A big broad peak shown in blank injection for RP-HPLC

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

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Hello,

I am running a RP-HPLC method for analysis of antibody, and i am seeing a big broad peak in mobile phase blank injection.

My system is UPLC, and

MPA: 0.1% TFA in Water
MPB: 0.1% TFA in Acetonitrile
Gradient:
Time Flow %B Curve
0 0.2 1 5
0.5 0.2 1 5
9 0.2 99 5
12 0.2 99 5
12.5 0.2 1 5
22 0.2 1 5

Column Temperature: 50C
Autosampler Temperature: 5C
UV: 280 nm and 214 nm.

Column of choice is Proswift RP-4H, 1x250 mm.

Here is my mobile phase injection screenshot.
Image

https://imgur.com/a/sQTlK6K

What could i be doing wrong?
Carryover? Can you performa a zero volume injection/system blank?
Looks like oligo-/polymeric compound.
E.g carry-over from your samples (sticky pproteins?) or contaminated water/solvent reservoirs (something has grown in there?)?
Hollow wrote:
Looks like oligo-/polymeric compound.
E.g carry-over from your samples...

...or from the polymeric stationary phase. Is the column new? Does the size of the hump decrease in a sequence of injections? The hump is in the end (the strong eluent part) of the gradient; therefore, it is unlikely to interfere with the components of the antibody sample to be separated.
vmu wrote:
Hollow wrote:
Looks like oligo-/polymeric compound.
E.g carry-over from your samples...

...or from the polymeric stationary phase. Is the column new? Does the size of the hump decrease in a sequence of injections? The hump is in the end (the strong eluent part) of the gradient; therefore, it is unlikely to interfere with the components of the antibody sample to be separated.


Hi, the column is new, and the size of the hump pretty much stays the same every blank injection. and you are right. the hump is at the end, so technically it does not interfere with the analyte of choice, but i wanted to know if there was any way to just have a flat baseline.

Does the gradient choice look okay in terms of eluting the antibody analyte? for the analyte injection, i see a single peak but it is broad, and small. what could be a better way to have a sharp and big single peak?

I usually dilute my sample with MPA(the non-organic water mobile phase aka 0.1% TFA in water.)
Purpose of the method?
Sample concentration?
Injection volume?
How "broad" and "small' is the peak? At which wavelength?
Up to 99 % ACN is not required to elute the MAb, but may be needed for column cleanup.
vmu wrote:
Purpose of the method?
Sample concentration?
Injection volume?
How "broad" and "small' is the peak? At which wavelength?
Up to 99 % ACN is not required to elute the MAb, but may be needed for column cleanup.


Purpose: to quantify and analyze the purity of mAb.
Sample Concentration: 1 mg/mL diluted in MPA (0.1% TFA in H20)
Injection Volume: 5 uL
It is pretty broad. I've attached what the peak looks like in my original post.
At 280 nm wavelength.
dlwngus810 wrote:
Purpose: to quantify and analyze the purity of mAb.
Please don't forget about aggregates (size-exclusion chromatography) and isoforms (ion-exchange chromatography).

dlwngus810 wrote:
It is pretty broad. I've attached what the peak looks like in my original post.
There is only a blank injection in the original post. No mAb peak can be found there.
vmu wrote:
dlwngus810 wrote:
Purpose: to quantify and analyze the purity of mAb.
Please don't forget about aggregates (size-exclusion chromatography) and isoforms (ion-exchange chromatography).

dlwngus810 wrote:
It is pretty broad. I've attached what the peak looks like in my original post.
There is only a blank injection in the original post. No mAb peak can be found there.


1. Hi, yes We have a SEC method, but due to the nature of our product, we are exploring RP-HPLC as well.

I know SEC can detect aggregation profile of a mAb, but will RP-HPLC also be
useful for detecting aggregates? What would be the crucial difference between
the 2 methods in terms of the target goal?

2. So our peak shape for the mAb sample improved a lot after changing the gradient method, and it looks like this. https://imgur.com/a/pHTN455

First picture is our mAb sample diluted in MPA, and shows a analyte peak around 1 minute at 40% MPB. and second picture is our MPA Blank injection. So, every one of our injection has that broad peak that starts to show up at 8 minutes.

I wanted to see if i can just have a flat baseline, and if the gradient method going from 40% MPB to 99% B is appropriate, knowing that the peak elutes at 40% MPB at 1 minute.
the rise in baseline seems really big, I suspect something with the TFA and/or acetonitril (or your water) and maybe not optimal system parameters (pump stroke volume, mixer, detector settings); see references below

check if TFA is fresh. Maybe use TFA in 1 ml ampulles to be sure it's fresh.
use fresh and clean glasware for solvent reservoirs; clean/replace solvent filters?
Filter your MPA through a C18 SPE cartridge.
Is the baseline much different at different wavelength?
Vary the length of the equilibration step, to see if the rise is volume dependent (so it's coming from the mobile phase or somewhere else)

look for these articles and similar on this topic:
https://www.researchgate.net/publicatio ... ng_eluents
LC-GC Europe, 2003, 16(2):96-105

cited in
Gritti F., Journal of Chromatography A, Volume 1633, 6 December 2020, 461605
https://doi.org/10.1016/j.chroma.2020.461605

both referenced by
Dwight R. Stoll
LCGC North America, February 2021, Volume 39, Issue 2, Pages: 52–55
https://doi.org/10.56530/lcgc.na.et5969b8
Hello,

This seems like a complicated issue, and before I offer any advice I'd like to ask if the column has been washed yet? Have you all attempted to wash the column at all during this process and gather the eluent to analyze? I find it very strange that a column (specifically the method) goes from 1.5% of pure water to 99% acetonitrile when it *seems* like the product elutes at around 40% MPB. What's the 99% or 1% for? Dewetting the column?

Now that I think of it: are you even sure that first peak in the second image is your antibody? The dead-volume is about 1 ml for that column size, and I'm not sure if you have any extra-column volume prior to increase that to around your RT. Plus, since you changed your starting mobile phase ie. 40% MPB, your MPA dilution of your anti-body WILL be different than the current mobile phase and should give you some disturbance. Overall, this is a pretty complicated issue and I'd recommend you to wash this column down, try and collect some of the eluent gunk and figure out what it is, and then run standards that should be comparable to what you got upon column arrival-- praying that that information is available!
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