Help with Injector Program on MassHunter

My LC is 1260 Infinity II with 100uL loop and MassHunter Acquisition 1.2.

I am working on a method that uses standard additions for quantitation. The column is HILIC but diluent is 80% aqueous, so dilution in acetonitrile is required along with the standard additions. It would be very nice if we could automate this part of the sample prep. I have tried doing dilutions/additions using the injector program both in a second vial and directly in the needle (examples below). In the examples below, p2-a1 is acetonitrile and p2-a2 is diluent. The sample is diluted 1/5 in diluent, and then diluted 1/5 in acetonitrile (or initial mobile phase which is mostly acetonitrile for the first example). The examples do not include standard additions yet, but they would be added during the first dilution.

The problem with the first example is that precision is VERY poor (%RSD > 15% for 5 reps). I am guessing that the sample is not being mixed thoroughly by bubbling air through the sample after everything is added to the vial.

Precision is better with the second example, but the injection volume is very large resulting in poor peak shape. I can't find any way to inject only a portion of what is in the needle, and can't decrease the total volume because 1.5 uL is already very small volume.

Any suggestions on how to make this work are greatly appreciated.

Dilution/Addition in Vial:

Dilutions/Additions in Needle:

Top Replies

  • Hi

    I assume that you're using low volume vials or inserts.  I have found in the past that drawing the liquid from the vial from a low position and ejecting back into the vial at a high position and at high speed mixes quite well.  I wonder if bubbling air leaves air bubbles at the bottom of insert vials?

    Kind regards

    Andy

  • Hello ,

    If you have not already, I would start by reading through this application note on using an injection program to perform dilution tasks. In a quick review of the program used in that note and what you have posted I would suggest adding an Eject step at the beginning so you can be certain you are always starting with the same state for the metering device. I would also avoid max speed ejects and draws until you have some baseline success with using the default speeds. Depending on sample volumes, vial material, sample viscosity, vial caps, ect. the maximum speed may be causing some issues with the prep and mixing of your solutions. Here is a screen shot/excerpt from that paper with the basic program and some explanations on each step.

    There is also a poster available that describes a similar technique for serial dilution that may also contain some useful tips for having success using the injector program for standard preparation. 

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