Moving peaks

[juddc]

Just because your excipients are soluble and pass through a filter doesn't mean that they won't foul your column...and I'd be willing to bet that many folks here have seen columns get fouled very quickly.

Was the other lab that you had try the analysis an outside lab or were they another lab "down the hall", if you will?

If the former, it seems to me that it's your sample (I'm making the big assumption that USP methods work at least for standard solutions).

If the latter, I'd look at sample and solvent (methanol, HOAc, and water) quality both.

For what' it's worth, sonication isn't the best way to keep your solvents degassed. For running an isocratic method with a premixed mobile phase it certainly can work, but it's not the best way to go in my opinion. Every Waters pump I've seen over the last 20 years has been equipped for a Helium sparge and I'd suggest that at a minimum. Be careful with a premixed MP, however - don't oversparge. You can also purchase an inline degasser as well.

If I was in your position, I'd do the following at this point:

1. Buy a tank of Helium or an inline degasser.

2. Measure the flow rate of your pump at 1 ml/min using methanol as a mobile phase with 500-700 psi backpressure (a column should give you that, though you might have to try a few). Use a stopwatch and a 10 ml volumetric flask and you'll know whether your pump is operating properly. Your flow rate should be within 5% of the set rate. (This is pretty close to Waters' OQ procedure). What's your pressure difference between the pump heads (ripple)?

3. Get fresh reagents for mobile phase preparation and make sure that your water is suitable. The water used to make mobile phase is critical and frequently overlooked. How do you know your water is pure enough for LC use? You can also get HPLC grade water from any mainline vendor.

4. Buy a guard column holder and appropriate cartridges from whomever supplied the columns.

5. Get a few solid phase extraction cartridges and try cleaning up your samples. Of course, you'll have to demonstrate recovery but if it helps stabilize your chromatography, it'll be worth it. If you call any major supplier and explain what you're up to they'll frequently send you half a dozen or so for free.

[unmgvar]

i am stepping in after i hope reading the thread carefully enough.

it looks to me like the problem is around 2 issues, as Hans and Juddc are suggesting.

you are getting different pressures and and different starting RT every day.
first problem is probably related to your mobile phase-composition at creation and during work. second, it can be that your product is slowly and surely leaving something on the column even after sample filtration.

make sure of the following:
1. for the coming days don't do a volume for volume mobile phase, extrapolate with density value and weight your mobile phase solvents.
This should decrease your margin of error, especially if you use plastic and not glass instruments. be precise, no 10% error of margin and stuff. be patient and precise.
2. make sure the solvents you use are freshly opened. use only closed sealed bottles of HPLC solvents
3. make sure that your bottles and utensils are dust/particle free. wash them a bit with your solvents/mobile phase prior to use.
3.use an appropriate glass bottle size. do not put 1 liter in a 5 liter bottles (exageration of course to make the point).
4. is your mobile phase bottle properly closed, with only a small opening to let the pressures equilibrate (make sure you do not have a fume-hood type of any sort right over your solvent bottle especially when it is not closed properly.
5. the oven temp, might be stable (this would mean that you have a temp. chromatogram over 24 hours to support the claim not the simple OQ procedure) but is the temp. in your lab? do you have A/C all the time? could it be that you have extreme temp. differences during the day, is your A/C closed at night? many labs do that
6. run only standards for 24 hours, no samples at all

tell us how it went

[mbicking]

To calculate "Change Ratios" simply choose one run as your reference, or "good" run. Then calculate the ratio of each "bad" value divided by the "good" value for retention time, peak area, and height.

There are several possible explanations, depending on the results, but here is the easiest one.

If you have a simple flow problem, then all the retention time ratios will be changed by the same proportion (i.e., all will be around 95%, or 102.5%, etc.); they won't be exactly the same, but should be very similar (+/- 0.5%). For the same run comparison, the peak area ratios should also be similar, and following the opposite trend from the retention time ratios (i.e., if RT ratios are less than 100% the area ratios will be more than 100%). (I can explain why in a later post.) The height ratios for this comparison should show no change (i.e., close to 100%).

If you do this comparison for runs with both shorter and longer retention times, we may get a clue about what is going on.

Regarding sonication, others have also echoed my earlier statements. I do not believe you need to do sonication at all with a pre-mixed mobile phase. And letting it sit overnight, even with a sealed container, works against anything you did with the sonication. Then, if you pour it into another container, or open the bottle in any way, you are just re-gassing the solution. Eliminate this step and see if some of the problem goes away. I think you have more than one problem at this point, but even solving one would help.

[mreyes]

Its a shot in the dark, but it seems to me that you need a stronger solvent to wash your column. Have you tried to inject your column with THF? Or even passivate your system with an acid or EDTA?

In one of your earlier posts you say that in the later injections your retention time shifts, have you checked the pH of your sample in the latter stages of your run to the ones you ran in the beginning. I have seen cases where an HPLC profile has changed due to the time it has been stored in your poly/glass vial.

As for the poster who said you received a bottle of ACN instead of MeOH. We must shop at the same place because we had the same thing happen. Luckily the person who found the problem thought the MeOH smelled funny and didnt use it.

[sgsnarayan]

Hi there,

I am just wondering if you are using any guard column. If you are using the guard column, then try to change it and see (perhaps you should do this before putting a new column). If you are not using a guard column, try using them for this analysis. My experience with tylenol and caffine analysis is that the guard coulmn does help to get good retention characteristics.

[gurkishan]

Hi Juddc
The lab was a professional lab in some other place. But my sample are also including some market formulations, so i dont think it is anything to do with them.
I will try the guard column next time and let u know what happens!

Hi unmgvar!
Well, i do measure out all my solvents separately to prevent contraction on mixing. Also, it has a column heater, so no temp variation (heater has been calibrated). All my solvents are from Fishers and i open them my self and make all solutions my self.
Will run the standars and let you know about it!

Hi mbicking!
Will get back to u with cahnge ratios soon.
As for sonication, it degasses and i do that in the final container. I dont think, that can be a large issue as my other studies have been done lie that. Only somehow, this study has become a pain in the ass.

Hi mreyes!
I have not washed with THF!
Will try it next time!

Hi sgsnarayan!
I plan to use the guard next time. Will keep u guuys informed!!
Thanks alot!!

[marino1129]

Hi,
Here's what I think your problem is: pH.

The mobile phase pH is 3.5, and the pKa of Aspirin is about 3.5 as well. The moving peak in your chromatogram is likely due to acetylsalicylic acid going through pH transitions. The problem you observed is typical when mobile phase pH is too close to the active's pKa.

What you need to do is to reduce the mobile phase pH down to 2 again. You can use phosphate buffer or formate buffer. As for the column, Phenomenex Germini-NX C18 has very good pH tolerence (between 1-11), or you could try Waters XBridge C18 column, which is also very good in tolerating extreme pH conditions. Otherwise, try Thermo's hypercarb or hypersil Gold columns.

Good luck.

[gurkishan]

This for the suggestion marrino!
But my column pH is like 2 already.
And i have already tried XBridge column but had the same problem

[mbicking]

If the acid is the issue (and lots of people think it could be, for several different reasons), try substituting it with something more benign.

I use 0.1% phosphoric acid in just about every method I develop. It is cheap, easy to prepare, relatively non-hazardous, and gives you a pH of around 2 (in water). Is compatible with most LC columns and equipment, and in my experience helps to minimize surface effects for some columns, without complicating the issue with ion pairing effects.

[mbicking]

No response for a while, so maybe your problem is solved? If so, write back and tell us about it.

If not, one of the few things left to try is to change the solvent inlet filter in the mobile phase reservoir (if you have one).

[gurkishan]

Sorry, was out of station for some time!!
Plan to put up a new column this week. Will let u know what ever happens!!
Thanks