9 Mistakes You May Make When Using the HPLC Columns

HPLC columns are an important part of the HPLC system and are being used more and more because of their speed, separation, and sensitivity. They need to be used with care and must be maintained after use to avoid problems with the next use. Frequent replacement of chromatographic columns is very expensive, so in order to save costs, the life of the column needs to be extended to a greater extent. At the same time, the column must be carefully maintained to maintain accuracy and consistency each time results are obtained. HAWACH has summarised 7 misconceptions about column use that will hopefully extend the life of the column.

Mistake 1: HPLC columns should not be back blown
In general, liquid chromatography columns are designed to withstand pressures well above the maximum working pressure. When replacing the column, back blowing can remove some adsorbed substances from the column head, and we flush out these residuals to prevent the pressure from rising. However, if you buy a column that uses a large particle-size packing in the column head, this packing will be flushed out during back-blowing. It is therefore important that you check the liquid chromatography column you are using before choosing whether or not to back blow.

Mistake 2: All C18 columns are the same
In the early days of HPLC systems, C18 was the bonded stationary phase for standard reversed-phase chromatography and therefore C18 was used as the standard for reversed-phase columns. However, times do not stand still. The pursuit and understanding of science became more sophisticated, more stationary phases emerged and many C18 HPLC columns were born. Although silica gel is also used as a matrix, each has its own specific filler bonding synthesis process and therefore different chromatographic performance. So just because the names are all C18 columns, it is wrong to assume that the performance is the same.

Mistake 3: Once air enters the HPLC column, it will be damaged
We all know that when the column is not connected to the chromatograph, it is important to ensure that the column is sealed tightly. However, in practice, even if a small amount of air enters the end of the column, it is not a major problem. When a column is used in connection with a chromatograph, the air is forced out by the solvent for a short time during the initial pressurization of the system. Therefore, do not assume that the column is damaged simply because a small amount of air has entered the column.

Mistake 4: Protective columns are not needed
HPLC often uses a variety of reagents in the mobile phase, with some insoluble or impure substances mixed in. Most insoluble material can be removed by filtering the mobile phase through a sieve or membrane filter, but not “filtering” very fine particles. In addition, after the mobile phase has been modulated, insoluble and impure substances may fall through air, containers, human bodies, etc. These insoluble and impure substances can contaminate the column, clog it, or have a poor effect on it. Effect on analysis.

In this case, by installing an analytical protection column in front of the column, they can be prevented and the column protected. Adding or replacing a protective column costs very little compared to replacing an expensive analytical column. Liquid chromatography guard columns have virtually no dead volume and are easy to replace. Suitable for high pressures in UHPLC systems, trapping compounds that are strongly retained and not adsorbable. Filtration of the sample and mobile phase maintains the ability of the guard column to adsorb chemical contaminants, maintaining column efficiency for longer and extending the life of the column.

Mistake 5: Reversed phase columns cannot be used with pure aqueous phases
Some chromatographers experience phase collapse when using low organic solvent content or pure water as the mobile phase for reversed-phase columns. As a result, some people believe that pure water cannot be used in reversed-phase columns. In reality, however, commercially available reversed-phase columns (such as polar-embedded and polar-capped columns) are hydrophilic and have surface properties that allow the use of pure water without causing collapse or retention time shifts.

Mistake 6: The smaller the particle size and the higher the column packing pressure, the better the separation
The performance of chromatographic columns cannot be evaluated simply by whether the packing is ultra-small in particle size and ultra-high in column pressure. Modern research into column properties is becoming more and more advanced and a number of methods have been developed to improve the efficiency of chromatographic columns. For example, the column efficiency of new surface porous material columns is very low compared to conventional columns, which have very low column pressures.

Mistake 7: Column pressure does not affect chromatographic separation
In recent years, the issue of HPLC column pressure has attracted the attention of many chromatographers. Many parameters of a chromatogram are affected by column pressure, including the molar volume of some solutes, retention volume, column porosity, retention factor, and mobile phase. If the column is operated at a pressure of 13.789 MP, even small differences in retention time are possible. However, as the column pressure approaches 2000 psi (13.789 MPa), the effect of column pressure may be more pronounced.

Mistake 8: Surface porous particles significantly reduce sample capacity compared to pure porous particles
The capacity of an HPLC column packing for a sample is proportional to its surface area, which is related to the amount of chemically bonded phase formed by the silanol group through monomer bonding. However, the results of the study show that the capacity of the sample is essentially the same for surface porous particles as for porous particles under the same test conditions.

Mistake 9: HPLC columns should not be reversed in order to flush out the particulate matter
In reality, HPLC columns are filled at much higher pressures than the maximum operating pressure (usually 2 times higher). A well-filled column can be used in both directions if the column is loaded with the appropriate homogenizer and the column bed is allowed time to stabilize.