Improper blank causing early elution

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

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I work on RP-HPLC, analyzing small peptides. Mobile phase A is water, 0.1% TFA and mobile phase B is 90% MeCN, 0.8% TFA. Before starting runs I will perform a blank using the same method set I will be using for the next sample. Each method typically ends with an organic wipe (20A:80B) for 5 min before re-equilibrating to aqueous conditions over the next 5 min followed by a 10 min delay between runs. In an attempt to save time I created a 20 min "blank" method which is: 5 min aqueous, 5 min organic wipe, 5 min to re-equilibrate to aqueous conditions; however, I noticed that this causes the material from my subsequent run to elute prematurely. Is this due to lingering organic solvent from the blank? Do I need to let the system equilibrate to aqueous conditions more slowly?
It sounds like you may be using HPLC for the first time. To help you, we need some very basic information about your method to understand it. For example: Please provide the dimensions of your HPLC column used (ID, Length, particle size) as well as the brand and name of the column too. What is the flow rate used? These two key bits of information are needed to start any HPLC discussion.

Next a few comments in no particular order:
(1) TFA is a VERY strong acid. Why are you using such an extremely high concentration in bottle 'B' (0.8%) !!! This may cause a number of problems.
(2) You use the word "wipe" several times. Do you mean that you FLUSH the mobile phase? Perhaps this is a language translation issue?
(3) Are you running in isocratic mode or gradient mode? Please provide us with the exact mobile phase composition used (%) at each time point. It is not clear from your statement what compositions you use, only that bottle 'A' has water, with 0.1% TFA in it and bottle 'B' has 90% ACN with water and 0.8% TFA. What is the mobile phase composition ?
(4) You state your analysis ends in 20% 'A' and 80% 'B', but we do not know what you started in.
(5) It sounds like you may be washing your column down with solution, but not allowing enough time for it to flush the column, then not waiting long enough for the column to re-equilibrate before the next analysis. However, you did not provide any information about the column size or flow rate used so it is not possible to know the answer or help you without this key information.

If possible, ask your teacher for help or find someone who has HPLC experience and is familiar with these basic concepts. Someone who is there with you, to look over things, can help you.
Multidimensional wrote:
(5) It sounds like you may be washing your column down with solution, but not allowing enough time for it to flush the column, then not waiting long enough for the column to re-equilibrate before the next analysis. However, you did not provide any information about the column size or flow rate used so it is not possible to know the answer or help you without this key information.


I also tend to think that you need longer equilibration. Keep in mind that equilibration is not TIME but volume/column volumes of initial mobile phase; so more is way better than less.

We used Agilent, and automatically programmed in organic wash followed by equilibration into the saved Method for gradient runs.

For isocratic - once we determined that we had the run time and/or delay time so that there were no late-eluting peaks in the subsequent injection/run, we washed with high organic after the sequence was completed and results reviewed.
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