High-Performance Liquid Chromatography (HPLC)

Last Wednesday (April 11th, 2013), Eun Ji showed me how a high-performance liquid chromatography (HPLC) work. A HPLC is a chromatographic technique used to separate a mixture of compounds in biochemistry or analytical chemistry to identify, quantify or purify the individual components of the mixture [1]. 

Simplified map of a HPLC
Retrieved from http://web.nmsu.edu/~kburke/Instrumentation/Waters_HPLC_MS_TitlePg.html

First, we connected the computer to the machine. We can control the flow rate by typing in the computer. Eun Ji had made two kinds of buffer (solvent) A and B for her proteins, each of which had a thin tube connected to the pump where the two buffers mix. We typed in 1.000 ml/min for the flow rate (*A+B = 1ml not 1ml for A and 1ml for B) Overtime, the concentration of A decreases while the concentration of B increased, but the flow rate remained the same. This means that [A] and [B] in the mix solution in constantly changing. Concentration of A and B manipulated the polarity in the column.

Next, Eun Ji injected her sample through injector. 

Together, the solution and sample traveled to the column. A column contained very small resins that formed a fine filtrate. The sample contained a mixture of proteins. However, according to the affinity of each protein, proteins gradually separated from each other as [A] and [B] changed and flew out the column at different times.

Black sample is separated into blue, red, and yellow (3 proteins)
Retrieved from http://www.waters.com

As the separated protein bands leave the column, they pass immediately into the detector. The computer then construct a graph that contained "peaks" in it. Each peak represented a protein, so by counting how many peaks were there, we were able to determine how many kinds of protein were present in the sample (*but cannot determine "what" proteins are they unless by using special HPLC or conducting further study).

Retrieved from http://www.waters.com

However, we there were something wrong with the machine when we ran our HPLC. The pressure of the tubes continued to rise (normal: A-60, B-100; ours: A-90+, B-140+) and that the pumps automatically stopped to prevent the tubes from bursting. We reset the machine again, but it did not help. In addition, pump B was making weird noise, so we stopped our experiment. Nevertheless, I thought HPLC is a really brilliant tool. Before I have read many papers containing HPLC in their methods, and I am very glad that I now know what it means!

Where does this fit on our map?