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Dear Forum Members
We want to analyze the brain dialysate samples using ISCO Model 500D syringe pump. We use Unijet reverse phase microbore columns of 150 x 1 mm dimension, packed with C18 material, particle size- 5um, pore size A* 100. A 14 x 1 mm guard column (packaging material specifications similar to main column) precedes the main column. The problem we are facing is rapid buildup of column pressure that reaches as high as 1100 psi after approx 30 - 40 dialysate samples are injects over the column. Sample volume injected is 5 ul using a fixed loop method. A new column typically delivers around 500 psi with the composition we use for the mobile phase. How many samples can one load on a microbore column in its life time with reasonable backup pressure? Is this normal with microbore columns and if not how to resolve this issue? I would appreciate if anyone could share their experiences.
Thanks
Manish Issar, Ph.D
Assistant Professsor of Pharmacology
College of Osteopathic Medicine of the Pacific
Western University of Health Sciences
309 E. Second Street
Pomona, CA 91766-1854
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40-50 samples is a bit short. Is the guard column doing anything? In most cases you should be able to replace the guard column and get back to the starting pressure. If you cannot, then the guard column is not doing a thing for you. And you can look for one that is exactly the same composition. What is the minimum sample volume you can use? What is the composition of the dialysate?
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Hi Ed
We are using the guard column that has the same composition as the main column. Since we are doing microdialysis the volume of the sample size is 5uL that is injected directly onto the column. I am suspecting that may be the culprit. A dialysate perse should not have any protein in it. The composition of the dialysate is NaCl, KCl and CaCl in water. We are injecting of 5ul of the microdialysate.
Manish
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What is your mobile phase? If you change your guard column do you get a return to your starting pressure?
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Pay attention to your mobile phase: is it compatible with stationary phase (i.e. pH >8)?, is it filtered? Daily filtering aqueous phase through 0.22 uM should avoid any microbial growth that could block the column frit.
On the other hand, pay also attention to the possibility of in column precipitation of the salts contained in the dialysate.
Best regards
manish Issar
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The following message was posted to: PharmPK
Dear Manish,
I do not use 1 mm column, but use 2mm column routinely. Did you confirm
that pressure buildup is due to column? I have observed many times,
pressure builds up due to inector clogging also. As you are using 1 mm
column, you must be using capillary connections, which may be blocked.
Regards,
Vinayak
Vinayak Nadiger
Manager, Bioanalytical
Forma Therapeutics(Singapore)
11,Biopolis Way ,Helios # 08-05
Singapore 1386607
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