Carryover problems

[Jeff35]

Hello,

I have a consistent problem with the waters UpLC H class systems. I've developed an impurity method and I'm looking to quantitate a known impurity at approximately 0.2% relative to sample concentration (500ug/ml). When I make an injection of diluent I get a small but significant peak at the retention time of my analyte. The pda spectra matches my analyte and is larger than my LOQ injection. I've tried washing the injector and flushing the system however the problem persists. I've had this problem multiple times in the past and have never been able to completely figure out where the problem is. I've read that a common problem is that the sample injector rotor can wear and buildup analyte that slowly blends onto the system with subsequent injections. Here are my chromatoraphic conditions

Mobile phase A: 0.1% tfa in water
Mobile phase B: 100% methanol
Gradient: 100%A for 1.5 min,92 %A at 8.5min, 80%A at 15 minutes, hold at 80% to 25 min, 40%A at 35 min, return to initial conditions at 36 and hold to 40
Column: 150mm x 2.1mm, 1.7um biphenyl
Column temp 35
Uv 214 and 271nm
Injection volume 2ul
Sample concentration: 25,000ug/ml and 500ug/ml
Standard concentration: 5ug/ml and 1ug/ml

Thanks

[mattmullaney]

Hi Jeff35,

What is/are the compositions of the Wash and Purge solvents you are using with this method? Are you priming both of these solutions prior to starting your blank injections?

I think Waters does allow for flushing the Acquity-H with 30% (v/v) phosphoric acid in water...this was a common practice with the Alliance. I'll give this a check and write back later.

[Jeff35]

I have my wash solution in approximately 50/50 methanol/water I didn't precisely measure it out. My diluent is 92:8 buffer:methanol ph2.35 with 0.1% trierhylamine.

The diluent is used from the assay diluent to match sample solutions for both methods for mass balance. Nevertheless I would think that if there would be carryover problems that the higher concentration component 25000 ug/ml would be giving me the problems.

Again I've had this problem in the past with other methods and never could figure out a good cleaning procedure to remove the contamination/carryover.

[mattmullaney]

Hi Again,

Somehow I lost my reply whilst you were typing yours. Okay, I'll try again. The Wash Solution isn't strongly-eluting enough--the Wash Solvent needs to be of stronger elution strength than the strongest-eluting mobile phase compostion in your solvent program. For your application, you'll Need something on the order of 90:10 MeOH/Water, as a guess. The Purge Solvent needs to be on the same order of elution strength as the weakest-eluting mobile phase compostion in your scheme--but contain some organic solvent. I'd try 90:10 Water/MeOH for this.

Use both of these...they are indeed different and serve different purposes.

This document,"Controlling Contamination in UltraPerformance LC®/MS and
HPLC/MS Systems, 715001307, Rev. F" recommends the use of 30:70 85% H3PO4/water to perform a system flush. Just run the system to waste container and not through the detector. After the flush, run dI water until the pH of the LC effluent matches clean dI water. This may take 12-16 hours.

Best Wishes!

[Multidimensional]

A few items appear to be absent from your description.
(1) NO mention of ever performing regular maintenance on the HPLC system including regular replacement of the injector rotor seal and possibly any isolation seals, these are normal wear and tear parts. HPLC injector valves should be opened up, cleaned, inspected for damage or wear, and worn or damaged parts replaced as needed or ~ 6 months. #1 carryover cause is a worn injector (followed by poor injector washing methods for Waters brand systems). "Washing the injector" does not fix the problem. Replacing the worn parts and cleaning the injector often does.

(2) Column wash methods to follow up after each analysis method. First, a proper column wash method should not be incorporated into the analysis method. The analysis method should contain only the analysis method and the wash method (wash plus return to initial conditions) should be a separate method. Column wash methods should always include a mobile phase which is stronger than the mobile phase solutions (stronger than as it enough to wash away everything).

(3) Is your sample dissolved in mobile phase or a weaker solution? It should be. If you use a stronger solution or different liquid, then it may contribute to peak shape problems or artifacts.

Troubleshooting possible carryover issues often turns out to result from a lack of maintenance or poor quality column cleaning. Here are some links to articles which you may find useful and provide you with additional LC training so you can quickly solve the problem.

Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems [https://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html]

The Three Most Common HPLC Questions and How To Solve Them [https://hplctips.blogspot.com/2017/03/the-three-most-common-hplc-analysis.html]

Troubleshooting HPLC Injectors (Manual and Automated) [https://hplctips.blogspot.com/2013/06/troubleshooting-hplc-injectors-manual.html]

Modern HPLC Method Development Tips (PART I) [https://hplctips.blogspot.com/2016/07/modern-hplc-method-development-tips.html]

Modern HPLC Method Development Tips (PART II) [https://hplctips.blogspot.com/2016/08/modern-hplc-method-development-tips.html]

[Jeff35]

Thanks guys for the info.

Something tells me that it is he sample rotate needs replacing or fixing. Is there a link to where I can do this myself? Is it an easy fix? Typically we have an outside company perform routine maintenance on our LC's

I don't know if this helps but I performed a 0ul injection of diluent and ran my gradient. I still saw the carryover peak and oddly enough I saw the solvent front (though the solvent front was very small compared to a normal injection). Doesn't this narrow down the source of contamination to the sample injector system and not a contaminated diluent?

I've seen this on two systems so far so it could be that two of our systems have worn injectors?

[mattmullaney]

You're Welcome, Jeff35,

Yes, that fix is easy. The injection valve cartridge may be replaced:

http://www.waters.com/waters/en_US/Syst ... =134739558

You can find a video describing the process above.

Now, the zero volume injection is a good diagnostic technique, yes. The indication is that the Acquity-H is suffering from contamination rather than from a contaminated diluent, yes again.

Does this mean that the injection valve cartridge requires replacement rather than a cleaning? Harder for me to tell--what normally is done regarding replacement of the injection valve cartridge? Typically this is an annual item for replacement...what is the approximate sample load per instrument? And, as mentioned above, what steps are used to maintain these systems in between chromatographic run?

Keeping to a proper Wash solution will also help going forward.