Selection and application of high performance liquid chromat

High performance liquid chromatography (HPLC) is the most widely used chromatographic analysis method, and its separation effect largely depends on the selection of chromatographic packing. However, the selection range of chromatographic packing is very wide, including polymer packing, silica gel matrix packing and other inorganic fillers. In order to select the appropriate chromatographic packing, we must have a certain understanding of this.
Selection of chromatographic packing
One
Silica gel matrix filler
Silica gel is the most common matrix for HPLC packing. In addition to the high strength shared by inorganic substrates, it also provides a surface that can be bonded to a wide range of ligands by mature silylation technology to form reversed phase, ion exchange, hydrophobic, hydrophilic or molecular exclusion chromatography. Silica based fillers are suitable for a wide range of solvents, from polar to nonpolar. Its disadvantage is that it is unstable in water-soluble alkaline mobile phase. In general, the recommended pH range for conventional analysis of silica based fillers is 2-8.
1. Normal phase chromatography
Silica and other polar functional groups, such as NH2, APS and CN, CPS, are usually used as stationary phases. Because of the strong polarity of SiOH or other polar groups on the surface of silica gel, the separation order is based on the polarity of each component in the sample, that is, the component with weak polarity is first washed the excellent column. The polarity of mobile phase used in normal phase chromatography is lower than that of stationary phase, such as hexane, chloroform, methylene chloride, etc.
2. Reversed phase chromatography
The packing materials used in reversed phase chromatography are usually silica gel, with relatively weak polar functional groups on the surface. The mobile phase used in RP-HPLC is a mixture of water, buffer, methanol and acetonitrile. The order of sample flow out of the column is that the component with stronger polarity is washed out first, while the component with weak polarity will have stronger retention on the column.
The commonly used reversed phase fillers are C18 (ODS), C8 (MOS), C4 (butyl), C6H5 (phenyl), etc.
Two
Polymer filler
Most of the polymer fillers are polystyrene divinylbenzene or polymethacrylate, which can be used in pH range from 1 to 14. Compared with the C18 packing, this kind of packing has strong hydrophobicity, and the macroporous polymer is very effective for the separation of protein and other samples. Its disadvantage is that the column efficiency is lower than that of silica gel.
Three
Other inorganic fillers
Other HPLC columns with inorganic packing have been commercialized. Due to their special properties, they are generally limited to special uses.
For example, graphitized carbon black is gradually becoming the packing of reverse chromatographic column. The separation of this kind of packing is different from that of alkyl bonded phase in silica gel matrix. The surface of graphitized carbon is the basis of retention and no other surface modification is needed.
The retention capacity of the column packing is generally stronger than that of the alkyl bonded silica gel or porous polymer packing. Graphitized carbon can be used for the separation of some geometric isomers, which can be used at any pH and temperature since it will not be dissolved in the HPLC mobile phase.
Alumina can also be used for HPLC. Alumina particles have strong rigidity and can be used in the mobile phase with pH up to 12. However, due to the strong interaction between alumina and basic compounds, the application scope is limited, so it can not be widely used.
The new Zirconia Matrix packing can also be used for HPLC. Only polymer coated porous zirconia microsphere chromatographic column was commercialized. The temperature could reach 100 ℃ with pH 1 ～ 14. Due to the fact that the research of zirconia fillers has only started in recent years, and the experimental difficulties are faced, its important uses and advantages are still in progress.
Selection of filler particle size
At present, the packing size of HPLC column manufacturers ranges from 1 um to more than 30 um, while at present, 3 um and 5 um packing are mainly used for analysis and separation.
The particle size of filler mainly affects two parameters of packed column, namely column efficiency and back pressure. The smaller the particle size is, the greater the column pressure is. The increase of column pressure limits the application of fillers with particle size less than 3 um.
Under the same selectivity conditions, increasing column efficiency can improve the resolution, but it is not the only factor. If the choice of stationary phase is correct, but the resolution is not enough, it is very useful to select a smaller size of filler.
The column efficiency of 3 um packed column was 30% higher than that of 5 um column under the same conditions, however, the back pressure of 3 um column was 2 times higher than that of 5 um column.
At the same time, the improvement of column efficiency means that shorter column can be selected under the same conditions, that is, the same number of plates or separation capacity, but shorter column length to shorten the analysis time.
In addition, low viscosity solvent can be used as mobile phase or increase the use temperature of chromatographic column, such as using acetonitrile instead of methanol to reduce the pressure of chromatographic column.
Use of liquid chromatography column
Before using the chromatographic column, it is better to test the performance of the column and keep the results as a reference for evaluating the performance changes of the column in the future. The column performance test should be carried out according to the conditions in the delivery report of chromatographic column (the condition used in the factory test is the best condition). Only in this way can the measured results be comparable. However, it should be noted that the performance of the column may be different due to the different conditions of sample, mobile phase and column temperature.
1. Sample pretreatment
It is better to use the mobile phase to dissolve the sample.
The pretreatment column was used to remove the impurities with strong polarity or irreversible adsorption with column filler.
A 0.45 μ m membrane was used to remove particulate impurities.
2. Preparation of mobile phase
Liquid chromatography is the separation of sample components by mass exchange between column packing and mobile phase
The flow relative to the sample has a certain solubility to ensure that the sample components will not precipitate in the column (or stay in the column for a long time).
The mobile phase does not react with the sample.
The viscosity of the mobile phase should be as small as possible in order to get a good separation effect, reduce the column pressure drop and prolong the service life of the pump (the viscosity of the mobile phase can be reduced by increasing the temperature).
The physicochemical properties of the mobile phase should be compatible with the detector used. If UV detector is used, it is better to use solvent with low UV absorption.
The boiling point of the mobile phase should not be too low, otherwise it is easy to produce bubbles, leading to the experiment can not be carried out.
After the mobile phase is prepared, degassing must be carried out. Removing the trace gas dissolved in the mobile phase is not only beneficial to the detection, but also can prevent the interaction between the trace oxygen in the mobile phase and the sample.
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