I have a bacterial metabolite. I want to identify both polar and non-polar metabolites present in the sample. Do I need to separate metabolites into polar and non-polar categories run separately such that polar metabolites through Hilic and non-polar metabolites through c18 columns. Or I can directly run the metabolites through Hilic and c18column without separating them to identify the metabolites.
If I need to separate the metabolites into polar and non-polar categories, how can I do that?
You can analyze your sample without any previous preparation, but keep in mind that this will impact on the lifetime of your column, if you have a pre-column installed this problem is reduced. but if you want to previously separate the polar and nonpolar fraction, I would advise using a C-18 SPE.
The polar fraction will be the flow-trough and the nonpolar fraction will be adhered to the C-18 and later washed out with acetonitrile or methanol.
No. You can use a gradient method. The most polar compounds are eluted first and the non-polar compounds stuck on the column. When the high organic mobile phase hits the column (I suggest IPA) these non-polar compounds are eluted.
Hilic is a good technique for analytes having a LogP lower than about 1, while reversed-phase for logP greater than about -1. In reversed-phase your sample must be ideally dissolved in an polar solvent or mixture (low strenght eluent for this mode) or in the initial mobile phase (e.g. water, water/methanol, water/ACN) while for hilic must be in medium or low polarity solvent or mixture (e.g. ACN, ACN/MeOH, or initial mobile phase). So, the dissolved sample you will use in hilic must be no optimal for reversed-phase, although in some cases it can be a good alternative. Running a sample that contain both polar and non-polar compounds in C18 or hilic is not an issue, polar compounds will not be retained in c18 and non-polar compounds will be not retained in hilic, but as these compounds pass through the column without being retained, they will not affect the chromatography. If your analytes have very different polarities, such as logP -1 and 4, you need to use both chromatographyc modes. If that is the case, is better to work with separated samples. You can partition your polar/non-polar analytes by means of liquid-liquid extraction or as mentioned by @Luiz Pollo through SPE as needed (e.g. C18, HLB, cation or anion exchange). Also you can exchange solvents after solvent evaporation.
The age old solvent extraction can help. But this too results in mixtures. another method could be gradient column chromatography. This is routinely performed in chemistry labs. But it is time consuming and requires sufficient quantity of the mixture. If you have prep-HPLC, with a universal detector like MS that is the best option.
Dear Mam,
You can prefer isolation methods like HPTLC or HPLC for separating non-polar and polar compounds.
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