Significant carryover in RP-HPLC

[NYYFan]

Hello All, I have been reading this website for a while and have usually found my answers just reading but finally found a reason to join and ask a question! So I'm trying to develop a RP method for a vaccine and am having significant carryover problems (2% in blanks after sample). I cannot solve the problem for the life of me, I've tried various needle washes and injector programs, various columns (C3, C4, C8, Phenyl, etc.) and a variety of mobile phases (H2O/ACN, H2O/IPA) with a variety of different modifiers (TFA, FA, HCl, H3PO4) and I still can't get rid of the carryover.

I've never had this much trouble developing an RP method before and was wondering if anyone had any similar experiences or any tips. Currently I'm using a C3 column as I can at least get no carryover on smaller injections (4 ug) but we really need to inject 10 ug per client request. The carryover is definitely in the column as well and not in the autosampler as a blank run after the sample on a different column came (just to clarify, the column was different, but all the specs were the same, i.e same packing material, manufacturer, size, etc.) up 100% clean!

Thanks in advanced!

[RobPelot]

I dont understand, one thing. You changed both the column and ran a blank at the same time and saw nothing? correct?

If this is so, how do you know which one was causing carry over? Should you not change the column, run a sample, then change the column back and run a blank?

Or is this what you did?

[Gerhard Kratz]

Beside the injection system many other sources can give you a carry over. Most common are the so called extra column effects, manly based on built in dead volume due to column connections, and there mostly between guard column and analytical column. Also possible is that your sample sticks to the stainless steel frits. I would recommend to check all connections first. If you use a guard column, disconnect it and run without it. Do one change at a time to see the effect. Good luck.

[HW Mueller]

I still have trouble finding a reason for carryover peaks which are not due to mechanical introduction of the substance which carries. Thus, it is easy to understand carryover peaks from analyte hung up in an injection valve, or if hung up elsewhere, if they form via a gradient. We do not have enough info to ascertain where your carryover comes from.

[DSP007]

Hi
Change columns will not help. This is system mistake . You just do not have time to wash the input device to a sufficient extent. The reason is that protein drugs usually have high viscosity and is easily adsorbed on glass and metal because of the isoelectric potential.

Wash the syringe / injector more carefully or do before a series of additional input samples, which need not be taken.


At the expense of the required number (multiplicity) washes, comes to mind anecdote is not the topic (as flood).
The Jews asked the rabbi, "the Rebbe, how do you brew a delicious tea" ?!
-Jews! Do not regert (feel sorry ) tea leaves!!!

[NYYFan]

Thanks for the input so far everyone. What I did was run a blank and sample on one column, remove that column, and proceed to run the blank after the sample on another column (of the same specs, on the same machine obviously, and same column line) and seen no carryover. If this were an injector/autosampler problem, wouldn't I have seen carryover on this other column then? I've tried a variety of washes on 1100, 1200, acquity, and h-class machines with no success... I like the idea of the stuff sticking to the stainless steel frits. I actually just orderd a C1 column to see if this would help as well (assuming stuff was sticking to the column). This is a gradient elution by the way, and I have two wash steps after my gradient with high organic.

[unmgvar]

what is the MW of your vaccine?
what exact type of buffer did you use, what concentrations?

what do you want to see in your MAB application exactely?
have you tought of going for IEX, HIC method more in specific WCX?

[NYYFan]

what is the MW of your vaccine?
what exact type of buffer did you use, what concentrations?

what do you want to see in your MAB application exactely?
have you tought of going for IEX, HIC method more in specific WCX?

MW is ~80 kDa. For buffers I've pretty much tried every iteration of ACN/H2O/IPA there is with THF, TFA, HCl, and FA as additives.

We actually have an IEX method that works fine, but our client specifically wants an RP method to use for stability studies, degradation studies, etc. and wants a method which basically gives separation between the main species and it's "shoulder" (which we have done) and "no carryover" which to me seems a bit impossible, but we're seeing significant amounts which I've stated above.