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- Posts: 32
- Joined: Sat Oct 27, 2012 8:10 pm
First, a bit description on how I prepared the smaples, then move on to GC.
A. Lipid extraction:
On 0.1ml animal blood cells
1. add 0.1 water, incubate for 15 minutes.
2. add 2.2ml isopropanol containing 0.005%BHT, place tube on shaker for 1h.
3. add 1.75 ml chloroform, votex and place tube on shaker for 1h.
4. centrifuge, take supernatant, and dry under nitrogen, reconstitute in 0.25ml hexane.
B. Tranesterification:
add 2.5ml freshly prepared 73%methanol:18%hexane:9%acetyl chloride (volume%), sealed, and heated at 100C for 1 hour.
Cool, add 4.5ml potassium carbonate, mixing and centrifuge.
C. GC:
inject 0.5 microliter to an Omegwax 250 column (30m x 0.25mm)
Now. I am seeing some peaks extremely late.
The 22:6n3 is supposed to be the last peak on this column with a retention time of 45 minutes. However, I am seeing a bunch of peaks after that, some eluted at 125minute!
If I run the sample for 70 minutes, these peaks would carry over to the next run.
What are these peaks? Are they free acids because of incomplete esterfication/transesterification? Are they artifacts? Do you ever see peaks eluting that late? By the way, I ran standards, solvent, and blanks, and these peaks were not there if there were no carryover from the previous runs. So these late peaks are from the real samples. In addition, the retention times of these late peaks were consistent in all samples, so they are real peaks.
I suspect that these are polar components because Omegawax has a polar stationary phase, but not sure. I may try extra extractions on the samples, which would get rid of the polar components.
I want to do a direct injection without purification. But if that is not possible because of the nature of samples, do you have a fast and easy purification method to recommend? I heard of passing sample through a silica gel bed in a disposable glass pipette, does it work well?
A. Lipid extraction:
On 0.1ml animal blood cells
1. add 0.1 water, incubate for 15 minutes.
2. add 2.2ml isopropanol containing 0.005%BHT, place tube on shaker for 1h.
3. add 1.75 ml chloroform, votex and place tube on shaker for 1h.
4. centrifuge, take supernatant, and dry under nitrogen, reconstitute in 0.25ml hexane.
B. Tranesterification:
add 2.5ml freshly prepared 73%methanol:18%hexane:9%acetyl chloride (volume%), sealed, and heated at 100C for 1 hour.
Cool, add 4.5ml potassium carbonate, mixing and centrifuge.
C. GC:
inject 0.5 microliter to an Omegwax 250 column (30m x 0.25mm)
Now. I am seeing some peaks extremely late.
The 22:6n3 is supposed to be the last peak on this column with a retention time of 45 minutes. However, I am seeing a bunch of peaks after that, some eluted at 125minute!
If I run the sample for 70 minutes, these peaks would carry over to the next run.
What are these peaks? Are they free acids because of incomplete esterfication/transesterification? Are they artifacts? Do you ever see peaks eluting that late? By the way, I ran standards, solvent, and blanks, and these peaks were not there if there were no carryover from the previous runs. So these late peaks are from the real samples. In addition, the retention times of these late peaks were consistent in all samples, so they are real peaks.
I suspect that these are polar components because Omegawax has a polar stationary phase, but not sure. I may try extra extractions on the samples, which would get rid of the polar components.
I want to do a direct injection without purification. But if that is not possible because of the nature of samples, do you have a fast and easy purification method to recommend? I heard of passing sample through a silica gel bed in a disposable glass pipette, does it work well?
