Chromatography Forum

I have a method using K2HPO3 (pH 8.9) and ACN as mobile phase. The compound ( small molecular) is very soluble in water. The carry-over is getting worse during the run. I can identify it is not from column by switching to a new system.

I used warm water, method, API to flush the detector without connecting column. No one works to remove the carryover.

Do you know any other solutions I can try?

Thanks a lot!

In the past I have used dilute nitric acid pulled through the cell via a plastic syringe.

HOWEVER:
1. check with the manufacturer *before* doing that to make sure there is nothing corrodable in the cell.
2. you want to *pull* the acid through, not push it (in case the syringe comes loose, you do not want to be squirting nitric acid all over the place!).

I am using Waters 2487 UV detector, the manual do suggest using 6M nitric acid ( is it too high?) to flush the flow cell. However I don't have nitric acid in hand, is there other acid can be used? what acid can remove --algae?

I don't understand, the compond is soluble up to 1g/mL, how come it can be trap into the flow cell?

In the past I have used dilute nitric acid pulled through the cell via a plastic syringe.

HOWEVER:
1. check with the manufacturer *before* doing that to make sure there is nothing corrodable in the cell.
2. you want to *pull* the acid through, not push it (in case the syringe comes loose, you do not want to be squirting nitric acid all over the place!).

I have used 30% nitric acid with success and confirm Tom's admonition to pull the acid through the flowcell. One other precaution is to be sure that all organic solvents have been flushed out of the flowcell before using the nitric acid. Nitric acid and organics don't mix

You've already got some useful tips regarding nitric acid cleaning. I've used it in the past successful with dirty detector flow-cells.
HOWEVER: You said, your problem is carry-over? I don't think the reason lies within the flow cell. Dirty flow-cells usually leads to higher noise and diminished lamp energies, but not to carry-over from previous injections. I'd suggest you should have a look to the autosampler's rotor seal instead...

HPLCAddict beat me to the reply while I was composing, but here it is anyway:

I don't understand, the compound is soluble up to 1g/mL, how come it can be trap into the flow cell?

Many compounds can adsorb to stainless steel. The nitric acid "passivation" presumably leaves behind a thin film of oxide (manganese?) which masks the underlying iron.

All of that said, as I re-read your original post, I doubt that the problem is in the flow cell. In fact, if you are seeing a distinct carryover peak for your compound, it is almost certainly *not* the flow cell. You have already exonerated the column, so the remaining possibilities are in the sampler/injector or the transfer lines and fittings between the injector and the column. It *is* possible to diagnose the source (I'll put in a plug for our HPLC Basics, Equipment, and Troubleshooting course on the web next month!). Here's what you do:

Inject a high-level standard followed by 2-3 blanks.

If the peak areas in the blanks decrease exponentially (e.g., the first blank shows 1% of the standard, the second shows 0.01% -- 1% of 1%, etc.) then the problem is probably "physical" carryover of sample trapped in poorly-flushed void spaces. Check for excess tubing, poorly assembled fittings, incomplete washing of the syringe, etc.

If the peak areas in the blanks decrease more slowly than exponentially (e.g., 0.1%, 0.08%, 0.06%, etc.) then you are probably looking at "chemical" carryover; adsorption of analyte to a surface somewhere in the system. This can be a real headache to track down. Remedies may include flushing with organic solvent, passivating the entire system with dilute nitric acid, or changing tubing from stainless steel to PEEK (or vice versa).

If the peak areas in the blanks stay roughly constant *and* you are running a gradient, the most likely cause is contamination of the "A" reservoir. You can confirm this with the "three blank gradients" test described in the mini-tutorial on gradient baseline issues that I posted at the top of the board (viewtopic.php?f=1&t=19085)

Thanks, how can I identify the contamination is from autosampler's rotor seal and how can I clean it?

You've already got some useful tips regarding nitric acid cleaning. I've used it in the past successful with dirty detector flow-cells.
HOWEVER: You said, your problem is carry-over? I don't think the reason lies within the flow cell. Dirty flow-cells usually leads to higher noise and diminished lamp energies, but not to carry-over from previous injections. I'd suggest you should have a look to the autosampler's rotor seal instead...

The carry-over peak is stay roughly constant when I inject several blank and the peak is getting biger run by run.

I am now watching at your mini-tutorial on gradient baseline issues ....

HPLCAddict beat me to the reply while I was composing, but here it is anyway:

I don't understand, the compound is soluble up to 1g/mL, how come it can be trap into the flow cell?

Many compounds can adsorb to stainless steel. The nitric acid "passivation" presumably leaves behind a thin film of oxide (manganese?) which masks the underlying iron.

All of that said, as I re-read your original post, I doubt that the problem is in the flow cell. In fact, if you are seeing a distinct carryover peak for your compound, it is almost certainly *not* the flow cell. You have already exonerated the column, so the remaining possibilities are in the sampler/injector or the transfer lines and fittings between the injector and the column. It *is* possible to diagnose the source (I'll put in a plug for our HPLC Basics, Equipment, and Troubleshooting course on the web next month!). Here's what you do:

Inject a high-level standard followed by 2-3 blanks.

If the peak areas in the blanks decrease exponentially (e.g., the first blank shows 1% of the standard, the second shows 0.01% -- 1% of 1%, etc.) then the problem is probably "physical" carryover of sample trapped in poorly-flushed void spaces. Check for excess tubing, poorly assembled fittings, incomplete washing of the syringe, etc.

If the peak areas in the blanks decrease more slowly than exponentially (e.g., 0.1%, 0.08%, 0.06%, etc.) then you are probably looking at "chemical" carryover; adsorption of analyte to a surface somewhere in the system. This can be a real headache to track down. Remedies may include flushing with organic solvent, passivating the entire system with dilute nitric acid, or changing tubing from stainless steel to PEEK (or vice versa).

If the peak areas in the blanks stay roughly constant *and* you are running a gradient, the most likely cause is contamination of the "A" reservoir. You can confirm this with the "three blank gradients" test described in the mini-tutorial on gradient baseline issues that I posted at the top of the board (viewtopic.php?f=1&t=19085)

hi

recently we also face Dirty flow cell problem in our ACQUITY and the Service Engg has clean it with 10 % phospheric acid, may be you can try it

Does your instrument have an injector flushing system? I have found a lot of carryover problems can be traced back to inadequate rinsing or solvent composition.

make sure there is rinse solvent in your bottle. (guilty of not checking!)
For my API's, I keep 50/50 Methanol/Water for the rinse. anything stronger can cause carryover. You may have a different ratio and solvent combo for your molecules.
Purge your rinse lines occasionally, and especially if you are seeing problems.

Good luck!
Wanda

Nitric vs phosphoric acid for passivation - good question

Waters also told me phosphoric for a couple reasons. the reason I liked the best is that phosphoric will achieve the same thing as nitric, and will clear the system quickly. I have pumped water for hours after a nitric passivation and still found the pH about 4. I used phosphoric last time, and I was done in an hour!

w

Wanda,

Could you clarify the importance of solvent strength in your rinse? Do the analytes condense when a weaker solvent is inevitably exposed to the loop and needle?

Ron J

Sorry, did not get chance to update, busy at heavy cleaning....

The nitric acid and phosphoric acid worked for cleaning, seems phosphoric acid is better since nitric acid leaves extra peaks at beginning.

the compound is very acidic, it might be the reason to attack the metal parts in the system.

Nitric vs phosphoric acid for passivation - good question

Waters also told me phosphoric for a couple reasons. the reason I liked the best is that phosphoric will achieve the same thing as nitric, and will clear the system quickly. I have pumped water for hours after a nitric passivation and still found the pH about 4. I used phosphoric last time, and I was done in an hour!

w

Flora, I don’t think you have a flow-cell problem/contamination – if you’re seeing peaks anyway.
When you say; you tried the method on another system, do you mean another system including the column or did you take mobile phase and column from the current system and installed it on the other system?
Also if you are correct in thinking the column’s not involved in the case, why don’t you remove the column and try to run the test/s with no column in the flow path?

Best Regards

Yes, I have set extended needle wash during the run.
Since acid works for the system clearn, i am monitoring phosphoric acid as needle wash solvent to control the carry-over.

Does your instrument have an injector flushing system? I have found a lot of carryover problems can be traced back to inadequate rinsing or solvent composition.

make sure there is rinse solvent in your bottle. (guilty of not checking!)
For my API's, I keep 50/50 Methanol/Water for the rinse. anything stronger can cause carryover. You may have a different ratio and solvent combo for your molecules.
Purge your rinse lines occasionally, and especially if you are seeing problems.

Good luck!
Wanda