Classification of liquid chromatography

Basic knowledge of chromatographic column
Chromatography is a kind of separation and analysis method, and separation is the core. Therefore, the chromatographic column responsible for separation is the heart of the chromatographic system. The requirements of chromatographic column are high column efficiency, good selectivity and fast analysis speed. Various kinds of microparticle fillers, porous silica gel and silica gel based bonding phase, alumina, organic polymer microspheres (including ion exchange resin) and porous carbon are commercially available for HPLC. Their particle sizes are generally 3, 5, 7, 10 um, and the theoretical column efficiency can reach 50-160 000 / m. For general analysis, the column efficiency of 5000 trays is only needed; for homologous analysis, it is only 500; for difficult separation material pairs, up to 20000 columns can be used. Therefore, the column length of about 10-30cm can meet the needs of complex mixture analysis.
The column efficiency is affected by the factors inside and outside the column. In order to achieve the best efficiency of the chromatographic column, in addition to the small dead volume outside the column, reasonable column structure (as far as possible to reduce the dead volume outside the packed bed) and filling technology should be adopted. Even with the best filling technology, the filling situation in the center of the column and along the pipe wall is always different. The position close to the pipe wall is relatively loose and easy to produce ditch flow. The flow rate is fast, which affects the manifold of flushing agent and widens the spectrum band. This is the pipe wall effect. The thickness of the wall area is about 30 times the material diameter from the pipe wall inward. In general liquid chromatography system, the effect of external column effect on column efficiency is much greater than that of tube wall effect.
1. Column structure
The chromatographic column is composed of column tube, pressure cap, ferrule (sealing ring), sieve plate (filter), joint, screw, etc. The column tube is mostly made of stainless steel. When the pressure is not higher than 70kg / cm2, thick wall glass or quartz tube can also be used. The inner wall of the tube requires a high finish. In order to improve the column efficiency and reduce the tube wall effect, the inner wall of stainless steel column is usually polished. Some people have applied fluoroplastics on the inner walls of stainless steel columns to improve the smoothness of the inner walls. The effect is the same as that of polishing. There are also fused silicon or glass lining used for thin tubular columns. A sieve plate is installed in the column joint at both ends of the chromatographic column, which is sintered stainless steel or titanium alloy, with a pore diameter of 0.2-20um (5-10um), depending on the particle size of the packing, so as to prevent the leakage of the packing. The inner diameter of the column is generally determined according to the column length, packing particle size and reduced flow rate, so as to avoid the pipe wall effect.
2. Development direction of column
A short column with length of 3-10 cm and packing particle size of 2-3 um was developed due to the emphasis on analysis speed. In order to improve the analytical sensitivity and connect with mass spectrometry (MS), narrow diameter column, capillary column and microbore with inner diameter less than 0.2mm have been developed. The advantages of thin tube diameter column are: 1) saving mobile phase; 2) increasing sensitivity; 3) less sample quantity; 4) using long column to achieve high resolution; 5) easy to control column temperature; 6) easy to realize LC-MS combination.
However, as the volume of the column becomes smaller and smaller, the effect of the out of column effect becomes more significant. It requires a detector with smaller cell volume (or even detection on the column), and a smaller dead volume column joint and connecting parts. The matching equipment should have the following performance: the infusion pump can precisely output low flow rate of 1 ~ 100ul / min, and the injection valve can accurately and repeatedly inject small volume samples. Because of the small injection volume, high sensitivity detector is required. Electrochemical detection band mass spectrometer has outstanding advantages in this respect.
3. Column filling
The performance of chromatographic column is not only related to the performance of stationary phase, but also related to the filling technology. Under normal conditions, when the particle size of filler is more than 20um, the dry method is more suitable; when the particle size is less than 20um, the wet filling is more ideal. There are generally four filling methods: ① high pressure homogenization method, mostly used for filling analysis column and small-scale preparation column; ② radial pressure method, waters patent; ③ axial pressure method, mainly used for filling large diameter column; ④ dry method, column filling technology is strong, most laboratories use filled commercial column.
It must be pointed out that the filling technology is an important link in the acquisition of high performance liquid chromatography column, but the fundamental problem lies in the performance of the packing itself and whether the structure of the matching chromatograph system is reasonable.
2、 Specification of liquid chromatography column
The chromatographic column can be divided into analytical column and preparative column
① Conventional analytical column (constant column), inner diameter 2 ~ 5mm, column length 10 ~ 30cm;
② Narrow diameter column (arrowbore) with inner diameter of 1 ~ 2mm and column length of 10 ~ 20cm;
③ Capillary column (also known as microcolumn), inner diameter 0.2 ~ 0.5mm;
④ Semi preparative column, inner diameter > 5mm;
⑤ The internal diameter of the column was 20 ~ 40mm and the length was 10 ~ 30cm;
⑥ The inner diameter of the column can reach tens of centimeters.
Understanding of chromatographic column
1) The smaller the particle size (DP), the higher the column efficiency (good mass transfer and small eddy diffusion), the higher the column pressure (poor permeability) and particle distribution
The wider the particle distribution, the lower the column efficiency (poor permeability)
Particle shape: high column efficiency, good reproducibility and uniform column bed structure
Amorphous: the structure of the column bed is not uniform, the linear velocity of the mobile phase is not uniform, and the spectrum band is extended
2) Bond phase chemistry
1. Affect the resolution of compounds
2. The separation of different compounds with different bonding is different
3. The separation mechanism may be different, such as C18, C8, CN
3) Carbon content
1. The higher the carbon content, the greater the K 'value (mass transfer effect of stationary phase increases)
2. High carbon content is conducive to the separation and hydrolysis stability of the compounds that are not easy to be retained, good reproducibility and tailing improvement of polar compounds
3. Low carbon content is beneficial to analyze neutral and basic compounds, reduce solvent loss, seal the end group of filler, seal residual silicon hydroxyl, reduce irreversible adsorption or tailing, and increase carbon content (0.1% - 1%)
4) End base sealing of packing
Sealing residual silicon hydroxyl, reducing irreversible adsorption or tailing, increasing carbon content (0.1% - 1%)
5) Activity of silica gel
The main factors affecting the retention behavior of basic compounds: K ', when producing silica gel, the activity of silica gel is also different, which is the main source of selectivity difference. The impurity content and heavy metal content of silica gel are low, the activity of silica hydroxyl is small, and the tailing is reduced. It is an important sign of the quality of chromatographic column. The stability of packing, silica gel packing, pH: 2-8, polymer packing, pH: 2-12, pH When the value is less than 2, the bonding phase is hydrolyzed. Whether a sample can be completely separated by liquid chromatography mainly depends on the selectivity and efficiency of the chromatographic column, which largely depends on the selection of the stationary phase.
6) Specifications of chromatographic column: inner diameter, detection sensitivity and sample capacity
Length, resolution, theoretical plate number, speed, sample capacity and detection sensitivity
7) Column performance evaluation: the purchased chromatographic column should be inspected before use, and it should be rechecked during use or after a period of storage. Column performance indicators include column pressure, theoretical tray height and plate number, repeatability of symmetry factor, capacity factor and selectivity factor, or resolution under certain experimental conditions (sample, mobile phase, flow rate, temperature). Generally speaking, the repeatability of capacity factor and selectivity factor is within ± 5% or ± 10%. When comparing the column effect, we should pay attention to whether there is any change in the external effect.
A qualified evaluation report of chromatographic column should give the basic parameters of the column, such as column length, inner diameter, packing type, particle size, column efficiency, asymmetry and column pressure drop.
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