KW-803 Question

Record: 805

Category: Column Problem
Product: KW-803
Sample:

Question
A customer is having a problem with a protein not eluting on his Shodex KW-803 SEC column. Here is what he has been doing:

“Lately, I have been working on developing HPLC methods for a client. The MW of the protein is about 75kDa, it is slightly basic (theoretical pI of 8.4) and has a few glycans attached, mainly consisting of mannose. The protein is highly pure and was polished using a preparative SEC step on a sephadex column. Also, the protein behaved as expected in an analytical SEC experiment on a superose column.

Method development has been successful for RP-HPLC and cation exchange chromatography but not successful for SEC. I have attached data from the Shodex column. In the excel sheet you will find chromatograms of HSA and lysozyme, both behaving as expected, and then of the protein sample.

A bit of information on the chromatography:

Column: Shodex KW-803

Guard: SecurityGuard Cartridges GFC 3000 4 x 3.0mm ID

Eluent: 100mM phosphate, pH 6.8

Flow: 1ml/min

Detection: 214 and 280 nm (214nm shown)

Oven T: room temperature

Amounts injected: 15-40 micrograms

A number of different eluents have been examined, mostly based on a phosphate buffer, neutral to slightly acidic pH. Additives such as NaCl, sulfate, arginine or small amounts of cosolvents such as ethanol and acetonitrile did not improve the results.

I would like to hear if you have any suggestions regarding optimization of the eluent composition – and we are mainly interested in conditions that can be expected to be non-denaturing. Also, I would like to hear if you have suggestions regarding an alternative matrix for HPLC-SEC.

Thank you very much in advance!”

The injection volume is 20 µl, injected amounts are comparable in the 3 attached chromatograms and injection solvent is the MP.

Answer
Since the IE method uses high salt solvent and it is working, it may require the high salt.
They mentioned that they tried adding NaCl. We were wondering if they have tried ~0.3 M NaCl + buffer solution, if not it might worth trying.
If this still does not work, we may recommend other base-material columns e.g. GS-HQ series.

 
 
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