Today was officially the last day of work in the lab, and it certainly was a busy one.
We have been rushing to get some last minute results with a new method. We have been methylating the fatty acids in hopes that they will separate better on the chromatogram. Our theory is that replacing the -OH group with an -OCH3 group will cause the fatty acid to not stick to the column as much, providing cleaner peaks on the chromatogram that do not have so long of a tail.
When we checked our results this morning, the internal standard was the best run we have had so far! We accidentally made the samples in the wrong concentration, so we had to rerun all of them, but we had high hopes. Late tonight, Neelam and I went back to check the GC/MS and found awesome results. There were few background peaks in the saliva/water sample, and fatty acid peaks in the oil/saliva sample look great.
Results like this are great to see and provide a promising future for the project. I really hope that I will have the opportunity to continue doing this with Neelam and Dr. Splawn because I feel that we can start quantifying our results and publish something official, which we hoped to do over interim but ran out of time.
Tuesday, January 28, 2014
Thursday, January 23, 2014
1/23
After examining the data from last night's sequence, we decided that a ramp of 15 degrees celsius per minute is the best. This method has a better peak shape and has a faster run time.
We decided to remove stearic acid from our research because we believe it is causing trouble in reading other peaks and we cannot get a good shape on the stearic acid peak.
We set up an experiment with different flow rates to see if that would help separate the oleic and linoleic peaks. We also changed our method to have a solvent delay of 8 minutes to create a better baseline in the chromatogram.
We also are running a sequence with different types of oil/saliva samples:
1. saliva + water
2. water + oil
3. saliva + oil
This is done with corn, olive, and mineral oil.
From our past runs on internal standards, we were able to calculate some peak areas and retention times for different acids that were broken down. With this information, we were able to calculate the response factor for the different acids.
We decided to remove stearic acid from our research because we believe it is causing trouble in reading other peaks and we cannot get a good shape on the stearic acid peak.
We set up an experiment with different flow rates to see if that would help separate the oleic and linoleic peaks. We also changed our method to have a solvent delay of 8 minutes to create a better baseline in the chromatogram.
We also are running a sequence with different types of oil/saliva samples:
1. saliva + water
2. water + oil
3. saliva + oil
This is done with corn, olive, and mineral oil.
From our past runs on internal standards, we were able to calculate some peak areas and retention times for different acids that were broken down. With this information, we were able to calculate the response factor for the different acids.
Wednesday, January 22, 2014
1/22
The first thing we did today was look at all of the data from the sequence that ran last night. None of the standards really turned out the way we thought they would, and they all really looked the same. All of the runs on the saliva looked good, however.
After looking at all of the data from the different methods, we decided that the method that ramped up the temperature at 10 degrees Celsius per minute looked the best. We think more experimenting with different methods could produce better results, so we started to prepare for new methods.
We created a new internal standard mix with a higher concentration (200 ppm) of nonadecanoic acid than before. We created five individual standards with each individual acid. A new saliva sample was created. We prepared samples for the GC/MS for each of the previously mentioned and additionally GC/MS samples of pure hexane and 2:1 chloroform:methanol.
We set up the sequence the run with ramp temperatures of 15, 20, and 30 degrees celsius per minute. Hopefully by tomorrow we will be able to tell which ramp rate is the best, and then we will work on figuring out the optimal flow rate of helium through the machine.
After looking at all of the data from the different methods, we decided that the method that ramped up the temperature at 10 degrees Celsius per minute looked the best. We think more experimenting with different methods could produce better results, so we started to prepare for new methods.
We created a new internal standard mix with a higher concentration (200 ppm) of nonadecanoic acid than before. We created five individual standards with each individual acid. A new saliva sample was created. We prepared samples for the GC/MS for each of the previously mentioned and additionally GC/MS samples of pure hexane and 2:1 chloroform:methanol.
We set up the sequence the run with ramp temperatures of 15, 20, and 30 degrees celsius per minute. Hopefully by tomorrow we will be able to tell which ramp rate is the best, and then we will work on figuring out the optimal flow rate of helium through the machine.
Tuesday, January 21, 2014
1/21
There were a few goals we had in mind to get accomplished for today:
1. make a new internal standard mix in hexane
2. run the internal standard on the GCMS, print to chromatogram, identify the peaks, calculate the peak area, and then calculate the response factor
3. run a blank hexane to clean the machine
4. make a fresh saliva sample and run it on the GCMS
5. clean the machine again with a sample of hexane
A new standard was created and ran under a few different methods on the GCMS. We are still struggling to find the best way to get a good separation of peaks. Eventually, we ran our first saliva sample on the GCMS. The peaks did not turn out very well.
Time is running out this interim semester, so we set up a sequence to run overnight with four different methods. Each method varied in different elements such as solvent delay and the speed it ramps up the temperature. We set up three runs of the standard for each method followed by a hexane run, then a run on the saliva, then a final hexane run to clean the machine.
Hopefully tomorrow we will see that one of these methods produces really good results and we will be able to run a lot of saliva samples tomorrow with it, finally collecting the data we need to get some results.
1. make a new internal standard mix in hexane
2. run the internal standard on the GCMS, print to chromatogram, identify the peaks, calculate the peak area, and then calculate the response factor
3. run a blank hexane to clean the machine
4. make a fresh saliva sample and run it on the GCMS
5. clean the machine again with a sample of hexane
A new standard was created and ran under a few different methods on the GCMS. We are still struggling to find the best way to get a good separation of peaks. Eventually, we ran our first saliva sample on the GCMS. The peaks did not turn out very well.
Time is running out this interim semester, so we set up a sequence to run overnight with four different methods. Each method varied in different elements such as solvent delay and the speed it ramps up the temperature. We set up three runs of the standard for each method followed by a hexane run, then a run on the saliva, then a final hexane run to clean the machine.
Hopefully tomorrow we will see that one of these methods produces really good results and we will be able to run a lot of saliva samples tomorrow with it, finally collecting the data we need to get some results.
Wednesday, January 15, 2014
Day 8
I think the biggest success of the day was the next attempt at saliva samples. My sample turned out the clear yellowish color we have been looking for, as did Neelam's! This is great news. Whenever we are finally ready to start running our own samples in the GCMS and HPLC, we will have mastered the technique to producing the saliva samples.
We also made another liter of the phosphate buffer (~pH 3) for the HPLC to use.
We ran a sample of the new 0.01 M internal standard on the HPLC. There were finally some peaks that showed up, but there was a bad baseline on the chromatogram. I am interested to see how the chromatogram of another sample of this standard will turn out.
Tomorrow, hopefully we will really get going on the GCMS. We are going to change our solvent for our standard to hexane. What we want to see with our runs is three good chromatograms with nicely shaped peaks and then we can move on to running our own samples. Hope we can knock that out tomorrow!
We also made another liter of the phosphate buffer (~pH 3) for the HPLC to use.
We ran a sample of the new 0.01 M internal standard on the HPLC. There were finally some peaks that showed up, but there was a bad baseline on the chromatogram. I am interested to see how the chromatogram of another sample of this standard will turn out.
Tomorrow, hopefully we will really get going on the GCMS. We are going to change our solvent for our standard to hexane. What we want to see with our runs is three good chromatograms with nicely shaped peaks and then we can move on to running our own samples. Hope we can knock that out tomorrow!
Day 7
The results on the HPLC showed that there are three consistent small peaks downfield. This means that something is stuck in the machine, but we may be able to just run our samples and subtract out those three peaks that keep showing up. We did run the other half of the sequence on the HPLC because the first half did not show expected peaks.
To try and get peaks to show up, we created a higher concentration internal standard: 0.01 M instead of 0.003 M.
We ran another sequence of 2:1 chloroform:methanol on the GCMS. We also got the older GCMS to work and successfully ran an instrument run (no blank or sample) and a blank run (2:1 chloroform:methanol) without getting any peaks. We ended our work on this GCMS by running a 11.46 ppm sample of our internal standard.
The last thing we worked on was our saliva samples. The first ones did not separate in the separatory funnel like they are supposed to. We believe this is because we agitated the solutions too much. The second ones we made separated much better and formed a clear layer at the bottom, which is what we are looking for. After filtering them, they are not as clear as what we would like, but sometimes they become more clear with time. Regardless, these samples were much better than the first ones we did, and hopefully we will continue to see improvement with them as we do more.
To try and get peaks to show up, we created a higher concentration internal standard: 0.01 M instead of 0.003 M.
We ran another sequence of 2:1 chloroform:methanol on the GCMS. We also got the older GCMS to work and successfully ran an instrument run (no blank or sample) and a blank run (2:1 chloroform:methanol) without getting any peaks. We ended our work on this GCMS by running a 11.46 ppm sample of our internal standard.
The last thing we worked on was our saliva samples. The first ones did not separate in the separatory funnel like they are supposed to. We believe this is because we agitated the solutions too much. The second ones we made separated much better and formed a clear layer at the bottom, which is what we are looking for. After filtering them, they are not as clear as what we would like, but sometimes they become more clear with time. Regardless, these samples were much better than the first ones we did, and hopefully we will continue to see improvement with them as we do more.
Monday, January 13, 2014
Day 6
We decided to recreate the internal standard because we decided that the solution might need to be refrigerated. We ran a sample of this internal standard in the HPLC, but just like last time, the peaks were not very clear. We wrote a procedure for the HPLC for future use.
Instead of getting more flustered about the HPLC not producing any good spectrum from our samples, we decided to move on to running a sequence of the individual standards of each acid. This sequence will be finished running tomorrow.
On the GCMS, a five sample sequence was run to try and clear out the machine. However, the concentrations of each acid increased with each run. This really makes no sense to anyone, unless the solvent is knocking off more and more of the residual acids. We may try to fix the leak in the other GCMS so we don't have to wait longer to start running actual samples instead of just solvent. A procedure was also written for the GCMS.
Instead of getting more flustered about the HPLC not producing any good spectrum from our samples, we decided to move on to running a sequence of the individual standards of each acid. This sequence will be finished running tomorrow.
On the GCMS, a five sample sequence was run to try and clear out the machine. However, the concentrations of each acid increased with each run. This really makes no sense to anyone, unless the solvent is knocking off more and more of the residual acids. We may try to fix the leak in the other GCMS so we don't have to wait longer to start running actual samples instead of just solvent. A procedure was also written for the GCMS.
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