1、 Basic knowledge of HPLC column HPLC column is a kind of separation and analysis method, separation is the core, so the chromatographic column responsible for separation is the heart of the chromatographic system.
The requirements for the HPLC column are high column efficiency, good selectivity, and fast analysis speed. The particle size of various kinds of silica gel used in HPLC column, as well as the bonding phase, alumina, organic polymer microsphere (including ion exchange resin), porous carbon, etc., which are sold on the market, are generally 3, 5, 7, 10um, and the theoretical value of column efficiency can reach 50000-160000 / m.
For general analysis, only 5000 plates are needed for column efficiency; for homologous analysis, only 500 plates are needed; for more difficult separation materials, up to 20000 columns can be used, so the column length of about 10-30cm can meet the needs of complex mixture analysis.
Column efficiency is affected by internal and external factors. In order to achieve the best efficiency of the column, in addition to the small dead volume outside the column, a reasonable column structure (to reduce the dead volume outside the packed bed as much as possible) and filling technology are also required. Even if the best filling technology is used, the filling condition in the center of the column and along the pipe wall is always different.
The position close to the pipe wall is relatively loose, which is easy to produce ditch flow, fast flow rate, affect the flow pattern of the flushing agent, and widen the spectral band, which is the pipe wall effect. The thickness of the tube wall area is about 30 times the material diameter from the tube wall inward. In general liquid chromatography system, the effect of external effect on column effect is much greater than that of the tube wall effect.
1.HPLC column structure The chromatographic column is composed of a column tube, pressure cap, ferrule (sealing ring), sieve plate (filter), joint, screw, etc. The column pipe is mostly made of stainless steel. When the pressure is not higher than 70kg / cm2, thick wall glass or quartz pipe can also be used. The inner wall of the pipe requires a high degree of finish. In order to improve the column efficiency and reduce the pipe wall effect, the inner wall of the stainless steel column is mostly polished. Some people also apply fluoroplastics on the inner wall of the stainless steel column to improve the smoothness of the inner wall.
2. The effect is the same as that of polishing. The column joints at both ends of the chromatographic column are equipped with sieve plates, which are sintered stainless steel or titanium alloy with a pore diameter of 0.2-20um (5-10um), depending on the particle size of the packing. The purpose is to prevent the packing from leaking out. The inner diameter of the column is generally determined according to the length of the column, the particle size of the packing, and the reduced flow rate, so as to avoid the pipe wall effect.
3. The development direction of HPLC column Due to the emphasis on the analysis speed, the short column has been developed. The length of the column is 3-10cm, and the particle size of the filler is 2-3um. In order to improve the analytical sensitivity and connect with MS, a narrow-diameter column, capillary column, and micro diameter column with an inner diameter of less than 0.2mm were developed.
The advantages of small-diameter columns are ① saving mobile phase; ② increasing sensitivity; ③ less sample; ④ using the long column to achieve high resolution; ⑤ easy to control column temperature; ⑥ easy to realize LC-MS joint use.
However, as the volume of the column becomes smaller and smaller, the effect of out of column effect is more significant. It needs a smaller cell volume detector (even on-column detection), smaller dead volume column joints, and connecting parts. The matching equipment shall have the following performance: the infusion pump can accurately output a low flow rate of 1-100ul / min, and the injection valve can accurately and repeatedly inject small volume samples.
Because of the small amount of sample and the requirement of high sensitivity detector, electrochemical detection assist mass spectrometer has outstanding advantages in this respect. The performance of the HPLC column is not only related to the performance of the stationary phase but also related to the filling technology. Under normal conditions, when the particle size of the filler is more than 20um, the dry method is more suitable for the preparation of the column; when the particle size is less than 20um, the wet method is more ideal.
Generally, there are four filling methods: ① high-pressure homogenization method, which is mostly used for the filling of analytical columns and small-scale preparation columns; ② radial compression method, waters patent; ③ axial compression method, which is mainly used for filling large-diameter columns; ④ dry method, which is highly technical, most laboratories use filled commercial columns.
It must be pointed out that the filling technology is an important link in obtaining HPLC columns, but the fundamental problem lies in the performance of the packing itself and the reasonable structure of the matching chromatograph system.