obtained through the analysis software depends not only on the quality of the image, but also on the ability of the analysis software to distinguish the image features from the provided data and to convert it into meaningful measurements. In this lab, the densitometry analysis of thin layer chromatography (that is the determination of intensity of specific spots) is analyzed to find the apparent percentage of amino acids present in the mixture. Here the analysis is done by using image analysis software, ImageJ. It is freely available via the internet, platform independent and is applicable to the biological researchers to quantify the results obtained in the wet laboratory techniques. The basic steps involved in densitometry analysis of amino acids are as follows:
- TLC plate images are imported into the software and the contrast is adjusted in such a way to obtain clear spots on the TLC plate.
- Using rectangular selection tool, area around each bands to be analyzed are selected.
- Subtract the background of the image, which helps to a create background uniformity for all the bands, to nullify the effect of background in each band and to normalize the value across the spots in the TLC plate.
- Analyze the spots using the software.
- The intensities of the spots are displayed and the relative concentration of amino acids in the sample is quantified.
Thin Layer Chromatography
Chromatography is by far the most useful general group of techniques available for the separation of closely related compounds in a mixture. Thin layer chromatography is a technique used to separate and identify compounds of interest present in a given mixture. A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for support. The silica gel acts as the stationary phase and the solvent mixture acts as the mobile phase. In the ideal solvent system the compounds of interest are soluble to different degrees. Separation results from the partition equilibrium of the components in the mixture. In the simplest form of the technique, a narrow zone or spot of the sample mixture to be separated is applied near one end of the TLC plate and allowed to dry. The strip or plate is then placed with this end dipping in to the solvent mixture, taking care that the sample spot/zone is not immersed in the solvent. As the solvent moves towards the other end of the strip, the test mixture separates into various components. This is called as the development of TLC plates. The separation depends on several factors such as:
- Solubility: the more soluble a compound is in a solvent, the faster it will move up the plate.
- Attractions between the compound and the silica: the more the compound interacts with silica, the lesser it moves.
- Size of the compound: the larger the compounds the slower it moves up the plate.
Amino acids have a carboxyl group and an amino group bonded to the same carbon atom (α- carbon). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge. The interaction of the amino acids with the stationary phase like silica varies depending on their 'R' groups. The amino acid that interacts strongly with silica will be carried by the solvent to a small distance, whereas the one with less interaction will be moved further. By running controls (known compounds) alongside, it is possible to identify the components of the mixture. Since amino acids are colourless compounds, ninhydrin is used for detecting them.
For more details refer Biochemistry Lab 1, Separation of Amino acids by Thin Layer Chromatography