By Krag Swafford on Tuesday, June 8, 2004 - 03:10 pm:
It is an isocratic separation with 0.5 mL/min flow with RI detector. Column dead time is 10 min and then solvent peak comes at 13 min and first peak of interest comes at 14min. And, the whole run takes 40 minutes. Where shall I start to look for shortening the dead time and/or the run time.
Thanks for the help.
By Einar Pontén - SeQuant AB on Tuesday, June 8, 2004 - 04:11 pm:
What column dimensions are you using?
Vo at 10 min corresponds to 5 mL total void volume. That seems to be large.
By Krag on Wednesday, June 9, 2004 - 07:18 am:
Cloumns are 87H ion exclusion with size of 300mm x 7.8mm and 100 x 7.8mm used in combination.
Thanks for your help.
By tom jupille on Wednesday, June 9, 2004 - 02:09 pm:
Column dead time depends on only two things:
- the column void volume
- the flow rate.
The easiest way to shorten run time is to increase the flow rate. My recolection is that those columns will go up to 0.9 mL/min or so, but check with BioRad to be sure (you can damage the column if you run it too fast).
For a given separation chemistry, the only way to decrease the void volume is to use a smaller diameter column, but this can result in more problems from extra-column band broadening (tubing, fittings, etc.), expecially where exclusion columns are involved. A shorter column will have less of an extra-column volume problem, but you will be giving up resolution (fewer plates).
You can try adding some acetonitrile to the mobile phase (do *not* use methanol; that will kill the column); that column should tolerate up to about 40%. That should shorten the retention of your late peaks, but it may also change selectivity.
By Krag on Thursday, June 10, 2004 - 01:07 pm:
Tom and all-
thanks for the help. When I add 10% AcN in the mp, RI detector gave a negative peak. any idea how to get rid of that?
Thanks
K
By Anonymous on Thursday, June 10, 2004 - 01:51 pm:
Krag,
To minimize the negative dip you should dissolve your sample in the mobile phase.
Regards,
Mark
By Anonymous on Saturday, June 12, 2004 - 07:59 pm:
Hi Krag,
RI detector allows you to change the polarity.
By Anonymous on Monday, June 14, 2004 - 03:37 am:
I'm working on isocratic separation of sugars (glucose, xilose, arabinose)with RI detector. In a few analysis the solvent negative peak disappeared. Anyone can tell me why? samples have all the same dilution factor and analysis conditions are the same. In calibration standard I have always solvent peak. How can I manage this fact? How can I perform a good peak integration?
By Krag on Tuesday, June 15, 2004 - 11:00 am:
yes but if i change the polarity all the rest of the peaks would be negative.
Krag