Chromatography Forum

I have some problems with Heparin LMW HPLC Distribution:
1) Which STDs can I use for the calibration of my System?
2) Did you have any problems using LiNO3 0.5M in your mobile phase and RID as a detector?? My detector always goes in "OVER"!!!

Stupid question nº 1: can you tell more about your method? (Column, flow rate, temperature, etc.)

RID are usually a PITA...

Excuse me!!! Could you please avoid using such adjectives to describe my question?

If you have never worked with LMWH you can't understand...
Who has tried to manage with this task knows what I'm saying?
Or you mean you already have the reference stds for the fractions of LMWH described in the PF but not yet for sales?

Don't reply back with "stupid" answeres..

This is only to tell you that it's better not to be offensive..

Just one more stupid question...
What do you mean with PITA?

Sorry, I didn't mean YOUR question was stupid, but MINE... I use this private joke with my friends and colleagues, everytime I want to ask something obvious... In theory you should be using a SEC-silica based column with an aqueous mobile phase... so, it is a bit stupid to ask that - get it?

My goal is to help, not to offend.

PITA was a term appeared in this forum - that I loved - and means "Pain In The A..".

By the way, I work with HPLC and GC since 1985... therefore, there is only a few terms I'm not acknowledged with... besides living in a small and medium-developed country...

ok, my friend...
No problem. I have no doubt you are very experienced in the matter..
actually I still don't know if I can tell openly the conditio we're using..
You know yourself that everytime I could have I did, in order to help my friends who work in chromatography...
I know that R.I.D. is a pain in the A**...
I need to have some information about buying the standards I mentioned..
You know, they're described in PF but not on sale yet...
Do you have a clue?
Thanks for your help.. I am not as experienced as you are...
Hopefully I 'll get as such..
And I ove the idea of this forum as I love the country you're from...

Zompa

To my knowlege there is no narrow fractions standards of heparin on sale. There are several "broad" standards which are somewhat described, but they are not suitable for the US Pharmacopeia draft article method.
EU Pharmacopeia uses other method with different calibration procedure and that method is a real PITA even with the standard available...

I do not know the particular requirements of the method that you are referring to, but it is quite common in SEC to calibrate your SEC columns with standards that are no identical to your analyte molecule. For example, you could calibrate your system with polydextrans.

Now for my stupid question (I ask this every time a RI around here seems to be stuck in "Over" mode) - has the reference cell been thoroughly purged with MP?

I've found that if I allow overnight equilibration of the RI to a temperature at least several degrees above ambient and I've purged the reference cell, the RI is seldom more than a minor PITA.