This guide will help you determine the best column to use when multiple compounds are in your mixture.
Choose the properties of each compound in your mixture, then a chart will show you the best column type with which to analyze it.
Please choose the properties of each compound in the drop down menus below.
Is your compound
hydrophobic or hydrophilic?*
Is your compound acidic, basic, zwitterionic
Is your compound strong or
Does you compound have one or
more acidic or basic groups?
*Hydrophilic compounds are compounds with a better solubility in water than in organic solvents, or completely non-soluble in organic solvents (et. acetonitrile, ethanol, hexane). Examples of such compounds are amino acids, sugars, peptides, nucleotides, protonated amines and any other molecules with one or several polar groups. Not every functional group makes the molecule hydrophilic. The presence of some functional groups does not make a molecule significantly polar in terms of LC hydrophobic interaction; there is a more objective characterization of hydrophobic/hydrophilic properties. Log P is a factor which represents the polar properties of the molecules. It is defined as a logarithm of the distribution coefficient of the compound in the liquid/liquid extraction system of water and octanol. The higher the Log P value, the more hydrophobic the compound. Generally compounds with logP > 1 are well suited for reverse phase chromatography and can be called hydrophobic. LogP for ionizable compounds is pH dependent. If the ionization state of the molecule is changed by the solvent's pH, then logP will be changed as well. The general rule is that lowering the pH increases logP for acidic compounds, and decreases logP for basic compounds. Many basic compounds become very polar at a low pH, and thus non-suitable for reverse phase separation. Acidic compounds, on the other hand, become more hydrophobic and retain better.
*Acidic compounds are negatively charged. Basic compounds are positively charged.
*Strong acids - compounds which are significantly ionized in solutions at a pH of 3 (pKa < 3). Weak acids - compounds that go through the transition from a negatively charged state to a neutral state within a pH range from 2 to 7 (pKa > 3). Strong bases - compounds which are significantly ionized in solutions at a pH of 6 (pKa >6). Weak bases - compounds that go through a transition state from positively charged to neutral within a pH range from 2 to 7 (pKa = 6).