Placebo Interference on SEC-RI HPLC Analysis

[CPR]

Hello Everyone,

I am currently trying to assay Dextran 70, a ~70kD sugar molecule by SEC with detection by RI. The standard has a small peak at ~5 min and one main peak eluting at ~7.5 min when dissolved in water. However, when analyzing a sodium chloride injection solution of Dextran 70 at the same concentration, there is a large fronting peak under Dextran 70 (approx. 10% of the STD area). The placebo is 0.15M NaCl and this peak is not present in water injections. Fresh preparation of the NaCl solution has eliminated contamination issues.

Here is a review of my system:

Thermo Separation Systems Pump/Autosampler
Waters 2414 RI (34C internal temp, 128 attenuation)
Column - Phenomenex PolySep-GFC-P4000 300mmx7.8mm
MP - Water
Flow Rate - 1.0mL/min
Injection - 20uL (200uL loop)



So does it sound like we have the wrong column for this analysis and neither of these are really being retained? Doing a rough calculation, the column should have approx. 7mL and that would equate to 7 min of mobile phase - close to my RT for both. Or am I missing something else? If it is the wrong column, what columns would work for this? Or could a guard column simply remove the sodium from the equation? Any help would be much appreciated, thanks!

CJ Rassbach

[Uwe Neue]

Looks fine to me. The column volume of such a column is 14.3 mL, the excluded peak should elute at about 5.5 mL. The dextran peak is in the separation range.

You should really run a calibration curve with dextran standards so that you get a handle on the MW distribution of the analyte, and if it really is 70 kD. I do not understand the shape of the placebo peak, if it really is only NaCl. Could be ionic interactions with the packing (???).

[tom jupille]

I'd vote for Uwe's speculation about ionic interaction (exclusion). The quick-and-dirty fix would be to add some salt to the mobile phase. In principle, 150mM NaCl would match your matrix and should eliminate the problem, but that might be a bit hard on the stainless steel. My guess is that you could use quite a bit less (just enough to swamp out any residual charge on the packing).

[CPR]

You guys are spot on as usual. I forgot to mention in the first post that my sample is diluted 1/20 in water, so the ending salt concentration is more like 7.5mM. Using that as my mobile has eliminated the placebo interference and should not be too tough on the stainless.

Thanks so much for your help!

CJ Rassbach