The common packing materials in liquid chromatography columns are C1, C3, C4, C8, C18, C30, amino, cyano, silica gel columns, etc. Classification of packing materials in common chromatographic columns.
a. Reversed-phase (with ion-pair) method
C18 (octadecyl or ODS): good generality; strong retention and versatile
C8 (octyl): similar to C18, but slightly lower retention value
C3, C4: small retention value; mostly used for peptides and proteins
C1 [trimethylsilane (TMS)]: smallest retention value; most unstable
Phenyl, phenylethyl: moderate retention value; different selectivity
CN (cyano): moderate retention value; both normal and reversed phases can be used
NH2 (amino): weak retention; used for hydrocarbons; less stable
Polystyrene-based: stable in mobile phases with 1 < PH < 13; good for some separation peak shapes and long column life
b. Orthophase method
CN (cyano): good generality; moderate polarity; Versatile
OH (diol group): more polar than CN
NH2 (amino): more polar, less stable
Silica gel b: good universality; inexpensive; less convenient to operate; used to prepare LC
c. Space exclusion method
Silica gel b: excellent universal applicability; used as adsorbent
Silylated silica gel: weak adsorption, good solvent compatibility; for organic solvents
OH (diol-based): less stable; used in aqueous SEC (gel filtration)
Polystyrene-based b: widely used in organic SEC (gel permeation); generally incompatible with water and polar organic solvents
d. Ion exchange method
Bonded phase: poor stability and reproducibility
Effect of packing on the performance of hplc chromatography columns
1, the effect of particle size liquid chromatography column packing is that the smaller the average particle size, the smaller the vortex diffusion, the better the mass transfer, and the higher the column efficiency. However, the packing particle size is small, resulting in poor permeability performance and higher column pressure. At the same time, the wider the packing size distribution, the worse the permeation performance and the lower the column efficiency. The common packing particle size of 3μm, 3.5μm, 5μm, 10μm, etc.
2, the influence of the shape of liquid chromatography column packing is divided into amorphous and spherical. The amorphous packing preparation of the liquid chromatography column bed structure is not uniform, and the mobile phase linear velocity is not uniform. The chromatogram peak shape is wider; the spherical packing preparation of the liquid chromatography column bed structure is uniform, so the column efficiency is high and has good reproducibility. Spherical packing is the most common liquid chromatography column packing, and this packing has better performance and reproducibility.
3, the influence of bonded phase with silica gel as the matrix, through the chemical bonding of C18, C8, amino, cyano, and other groups bonded to the matrix, as the packing in the liquid chromatography column. By bonding different chemical groups, liquid chromatographic columns with different performances are obtained due to the different separation mechanisms of different bonding groups, affecting compounds’ retention and separation. The bonding of fillers makes the stationary phase of liquid chromatography columns relatively more stable and less susceptible to lose while largely eliminating the adverse effects of silicone hydroxyl groups, which can be applied in a wide range of mobile phases and widely used. However, the bonded filler is poor acid resistance, and the pH value can not be less than 2. When the mobile phase pH exceeds the acidic range, the bonded phase is easy to lose, and durability and stability will worsen.
4, the effect of end group sealing silica gel is widely used as a matrix material for liquid chromatography column packing because of its special surface chemical properties. The surface of silica gel has silicone hydroxyl groups, the density, distribution, and chemical properties of silicone hydroxyl groups will have different degrees of influence on the behavior of various types of chromatography. When silica-based HPLC columns, especially in reversed-phase columns, non-specific adsorption due to free silicone hydroxyl groups (or silanol groups) is often encountered. For some more polar solutes, such as alkalis, chromatographic summits can be severely trailed or even fail to elute due to strong adsorption. The effect of the silicone hydroxyl group is usually reduced by tailing the filler with a small silane (e.g., trimethylchlorosilane) to increase the surface coverage by reacting away some residual silicone hydroxyl group. This approach not only reduces irreversible adsorption or tailing but also increases the carbon content. However, this method does not completely react off the residual silicon hydroxyl groups, and 50% of the silicon hydroxyl groups are still left unreacted.
5, the effect of the carbon content of liquid chromatography column packing, especially the carbon content of reversed-phase packing, is often used to characterize the degree of surface chemical modification. Usually, carbon chains are introduced into the packing through bonding, and the higher the carbon content of the packing, the higher the density of carbon chains. The longer the carbon chains, the larger the capacity factor and the stronger the hydrophobicity. In reverse-phase conditions, the growth of carbon chains means that the filler has a larger specific surface area, the bonding is enhanced, and the retention of the material to be measured increases. Therefore, the liquid chromatography column with high carbon content packing has good stability and reproducibility, which is conducive to the separation of poorly retained compounds and can improve the trailing of polar compounds; the liquid chromatography column with low carbon content packing is conducive to the analysis of neutral and basic compounds and can reduce solvent loss.
6, the impact of other factors in addition to the above, the activity of silica gel packing, impurity content, pH stability, thermal stability, etc., will also affect the performance of the liquid chromatography column. When producing silica gel, the processing temperature and the silica gel activity are also different. The activity of silica gel is the main source of selectivity differences, mainly affecting the retention behavior of basic compounds; impurity content of silica gel is an important sign of the quality of the column, low content of heavy metals, silica hydroxyl activity is small, dragging the tail is reduced.
Article source: “Liquid Chromatography in Action
Reprinted from: “Liquid Chromatography Practical Treasures
Post time: Nov-05-2022
