Stationary phase parameters: matrix type (such as silica gel or polymer); Jianhe phase-type (such as C18 or CN); particle size (such as 3 um, 5 um); specific surface area of the stationary phase (such as 130 m2 / g ); The pore size of the stationary phase [such as XX? (This unit reads ai)]; Uniformity (this can emphasize the quality of the adsorbent OK, but customers will not put forward specific parameters when purchasing), stationary phase purity (this can emphasize the quality OK, general customers will not ask for specific parameters when purchasing). The shape of the stationary phase particles (eg, irregular or circular, this point is not high, so customers may not write specific requirements when buying).
Because HPLC has the advantages of high speed, high separation effect, high sensitivity, etc., it has wider and wider applications. HPLC chromatographic column is an important part of the HPLC system. It needs to be used carefully when used, and it must be maintained after use to avoid problems in the next use. Frequent replacement of the chromatographic column is very expensive, so in order to save costs, it is necessary to extend the service life of the chromatographic column to a greater extent. At the same time, the chromatographic column must be carefully maintained to maintain accuracy and consistency every time the result is obtained.
HPLC columns are prone to blockage and damage, so some laboratories choose to use a guard column before the HPLC column to extend the service life of the HPLC column. Guard columns generally include ferrule + core + peek tube + peek connector.
HAWACH editor has summarized 10 misunderstandings about the columns hoping it would extend its service life.
Error 1: HPLC column cannot be recoil
In general, the pressure value of the liquid chromatography column is much higher than the maximum operating pressure (about 2 times). Therefore, for a stable packed bed, if a suitable mobile phase and a reasonable time distribution are used, a well-filled column can bow from left to right. In turn, the reasons for using the HPLC column are as follows: recoil when changing the column, clean the substances strongly adsorbed on the column head, and wash out the residual substances to prevent the pressure from increasing.
Error 2: All C18 (L1) columns are the same
In the early HPLC system, C18 was the only binding stationary phase of reverse chromatography, so C18 was used as the standard of reverse chromatography column and many pioneers were willing to use it. Because HPLC was first used in the pharmaceutical industry, and the management organization was not willing to make new designs. FDA and USP also provide the classification of analysis methods designed when submitting new drug applications. The HPLC column given C18 is classified as “L” because most of the C18 columns are used as the method columns for submitting new drugs, so C18 becomes L1, which is the first standard column. But more fixed phases appear, named after the new L value. (L7 = C8, L10 cyano, L11 phenyl).
Error 3: Protecting the column does not affect the separation effect
If the selection of the stationary phase is wrong, the protective column has an effect on separation, but the protective column is used to protect the analytical column from being polluted by high residual components. Therefore, if the retention of the stationary phase is strong (such as high carbon load and mixed-phase), it can make the retention cheap and affect separation, even lead to different selectivity. If the retention is weak, the problem is not so obvious unless the stationary phase affects the selectivity of the whole system.
Error 4: HPLC column cannot be backflushed
Under normal circumstances, the designed pressure resistance value of the liquid chromatographic column is much higher than the maximum operating pressure. When replacing the column, backflushing can clean some substances with strong adsorption properties at the head of the column, and we flush out these residual substances to prevent pressure increase. But if the chromatographic column you buy uses a large particle size packing at the column head, this part of the packing will be flushed out when you backflush. Therefore, you have to check the liquid chromatography column you are using before choosing whether to backflush.
Error 5: All C18 columns are the same
In the early HPLC system, C18 was the bonded stationary phase for standard reverse chromatography, so C18 was used as the standard for reverse chromatography columns. However, the times will not remain stagnant. People’s pursuit and understanding of science has become more and more profound, more stationary phases have appeared, and many C18 chromatographic columns have been born. Although silica gel is also used as the matrix, each has its own specific filler bonding synthesis process, so the chromatographic performance is different. So simply because the names are all C18 columns, it is wrong to think that the performance is the same.
However, the times will not remain stagnant. People’s pursuit and understanding of science has become more and more profound, more stationary phases have appeared, and many C18 chromatographic columns have been born. Although silica gel is also used as the matrix, each has its own specific filler bonding synthesis process, so the chromatographic performance is different. So simply because the names are all C18 columns, it is wrong to think that the performance is the same.
Error 6: Once air enters the chromatographic column, it will be damaged
We all know that when the column is not connected to the chromatograph, it is necessary to ensure that the column is tightly sealed. However, in practical applications, even if a small amount of air enters the end of the chromatographic column, it is not a serious matter. When the chromatographic column is connected to the chromatograph for use, the air will be squeezed out by the solvent in a short period of time during the initial pressurization of the system. Therefore, do not assume that the column is damaged just because a small amount of air has entered the column.
Error 7: The guard column is unnecessary
In fact, there are many advantages to using a guard column. Various reagents are often used in the mobile phase of HPLC, and some insoluble or impure substances are mixed in it. Most insolubles can be removed by filtering the mobile phase with a sieve or membrane filter, but very fine particles cannot be completely filtered. In addition, after the mobile phase is modulated, there is a possibility that insoluble and impure materials may fall through the air, container, human body, etc. These insoluble and impure substances can contaminate the column, block the column, or have an adverse effect on the analysis.
At this time, by installing an analytical guard column in front of the chromatographic column, it can prevent its generation and protect the chromatographic column. Compared to replacing an expensive analytical column, adding or replacing a guard column requires very little expense. The HAWACH liquid chromatography guard column has almost no dead volume and is easy to replace. It is suitable for the high pressure of the HPLC system and intercepts those strongly retained and non-adsorbable compounds. The filtration of sample and mobile phase can maintain the adsorption capacity of the guard column for chemical contaminants, maintain column efficiency for a longer time, and extend the service life of the chromatographic column.
Error 8: Reversed-phase chromatographic column cannot use pure water phase
Some chromatographers experience phase collapse when using low organic solvent content or pure water as the mobile phase of reversed-phase chromatography columns. Therefore, some people think that reversed-phase chromatography columns cannot use pure water. But in fact, the reversed-phase chromatography columns sold on the market (such as polar embedding and polar capped columns) are all water-wettable, and their surface characteristics allow the use of pure water, which will not cause collapse or retention time shift.
Error 9: The smaller the particle size and the higher the pressure of the column packing, the better the separation effect
The performance of a chromatographic column cannot be evaluated solely by whether the filler is ultra-small particle size and ultra-high column pressure. Modern researches on the column characteristics of chromatographic columns have become more and more in-depth, and some methods have been developed to improve the efficiency of chromatographic columns. For example, the column efficiency of the new surface porous material column is as good as that of the sub-2µm UHPLC column. It’s column pressure is very low, compared with the conventional column.
Error 10: Column pressure will not affect the chromatographic separation effect
In recent years, the problem of column pressure has attracted the attention of many chromatographers. Many parameters of chromatogram are affected by column pressure, including the molar volume of some solutes, stagnation volume, column porosity, retention factor and mobile phase etc. Our attetnion will not be attracted, if operating the column at a pressure of 13.789MP, even with small difference in retention time. However, when the column pressure is close to 2000psi (13.789MPa), the effect of column pressure may be quite obvious.
Conclusion In recent decades, high performance liquid chromatography has been widely used in the fields of life sciences, drug analysis, food analysis, environmental monitoring and other fields. People’s continuous research on liquid chromatography has also produced more liquid chromatography applications, solutions and methods, etc. It is believed that the application field of liquid chromatography will be broader in the future.