To have a deeper understanding of HPLC column
Column structure: plastic protection head of the column; stud screw; blade ring; seal ring; filter (sieve plate); column (stainless steel tube) and column packing.
The sieve plate of the column is usually made of stainless steel or titanium alloy. Its pore diameter is generally between 0.2-20μm. The pore size depends mainly on the particle size of the column packing. It is a very important part and it can prevent the packing from leaking out. At the same time, it is to prevent the impurities from entering the column during operation to reduce the separation effect, and even the column is easily blocked.
The connection between the column and the infusion tube generally requires the use of a stainless steel ferrule and stainless steel fastening the joint screw. They are also very important, otherwise, the column system will leak, resulting in a drop in column pressure, a mobile phase or a sample leaking, which affects the analysis of test results, even the analysis, and testing work cannot be done.
HPLC column classification
In terms of type, the column can be divided into two types: preparative type and analytical type. Generally, the analytical type column is used most. If subdivided, the column can be further divided into a conventional column, a narrow column, a capillary column, a preparative column, and a semi-preparative column.
In order to improve the speed of analysis, short columns are often used, the column length is 5-10 cm, and the particle size of the filler is about 3 μm. Many analysts have emphasized analytical sensitivity, so narrow-diameter columns, capillary columns, and micro-diameter columns have been developed.
HPLC column efficiency
Column efficiency is the most important key indicator and generally depends on the performance of the stationary phase and the packing technology. There are roughly three types of column stationary phases (packings) for general HPLC: silica gel or silica-based fillers, polymer fillers, and inorganic fillers.
Most of the normal phase chromatography columns use silica gel fillers, while the reverse phase chromatography columns mostly use silica gel as a matrix functional packing. The most outstanding feature of the polymer-based column is that the pH can be used between 1 and 14 and the hydrophobicity is strong. However, inorganic packing columns are generally limited to special applications and are less used.
Because the HPLC samples are all solutions, and the solution is greatly affected by temperature, for example, the solubility of the solvent on the solvent, the efficiency of the column, and the viscosity of the mobile phase all have an effect.
If the temperature rises, the solubility of the solution in the mobile phase may be increased, and the partition coefficient K may be lowered; lowering the viscosity of the mobile phase may lower the column pressure, which is beneficial to protecting the column and prolonging the life of the HPLC column. However, the temperature should not be too high, otherwise, bubbles will form.
HPLC workers must also pay attention to different temperatures, which will have an impact on the retention time of the column, the separation effect, and the sensitivity of the detector, especially the sensitivity and minimum detection limit of the refractive index refractometer. Therefore, the design of constant temperature systems is the issue that HPLC designers and users should pay attention to.
In addition to the structure of the column, it is related to the performance of the stationary phase and to the packing and filling technology during column packing. Some users install their own columns according to their requirements. Most users use the already installed commercial column. Regardless of the type of column, it must be carefully examined before use; it should be re-examined and tested during use or after a period of time.