Chromatography Forum

Hi All,

Any

We are having a very peculiar problem and request your help & suggestions to find an amicable solution to this problem.

Customer Sequence of sample is: Blank followed by a sequence of six standard injections
Problem: Always his first injection Area from the same Vial (6-replicate injections from the same Vial) is giving less or variable response leading %RSD of 4.6 (if we remove the first injection and consider the remaining injections the %RSD is around 1%) We also tried to remove the Septa assuming this to be the case of some Air getting sucked from the vial during the first injection but even this didn't workout.
Also repeated this set of sequence samples more than five times and each time the results from the first injection are the cause of %RSD failure

Please let us know what else needs to checked to find a solution.

Does FDA allow the enduser to deselect the first injection and consider the remaining 5-6 runs only for the %RSD calculations ??? if yes is there any documented statement which can be provided to circumvent the current situation.

Await for your further reply.

warm regards,
gsck9

Could it an equlibration problem? Have you tried running few more blanks, before going to standards?

What about running each six std injections from different vials? You may have created a vacuum into the vial by injecting many times from the same one.

What about carryver? Have you tried injecting a blank after standard?

What about backpressure? Is it constant from run to run?

I vote for carryover from the blank diluting the first injection. Even the "good" data seems worse than this autosampler is able to do. Did you try MK's suggestion of testing a blank after the standards?

I admit it is hard to imagine what could cause this much carryover, given the design of this autosampler, but that explanation fits the symptoms.

I agree with Mark's assessment that the problem might be carry-over.
I would investigate possibilities for the carry-over, looking at the flow path, but also including adsorption of the analyte to seal material in the injector and the type and nature of wash solutions.

Thanks for all the recommendations and advice.

What is the most surprising fact to us at this customer site is this particular application giving this problem only for the first injection. So in order to get ourselves convinced before even posting this to the attention of you all, we had carried out a few other studies on the same system using Injection Prescision Carry Over test which Passed, then we had other samples run on the same system with UV as detector instead of the Refractive Detector and this too didn't give any problems and all the results passed especially the first injection never gave this problem.

Hence we donot suspect any carryover on this system.

So request you all to rethink and help us understand as to how this could e happening, has this anything to do with concentrating the system ALS with high concentrated sample before actually injecting the sequence of customer analysis..will this help if Yes then why???

Hope one of you will comeup with a better idea

Await for your replies...Thanks once again to all of you who replied and gave us diversity into the problem resolution

regards,
gsck9

How about following the injection sequence with a blank injection. This is the simplest way to test for carryover. Carryover may be specific to this sample, and if this is the case, you won't detect it with a standardized test.

Well, there are some more exotic possibilities:

1. Adsorption in the ALS. Of course, what goes on will come off eventually, and this will have the appearance of carryover, but will be compound-specific. Here your hardware will test OK, but perform as if it carrys over. Adsorption could also occur on a dirty frit.

2. A faulty instrument program. If the sample valve does not remain in the inject position long enough to sweep the loop, you will get carryover. Again, the standard test will show OK, but it will carry over only for the faulty method file.

3. Mismatch between the sample loop volume, syringe volume and the specified sample draw volume. Not all ALS designs are vulnerable to this. A standard carryover test might not catch this, and the problem would be specfic to a method.

4. Siphoning of the sample due to a misplaced waste line. Not all ALS designs have this vulnerability.

In all the above cases, you must test for carryover-like behavior using the sample and program that are causing trouble. Try an alternating sequence of high-level standards and blanks.

By the way, another symptom of carryover is nonlinear calibration. See any of that?

5. Mini-sample vials sometimes trap a bubble in the bottom. This would be more sporadic in occurrence.

Please let us know how you solve this problem. We might all learn something.

What type of autosampler is it (Dionex, I would assume, but is it one where the needle & loop spend the majority of their time in the high pressure path and there is little or no over draw, or is it the kind where there is at least 50µL of overdraw and the needle doesn't ever see the same pressures that the rest of the lines do)?

gsck9,

You are getting a lot of useful suggestions for your problem. I'll address one of your other questions. You asked:

Does FDA allow the enduser to deselect the first injection and consider the remaining 5-6 runs only for the %RSD calculations ??? if yes is there any documented statement which can be provided to circumvent the current situation.

The answer is yes. It is common practice to use one or more test injections of a system suitability standard at the beginning of a run (at least in the pharmaceutical industry). These injections can directly precede the actual system suitability injections but not be included as part of the system suitability. One recommendation: label these injections as "Test"; "Test injection", or something along these lines. This way you are clearly noting that these are test injections.

As to documentation for this practice, I know of none.

However, please be aware that this is only something to "circumvent the current situation" (as you stated). It is a 'work-around' that is, as I said, in common use.

So, I recommend that you follow the suggestions others have posted and try to identify the problem. Failing a resolution of your problem, go ahead, use test injections.

Regards,
Dan

A couple of things come to mind. One: Often the first sample will load portions of the valve/tubing and column. I used to call it marination when I worked at Rheodyne. Second: Depending on the type of autosampler; you may need 10 or more inj loop volumes to fully fill your loop with 100% sample.

As a first guess I would suggest changing the amount of solution you dispense with your autosampler.

Hi gsck9
All the sugestions were great, but maybe the equipment is not your problem. Many of us forget that the column is part of the system.
There are some other considerations, like:

1. Do you dilute the standard with the same solution that you use as the BLANK injection?
For example if you inject 100% Acetonitrile as blank followed by six injections of Std. (dissolved in water) you may always have problems with the first std. injection.

2.What kind of detector are you using?

3.Have you tried to perform this test in other equipment?

I really dont think your LC is the problem.