By Anonymous on Friday, December 5, 2003 - 12:36 pm:
I have run a method on three different instruments (2 water's alliance instruments and an agilent 1100 series)and have experienced carryover on all three instruments. I am currently running on the agilent. The first time I set up on the agilent I equilibrated my column and injected a blank which consisted of 30% water and 70% methanol. A peak appeared at the retention time of my sample peak. Since my sample has never been injected on this instrument, I know it is not due to the injector. If I run a cleaning gradient, my blank comes out clean. Once I start injecting sample, the carryover returns. I am running a gradient method consisting of methanol and phosphate buffer. My gradient scheme is the following:
time (min) buffer methanol
0 30 70
1 30 70
25 2 98
30 2 98
32 30 70
45 30 70
My column is a Supelco, Discovery RP-Amide C16, dimensions 4.6 mm x 250 mm. My sample is dissolved in methanol and then diluted to volume with a 30% water and 70% methanol solution. I have tried extending the time at 98% methanol to 17 min and 27 min to wash off my sample, and I still get carryover. Any suggestions would be helpful.
By Russ on Saturday, December 6, 2003 - 07:22 am:
1. Have you tried running the gradient without making an injection? If so, do you still see the peak?
2. Are you sure your buffer is soluble in 98% methanol?
By Anonymous on Saturday, December 6, 2003 - 09:09 am:
This is not carryover, you said it youself, the sample has never been on the instrument. In these cases it is almost always your water quality or, in some cases, dissolved gas that can generate a peak. I have also seen poor quality vials (dirty) and poor quality tansfer pipettes make peaks as well. Some amide columns can shed bonded phase at high organic and in some detection schemes this can be seen as a large peak or sloping baseline.
By Anonymous on Saturday, December 6, 2003 - 03:08 pm:
I have run the gradient without making an injection and I still see the peak.
By Anonymous on Sunday, December 7, 2003 - 07:22 am:
If you have run the gradient without making an injection, the problem is your water.
By Tim on Monday, December 8, 2003 - 02:53 am:
Run your gradient with just water in place of buffer on the same column, or try injecting just water and compare with the "blank" gradient, to see if there is an increase in peak size. Water is the most probably cause, but it could be from your buffer salt or acid used to pH the buffer. Check all three (i.e. use brand new containers of buffer salt and acid - they could be contaminated by other analysts).