Chromatography Forum

Hi all ,
I am using XDB 50 mm column with a mobile phase (30:70 )ACN:H2O.
I am injecting 3 Nitrotoluene 10 ul.But the problem is when I do replicates of my standards I am not getting similar or somewhat similar peak areas.The same with peak heights.
They differ significantly and cannot be considered as data for quntification!
Can anybody suggest me what can I try?.The peak has also started tailing a bit.
please help me.I am using perkin elmer's HPLC.
thank you

You should try to find the source of the variation.
Do you have some in house procedure for testing an HPLC?
Or you can try to add an internal standard and see if this solves your problem (in case of an autosampler with a variation that is too high)

Ace

I suggest you start by checking your injection repeatability.


I bet you have already done the paragraph below and that is why you wrote.

I want to ensure your problem is not in sample preparation. First, ensure that you have a large (5 mL) quantity of fully dissolved, well mixed sample solution. Inject the same sample 2 or 3 times to check repeatability.

It is probably helpful to check with your injector/autosampler supplier for advice. Here are some problems I have had or seen in the past.

1. Injecting too little material into a manual injector. Inject 3-5 loop volumes (Rheodyne discusses this quite nicely in their manuals).
2. Autosampler needle is hitting the bottom of the vial. Change the autosampler settings to that the needle does not go down too far.
3. Autosampler is drawing less material because no air is replacing the liquid drawn up by the needle. Removing the septum from the autosampler vial fixes this, but I agree it is unsatisfactory.

Once you have repeatable injections, you can try making the same sample multiple times to see if there is an issue with solution concentration repeatability.

post results please

First of all thank you all for replies!
I am using inhouse method.I am shooting replicates of stanard(15ppm).
Here posting results,
peak area height
7416530.37 1.04E+06
7786459.2 1.11E+06
8439518.21 1.18E+06
So,this is what happening!..getting indifferent results for three consecutive injections, of a single std.vial.
I have made sure that the needle is not touching the bottom if vial.

thank you

"1.04E+06" - does it mean absorbance is about 1.04?

peak area nad height is rising, do you purge(wash?) injector between injections?

Thanks.
No, I don't wash injector before every injection.When I make my system ready I do wash it,flush autosampler,and then start a whole sequence when I am injecting a single sample or series of stds.of same conc.
So,do you think I need to flush it everytime I make a single injection?
really appreciate your responce!

well I use HPLC with autosampler, I can specify how long injector is washed between injections, I can change wash solvent if I get carryover which helps, newer systems can have more than one solvent to wash injector

This is washing between injections not purging of course
If you do not wash than you should

Could it be that your solvent is evaporating?

I agree with grzesiek regarding the wash between injections. That feature was added to autosamplers to eliminate injection port/needle carry-over.

Thank you all for your kind replies!
I will make sure injector wash before every injection!!
will let you guys know when I fix this problem.
Thanks!!

We might be jumping to conclusions based on a small data set (there is, after all, a 50% chance that a third measurement will trend in the same direction as the second). The key would be to run a larger number of replicates (maybe six?) to confirm that the trend *is* in fact that your peak size increases continuously.

Hi all,
Actually ,I am using MeOH as a diluent (for standard dilution here).
I am not sure if MeOH evaporates so fast within a five minute run time.
so,I don't know but there might be a chance of increase in peak area?..do you think it is possible..or else I have to blame the machine..!
So, I also have tried this dilution with N-hexane..but results are still the same!
STILL THINKING OVER THIS PROBLEM!

Could you post results of 6 injections in a row?
And if possible 6 injections with an injection of pure methanol inbetween.

Ace

Well, if it is carryover then a blank injection should show it.