How to adjust mobile phase gradient?

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

6 posts Page 1 of 1
I reran a vendor's method for identity and purity of a polypeptide. Invendor's method, they use Agilent HPLC. However, I used Perkin Elmer HPLC. I follow the vendor's method exactly: the gradient, column type, column temperature, injection volume,etc. My system suit failed. I think the failure maybe due to the gradient since different instrument has different dwell volume. So, I went on and measured the dwell volume of my instrument. I don't know how to adjust the gradient if the dwell volume is known. Can you please help! Thank you,
The dwell time (dwell volume divided by flow rate) is effectively an isocratiddc hold at the beginning of the gradient. All you need to do is to purposely add an isocratic hold or a delay to injection to make the total delay the same on both instruments. That said, there may be other factors such as mixing volume, proportioning accuracy, or column chemistry involved.

Knowing what part of system suitability failed might give us more to go on.
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374

If you have 2 different pumps (quat vs. binary), different tubing the total system volume will be different. So in my opinion the best option is to adjust gradient manually just to achieve correct parameters (resolution between peaks, retention time range).


Tomasz Kubowicz
What is the QA used by the vendor? Metric for performance?
Is anyone aware how to create an injection delay to an Agilent 1260 running chemstation? The injector program function eludes me. Much more familiar with MassLynx.

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6 posts Page 1 of 1

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