Today marked the end of the first week in the lab.
We attempted to shorten the gradient on the HPLC after looking at the previous day's run. We started from the conditions at time=30 minutes, where methanol and buffer concentration were both 50%. The run was also extended to 70 minutes. After letting these settings run, the resulting graph showed that we pretty much messed up. The peaks were not clear like the last run.
We tried attempting a few different gradients with different conditions, but each time, the graph seemed to show worse and worse results.
On the GCMS, we ran another sample of 2:1 chloroform:methanol to clean out the machine. The peaks decreased from the previous run, so it seems like the machine is getting clean!
We ran another sample on the GCMS, and, unfortunately, the peaks were higher, indicating that the machine was not clean.
Today was a little disheartening because we could not get any consistent good results, but hopefully the next samples will produce better results!
Monday, January 13, 2014
Thursday, January 9, 2014
Day 4
I feel like today good progress was made for our research.
We made an internal standard solution with oleic, nonadecanoic, stearic, palmitic, and linoleic acid. Each acid had a concentration of 0.003 M in 100 mL of solution.
The internal standard was run in the HPLC. Ideally, we would get five peaks showing the five different components we put in the solution, but we actually got seven peaks. Some of the acids may be contaminated. To be able to determine which peak is which, individual solutions of each acid needs to be prepared and then run through the HPLC. Only the peaks from that acid will show up on that run.
We made the standards for each individual acid today and will run them tomorrow in the HPLC.
We also discussed what our goals were for tomorrow and how we would accomplish them, including shortening the gradient time on the HPLC, how to calculate the response factor for each acid, and how to calculate the concentration of each acid in a saliva sample.
We made an internal standard solution with oleic, nonadecanoic, stearic, palmitic, and linoleic acid. Each acid had a concentration of 0.003 M in 100 mL of solution.
The internal standard was run in the HPLC. Ideally, we would get five peaks showing the five different components we put in the solution, but we actually got seven peaks. Some of the acids may be contaminated. To be able to determine which peak is which, individual solutions of each acid needs to be prepared and then run through the HPLC. Only the peaks from that acid will show up on that run.
We made the standards for each individual acid today and will run them tomorrow in the HPLC.
We also discussed what our goals were for tomorrow and how we would accomplish them, including shortening the gradient time on the HPLC, how to calculate the response factor for each acid, and how to calculate the concentration of each acid in a saliva sample.
Wednesday, January 8, 2014
Day 3: First Troubles
The first thing we did today in lab was go check the results of the series of GCMS runs that we created at the end of the day yesterday. When we got to the machine, there was an error message from the fourth sample of the series, so it did not get even close to being finished. We couldn't even log onto the computer, so there was some real trouble. Hopefully, the computer will be fixed by another professor in the chemistry department by tomorrow.
We then made a phosphate buffer for the HPLC to use, since the amount of buffer in the machine was low. We made a solution by diluting phosphoric acid in water, then adding concentrated sodium hydroxide to get to the desired pH of 3. The buffer was added to the HPLC.
Tony Le, who did research with Dr. Splawn this past semester, came to the lab to show us how to create a saliva sample. He mixed together 11 mL of chloroform, 5.5 mL methanol, and 0.5 mL of water. Three mL of oil is injected in the mouth, swirled around for 3 minutes, and then spit into a small beaker (you want to generate around 10 mL of saliva). Two mL of ethanol is added to the sample to kill any enzymes.
The saliva sample and the chloroform, methanol, and water mixture are combined into a separatory funnel. The flask is then inverted for 3 minutes and then is allowed to sit to let the layers separate. The bottom layer, which contains the fatty acids, is the one we want to collect. The sample is then ready to be put into a machine.
Neelam and I tried to create our own sample after Tony showed us, but our layers would not separate. Hopefully tomorrow we can create some successful samples!
We then made a phosphate buffer for the HPLC to use, since the amount of buffer in the machine was low. We made a solution by diluting phosphoric acid in water, then adding concentrated sodium hydroxide to get to the desired pH of 3. The buffer was added to the HPLC.
Tony Le, who did research with Dr. Splawn this past semester, came to the lab to show us how to create a saliva sample. He mixed together 11 mL of chloroform, 5.5 mL methanol, and 0.5 mL of water. Three mL of oil is injected in the mouth, swirled around for 3 minutes, and then spit into a small beaker (you want to generate around 10 mL of saliva). Two mL of ethanol is added to the sample to kill any enzymes.
The saliva sample and the chloroform, methanol, and water mixture are combined into a separatory funnel. The flask is then inverted for 3 minutes and then is allowed to sit to let the layers separate. The bottom layer, which contains the fatty acids, is the one we want to collect. The sample is then ready to be put into a machine.
Neelam and I tried to create our own sample after Tony showed us, but our layers would not separate. Hopefully tomorrow we can create some successful samples!
Tuesday, January 7, 2014
Day 2: Learning the Lab
Today, Neelam and I received an introduction to the instruments that we are going to be working with over the month: the GCMS and the HPLC.
Unfortunately, from past research, the machines are a little clogged up with residual fatty acids. The first thing that needs to be done over interim is get rid of those fatty acids. We prepared a 2:1 solution of chloroform to methanol, respectively. We then ran samples of this solution in both machines.
In the GCMS, after the first run, there were still peaks indicating the presence of fatty acids. To hopefully fix this problem, we set up a series of 21 measurements to run overnight where the machine will keep taking more of the chloroform and methanol solution to clear it out.
In the HPLC, after the first run, the machine seemed to be cleared out. The spectrum that resulted after our run showed a peak, but its absorbency was only 5 MAU, where a peak for fatty acids or other compounds would be expected to be in the hundreds. We can basically assume this very small peak is zero and the machine is ready for other samples.
Unfortunately, from past research, the machines are a little clogged up with residual fatty acids. The first thing that needs to be done over interim is get rid of those fatty acids. We prepared a 2:1 solution of chloroform to methanol, respectively. We then ran samples of this solution in both machines.
In the GCMS, after the first run, there were still peaks indicating the presence of fatty acids. To hopefully fix this problem, we set up a series of 21 measurements to run overnight where the machine will keep taking more of the chloroform and methanol solution to clear it out.
In the HPLC, after the first run, the machine seemed to be cleared out. The spectrum that resulted after our run showed a peak, but its absorbency was only 5 MAU, where a peak for fatty acids or other compounds would be expected to be in the hundreds. We can basically assume this very small peak is zero and the machine is ready for other samples.
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