by
crong » Wed Jun 06, 2018 8:03 pm
Multidimensional wrote:
(1) "90% water rinse" is NOT a column wash method. You should be "washing" the column after each run using a solution which is stronger than the mobile phase, not weaker than the mobile phase. Water would not wash anything off, and in fact, it probably would assist in retaining compounds on the column to foul it.
(2) Get rid if the probably worthless guard columns. They primarily exist to make money for the column vendors. *Filter your samples! **Always, always make sure your samples are 100% dissolved in the mobile phase before injecting them!
(3) Find and use an experienced chromatographer at your school and have them review your actual method. They can probably identify a number of areas (like the lack of column wash) where you can improve how you are doing things. You are just starting out with HPLC and amino acid analysis takes years to be proficient in (that is a bad method to start out with for a beginner). I doubt you have the available time just to learn this one technique. Instead, invest the time in having someone experienced supervise the analysis (or just pay them to run the analysis for you so you can work on whatever your primary project is).
(4) Depending on the source of the samples (matrix) and its complexity, your column may last from 200 to 2,000 samples. Columns are consumable items. Their lifetime is a function of the purity and cleanliness of the samples, the quality of the wash solution used, the strength/types of the solvents it is exposed to and to some extent, the pressures it is exposed to (though, pressure is less important than people think).
Thank you for your advice.
(1) The water is used to rinse off any buffer in the column, not to clean the column. I should clarify that this is done at the end of the sequence prior to shutting down the instrument (not at end of each sample). Before adding the water rinse, the pressure was increasing with each new sequence, hence my suspicion that buffer was still left in the column and precipitating. Since adding the water rinse, the pressure has stopped increasing, however that did not stop the continued loss of resolution. So to me that means the loss of resolution isn't related to any kind of clogging or precipitation.
(2) Samples are all filtered to 0.45 um or 10kDA through microfiltration. They are aqueous samples to start with so no issue with dissolution I would think.
(3) The method is an exact transfer from an application note provided by the instrument manufacturer (Thermo Scientific) only with a different column and flow rate and gradient changed to accommodate my column size.
(4) Well this is part of my question, for those who have experience with amino acid analysis, whether it is normal to have such a short life time and if not, what can be done to improve it? I'm no expert but I have never experienced this short of a life time on any kind of column.