Summary
The main separation theory of chromatography is lying in that when a substance dissolved in the mobile phase pass through the stationary phase, it will react with the stationary phase. And then researchers can separate them according to the different sizes, strength and retention time.
Body
This post will give us a clear view of the workflow of HPLC in detecting amino acid.
Theory
After hydrolyzed in acid and base, the protein sample will dissolve mobile phase. And then we can use the RP-HPLC with C8 reversed phase column fluorescence detector to detect the content of all kinds of amino acid.
Instruments and reagents
Here we mainly use the high performance liquid chromatography apparatus. As for reagents, we choose acetonitrile, acetone, acetone solution of dansyl chloride, buffer solution, Sodium hydroxide solution 0.5mol/L, hydrochloric acid solution 0.01mol/L and 6mol/L and barium hydroxide solution 6mol/L and Acetic acid, phosphoric acid, amino acid mixed solution, internal standard solution, mobile phase solution. Some solution should be prepared when we are doing the experiment.
Sample preparation and measurement
A. Protein hydrolysis
Putting protein sample about 1 mg into the glass tube with 6mol/L hydrochloric acid 1 ml. And then sealing the opening. And heated for 16 hours in the 110 celsius incubator, it will be cold outside. And then putting hydrolyzed sample solution into a glass tube and using the norleucine as an internal standard solution, and then it should be steamed.
B. Preparation and determination of derivatives
Adding buffer solution about 40 microliters into the sample we made above. And then we can add dansyl chloride solution 100 microliters and shack solution for 15 minutes. Next the sample tube should be heated in the 100 celsius water for 2 minutes. And then we add the two solutions into a sample tube to shake for 15 seconds. The last step is to add 10 microliters sample solution into the HPLC columns and note the chromatogram.
These are main tips for hplc used in detecting amino acid. As for the final steps are to calculate the data. We hope these tips can meet your demand.