How to Choose the Right Liquid Chromatography Column, SPE, FALSH, or HPLC?
How to understand the chromatographic separation? Put it in short, the more similar the separated substance to the stationary phase, the longer the residence time on it. In this way, different substances in a complex mixed solution can be separated according to time, and the separated substances are sequentially entered into the detector to be detected by the detector, and then the scientific researchers can use these detected signals (chromatographic peaks) to calculate the concentration and content of a substance.
To choose the right liquid chromatography column, firstly we should know the packing matrix and its influencing factors.
Packing matrix
1. Silica gel matrix-pH 2-8 —————— At high or low pH, silica gel will dissolve
2. Al2O3. NH2O-pH1-14 ————- Chemical modification is difficult
3. Polymer matrix-pH1-14 ————- Complex pore structure, uneven pore size results in insufficient column efficiency, organic solvents may cause swelling and damage to the polymer matrix.
Influencing factors
1.Physical factors
Purity of silica gel —Purity of filler silica gel and residual metal ion concentration
Column size — length and inner diameter of packed bed
Particle shape —-spherical or irregular
Particle size — average particle diameter
Surface area—the sum of the outer surface of the particle and the inner pore surface, expressed in m2 / gram
Pore size— The average size of the pores or cavities of the particles, ranging from 80-300 & Aring
2. Chemical properties
Bonding type — monomer bonding-the bonding phase molecule and the matrix are connected at a single point
Carbon coverage—the amount of bonded phase connected to the matrix material
Capping—After the bonding step, use a short chain to bond the exposed silyl hydroxyl groups and block them.
About the samples and impurities’ type structure, polarity, acidity, molecular weight
Before choosing a column, learn more about your samples and impurities, the type structure, polarity, acidity, molecular weight, etc.
1. The sample is polar and weakly acidic: you can choose C18 for detection under the condition of 100% acidic aqueous solution, that is, select a chromatography column that withstands 100% pure water and retains polar compounds well.
2.Too polar or acidic sample: silica gel column, CN, NH2, HILIC (hydrophilic chromatography) can be adopted. The disadvantage is that the ion-pairing reagent has a long equilibration time, and the pH of the mobile phase is relatively precise. Otherwise, it is difficult to repeat the experiment. In addition, the ion-pairing reagent is difficult to wash off.
3.If the sample is alkaline: choose a high-purity silica gel column (high-purity silica gel lacks metal impurities and the end-capping of the silica gel) or some modified C18 columns (such as polar embedding technology or alkaline deactivation technology), which will reduce the tailing of alkaline compounds. So generally we choose to do under neutral or alkaline conditions, because this can increase the retention of alkaline samples.
4. If the polarity of the basic compound is too strong, or the basicity is too strong: a wide pH C18 column can be selected for high pH detection. The advantage is that the method is simple to develop, and the disadvantage is fewer supplier and high price or choose HILIC column (silica gel column is used under reversed-phase conditions, a classic method to detect alkaline samples).
Selecting strong ion exchange column’s disadvantage is that it cannot be used to analyze other samples, more precise requirements for mobile phase pH, otherwise it is difficult to repeat the experiment. There are also C18 + strong anion pair reagents or strong anion exchange columns.
