Column Care and Maintenance
Maximizing HPLC column lifetime and performance is best achieved through a program of preventive care and maintenance. The requirements of such a program vary depending upon the specifics of the HPLC method and the nature of the samples being analyzed. Because more than 90% of analysts use reversed-phase chromatography, this section will focus primarily on this mode. An HPLC column that is performing well offers: 1 Satisfactory peak shapes 2 Satisfactory peak widths 3 Reproducible retention times 4 Reasonable operating back-pressure 5 Satisfactory resolution of analytes of interest 6 Stable detector baselines Deterioration in any of these parameters is indicative of a column problem. A reduction in column performance may also result from other system problems.
1 If an arrow is inscribed on the column, install the column so the flow direction matches the arrow. Most columns have a preferred direction of flow designed to avoid column problems and failures.
2 Column lifetime can be extended by filtering all eluents and samples. This should be done offline using a 0.5 μm chemically compatible membrane filter. In addition, an in-line filter between the solvent delivery system and the injector will trap debris from mobile phase components, pumps and mixing chamber, protecting both the injector and the column. A column inlet filter (Grace Vydac Cat. No. CPF10) will help to protect the column from particles generated by the injector or in samples. A high-performance guard column containing adsorbent similar to that in the column will remove insoluble and strongly adsorbed materials that may clog the column.
3 After installation, the column must be equilibrated with mobile phase. Ensure that the mobile phase is miscible with the storage solvent in the column. (Isopropyl alcohol is a convenient intermediary solvent.) Equilibrate the column by pumping a minimum of 10 column volumes of mobile phase through the column before making a sample injection. When using buffers, rinse the column with mobile phase without the buffer before instroducing the buffered mobile phase. This prevents buffer precipitation in the column or the HPLC system. Also when using buffers and ion pairing reagents, allow longer equilibration times.
4 Make sure that column end fittings, tubing nuts and ferrules are compatible. Connecting incompatible fittings can result in leaks, added dead volume and damage to column threads.
5 Do not overtighten end fittings. This can deform a ferrule and cause a leak or add system dead volume. Install fittings finger tight, then tighten using a wrench an additional 1/4 to 1/2 turn.
6 Avoid sharp blows or dropping the column. Physical shock can create voids and channels in a column that cause peak tailing and loss of efficiency.
7 Avoid abrupt changes in flow rate (pressure), eluent composition and column temperature. Such changes can physically or thermally disrupt the column bed, create channels or voids, and cause peak tailing and loss of efficiency.
8 Avoid operating the column at excessive back- pressure. This can create channels or voids. Check the column manufacturer’s specifications for maximum recommended pressure limits.
9 Before storing a column, thoroughly rinse out buffered mobile phases or ion pairing reagents with water followed by a water/organic solvent mixture. This will avoid adsorbent degradation or buffer precipitation in the column. Store the column in the shipping solvent, or use a water/acetonitrile mixture with at least 50% acetonitrile for a typical reversed-phase column. Make sure the storage solvent is miscible with the mobile phase solvent. Label the column with the storage solvent used, date stored, and the last method on which the column was used. Maintaining a log for each column in the laboratory is good practice.
10 Check the column end plugs to ensure they are fitted tightly to prevent solvent evaporation when the column is not being used. Some columns do not tolerate drying and may not perform satisfactorily even after re-wetting, although most standard HPLC columns do not suffer from this problem.
Mobile Phase Considerations
1 Use only high-quality HPLC-grade solvents free from impurities. Solvent impurities can negatively affect the resultant chromatography and irreversibly contaminate the column.
2 Use only mobile phase solvents that are compatible with the adsorbent. For silica based adsorbents, this typically means buffered solvents between pH 2.0 and 7.5. Some new-generation silica materials, such as GENESIS, are capable of working outside this range, and the C18 phases are stable from pH 1 to 10. Note that temperature, buffer type and concentration, and organic content all affect the pH stability range. If the analysis requires operating outside this range, consider using a specialized phase stable to extremes of pH. A pre-saturation column of the same phase as the analytical column may also be used. A pre-saturation column, located between the pump and injector, saturates the mobile phase with dissolved silica thereby protecting the analytical column from solvent degradation.
3 Filter all mobile phase solvents through a 0.5 μm or 0.2 μm filter prior to use. Aqueous buffers should be prepared or re-filtered daily to avoid column contamination by bacterial growth. Use a 0.5 μm inline filter between the injection valve and the column to trap any remaining particulates. Filtration prevents particulate build-up on the column frits and packing. Such build-up can increase column back-pressure.
4 When changing the mobile phase from buffer to a high organic concentration, use an intermediate aqueous flush solvent without buffers prior to conversion to avoid buffer salt precipitation. Buffer precipitates can destroy the column and damage the HPLC system. 5 Ion-pairing reagents such as tetra-butyl-ammonium chloride and octanesulfonic acid tend to adsorb permanently to reversed phase stationary phases, modifying their chromatographic behavior. Once these reagents have been used, it is recommended that the column be dedicated to ion-pairing separations only.
1 Filter all samples through a 0.5 μm or 0.2 μm filter prior to injection. When working with biological samples, interfering proteins should be removed by precipitation and filtration or centrifugation, or by alternative techniques such as solid phase extraction (SPE). Proper sample preparation techniques such as SPE will extend column life, provide better chromatography and increase assay sensitivity.
2 Place a guard column between the injector and the analytical column to remove particulates and prevent sample contaminants from reaching the analytical column. This extends column life and prevents impurity peaks.
3 The sample should be dissolved in a solvent that is miscible with the mobile phase, and preferably in a solvent that is weaker (less eluotropic) than the mobile phase.
Due to the nature of the reversed-phase surface, column performance (resolution, retention) may change slightly during the first few injections of highly retained compounds such as polypeptides. This so-called "column conditioning" occurs for most polypeptides larger than 10,000 molecular weight. A column can be conditioned by repeated injections of a polypeptide until column performance remains constant. This typically requires injection of about 100 μg of polypeptide for a 4.6 mm i.d.x 250 mm column. Alternatively, column conditioning can be accomplished by injecting a commonly available proetin such as ribonuclease followed by elution of the column with a typical acetonitrile gradient with 0.1% TFA.
Pressure and Temperature Limits
Grace Vydac silica-based HPLC columns are stable from 0-60°C and at pressures up to 5000 psi (335 bar). The upper pressure limit of the HPLC system used should be set to a maximum of 5000 psi (335 bar). Back-pressure of Grace Vydac silica-based reversed-phase columns under standard test conditions (50-70% acetonitrile in water) is initially in the range given below:
|Column Size (mm i.d. x mm)
||Particle Diameter (μm)
||Flow Rate (mL/min)
||Back Pressure Range (psi)
|2.1 x 250
|4.6 x 250
|4.6 x 150
|10 x 250
|4.6 x 250
|10 x 250
|22 x 250
VYDAC 259VHP polymer reversed-phase columns are stable from 0-80°C, but pressure-limited to 3000 psi (200 bar).
Each lot of Grace Vydac reversed-phase material is tested for selectivity with an appropriate set of standards. Individual columns are tested for efficiency. Test conditions and results are included with the column documentation. If you would like to test or verify column performance, we recommend repeating the selectivity or efficiency test according to the supplied documentation.
Grace Vydac reversed-phase columns are stable in most common organic solvents including acetonitrile, methanol, ethanol, isopropanol, dichloromethane and chloroform. When switching solvents it is important to verify that subsequent solvents are miscible with the previous solvent used. Follow the recommended pH range for the column you are using. Extremes of pH, especially values outside the recommended range, are likely to reduce column life.
Recommended Elution Conditions
Grace Vydac reversed-phase columns are typically eluted with an increasing gradient of organic solvent in the presence of an ion-pairing agent. The most common organic solvent used is acetonitrile due to its low viscosity, good UV transparency and high volatility. Methanol, ethanol and isopropanol are also occasionally used. Trifluoroacetic acid (TFA) is the most commonly used ion-pair reagent and is usually present at concentrations of 0.05 - 0.2% (w/v). However, VYDAC LC/MS grade columns produce excellent separations with TFA concentrations as low as 0.01% (w/v) or no TFA and one of the following ion-pairing reagents: acetic acid, formic acid, heptafluorobutyric acid (for basic polypeptides), triethylamine phosphate (TEAP), phosphoric acid. Typical flow rates for Grace Vydac reversed-phase columns are:
Detergents and Other Chemicals
Common protein detergents such as sodium dodecylsulfate (SDS) can be used without harm to columns and may be removed by rinsing the column with acetonitrile or isopropanol. However, detergents are likely to affect the resolution of the column during the run in which they are present. Oxidative eluents or sample additives should be avoided.