Retention time reduced by 75%

[vliu]

I am trying to work out why my CuroSil-PFP 5µ column retention time has reduced dramatically, from 16 mins to 5 mins but the response and area is quite close to the last run ~90%. I tried to troubleshoot without success, until now I've tried the following but did not change A THING...

1. Using the Mobile Phase that was used for the last run
2. Sonicated the nebuiliser as I'm using a light scattering detector
3. Flush the column with 100% ethanol for 2 hours. (Mobile Phase is a mixture of ethanol and water)
4. Equilibrate the column overnight
5. Injecting different samples and stanards

I am about to try to run the same components and condition as given on the COA.

Please help!!

[laserman]

I think you have to identify the problem caused by the column or HPLC.

1. Did you check pump pressure and flow rate? I am wondering if you are having accurate flow rate. Sometimes pumps are stuck, or tube joint leak. For example, set flow rate 1 mL/min, and measure the volume. The volume should be 10 mL after 10 minutes.

2. According to 5 in your post, you have different samples analyzed before. Is it same compound? If you do not get similar data using different column and same detector, the detector has problem.

3. If the results of 1 and 2 are fine, your column might be deteriorated. You’d better check your analysis method and sample. I also recommend checking how many times you injected to the column, possible pH range, and pressure.

Best Regards,

[Bruce Hamilton]

Assuming your column pressures haven't increased to several times previous, I would suspect...

1. The column, what did your replication of manufacturer's test show?. Note that if your pump mixer is leaking, you could get early peaks. The column could also have died if stored/used incorrectly.....
2. Mobile phase - could be composition, or could be that the pump mixer is leaking from another channel. I suggest filling any other channels with water if a mixer problem is suspected..
3. The system was leaking last time you performed the test, particularly if last time was also the first time

I'm less like to suspect the flow rate, as pressures should have increased greatly, also injector and detector would be unusual causes.

Please keep having fun,

Bruce Hamilton

[syx]

how do you mix the mobile phase? manually or by proportioning valve? if you use the last, you may try to mix mobile phase manually and put all your inlet tube to this mixture's reservoir.

[HW Mueller]

How can a detector reduce the retention time by a factor of ~3??

[mohan_2008]

Switch to instrument#2 if you have an extra one, and perform the analysis. (Did you check if your column doesn't leak?)

If the retention drift is corrected, it definitely is your Instrument #1 problem. You can run your experiment on the Instrument #2 or fix #1, but either way.

If not, then it is a bigger problem. Change the column with a similar one (or new) and evaluate if your earlier column has voids.

You should be able to figure it out by then.

[vliu]

Thank you all for your replies. That's only limited troubleshooting I can do as I only have 1 system and 1 column.

Bruce hamilton:
I was going to run uracil injection and compared it to the COA (COA ran 4 components but I can only locate uracil in the lab. Will that be sufficient to determine the efficiently of my column? I made up a 0.15µg/ml uracil solution and about to perform an injection. Is this concentrated enough?? What is the standard uracil concentration used for qualifying the column.

Laserman:
-Pump pressure was comparable to the last run
-checked flowrate, was fine-10ml in 10 min
-same std analysed on the same column and detector=>does it mean my detector is blocked?? what can I do to unblock it??

[Bruce Hamilton]

Sadly, Uracil is very, very poorly retained, and is used by most manufacturers as the void volume marker because it's easily detected. It would not be a good compound to test the column's condition, and you have confirmed your instrument's flow rate is OK ( which void volume might confirm ).

I would suggest that if you can't find any of the retained components of the manufacturer's test mixture, then try find any sample that was analysed using a similar method before the first sample was analysed, and repeat that analysis. Also repeat the first sample analysis just to ensure that sample also now elutes early.

One mistake I routinely make following published methods is that I prepare 60:40 CH3CN:H2O instead of H2O:CH3CN, however the pressures would be significantly different.

I suspect that your column may have died if the pressure are similar to the first run, because pressure is usually a good indication of wrong solvent or precolumn leaks. If you had a leak between the column and detector the first time, you would get longer retention without a noticeable change in pressure.

I'd definitely really want to try and obtain at least one retained component of the manufacturer's test mix, because that remains the fastest way to confirm the column has changed/died since manufacture.

However if you are desperate, there are some published retention volumes for acetone, heptanone and benzene on a Curosil PFP column in a published thesis ( Fig 3.37 p152 onwards ) if your eyes don't glaze over finding that data at the location below. Link may not work, then search using Google.

http://domapp01.shu.edu/depts/uc/apps/l ... 5.pdf?Open

Please keep having fun,

Bruce Hamilton

[vliu]

I flashed the ELSD with 100% HPLC grade water for 10mins at 3mins per/min (without column), and I seemed to be getting retention times similar to my last run, great news!

However, when I put an overnight run on last night, the peaks are split, for both component of interest and retention time seems to vary alot from 12-15min, due to my mobil phase? It's not due to column temp as I have the settings on. The split peaks might be due to a blocked frit or void?? How should I troubleshoot this problem?? Does it mean my previous samplerun is not reliable?

[Bruce Hamilton]

It appears that your method is not robust, and needs more development.

If your peak is jumping around with regard to retention time, you have to fix that problem. Common causes are inadequate control of pH, sample solvent different to mobile phase, flowrate variability, poor mixing of gradients.

If your peaks are split, common causes are sample solvent different to mobile phase, void in front of column, injector failure, gap in column connection joint. Blocked frits are unlikely to split peaks.

I still recommend getting one of the column retained test compounds, as that will quickly help eliminate the column-related causes.

Bruce Hamilton

[rhaefe]

I have seen my share of split peaks due to partially blocked frits (=no or no significant pressure increase observable) but I agree that shouldering peaks are more likely with that cause.
I agree with Bruce, try to get a retained compound and run it to determine retention. I see your mobile phase is water/ethanol. Are you using the identical brand/ part number of ethanol? There is a large number of denaturing recipes for ethanol out there. Make sure you use the identical EtOH every time.

[vliu]

Thank you all for your advices and recommendations.

I was checking out different catalogues for std mix but I am unsure of which one to get. Can someone please recommend a commonly used std mix for most columns and for my suspected blocked frit Curosil PFP column (phenomenex). Can frit be replaced? If so, how? How do I find out which one to purchase?

Rhaefe:
I am using an identical Ethanol as the method states.



Bruce Hamilton:
My method require no adjustment of pH. It is a validated method and i assume inconsistent retention time was not observed during the validation process.

The only thing I am doing differently to the method is that I am only filtering the pre-mixed mobil phase through a 0.45µm filter rather than filtering under vaccum as in the method, but I didn't think this could cause noticable difference in the chromatogram.

[rhaefe]

I would use the same test mixture the column was tested by the manufacturer. If you don't have them at hand order them: they are usually cheap.

I am not familiar with this columns hardware configuration so I cannot comment on the possibility of replacing the frit. Column manufacturers usually do not condone the opening of columns and replacing parts of it.

Just to clarify: do you say that the original method required the vacuum filtration of your pre-mixed mobile phase (which contains ethanol)? Is this correct? If so this is usually a very big no-no since during vacuum filtration you can loose a significant amount of organic solvent due to evaporation and thereby change the composition of your mobile phase.

[Bruce Hamilton]

I agree with rhaefe, if you have changed to pressure filtration from vacuum filtration, you could now have a different mobile phase composition.

If the vacuum filters were partially blocked, or of unsuitable type or pore size, and a high vacuum was used, the ethanol may azeotrope off, and the retention times will increase - exactly what you are seeing.

If you are using HPLC grade solvents, just mix, wait to come to ambient temperature, and use, provided your instrument has a degasser.

The filtration would not explain retention times during a sequence jumping around, unless you were adding mobile phase during the sequence, which you should not do for validated methods. Another possibility is that your instrument temperatures are varying.

Are you using a guard column or prefilter, if so they are easy to change. Changing the main column frit is more complex, and a backflush may be the preferred first option - depending on what the column manufacturer suggests..

You should be able to obtain sources for the test mix from the column supplier. You want to compare the retention times against the manufacturer's CoA.

Please keep having fun.

Bruce Hamilton

[vliu]

I think the jumping of retention time is due to the system not able to accurately deliver the required mobile phase composition, please let me know if I am on the wrong track. What I did was, I did 40 blank injections using the same method but alter the moile phases,

Mobile phase A=100% MilliQ water
Mobile phase B= 1% acetone in water

The method as follows

0-19min- 80%A;20%B
20-34min-60%A;40%B
35min-52%A;48%B
36-38min-0%A;100%B
39-45min-80%A;20%B

So in theory I should see a nice slope which plateau at ~37min. This is indeed observed, but only for the first 7 injections then flat baseline observed after that (however void peak was still observed). So is my assumption correct? If so, how can the problem be fixed?

Thanks in advance.