Chromatography Forum

Virtual Colleagues,

A co-worker is trying to develop a SEC method (at which we, the analytical group, are novices) to detect/quantitate a PEG acid molecule. The compound consist of an acid group at each end of two cross bar PEG units. i.e., an X-shaped molecule. The MW is ~ 40K.

The LC conditions reported to me are:

GFC BioSep-SEC-s3000 column, 300 mm x 7.8 mm
Mobile phase: 54 mM HOAc pH 3 (checked w/ pH meter)
Flow rate: 1 mL/min
Inj volume: 50 uL
Col temp: 40 C
Sample conc.: 200 ppm
Sample diliuent: water, or mobile phase
Detector: ultra CAD; neb temp: 35 C, Range: 100 pA, Filter: none

Issues: inconsist chromatography for same sample on consecutive injections, small negative spikes in the baseline, loss of peak response over time (co-worker states it is not a degradation issue).

Not sure if these are instrument or chemical related.

Thanks in advance,
R.J.

Did your co-worker check the pH of mobile phase at room temperature or at 40°C? Could be that your GFC column is loosing some endcapping. It is not a temperature problem, I guess it is the pH at that temperature. Maybe the manufacturer has other ideas.

what is the exclusion volume? Inclusion? what is the sample retention volume?

200ppm= 200ug/mL ?

if yes, 200u/mL for 40K molecules can be very dilute(I am not sure). Can you try 2mg/mL or 20mg/mL for repeatability?