In high-performance liquid chromatography (HPLC) systems, the column is one of the core components. Proper use of the column ensures that the analysis is carried out efficiently and sensitively and plays an important role in extending the column’s life and maintaining long-term stable working conditions. As chromatographic columns are expendable components with a certain lifetime, they are expensive. For example, a chiral analytical column usually costs several thousand or even tens of thousands. Therefore, extending the column’s life is vital to reducing costs. So how do you ensure the longevity of a column by using it in a standard way?
PART 01 Preparation before use
1, read the column manual carefully before use. To understand the type of column, choose the appropriate column.
When choosing a column, the polarity of the sample to be analyzed, the number of compounds, and their structural characteristics should be fully considered. Choose a suitable column and analytical conditions according to the nature of the compound. Different columns use different mobile phases, and using the wrong mobile phase can reduce column efficiency and damage the column. For example, a hydrophilic reversed-phase packing should be used to analyze polar polysaccharide components. The column’s first use should also follow the manufacturer’s factory instructions on the column for low flow rate rinse activation, chromatographic packing covalent bonding force, column efficiency, and life extension.
2, sample preparation and pretreatment
Our experience is that the cleaner the sample is purified, the longer the column’s life. Many samples, especially biological samples, are very complex and damaging to the column, and direct analysis of samples without pretreatment can seriously shorten the column’s life. Therefore, samples must be pre-treated during sample preparation, including selecting preparation solvents and sample filtration.
2.1 Selection of sample preparation solvents
The solubility of the sample, its compatibility with the mobile phase, and the suitability of the chromatographic packing material are all aspects that need to be considered. Such solvents need more soluble in the sample than in the mobile phase. The elution strength should be lower than that of the mobile phase or the starting mobile phase in gradient elution to avoid affecting the sample separation. Many chiral columns currently prohibit the use of DMSO, tetrahydrofuran, and chloroform to dissolve samples; these solvents can destroy the structure of the stationary phase and thus shorten the column’s life. In addition, the sample preparation solvent should also be compatible with other components of the chromatography system, such as high-pressure pumps and injectors.
2.2 Sample preparation solvent filtration
The sample solution needs to be filtered before feeding, e.g. using a 0.22 μm microporous filter membrane to remove insoluble particles so as not to block the column cap frit and the packed bed inside the column. Where conditions permit, it is preferable to filter the feed solution using a solid phase extraction column with the same packing as the column, reducing the amount of dead adsorbed material on the column or large molecules that can easily clog the sample. For example, small polar oils and fats in biological samples are easily precipitated and dead-sorbed in the C18 reversed-phase column, resulting in lower column efficiency and higher column pressure. SPE column filtration can effectively reduce the dead adsorbed components deposited in the column, protect it from contamination and ensure its service life.
2.3 Others, such as solution concentration, injection volume, etc.
Certain properties of the analyte 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, ultimately leading to separation failure. In addition, excessive sample feeds and overloads can affect the performance and lifetime of the column.
PART02 Maintenance during use
1. Use of mobile phase and choice of analytical method
The purity of the mobile phase, the choice of solvent, and the use of appropriate analytical methods are closely related to the performance and lifetime of the column.
1.1 Selection of mobile phase
The mobile phase chosen is compatible with the column, and the sample to be analyzed, i.e. the sample, sample solution, and mobile phase are mutually soluble. The mobile phase is capable of dissolving the sample and avoiding the precipitation of the sample; it is also required that the mobile phase does not react chemically with the sample and cannot dissolve or react chemically with the column.
For chromatographic analysis, a chromatographic-grade mobile phase should be selected. Often analytically pure solvents contain traces of impurities, such as polyethylene glycol in organic solvents and inorganic iron ions (Fe+), which can cause changes in column performance when used in large quantities as a mobile phase. It is best to use chromatographically pure grade or higher purity reagents to minimize the damage caused by impurities in the solvent.
1.2 Mobile phase filtration
The mobile phase should be filtered through a 0.45 μm or smaller pore-size membrane and degassed by ultrasound before use to reduce blockage of the column by dust, microorganisms, and other impurities, especially water-soluble mobile phases that can cause microbial growth and blockage of the column. The mobile phase should be ready for use and not be left for more than 2 days.
1.3 Selection of mobile phase pH and buffer salts
The extreme pH of the mobile phase can break the covalent bonds within the packing, “dissolve” the silica gel and cause loss of stationary phase, thus reducing the efficiency of the column and shortening its life. Stationary phases using silica as a matrix generally require a pH range of 2.5-7. When used at pH >7 or pH <2 for long periods, the silica gel will gradually dissolve, or the functional groups bonded to the surface will gradually be lost. If a mobile phase with a high or low pH must be used, it is best to use a compatible chromatographic packing.
1.4 Control of flow rate
The current particle size of 1.8μm UPLC flow rate standing for 0.3~0.5mL/min, the particle size of 5μm HPLC analysis flow rate is not more than 1.5mL/min, the particle size of 10μm semi-preparative column flow rate control in 3mL/min. The flow rate is too large, and the pressure rises, which will cause the chromatographic packing to wash down, and collapse.
2, the operation of chromatographic instruments
Every time you start using the analytical instrument, the pump starts too fast, and the flow rate and the rapid rise in column pressure, the column bed, is impacted, causing turbulence, generating voids and affecting the service life of the chromatographic column. Therefore, at the beginning of the operation experiment, the flow rate and column pressure should be gradually increased.
3, the use of a guard column
The “guard column” is the same packing as a liquid chromatography column short column, which can effectively block damaged columns of large molecules and insoluble particles easily, filtering easy-to-precipitate chromatography column dead adsorption of substances and extending the service life of the column.
4, the control of the column temperature
Different types of chromatographic column temperature tolerance vary. Usually, chromatographic column temperature is maintained between 10 ~ 40 ℃, which can fully and optimally play the performance of the column. Beyond the temperature range of the column, especially above the range of column temperature, will increase the adsorption of chemicals in the mobile phase, causing changes in the stationary phase structure of the column; in addition, it may also cause the collapse of the column bed, change the peak shape, reduce the column efficiency, irreversible damage.
PART03 Cleaning and preservation after use
After the column has been used for some time, there will always be some impurities accumulated in the column. The weaker retention value of the material generally can be quickly flushed out from the column, without interference; medium retention strength of impurities can be slowly flushed out but produce some interference to the analysis; strong retention impurities usually gathered in the column head or column, difficult to be eluted, and may even interact with the packing material, the formation of a new pseudo stationary phase, The separation performance of the column is altered. This usually manifests as increased column pressure, uneven baseline, double peaks, and reduced separation performance. These contaminated columns can recover most of their separation capacity after cleaning. Therefore careful and regular cleaning after use will not only extend the column’s life and save resources but also greatly reduce the analysis cost. To our commonly used silica matrix chromatographic column as an example, briefly explain the common column cleaning and regeneration.
1, Column cleaning and regeneration
Chromatographic column before and after the use of a strong mobile phase rinse. Usually, in the use of silica, alumina, and polar bonded phase chromatography column, each time after the use of dichloromethane or hexane and other solvents can be used for a long time with a low flow rate rinse; bonded reversed-phase silica column, ion exchange column and gel chromatography column can first use a high proportion of water (methanol-water mixed solvent) rinse, and then 100% methanol rinse. In addition, reversed-phase rinsing of the column at low flow rates effectively removes impurities clogging the column head or sieve plate. It cleans stronger adsorbed material collected in the column header area. Some columns are used in reverse after many methods have failed to treat the contamination. The column pressure drop becomes smaller, and the column efficiency can be restored, extending the column’s life.
Suppose the above conventional cleaning method can not remove contaminants. In that case, it is necessary to use a stronger eluent cleaning, such as the reverse-phase material rinsing sequence: 100% methanol → 100% acetonitrile → acetonitrile: isopropanol (75:25, V/V) → 100% isopropanol. Alternatively, a lower concentration of dilute acid or dilute base can remove contaminants that cannot be eluted by organic solvents. For example, a 0.05 mol/L solution of sulphuric acid and mobile phase can rinse the column with good results, or a 1% aqueous solution of ammonium hydroxide or 50% dimethylformamide can be used to provide good cleaning of contaminants that have collected at the column head.
If the mobile phase contains a buffer (usually a salt solution), it is advisable to rinse the column with water instead of the buffer mixed with the organic phase (20 times the column volume); then rinse with 100% organic solvent. A direct rinse with 100% organic solvent can cause buffer deposits to precipitate, which can damage the quality of the column. Similarly, if acid or base solutions are added to the mobile phase, a high ratio of water (water: methanol 10:90) should be used to rinse 20 times the column volume first, as described above, to prevent strong acid and base solutions from causing dissolution of the silica matrix packing.
Contamination of reversed-phase columns by proteins has become a common problem, especially in separating biological samples such as untreated animal tissues. Pure organic solvents such as acetonitrile or methanol do not clean the column very effectively, thus requiring some special cleaning methods. First, try washing with a mobile phase with a high proportion of a strongly polar solvent, e.g. acetonitrile: isopropanol (1:2, V/V); or use 0.1% aqueous trifluoroacetic acid or 0.1% aqueous acetic acid. Alternatively, a 1% sodium dodecyl sulphate (SDS) followed by a 5% to 95% acetonitrile/water (with 0.1% TFA) gradient rinse can be used to remove protein contaminants more effectively.
Suppose the column does not achieve the desired results after cleaning using the above conditions. In that case, it is necessary to remove the stationary phase from the column, clean and regenerate it and then reload it. The stationary phase is removed from the column by flotation with methanol to remove the fine broken particles, followed by ultrasonic cleaning with dimethylformamide, acetone, and methanol, and finally drying the stationary phase reloading the column. The performance of the column treated by this method can be significantly improved.
2, the preservation of chromatographic column
The chromatographic column should be stored in 100% organic solvent as far as possible, following the solvent specified in the instruction manual for filling. The chromatographic column can not be stored in water, or the high water content of the solvent will cause microbial growth, affecting the column’s life. If the reversed-phase column is not used for a long time, it is best to store it in an aqueous mixture of 90% to 95% organic solvent to prevent the column from drying out at both ends due to poor sealing and shortening the life of the column due to fracture.
In addition, the column should also be handled gently to avoid collapse and fracture of the column packing caused by violent collision, shortening the service life.
Post time: Dec-29-2022
