Many questions come to mind when putting together a development kit in HPLC. Why is it so hard to predict chromatographic separation? Will I be able to achieve my separation on a C18 column? Or do I need to go on a different chemistry? If so, which one? And why?
Developing a chromatographic method can be very complicated! For this reason, we have introduced a selection of kits alongside AMT that will aid you. Choose up to three chemistries and define your own Method Development Toolkit. To help you choose the right chemistries, let's consider the different retention mechanisms involved in reverse phase chromatography and what column chemistries should be associated.
First, A Quick Recap on Reverse Phase Chromatography
The hydrophobicity of an analyte molecule will be the primary indicator as to the retentivity in reversed phase HPLC.
Hydrophobicity is often expressed as log P which is a measure of the way an analyte (in its neutral form) partitions between two immiscible solvents (usually octanol and water) under standard conditions (Equation 1).
The higher the value of log P, the more hydrophobic the molecule.
Polar analytes interacting with silica surface silanol groups undergo an adsorption type interaction as well as their partitioning behaviour - this can lead to detrimental peak shape effects along with an increase in retention time.
The structure of the sample molecules will also give clues as to their elution order. The elution order is governed by the water solubility of the molecule and the carbon content for an analogous series. Some observations governing sample elution order include (again, these assume compounds are of analogous series):
The less water-soluble a sample is, the more retention
Retention time increases as the number of carbon atoms increases
Branched chain compounds elute more rapidly than normal isomers
Unsaturation decreases retention
Neutral polar and charged species typically show the least retention followed by acid then basic compounds all eluting early
How Do I Select My Column(s)?
There are many parameters to consider before making the correct selection:
Particle size and particle technology: The particle size or particle diameter (dp) is the average diameter of the column packing particles. This should be selected based on the instrumentation you are using (HPLC or UHPLC) and also the objective of your analysis (analytical or preparative chromatography). If you are only using HPLC system, you can also look at improving your column efficiency by selecting coreshell column, which are available both for analytical and preparative chromatography.
Pore size: The pore diameter chosen is directly related to the hydrodynamic volume of the molecules in the sample. Larger pores allow large molecules to access the bonded phase found within pores for maximum separating ability. Select pore sizes of 150 Å or less for small molecules. Large pores, 300 Å or greater, are used for samples having a molecular weight greater than 2,000 Da.
pH range: Very important parameter, the pH range gives the pH values at which the HPLC column can be used without degradation of the solid support and stationary phase which will ultimately result in deleterious chromatographic results.
Traditional silica has a working range of pH 2.5-7.5. At low pH acidic hydrolysis of the silyl ether linkage between the bonded phase and silica surface will occur, resulting in column bleed (loss of stationary phase), poor peak shape, and loss of efficiency. At high pH the silica surface itself is at risk of basic hydrolysis, sometimes referred to as silica dissolution, whereby, the solid silica support is cleaved apart and fines are created which block the support material pores, interstitial gaps between the particles, and the column outlet frit resulting in the system over pressurizing and shutting down. Ensure you are using your columns within the recommended pH range and select your columns based on the parameters of your analytes you wish to separate.
Selecting The Relevant Chemistries
There are a few things you need to consider when developing a new method. What is your matrix? How many analytes would you like to analyse? What are the other components present in your sample? And what sort of molecule are you dealing with?
If you are dealing with a traditional, small and hydrophobic molecule, then C18 or C8 columns are the standards to achieve fast, robust, and reproducible separation. These columns will show excellent performance for a broad range of analyte polarities. Separations are due primarily to hydrophobic interactions and differences in hydrophobicity between analytes.
A great addition to these 2 standard columns is a phenyl column. These columns are great to introduce shape selectivity in your chromatographic separation. It will separate analytes via a combination of hydrophobic and π-π interactions. Also, phenyl columns show enhanced retention and selectivity for aromatic and unsaturated analytes, especially those containing electron-withdrawing groups and halogens.
After, you can start looking at orthogonal type retention where, for example, an RP-amide column will offer complimentary selectivity to a C18 and a C8 and are recommended for samples containing acidic and basic compounds that require high aqueous eluents. Separations are influenced by both hydrophobic and hydrogen bonding interactions.
Another orthogonal column in your toolkit could be a CN column, which provides strong dipole-dipole interactions with analytes and weak hydrophobic interactions. It is suitable for use in reversed-phase, HILIC and normal-phase modes.
Finally, in the reverse phase mechanism, PFP columns are particularly well suited for the separation of halogenated compounds, nitro-aromatic compounds and polar bases, with separations being primarily influenced by hydrogen bonding and dipole-dipole interactions. It's worth noting that this phase can also be used in HILIC mode with mobile phases containing >80% acetonitrile.
If you need more information on your next method development kit for HPLC, then get in touch: