In my LC-MS experiment, I am utilizing an Agilent Polaris NH2 column to separate a mixture of glucose, fructose, and sucrose. I am using water and acetonitrile as A and B mobile phases respectively, both with 0.1% ammonium hydroxide. I have employed a gradient elution, starting with 90% B, gradually decreasing to 0%, and then increasing back to 90% B over 17 minutes. Initially, I did not face any issues and the separation was satisfactory. However, lately, I have been encountering problems. Upon analyzing a large number of samples, I could only detect the sucrose peaks in a few standards. Additionally, I noticed a white substance on the ESI source, and I had to clean the spray and ion capillary extensively to regain sensitivity. I am now concerned whether my mobile phase condition can damage the HILIC column.
Hello. My main guess would be that the column phase is degrading/dissolving. The silica packing that carries the modification (NH2) is what's failing. The manufacturer states pH stability of 2.0 - 8.0 at not more than 40oC, which is typical for silica containing HPLC columns unless specifically modified/stabilized. Higher temperature and salt will speed up the deterioration. The pH of your 0.1% ammonium hydroxide would be close to 11.
Reference: Agilent's Column Care Manual
https://www.agilent.com/cs/library/packageinsert/public/820000-998_LR.pdf
page 7/110
Alkaline modifiers in HILIC conditions aid in separation but decrease the column lifetime indeed. It is better to use amide phases instead of amine ligands for carbohydrate analysis. Silica hydrolysis under alkaline conditions but also the Schiff base formation is not a negligible drawback in a high pH environment which results in as cleaved amine ligand and decreased retention time along with resolution loss...Amide phases are not susceptible to these kinds of harsh conditions. Either change your buffer system (try NH4bicarbonate-ammonium acetate modifiers, not the first choice surely) or your column of choice (best option).
"HILIC" is not a column, but a MODE of chromatography. *It is sometimes erroneously used as a marketing name by sales people to sell existing products under other names, iow "Columns" labeled "HILIC", which is misleading.
You are using an amino column (labeled "Amino", "NH" or "NH2) and most of these columns are based on silica which usually dissolves when in contact with strong bases (pH > 8). The instruction manual/guide that came with your specific HPLC column should list the usable pH range for your column. 0.1% ammonium hydroxide may be too strong to use (?). It may have already damaged your column (if so, dispose of it so someone else does not try and use the column). *Perform a column test to determine the condition of the column before use.
If you would like to learn more about this unusual mode of chromatography, then more info can be found at this link:
- "Hydrophilic Interaction Chromatography (HILIC)"; https://hplctips.blogspot.com/2012/03/hydrophilic-interaction-chromatography.html
To address the issues you are experiencing with the Agilent Polaris NH2 column, such as the disappearance of glucose and fructose peaks and the appearance of a white substance on the ESI source, you should consider reducing the concentration of ammonium hydroxide in the mobile phases from 0.1% to 5-10 mM, as the high concentration of this highly basic additive may be causing degradation of the column material or the analytes. Additionally, you should adjust the mobile phase gradient to reduce the amount of time the column is exposed to the high concentration of acetonitrile, and regularly monitor the column performance by analyzing standards to ensure it is not degrading over time. Thoroughly cleaning the column before use and ensuring the samples are properly prepared can also help resolve the issues you are facing.
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