Chromatography Forum

Hello,

I would like to separate proteins (three) in reverse phase with UPLC or HPLC but for the moment no results.
We try to separate with C4 , CN, C8, C18 columns in ACN+TFA or MeOH, IPA.
Iso electric points are similars.

Someone has a good idea ?

I presented at the last HPLC conference on the use of the ACQUITY UPLC 300 C4 packing, which is designed for protein separations.

Not all packings are behaving equally well for proteins. You need to use one that has been specifically designed for such separations. The larger pore size is needed due to the size of proteins. C4 is preferred over C18. Acetonitrile/TFA gradients are the standard approach. If you ask more specific questions, I can help you further.

We have aso tested BEH 300 A C4 from Waters with ACN/TFA elution.

Can i have more informations about your processing method ?

Temperature oven, gradient, time, ...

Thanks a lot

Can you post a chromatogram on here with experimental conditions?

No,

But i send you an e-mail with conditions and chromatogram.

Thanks in advance

We did not do anything unusual. Here are two rather standard gradient conditions:

This was for a run on a 1.7 micron 2.1 mm column:
Gradient: 28% B to 100% B in 25min, 2 min hold in B, 28% B for 18 min
A: 0.1%TFA in 100% Milli-Q water, B: 0.075% TFA in 71.4% ACN (v/v)
Column Temperature 40°C, 0.2mL/min

This was for a run on a 3.5 micron column, 4.6 mm i.d.:
Flow rate: 0.96 mL/min
Gradient: 14 to 86% B in 25 minutes
A = 0.1% TFA in 100% Milli-Q Water,
B = 0.075% TFA in 71.4% MeCN

Send me a copy of your C-gram too, with conditions

Thanks,

I send you conditons and chromato.
For UPLC conditons, lenght of column is 5, 10 or 15 cm ?

separate proteins by using the PolyWAX LP in anion exchange mode. You can find quite a lot of application notes on NestGroup homepage or at www.polylc.com . for me it works great.

For a protein separation in UPLC, you do not need a long column. Usually, a 5 cm column is the best solution.

I don't work tomorrow but testing are for friday or next week.

Unless your proteins require a hydrophobic surface to stick to, try separating them using IEX columns and our pH gradient system. Since we can create and control gradients over a wide pH range (2-12), you can run very flat gradients (~.01 pH units/column volume) as segments, say, tandem to steeper gradients and you can separate isoforms differing in electrophoretic pI by as little as 0.01 pH unit

Allen, I checked that Cryo.... homepage. Links didn´t work, so I did not see anything that showed what is new nor why it is new. Especialy I didn´t see any re-inventions. Do you have a ref. in a refereed journal that supports those claims with something of the sort of proof for the newness, uniqueness, advantage over similar techniques?

Allen, I checked that Cryo.... homepage. Links didn´t work, so I did not see anything that showed what is new nor why it is new. Especialy I didn´t see any re-inventions. Do you have a ref. in a refereed journal that supports those claims with something of the sort of proof for the newness, uniqueness, advantage over similar techniques?

I don't know why the links don't work for you. To be frank, we have never had anyone else tell us that the links don't work, but I apologize for your difficulties. We have published two papers on the method in the last 6 months. They are:

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases Journal of Chromatography A, 1200 (2008) 166–182 and Application of Well-Controlled pH
Gradients at Variable Isocratic Salt Concentrations to IEX Chromatography in American Biotechnology Laboratory Oct 2008.
The first is an extensive in depth description with considerable new physics in it, but plenty of exciting experimental results as well. The ABL paper focuses on controlled gradients in the presence of variable levels of NaCl and has very little theory. I think you will find both very informative.