Chromatography In Sequencing A Hexapeptide

Chromatography is a separation technique between substances in a solution in which for the results you will get different components that were present in the solution. According to the Biochemistry 313 Lab Manual 3rd edition, the three steps of purifying a protein is cell lysis, sample clarification and lastly chromatograph [1]. There are many different chromatography procedures but today we will focus on TLC (thin layer chromatography) in sequencing a hexapeptide. Given two set of tubes that has our controls and the unknowns, we have to determine the unique sequence for the pertaining hexapeptide each of the unknown tubes. This technique is very helpful when separating and identifying compounds is needed.

Introduction

In a thin layer chromatography procedure, there are two phases; a stationary phase and a mobile phase. The stationary phase in this lab would be the silica gel that is present on the TLC plate. On the other hand, the mobile phase would be the molecules that are being separated. According to the Central European Journal of Chemistry, it states multiple of advantages in using this technique. It states that TLC is very convenient and simple to perform, it consumes small portion of solvents, along with being flexible with using stationary phases and mobile phases and has many more advantages [2]. Performing TLC lab is also very useful in ruling out the polarity of the components. According to the polarity rule, molecules that have a low polarity will often have a weak affinity towards the gel and then will migrate upwards the plate towards the solvent front. In the lecture that Sharmaine S. Cady wrote, she stated as the polarity increases, it decreases the attraction of the polar solutes and thus increases the distan...

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... to modify a reagent for the amino groups were FDNB (fluorodinitro benzene) which we then used a acid hydrolysis which then yielded DNP-Lys and DNP-Ala. In TI and TII, the trypsin was split into two different fragments which was then purified and separated from each other. Then the treatment to separation the different portions of hexapeptide follow. The hexapeptide that consisted of chymotrypsin yielded three different fragments which were very large after it was purified and also hydrolyzed entirely. The amino that were released are present in CIII tube. Finally, two peptide bond cleavages happened because of the limited amount of acid hydrolysis of the hexapeptide which caused it to yield a free amino acid, a dipeptide and also a tripeptide. We took the tripeptide and purified and hydrolyzed it into different components of amino acids which are found in tube HIV.