Using and then different amounts of BSA was
Using Chromatography and Spectrophotometry to Analyze and Interpret Amino Acids and ProteinsHypothesis: If amino acids are spotted onto a polar chromatography paper and sit in a nonpolar solution for one hour, the more hydrophilic amino acids will have a lower Rf value due to their tendency to adsorb to the matrix and travel a shorter distance. Introduction: There are 20 naturally occurring amino acids, and they are either classified as polar or nonpolar (Freeman 78-79). Polar amino acids are hydrophilic, and nonpolar amino acids are hydrophobic. Characteristics of amino acids are used to differentiate and establish the different amino acids in a process called chromatography (Lombard 18). In this lab, different amino acids were spotted onto a matrix with a micropipet and put into a non polar solvent for one hour. After it was taken out and dried in a fume hood, the distance each amino acid traveled was measured, and the Rf values were calculated by dividing the distance traveled by each component by the distance traveled by the solvent (Lombard 19). The amino acids that were more hydrophilic adsorbed into the paper, in contradiction with the hydrophobic. Higher Rf values were obtained by the more nonpolar, hydrophobic amino acids. Paper chromatography is useful for determining unknown substances when the known substances and unknown substances are spotted onto the same matrix (Kuwata 1). Spectrophotometry is an examination of substances based on their ability to absorb specific wavelengths of light (Lombard 22). In this lab, a spectrophotometer was used to detect and measure the amount of light absorption at a specific wavelength (Lombard 22). A standard curve can be created using the absorbances of different concentrations. In this lab, absorbances were tested using a spectrophotometer from wavelengths of 490-570 nm, increasing by 10 nm each time. A biuret reagent was mixed with distilled water, and then different amounts of BSA was added to different test tubes, including a blank test tube and an unknown sample.Materials and Methods:The lab manual and procedure were strictly followed during this experiment (Lombard).Results: In the experiment involving the paper chromatography, the six given amino acids and their respective Rf values were aspartic acid; 0.16, histidine; 0.33, leucine; 0.71, lysine; 0.244, proline; 0.488, and valine; 0.622. The colors of each of each of these amino acids that appeared on the matrix were dark blue, dark purple, light purple, dark purple, yellow, light purple, and light blue, respectively. There were also two unknown amino acids, Unknown X and Unknown Y. They each had two different colors and Rf values, as they were each a mixture of two amino acids. Unknown X had colors of light blue and yellow, with Rf values of 0.15 and 0.53, so were expected to be the amino acids aspartic acid and proline, respectively. Unknown Y had colors of light purple and dark purple, with Rf values of 0.622 and 0.280, so were expected to be the amino acids valine and lysine, respectively. The spectrophotometry experiment tested absorbances at a wavelength of 550 nm, as that had the highest absorbance for the presence of albumin in test tube 5. The blank test tube had 3.0 mL of distilled water, and the rest of the test tubes successively 0.5 mL less, except the unknown test tube had 2.0 mL of distilled water. Every test tube had exactly 2.0 mL of biuret reagent, including the unknown test tube. Each test tube had 0.5 mL more of 2.5 mg/mL BSA stock than the one before it, starting with the first test tube at 0, and excluding the sixth test tube with 1.0 mL of the BSA stock.The first test tube was a blank, so the absorbance was 0 at 550 nm. Test tubes 2-5 followed with absorbances of 0.067, 0.145, 0.222, and 0.293. The unknown test tube was filled with an unknown BSA, and at 550 nm, had an absorbance of 0.066. Conclusion:The results of the experiment done with chromatography supported my hypothesis, as the more polar amino acids adsorbed to the paper, and crawled a shorter distance than the hydrophobic amino acids.This then means that the nonpolar amino acids had higher Rf values than the polar ones, because they were absorbed to the matrix. This is because of the “like likes like” theory, the polar amino acids adsorbed to the polar chromatography paper. Because of the ability of amino acids to react or not react with the chromatography paper, it was possible to identify the unknown X and Y absorbances. The Rf values of Unknown X (0.53 and 0.15) were very similar to those of proline and aspartic acid (0.488 and 0.16). The Rf values of Unknown Y (0.622 and 0.280) were very similar to those of valine and lysine (0.622 and 0.244). Potential drawbacks of the chromatography experiment that could have skewed results could have been the placement of spots on the matrix, whether exactly on the X’s drawn exactly on the line, or slightly above or below. For the spectrophotometry lab, the top to the spectrophotometer may have not totally closed over the test tube, skewing results as well. The results of the spectrophotometry lab made it possible to find out the final concentration, in mg/mL of the BSA in the Unknown A sample. After making the standard curve graph, it made it easy to tell what the concentration of the Unknown A due to the absorbances and BSA concentrations of the other 5 test tubes. A line of best fit works to see where the unknown BSA concentration is met with the 550 nm absorbance of 0.066.