It's officially half way through the program and I am making significant progress on my project. I feel like every couple days I'm taught a new technique or tool to add to those that I'm already using. I've been very busy the past couple of days and it's great because it makes the day go by a lot quicker. Being busy also helps me make it to lunch, which doesn't come until about 2:30 or 3 in Spain. My stomach is used to it now, but I can't wait to eat lunch at noon again. Anyhow, in lab I now run TLC plates, use the HPLC, and today I used the NMR (nuclear magnetic resonance) for the first time.
Until now, I have been purifying a compound and checking the number and purity of compounds in my extract through TLC plates. These are good to check the purity of your sample quickly and compare it to other compounds in a couple minutes. However, in order to really see exactly what compound you are working with, an NMR is the best.
The University of Cadiz has three NMR instruments, 300, 400 and 600 Hertz. Each one offers a different level of resolution - the one used most often in the types of purifications we are doing is the 400 Hertz. This resolves very well between our peaks of interest, which tend to be more spread out. One of the PhD students working in my lab, Andy, uses the 600 Hertz NMR because he is working with sugars whose peaks all fall within a few parts per million (ppm) of each other.
After we ran NMRs on four of the samples I purified, I found that they are in fact all distinct compounds and pure! They are also all steriods, because of the large number of peaks at a low frequency. Reading an NMR takes lots of practice and memorization, but my mentors have been working with these molecules for years and know exactly what to expect. They explained, and I took notes ... in Spanish.
I will now continue running HPLC on isolated fractions in hopes of finding more compounds that are present in large amounts and can be purified. In this case, a large amount is anything more than 1 milligram. In a couple weeks, my mentors say I will be ready to run a bioassay. The bioassay involves using specific concentrations of the isolated pure compound to affect the growth of a plant seed. A bioassay can be done with pretty much any kind of seed (lettuce, tomato, wheat, onion). We will be using wheat. Once the seeds are in a petri dish with a certain concentration of the compound of interest, they are left for two or three days. After this time, the primary shoot coming out the seed is measured and compared to that of an untreated seed. The inhibition or activation of growth is quantatitively measured, and you can see if the isolated compound if bioactive. That is where our summer work will end (hopefully we make it that far). In the future, the mode of action for the bioactive molecule can be studied... but that gets really complicated.
As far as outside of lab news, this weekend is the 4th of July, and I will definitely be missing the fireworks. However, Joanne and Micheal are cooking another dinner for us on Saturday and it will be a big party (their kids and friends are coming, as is Dr. Casteel and her husband from Bucknell). It won't be the traditional 4th of July, but I'm really looking forward to it!
