Contamination Peak HPLC

[KVargas22]

I need help troubleshooting a contamination peak in my Hitachi HPLC system. My system consists of a D-7000 Interface unit, L-7100 pump, L-7200 Autosampler, L-7455 Detector, L-761 Degasser. The method that I am running is a gradient that is specific for carotenoids and is as follows:
Solvent A (81% Methanol, 15%MTBE, 4% Water)
Solvent B (88% MTBE, 8% Methanol, 4% Water)
Time A B
0 100 0
30 33 67
31 0 100
34 0 100
35 100 0
39 100 0
The contamination peak first appeared in a blank after injecting a Beta-Carotene standard. The contamination peak has the same retention time as what I have been seeing in the Beta-Carotene standards, so this leads me to believe it is sample that has gotten trapped in the system. Note that I have ran these standards with blanks with this method without issue at least twice previously. This lead me to believe that it could be the rotor seal, so I have changed that along with the isolation seal. When changing the rotor seal I submerged the stator in 100% methanol to clean for 20 minutes then rinsed with 100% HPLC grade water, followed by 100% Acetonitrile and again followed with 100% HPLC grade water. I then ran a blank 2-propanol injection that still showed a contamination peak. To troubleshoot this peak I have removed the guard column as well as the carotenoid column and replaced with a union. With the union on I tried running a double gradient where I did an injection with 40uL 2-propanol and still saw contamination peaks coming off where the Beta-Carotene standard is expected to come off with peak areas being fairly consistent with each other. I followed this by running a double gradient with no injection and found that the contamination peaks still showed, leaving me to believe that the issue lies within the system. I have a 6 port HPV stator that contains 4 PEEK tubing and 2 stainless steel connections. I changed all of the PEEK tubing as well as the fittings. I have not changed the stainless steel fittings. I did not change the stainless steel fittings or any parts involving the injector because the null injection rules out that the injector and its pieces are not the issue. I pushed a cleaning solution through the system for 2 hours that consists of (25% methanol, 25% Acetonitrile, 25% 2-propanol, 25% HPLC grade water). When running my next gradient, I still saw a contamination peak. I removed the flow cell from the detector and checked for scratches on the outside and rinsed the outside with 100% acetonitrile followed by 100% HPLC grade water, 100% methanol, 100% acetonitrile, 100% HPLC grade water respectively. At this point I have ran many 2-propanol, solvent A , and null injections through the system (At least 15). The peak area seems to vary slightly, but is fairly consistent throughout each run. If helpful, my contamination peak is followed by a negative peak as well.

I have read the references below to help me:
1. https://www.sigmaaldrich.com/US/en/tech ... V4QAvD_BwE
2. https://www.ssi.shimadzu.com/service-su ... index.html
3. https://hplctips.blogspot.com/2015/02/c ... on-in.html
4. https://www.labmanager.com/product-focu ... yover-1850
5. https://alfresco-static-files.s3.amazon ... -18135.pdf
6. https://www.chromatographyonline.com/vi ... -lc-system

[TylerSmith123]

Hi KVargas,

"To troubleshoot this peak I have removed the guard column as well as the carotenoid column and replaced with a union. With the union on I tried running a double gradient where I did an injection with 40uL 2-propanol and still saw contamination peaks coming off where the Beta-Carotene standard is expected to come off with peak areas being fairly consistent with each other"

I'm not going to lie to you, I think the issue that you are seeing is simply because when you're injecting a different solvent you will see a spike, and sometimes a negative peak, when you inject in something other than mobile phase (especially if it's a lower-quality solvent). It simply does not make sense to me that a contaminant would have identical retention with and without your column, that would indicate that your column has absolutely no affects on this contaminant-- even volume-wise (and potentially your beta-carotene as well)! What is the retention factor of this contaminant and your standard as well?

Have you injected with your mobile phase? Do you still notice this peak?

I don't use MTBE as a MP myself, but from what I've just recently read on the forum that MTBE has a horrible miscibility with water and your MP seems to have a 4% water additive, would it be extreme to think this solvent incompatibility could be causing some of your issues? But take that with a grain of salt.

Regardless, this impurity seems to originate past the column as even the column's volume does not seem to slow-down this peak's elution so I would typically point to something like the detector.