Selection and Use of Packing for HPLC Columns

HPLC is currently the most widely used chromatographic analysis method, and its separation effect depends largely on the choice of chromatographic packing. However, there are many chromatographic packing choices, including polymer, silica gel matrix, and other inorganic, as many of types of packing used for gravity or low-pressure chromatography cannot stand the high pressures which are used in an HPLC separating system.

Due to the increased surface area, smaller-diameter beads can improve the sensitivity of separation. For an analytical HPLC column, media bead diameters are usually in the range of 1.8–5 μm. We should set the lower limit of bead size, as column pressure increases and bead diameter is reduced for a given flow rate, at the same time.

In the early time, we pack early HPLC columns with irregularly shaped silica particles to increase surface area. When scientists found that the spherical shape could provide increased efficiency and the porosity of the surface area, irregular silica was replaced by spherical porous silica. Hawach discusses how to choose a suitable chromatographic packing. You also can choose SCX HPLC Columns, NH2 HPLC Columns, C18 Alkaline HPLC Columns, and other HPLC Columns using.

The choice of HPLC chromatography packing

1. Silica gel matrix packing

Silica gel is a common matrix for HPLC column packing. In addition to the high strength common to inorganic substrates, it also provides a surface that can bond multiple ligands through mature silanization technology for reverse phase, ion exchange, hydrophobic interaction, hydrophilic interaction, or size exclusion chromatography. Silica gel packing is suitable for various solvents from polar to non-polar. The disadvantage is that it is unstable in the water-soluble alkaline mobile phase. Generally, the recommended routine analysis pH range for silica-based packing is 2-8.
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1.1 Normal phase chromatography

The stationary phase of normal phase chromatography is usually silica gel and other bonded phase fillers with polar functional groups such as amine groups (NH2, APS) and cyano groups (CN, CPS). Since the silanol (SiOH) or other polar groups on the surface of silica gel are more polar, the order of separation is based on the polarity of the components in the sample, that is, the less polar components are washed out of the chromatogram first column. The mobile phase used in normal phase chromatography is relatively low in polarity than the stationary phase, such as Hexane, Chloroform, Methylene Chloride, etc.

1.2 Reversed phase chromatography

The packing used in reversed-phase chromatography is often silica gel as a matrix and a bonded phase with relatively weakly polar functional groups bonded on the surface. The mobile phase used in reversed-phase chromatography is very polar, usually a mixture of water, buffer, methanol, acetonitrile, etc. The order in which the sample flows out of the chromatographic column is that the more polar components are washed out first, and the less polar components will be more retained on the chromatographic column. Commonly used reversed-phase packings are C18 (ODS), C8 (MOS), C4 (Butyl), C6H5 (Phenyl), and so on.

2. Polymer packing

The polymer packing is mostly polystyrene-divinylbenzene or polymethacrylate, etc., and its advantage is that it can be used at a pH of 1-14. Compared with silica gel matrix C18 packing, it has stronger hydrophobicity, and the macroporous polymer is very effective for the separation of samples such as proteins. The disadvantage is that the column efficiency of the chromatographic column is lower than that of the silica-based packing.

3. Other inorganic packing

Other HPLC inorganic packing chromatographic columns have also been commercialized due to their special properties and are generally limited to special applications. For example, graphitized carbon black is gradually becoming a reverse HPLC chromatographic column packing. The separation is different from the silica-based alkyl bonded phase. The surface of graphitized carbon is the basis for retention and no other surface modification is required.

The HPLC column packing generally has a stronger retention capacity than alkyl bonded phase silica gel or porous polymer packing. Since it will not be dissolved in the HPLC mobile phase, this type of column can be used at any pH and temperature.

Alumina can also be used in HPLC. However, due to the strong effect of alumina and basic compounds, the scope of application is limited to a certain extent, so it has not been widely used.