How to choose the column of liquid chromatography

1、 Chromatographic column parameters and their significance
The influence of different column parameters should be considered in the selection of chromatographic column. The parameters of chromatographic column are analyzed in the following contents.
1. Column size
The longer column has stronger retention, longer operation time, higher column efficiency and better resolution. In practical work, the running time and column efficiency should be comprehensively evaluated, and the column with suitable length should be selected. For example, the requirements of content determination and dissolution detection for separation are low, so shorter chromatographic column can be selected to reduce the operation time; the longer chromatographic column is generally selected for related substances to improve the separation effect.
The chromatographic column with small inner diameter has higher sensitivity, narrower peak shape, but smaller sample load. If the sample load meets the requirements, higher and sharper peaks are needed to improve the sensitivity, and smaller inner diameter can be selected. However, if the injection volume exceeds the column load, the chromatographic peak will be greatly widened. Therefore, the sensitivity and sample load should be measured comprehensively.
The smaller the particle size, the better the resolution and the greater the column pressure. If the resolution of chromatographic column with 5 μ m particle size is slightly insufficient, the resolution of the same type of 3 μ m column will generally provide satisfactory selectivity.
If a smaller size chromatographic column is selected, the influence of dead volume outside the column on the column efficiency should be measured. The dead volume outside the column includes the connecting pipeline, the injection volume, the gap between the column joint and the column, etc. Therefore, the chromatographic column commonly used in ultra-high performance liquid chromatography is not suitable for conventional liquid chromatograph. It is better to use the same brand of instrument for small size chromatographic column to reduce the dead volume introduced by the gap at the junction. Digression: the mismatch between the instrument pipeline joint and chromatographic column between Shimadzu, Agilent and waters, and the inconsistency of instrument and chromatographic column brands will inevitably lead to large connection gap. Attention should be paid to the influence in this respect.
2. Other parameters
High specific surface area has strong retention capacity, column capacity and resolution, and low specific surface area can reach equilibrium state more quickly, which is of great significance in gradient leaching.
Large pore size packing can prolong the retention time of macromolecules in the packing, achieve full separation and improve the peak shape. Large pore size chromatographic column should be selected for macromolecular samples, such as proteins and polypeptides.
The results showed that the equilibrium of single tooth bonded column was faster, the stability of double tooth bonding column was better, and the column capacity was larger.
The results showed that the amount of carbon planted was large, the retention was strong, and the separation effect was good. On the contrary, the retention is weak and the separation effect is poor.
The end capped column can reduce the tailing phenomenon caused by the interaction between the basic component (cation) and the residual silica anion on the surface of silica gel, and the selectivity of the column with and without tail sealing treatment is also different for polar samples. Generally, the chromatographic column with tail sealing treatment should be selected for basic compounds.
2、 Analysis of common stationary phases
1. Straight chain alkanes (C18, C8, C6, C4, C3...)
This kind of stationary phase column is often used for non-polar and weak polar compounds. Generally speaking, the longer the chain length, the larger the column capacity, the longer the retention time, the better separation effect and column stability. Therefore, C18 is the most commonly used reversed-phase chromatographic column, but some components such as macromolecular samples are difficult to elute because of their strong retention, so shorter chain stationary phase columns (such as C4, etc.) will be used. In the development of the method, if C18 column is used, the most difficult elution component remains too strong under the highest proportion of organic phase allowed by fluidity, resulting in too long running time. Therefore, C8 column can be considered.
The retention mechanism of this kind of stationary phase is "similar phase soluble" liquid-liquid partition chromatography. The stationary phase is of small polar carbon chain. The retention of components varies with the polarity. The smaller the polarity, the stronger the retention. The elution order of different components can be determined according to the relative polarity of the column. For example, benzene and phenol, phenol because of the addition of a hydroxyl group, the polarity of phenol becomes larger, and elutes faster in the C18 column.
One of the greatest advantages of this kind of column in method development is that the retention can be predicted, for example, generally reducing the organic comparison will increase the retention, increase the resolution and so on.
2. Phenyl
The retention mechanism is the same as that of straight chain alkanes. It has good resolution for compounds with benzene ring structure, especially for the subtle differences in benzene ring, such as whether there are halogen in the structural formula, the position of functional groups in the benzene ring is different, etc.
3. Cyano group
CN (cyano) packing can be used in both normal and reversed phase chromatography because of its medium polarity cyano bonded stationary phase. When it is used in normal phase chromatography, it can be used as a low polarity mobile phase such as n-hexane; when it is used in reverse phase chromatography, it can be used as a strong polar mobile phase of methanol or water.
The hydrophobicity of CN column is relatively low in RP-HPLC stationary phase, and it shows different selectivity compared with ODS due to its π - electron interaction with nitrile group. Cn column is suitable for separation of components with long retention time on ODS, and it is very difficult to optimize chromatography on ODS. Cn column can also be used for the detection of strong polar compounds with too small retention on ODS. If the polarity difference between the components is too large, the CN column may provide a satisfactory result. For example, in a dissolution methodology study of a project, I found that the main peak deformation of C18 column specified in the Pharmacopoeia method after repeated analysis of the dissolved samples was caused by the inability to elute the strong retention component in the capsule shell, so the CN column was selected to continuously analyze thousands of samples, and the column efficiency was still good.
In some cases, the change of retention time of CN column is not as obvious as that of straight chain alkane column. In some cases, the change of organic phase is dozens percent, and the retention time does not change significantly. This should be noted.
4. Amino group
Amino column is a polar bonded chromatographic column formed by the polar group aminopropylsilyl bonded on silica gel. Generally, it can be used in normal phase or reverse phase. It is generally used for the analysis of sugars and carbohydrates.
At present, the separation principle of amino column is mainly based on hydrogen bond force and van der Waals force. It can be seen that amino column is generally not used for the analysis of easily dissociated compounds. There are two reasons: one is that the amino column is very unstable, and buffer salts are usually used for the easily dissociated compounds, which accelerates the destruction of the amino column stationary phase; the other is that the retention mechanism of amino column for easily dissociated compounds is very complex, in addition to the hydrogen bond and van der Waals force mentioned above, there are also ion exchange chromatography mechanisms, such as mutual ion repulsion between basic compounds and amino stationary phase, acid It is difficult to predict the chromatographic behavior because of the mutual attraction between the amino group and the natural compound. At present, amino acid column is most commonly used for carbohydrate analysis, and water organic phase system is used for fluidity.
The biggest disadvantage of this kind of chromatographic column is its poor stability and short service life.
5. HILIC (hydrophilic interaction chromatography)
This column is a stationary phase of zwitterions and covalently bonded to porous silica gel. It is suitable for the detection of strongly ionized samples with weak or no retention in reversed-phase chromatography, which solves the problem that the samples in this field are difficult to dissolve by normal chromatography. This is a relatively new type of chromatographic column, and there is little research on it at present.
The separation mechanism of HILIC column is very complex, mainly including the following aspects: liquid-liquid distribution, ion exchange, dipole dipole interaction, hydrogen bonding, etc. many experiments show that the retention mechanism of HILIC is the joint action of the above mechanisms.
The typical HILIC mobile phase is water or buffer containing 40% - 97% acetonitrile, so pure acetonitrile should not be used. The retention time of the components generally increases with the increase of the proportion of organic phase and decreases with the increase of the proportion of water. Therefore, gradient setting is the opposite of RP-HPLC, which is a process in which the proportion of organic phase decreases gradually.
The sample should not be dissolved in pure water. The best proportion of water is the same as the initial proportion of mobile phase. The principle is the same as the common "solvent effect" of reversed-phase chromatography.
6. Ion exchange column
It is divided into anion exchange column and cation exchange column. It is mainly used for the detection of strong ion components, such as metal ions, acid radicals, etc. Most commonly used in ion chromatograph, if the component has UV response, it can also be used in general liquid chromatograph. Its principle and application have been studied in many aspects, so this paper will not introduce it in detail.
7. Others
The above six types of chromatographic columns have included most of the common chromatographic columns in the laboratory. Other chromatographic columns such as chiral column or special packing column have limited space due to their different properties or less application. This paper will not give a detailed introduction.
3、 Some other contents related to chromatographic column
1. Flow direction of chromatographic column
In general, the direction of the arrow on the column should be installed to set the direction of the mobile phase. If the pressure of chromatographic column is obviously increased, the chromatographic column can be disconnected, and the detector can be turned around for recoil. After this operation, the chromatographic column is best used in reverse. Avoid frequently changing the flow direction of the mobile phase in the chromatographic column, because the bonding phase of the chromatographic column has an arrangement direction. Frequent change of the flow direction of the mobile phase in the column will destroy the arrangement and reduce the performance of the column.
2. PH stability
As mentioned above, the longer the carbon chain of the bonding phase is, the more stable it is, which means that the longer the carbon chain is not easy to hydrolyze at low pH value. Therefore, it can be concluded that the chromatographic column with short chain tail sealing should not be used under the condition of low pH value (< 2). At high pH value, silica gel will be dissolved because of silica anion on silica gel. Therefore, it can be concluded that the column after tail sealing has better stability at high pH. In the process of method development and sample detection, the chromatographic column can be selected according to the above conclusion, and the chromatographic column life can be improved.
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