General HPLC questions

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Dear all,
I hope it is alright to not open a new topic for every minor question. So I am doing it in this thread.

I am using a waters alliance 2695 coupled to a DAD coupled to a mass spectrometer equipped with an ESI source.

1) My gradient delay time is around 6 min. This seems to be very long. I don't know if it is correct but I once read that HPLCs with a gradient proportioning valve generally have higher gradient delay times. Is is true? What could I do to decrease the gradient delay time? I thought about decreasing the inner diameter of the tubings and also about shortening them.

2) When exactly do you run a blank? Right now I do it in the beginning, before every new set of samples and after all samples. Obviously if I have a sample with profound carry over I mix blanks in between. What is your advice?

3) Where excatly does an HPLC measure the pressure? If my HPLC for example says 100 bar it only means that this pressure exists in the tubing before the column, right? Downstream of the column is not a 100 bar, is it?

4) Would you ever run an isocratic method for an analyte in a biological matrix i.e. cell remnants? The reasoning behind my question is the following: In my opinion the isocratic method could lead to hydrophobic compounds accumulating on the column leading somewhen to a sudden elution. Do you agree?

5) I recently measured an API in a biological matrix. In certain cases the compound displayed severe peak tailing and reduced peak height and in other cases the peaks looked "perfect". What could be the reason?


Thanks in advance people!
1)
you don't tell what your flowrate is.
The 2695s I came across so far, all had gradient delay volumes of about 1-1.3 ml. So if your flow is 0.2 ml/min, then 5-6 min of gradient delay seems plausible.
Guess on the 2695 you could not do much to reduce this volume, because of the design. Maybe there's the possibility to install a smaller injection "loop" tubing (together with smaller syringe to stay safe). Don't know if smaller pump heads exists too.

yes, low pressure mixing systems have l'bigger dwell-volumes because the mixed eluent needs to go through the pump heads
too. That's why even Waters low-pressure mixing UPLC "H-class" has about 600 ulof delay volume.

2) no clear rules. Normally I do some before my references at the beginning and end of the sequences

3)
yes, there's a steady decrease of the pressure from the pump to the end of the last tubing. The main resistance is the column. After the column only tubings and detector cell add up.

4)
agree, but sometimes isocratic is needed for separation. We then add a column flush step after the peaks of interessts are out. Depending on the time needed for reequilibrating the column [(VDwell+ca. 5-10 VColumn)/Flowrate], do it each injection or after several samples or at the end of the sequence

5)
have to ask the crystal ball, but could be a pH issue. Did you control/check the pH of the matrix or injection sample? What about the eluents? Are they acidified or even buffered?
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