WCX Early Eluting Mystery Peaks

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

4 posts Page 1 of 1
Hello,

Column: Weak Cation Exchange (COOH-)
Buffer System: Sodium Acetate and eluting with calcium chloride
Column Cleaning: Elution Buffer->Water->Phosphate Buffer (Storage buffer)

Over the past year and a half I've gone through about 5 columns that all eventually show a growing peak at about 7 minutes into a method. This peak only shows up when an actual sample is run. The particular profile of the peaks in this area are dependent on the column. It is present in both the standards and the test samples, all of the same protein, but not in the blanks. Also the time in which this peak appears varies. I had two columns that produced this in about 4-5 months where another produced it in only a matter of a couple weeks.

Image

I have tried cleaning the column via forward and back flushing in this progression:
Water (RT)
Hot Water (~40C)
Elution buffer (Acetate buffer with .2M CaCl2)
Stronger Elution Buffer (Acetate with 2M CaCl2)
Acetate with 2M NaCl
guanidine hydrochloride, 1M

However I have had no luck in getting rid of these mystery peaks.

I've managed to minimize it (so far) happening on a new column by 1. adding sodium azide to the storage buffer, 2. adding a guard column, 3. adding a pre column filter (samples are centrifuged as well)

I would like to have a better understanding of what this is so I can maybe reverse it to recover my old columns. the only other thing I've noticed really is that i will see jumps in the size of this peak after storage.

Any Ideas?
Can you scan the full UV abs spectrum for the mystery peak?
Maybe sample matrix? Why 2M NaCl? How many injections you did before you saw the mystery peak?
Gerhard Kratz, Kratz_Gerhard@web.de
The UV absorption is the same as my main protein. I am running the method at 280. I have run the same sample on a good as well as a "bad" column and the mass balance says it is from my main peak. So I am pretty sure it is my protein, but what has changed is how it interacts with the column.

The sample matrix is the same as the running buffer. It gets diluted about 200 fold from the neat sample and the neat sample is in an acetate buffer as well. Nonetheless, I've run the exact same sample on two columns and seen different profiles in this area. One where the peak is present and one where it is not.

The number of injections has varied. In some cases I've run a couple hundred injections and the area percent of the mystery peak has stayed below 0.1%. Other times I've injected less than 50 samples and it can get as high as 2-3%.

I used NaCl because I wanted to try a different salt. I understand it is lower on the Hofmeister series and a weaker elution species than what I normally use but I was just trying different options.
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