Help on: Selecting Columns for HPLC

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Question:
“Can you advise me on how to select a column for a particular molecule?
e.g. Molecule is acidic in nature; Molecule is basic in nature; Molecule is neutral in nature; In which cases would we prefer cyano and amino columns; When we should use an amino column for the analysis of sugars on a Refractive Index detector; Why a lot of noise is produced which is not produced while using a union; What is the best solvent for preservation of amino columns, excepting hexane?"

Answer:
“The process of selecting a column for a particular analysis is quite complex and requires a good knowledge of HPLC method development. I recommend the book ‘Practical HPLC Method Development’ by Lloyd Snyder, Joseph Kirkland and Joseph Glajch or, if you can, attend a good training course on HPLC method development such as those offered by MTS. As a general rule the type of column that you select will depend on the type of HPLC that you are using and the selection of the type of HPLC is based on the polarity and molecular weight of the molecule you wish to analyse. Types of HPLC include: partition, adsorption, size exclusion chromatography (SEC) and ion exchange chromatography (IC). Of these partition in the reversed phase (polar mobile phase) is the most commonly used. Acidic, basic and neutral molecules may be analysed using all these types of HPLC and so many columns can be used. Sorry that my answer cannot be more precise but the fact is that it is not just the acidic or basic nature of the molecule but also its other features that dictates the choice of column. My first choice with a new molecule is reversed phase partition HPLC if possible, using a C18 column and only move to other phases if necessary.

Both amino and cyano columns may be used in reversed phase and normal phase modes. Amino columns are extensively used for the analysis of sugars and saccharides. Cyano columns can be used for a range of molecules. Cyano offers an alternative selectivity to alkyl columns such as C18 and C8 but in past it has been difficult to obtain reproducible chromatography using cyano columns. Recent developments in column technology have improved this phase. You might try a cyano column at the outset of a method development to investigate which phase gives the best separation for a particular set of analytes. There is no specific best use for cyano columns in the way that amino columns are used for sugars.

I would agree that your problem with detector noise appears to be related to the column since it is absent when the column is absent, however the union does not provide back pressure comparable to a column and it may be that the problem is in the HPLC system. Can you try other columns? If it is due to your column then it may be contaminated and need cleaned, since RI detection is universal it is very sensitive to any compounds in the mobile phase. Unfortunately RI detection is very prone to baseline instability, controlling the temperature of the column may help.

I suggest isopropanol for the storage of your amino columns.”