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Posted: Fri Mar 04, 2005 12:04 am
by jzt
syx,
I am having a problem with baseline (sudden drop during a gradient run). I would like to post it on this forum but it's hard to describe the baseline without a picture. could you let me know how you got the picture in your posting? Thank you in advance!
Regards,
JZT
Posted: Fri Mar 04, 2005 12:34 am
by syx
Ok, seeing the chrom now: It really looks like what I have seen, sometimes, when air was co-injected, though I don´t understand why your blank doesn´t do this (why don´t you inject H2O?). Do you concentrate the samples with a stream of gas?
I will try to inject H2O as you suggest. I think the blank is mobile phase or sample diluent, and I didn't use water as the diluent.
I do not concentrate samples.
I would like to post it on this forum but it's hard to describe the baseline without a picture. could you let me know how you got the picture in your posting?
Jzt, open <http://imageshack.us/>, register, hosting an image, and copy-paste the code they give to this forum.
Posted: Fri Mar 04, 2005 2:12 am
by Uwe Neue
The "active substance solution" contains other stuff than just your active ingredients. A polymer, maybe...?
Posted: Fri Mar 04, 2005 7:27 am
by syx
hmmm... I don't think it has a polymer inside the bulk. I will try pH effect next week. I will send the report here.
Posted: Sat Mar 05, 2005 11:31 pm
by Uwe Neue
Is there anything else in your active substance solution? Some polymeric ingredient?
Posted: Mon Mar 07, 2005 7:30 am
by syx
... maybe its impurities. Can it be a polymer?
Posted: Thu Mar 10, 2005 1:21 am
by syx
I have tried pH effect to separation. In lower pH (I tried pH 4.5 and 2.5) the main peak will loose its retention with shallow tail.
The problem is reduced when I try to inject lower concentration of the substance.
Posted: Thu Mar 10, 2005 12:14 pm
by FARD
What diluent are you using for Sample preparation?
Posted: Mon Mar 14, 2005 12:50 am
by syx
I use mobile phase.
Posted: Mon Jun 06, 2005 8:59 am
by syx
Thank you Mr. Tom.
I try to use higher pH value of mobile phase.
The pKa value of the substance is 8.91.
The maximum pH can be used for LC system and the column is 10.
Based on those 2 facts, I decided to use ammonium buffer adjusted to pH 9.0 - 9.5 and found three peaks with no baseline problem.
First peak is related to degradation product of the main substance in basic circumstance. So,. I use the mixture of water – acetonitrile instead the mobile phase, but still in the same strength.
The last peak (the smallest) is unknown. It has same spectra with the main peak. Is it probably the enantiomer?
Posted: Mon Jun 06, 2005 10:19 pm
by tom_mizukami
Ok, here is my wild guess. You have a degradant or impurity in your TEA that is normally at equilibrium. Your injection is disrupting this equilibrium and what you are seeing is the system returing to equilibrium.
What is the size of your injection? Have you tried without TEA, etc. I would try switching out your reagents individually with fresh high purity ones and try to locate the problem. Good luck and let us know how it turns out.
Posted: Tue Jun 07, 2005 12:40 am
by syx
What is the size of your injection? Have you tried without TEA, etc. I would try switching out your reagents individually with fresh high purity ones and try to locate the problem. Good luck and let us know how it turns out.
Injection volume: 20 uL.
If we did not use TEA in lower pH of mobile phase (as we use in first post), the peak would be shallow with long tail.
In pH 9 or greater, we do not need TEA to get a sharp-thin peak.
Condition:
Column: 250 x 4.6 mm, contains 5-um packing L1
Temperature: 25 C
Mobile phase: mixture of acetonitrile and 0.05M ammonium acetate buffer pH 9.0 (55:45)
Main peak is eluted in 27.2 minutes after injection.
Asymetry: 1.2; Theoretical plate over 9000. Resolution of the last peak (Rt 30') to the main peak is 2.38.