It can be said that the chromatographic column is the most important part in the chromatographic analysis process, which is equivalent to the “heart” of the human body. To select the appropriate liquid chromatographic column according to the samples to be analyzed, it is necessary to consider the impact of different parameters. For example, chromatographic column size: column length and inner diameter; Chromatographic column packing: particle size, pore diameter, specific surface area, matrix and its surface characteristics, carbon load and type of bonded phase.
In daily liquid phase analysis, correctly selecting the chromatographic column suitable for your own experiment will make your experiment twice the result with half the effort! Today, we will talk about how to select liquid chromatographic column in liquid phase analysis.

Chromatographic column parameters – column length

For two chromatographic columns with the same packing, the longer the column length is, the longer the separation time is. However, the longer the column length is, the better the resolution will be. At the same time, the greater the column pressure will be. The appropriate column length can be selected according to the complexity of the sample and the desired separation time, generally 2.0~250mm.

Column parameters – inner diameter

Narrow diameter column can improve the sensitivity of the column, and can significantly reduce the amount of solvent used;
Wide diameter column can minimize the influence of dead volume of the system, with high sample load and anti pollution.

Chromatographic column parameters – particle size

Example: 1.8 μ m、3 μ m、5 μ m、10 μ M, the smaller the particle size, the faster the separation and the higher the column efficiency. However, the higher the column pressure is, the easier the column is to be polluted and the lower the service life is.

Column parameters – pore diameter

If the pore diameter is small and the porosity is high, the specific surface area is large and the carbon load is high.
Column parameters specific surface area
For example: 180 ㎡ /g, 320 ㎡ /g, etc. (the ultimate series is 320 ㎡ /g). Select a specific surface area suitable for the target object. The surface area per gram of filler (related to particle size and porosity). A large specific surface area will increase the reaction between the sample and the bonding phase, increase retention and sample loading. A small specific surface area can shorten the analysis time and the equilibrium time in gradient.

Column parameters – carbon loading

For example: 10%, 14%, 17%, 23%, etc. the carbon loading usually affects the selectivity. The proportion of bonding phase on the surface of silica gel is related to the specific surface area and bonding coverage. If the carbon load is high, the retention increases, which is suitable for the analysis of non-polar compounds. The loading amount of preparative chromatography can also be increased.

Column parameters – silica gel matrix

  1. silica gel matrix column has higher column efficiency than columns made of other materials;
  2. good mechanical strength of silica gel;
  3. the surface of silica gel can be chemically modified to form a bonding phase with various functional groups;
  4. the silica gel will not change in volume;
  5. silica gel will dissolve at high pH value;
  6. the surface of silica gel is acidic, and it is easy to produce tailing when separating alkaline compounds

Column parameters – polymer matrix packing

  1. cross linked styrene / divinylbenzene, methyl phenylenoate, etc;
  2. good pH stability: pH 1-14;
  3. surface chemical treatment can be carried out in a wide range to meet the needs of reversed-phase chromatography, ion exchange chromatography, hydrophobic interaction chromatography and volume exclusion chromatography;
  4. it has a better peak shape for strongly alkaline substances at medium pH;
  5. the volume of packing materials changes with different mobile phases, such as expansion or contraction;
  6. the separation efficiency is lower than that of silica gel;
  7. poor reproducibility;
  8. poor mechanical strength

Column parameters – silyl alcohol

The silanol group is more acidic and can be firmly combined with alkaline compounds due to electrostatic force. Therefore, packing materials containing more residual silanol groups are prone to tailing, widening and retention extension when separating alkaline compounds.

How to shield the influence of residual silicon hydroxyl: adding appropriate end-capping agent such as triethylamine can mask the residual silicon hydroxyl and improve the peak shape of basic compounds; Reducing the pH value of mobile phase less than 3 can reduce the reaction of silyl alcohol group, thus reducing the tailing phenomenon. Or choose to use the chromatographic column with double end-capping preferentially.

Chromatographic column parameters – End-capping

In order to reduce the influence of silanol group, the chromatographic column needs to be end-capped. Usually, the bonded packing material reacted with small silane (such as trimethylsilane) to shield some residual silanol groups.
Purpose of end-capping: 1 Shielding the residual silanol group; 2. improve selectivity.

Column parameters – Classification of bonded phases

Bonding phase: reversed phase (70%); C18(65%); C8(15%); Cyano(5%); Phenyl column (<5%); Normal phase (20%; silica gel, -NH2, -CN); Others (10%; chiral column, ion exchange column).

For the reversed-phase column with the same silica matrix from the same manufacturer, the retention value increases with the increase of carbon loading; The longer the bonded carbon chain, the greater the retention. C18 ≈ C8 > C4 > C1, there is little difference between the long chains. According to the properties of the substance to be analyzed, select the appropriate bonding phase and separation mode.
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