How to Extend the HPLC Column Service Time?
HPLC is a separation and analysis technique developed in the late 1960s and is an important tool for modern separation and determination. Since its introduction, it has been widely used in biochemistry, pharmaceutical and clinical analysis because of its high separation efficiency, fast analysis speed, good detection sensitivity and the ability to analyse thermally unstable physiologically active substances that have high boiling points but cannot be vaporised.
The chromatographic column consists of a column tube, pressure cap, ferrule (sealing ring), sieve plate (filter plate), joint, screws, etc.. The column tube is mostly made of stainless steel, when the pressure is not higher than 70 kg/cm2, thick-walled glass or quartz tube can also be used, the inner wall of the tube requires a very high degree of finish. In order to improve the column effect, reduce the wall effect, stainless steel column wall after polishing. There are also people in the stainless steel column wall coated with fluoroplastic to improve the inner wall finish, the effect is the same as polishing. There is also the use of fused silica or glass lining for fine tube columns.
The chromatographic column is the heart of liquid chromatography. In the use of liquid chromatography, it is very important to maintain the column efficiency, capacity and permeability to powder and to extend the life of the column. By taking the time to maintain your columns, you will save more time in dealing with column breakdowns and your columns will last longer. The service life of a column, apart from the sample and mobile phase analysed and the frequency of use, is mainly related to the daily maintenance.
Before HPLC column use
Because of the complexity of the organisms, the higher the degree of sample purification, the longer the life of the column and therefore the pre-treatment of the samples is very important. For example, the choice of solvent, sample filtration etc. We can choose pure water for chromatography as the solvent and use a solvent inlet filter to filter out impurities, particulates etc. This will make the sample more “pure” and will help to extend the life of the column.
Most of the commonly used HPLC columns are silica-based packed, and are divided into two categories: normal phase columns and reversed phase columns. When performing preparative HPLC, it is necessary to select columns that are as pure as possible and that allow a large number of compounds to be obtained simultaneously. Silica matrix filled preparative HPLC columns are widely used in synthetic chemistry, natural substance chemistry and other fields. Reversed-phase preparative HPLC columns use different chemically bonded phases to maintain peak patterns and achieve high purity refinement. In addition, the choice of eluent is important when performing preparative HPLC in relation to the size of the sample load, shorter column wash times, faster re-equilibration times, speed of solvent removal, etc.
HPLC columns can be prepared with internal diameters ranging from 6.0mm to 100mm, depending on the purpose of use and the set flow rate range of the device being used. If all conditions other than internal diameter, such as packing and column length, are the same, the flow rate and sample loading can be adjusted in proportion to the cross-sectional area of the column to give almost identical chromatograms.
There are other influencing factors to consider before using a column, such as certain properties of the analyte that can also affect the life of the column. Strong acids, strong bases and protein-based biomolecules, which can interact with the stationary phase packing or generate irreversible adsorption layers, change the surface characteristics of the packing and cause changes in the performance of the column, eventually leading to separation failure.
During HPLC column use
Avoiding mechanical shocks, which can cause damage to the column if the pump is started too quickly and the column bed is shocked by the instantaneous increase in flow rate and column pressure. Use the column chamber to keep the column temperature constant. Higher temperatures than the column can withstand will increase the adsorption of chemicals in the mobile phase and may also change the peak shape, reduce column efficiency and cause irreversible damage.
Change the operating flow rate and pressure gradually, never make abrupt changes. Excessive flow rates and pressure increases can cause the chromatographic packing to collapse and collapse. Operating within a suitable pH range, stationary phases using silica gel as a matrix generally require a pH range of 2.5 to 7. Prolonged use at pH > 7 or pH < 2 will result in the gradual dissolution of silica gel or the gradual loss of functional groups bonded to the surface. Avoid overloading the column by large volume injection and do not over-torque the column as this may cause damage.
Cleaning and storage after use
After a period of use, the column is often contaminated and the efficiency of the column decreases to a certain extent, so using appropriate methods to protect the column can extend the life of the column. Pay attention to the cleaning of the chromatographic column after use, rinse with a stronger mobile phase, if it still cannot be cleaned, it can be cleaned with a stronger eluent.
After cleaning, do not store the column in a refrigerator or hot air oven as this may cause irreversible damage to the stationary phase. Handle the column gently before and after use and take care not to knock or hit it hard enough to cause damage. Even after the recommended precautions have been taken, the performance of the column will be affected due to its limited life span. You should regularly monitor the performance of the column and take effective measures to observe decreases in peak shape, changes in retention time, poor separation or the appearance of unwanted peaks.
These are some of the considerations on how to extend the life of the column, but the above is one of the factors that can affect the life of the column. Understanding the column before use, reading the column manual, operating with care during use and having experienced people guide the use can all prevent damage to the column.