Syringe. Clean. Aspirate. Inject.

In four actions, that is essentially a day at work for me. Let me clarify, that’s just the series of actions you go through when you’re running samples through High Performance Liquid Chromatography (HPLC). I’m essentially a lab assistant and in a day of work I help around lab, clean, and run samples through HPLC. I’ve also helped in lab with an unexpectedly biological aspect: the sacrifice and dissection of rats and rat brains, respectively. I want to talk about the two main contributors to my day in lab but also discuss how I’m still growing by working in lab.

HPLC is an analytical chemistry technique used to separate analytes of interest in a solution and analyze them. Paired with an electrochemical detection (ED) system and computer software it can visualize the presence of your analytes as peaks on a chromatogram and estimate concentrations. This technique is simply column chromatography performed under pressure. Simple column chromatography is a column with some sort of “stationary phase” particles through which a liquid mobile phase is passed through at the same time a sample is in the column. A combination of the interactions of the analytes with the stationary and mobile phases will determine the order in which the analytes are eluted, or released. HPLC is this in essence except in order to achieve effective detection of little analyte, it is run under pressure. The applied pressure pumps the mobile phase through the column of stationary phase and a sample is ejected in. This is the most tedious part of the day for me. The apparatus just runs for hours when the pump is turned on and it is just a matter of constantly taking a syringe, cleaning it, aspirating sample, and injecting it. Though tedious, it is a necessary process. Further, the interesting components of HPLC is the ED system.

The ED system works through an electrochemical cell with working, auxiliary, and reference electrodes. As analytes pass through the column they diffuse through into the cell and undergo an electron transfer reaction, an oxidation or reduction reaction, electrons are transferred and detected by the working electrode, and the signal appears as a peak on a chromatogram through a computer program. Almost exclusively because of the electron absorbing properties of the working electrode, an oxidative reaction is the preferred electrochemical reaction to occur after diffusion of analyte into the electrochemical cell. HPLC conditions such as the pH of the mobile phase and the temperature used to run the apparatus are tailored to favor oxidative reactions. The peaks are further processed using computer programming to determine concentration of analytes by comparison with a pre-made standard curve. This final step wraps up of the process of HPLC paired with an ED system. What is left to be discussed is the animal handling part of my work before moving onto what I have gained from these experiences.

A total of three times, I have helped in the process of harvesting blood and brain samples from male and female rats. We begin by weighing the rats and performing one of three treatments on them: 1) gavaging them with the RTD (Rapid Tryptophan Depletion) amino acid (AA) solution, 2) the BAL (balanced AA solution with all essential AAs including tryptophan), or 3) treating them with neither solution. We euthanize the rats in a bell jar to the point that they will not suffer during the sacrifice. The brain is extracted from the cranium and dissected for predetermined brain parts. The blood is also centrifuged for the extraction of plasma and the brain is stored before being sonicated later and centrifuged for analysis. Both the plasma and brain samples are run by me and my lab supervisor, Zack Reavis, through HPLC for sample analysis.

These two processes are essentially tedious yet necessary for the continuation of progress in lab. Sometimes it does become quite drab and it really starts to feel like I’m working a glorified 9-5 job. However, I quickly correct myself. I cannot take for granted just how much I have learned in lab, how many problems I’ve encountered that I’ve had to actually think about to solve. My PI was so particular about designing a project that would somehow allow me to encounter a problem essentially chemical in nature for me to solve and actually do some chemistry! I helped adjust a protocol such that it was tailored for our needs during sample analysis: adjusting the pH of the mobile phase such that the eluted analytes came at a rate that would produce a clean chromatogram with nicely separated peaks. I need to appreciate that even though I am completing a project here that will allow me to experiment to answer a question I’m mostly here this summer to learn what it is like to work in a lab environment, learn some lab techniques, and just get a feel for the life of lab work. This is exactly what I have gotten to do and I’m getting quite a good feeling about what it’s like working in the lab. I’m beginning to think this is something I might want to do for the rest of my life.

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