Hi,
I have been trying on a USP method regarding Glucosamine Sulfate Potassium Chloride. The assay is rather simple. It requires the use of amino-phase column to be used in reversed phase mode (which some may call HILIC?). The column i am using is Phenomenex Luna NH2 column (150 x 4.6mm, 5micron). The mobile phase used is premixed 75% acetonitrile: 25% phosphate buffer.
After about 20 injections, i discovered that the retention time shifts forward (earlier elution). Initially, it was around 10min (as indicated in the monograph), later shifted to about 5min and stabilised. This is on of the issues. However, a second issue later arises. After about 140 injections, the theoretical plate numbers fell steadily from 2400 to 1200. Then, i tried washing the column as instructed by the manufacturer with EtOH, followed by Hexane for 20 column volume. Unfortunately, the efficiency fell sharply to 700 (i might have killed the column?).
Here are some of the things that i have been doing:
There are actually a few questions on my mind right now:
Your help and sharing is much appreciated! Please let me know if i have left out any info to allow you to better understand my situation.
Thanks!!
Most amine columns are known to degrade faster than other column types. They are particularly susceptible to Schiff base formation in the presence of carbonyl or hydroxy groups. I've worked for some time with different amino columns, including the Phenomenex Luna NH2, for sugars analysis, and I've also experienced this issue, even though diluting the sample and buffering pH around 7 helped contain the problem.
I would also be wary of increasing pH too much since your column is still silica based.
Please look at the following below links which may help you in your analysis:
https://www.dia-m.ru/upload/iblock/13c/262-machereynagel.pdf
https://www.waters.com/webassets/cms/library/docs/lcAC_new.pdf
http://www.revodix.co.kr/superboard//data/board/cataloguelab/237106713_fIXao0Vp_2ca15ffce9db6e862a64901f8fda56f555e13a58.pdf
Thanks
Davide Refosco Thanks for your reply.
About the Schiff base formation, it seems like there is no way to prevent that from happening.
About the pH, i also just realised that the pH increased to 8.8 after mixing. Maybe i will try and bring the pH back down to 7.5 after mixing and see how it goes. However, i dont have a new column to try that one out at the moment. Because it seems like my current column is done for. I will let you know more about it after trying it in the near future.
Do you think my current column is salvageable?
Other than that, may i know which column brand you are using right now? Thanks.
Isam Eldin Hussein Elgailani Thanks for your suggestions. I will read into them.
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