Spring SAVY 2017, Day 5- Chromatography 101
We began this week by reviewing our high-performance liquid chromatography (HPLC) lab results from last week. The students explained that they were able to identify which water contaminant was which by comparing retention times (the amount of time a compound in the mixture stayed on the column) with that of a standard compound. Then they were able to determine the amount of a given compound in the sample by comparing the peak area on the chromatogram with those of known concentrations of each compound (plotted as a standard curve on a graph). We noted that there are several advantages of HPLC over the previous types of chromatography that we have examined; for example, HPLC is usually much quicker, requires less active monitoring than column chromatography, allows for easier determination of when all of a given compound has eluted from the column, and enables quantification of a compound (as opposed to just detection). I then asked the students what they would do if they detected a peak in their chromatogram that didn’t match any of their standards. Suggested ideas included collecting the compound for visual analysis or comparison of the compound’s retention time with some sort of database. While these suggestions might be viable in some cases, if the compound was clear in solution or wasn’t previously analyzed under the exact same conditions, we would probably be left speculating as to the compound’s identity.
This led us to our new technique for this week: LC-MS/MS (liquid chromatography mass spectrometry). This technique is very similar to HPLC, except instead of a UV detector for detecting the presence or absence of a compound, it employs a mass spectrometer as its method of detection. A mass spectrometer enables determination of a compound’s unique mass, and in the case of a MS/MS set-up, it allows for determination of a compound’s mass and the mass of one or more fragments of the compound that form when the compound is “smashed” to pieces. Thus, if one was to detect an unknown compound eluting from the HPLC column, one could at least determine its mass, which might aid in its identification. When I asked the students to brainstorm a list of possible uses for this technique, they came up with a wide range of ideas—everything from pharmaceutical scientists analyzing drug purity to clinical scientists interrogating samples for the presence of drugs-of-abuse to biochemists quantifying compounds of interest to synthetic chemists determining reaction products.
In our lab experiment this week, we took on the role of forensic scientists looking for the presence of a drug at two potential crime scenes. (Another advantage of mass spectrometry is that it can be used to detect extremely low levels of analytes, making it ideal for trace evidence analysis!) Neither crime scene showed obvious evidence of drug presence, so we swiped down each surface with a cotton swap dipped in methanol and placed the sample in a mass spectrometry vial. We then took a field trip to the Mass Spectrometry Research Core at Vanderbilt and analyzed our two samples, as compared to a standard solution of the drug. Ask your student to share our results with you.
I hope I have the opportunity to meet with you next week from 11:15-11:45 during our Parent Open House.