High-Performance Liquid Chromatography (HPLC) is a cornerstone in analytical laboratories, empowering scientists to separate and analyze complex mixtures with precision. The HPLC column, a critical component, is subject to wear and tear over time, impacting the quality of results. In this blog, we will explore five proven techniques to extend the lifetime of your HPLC columns, ensuring sustained efficiency and reliability in your chromatographic analyses.
It is necessary to read the manufacturer’s literature regarding recommended pressure, eluent pH, and temperature operating ranges for the column and adhere to these ranges
Note that higher operating temperatures often go hand in hand with reduced pH operating ranges. At low pH, the main symptom of column degradation is usually loss of column efficiency (peak broadening), whereas at high pH, peak tailing and increased column back pressure occur.
Each time you start the eluent, avoid mechanical shock to the column bed (dropping the column) and slowly increase the pressure and flow rate (1mL/min/min is ideal)
Most modern HPLC equipment is capable of automatically achieving this flow-pressure ramp through secondary instrument settings. Bed voiding due to pressure shock often manifests itself through splitting or very severe tailing peaks. If alternatives are not readily available, the column can be inverted for analysis; however, the efficiency of the column may decrease more quickly because the bed will eventually collapse in the opposite direction, causing the same chromatographic symptoms.
If the column dries out, start the flow very slowly (0.1mL/min/min) using an eluent containing at least 50% acetonitrile
If a “standard” (non-“aqueous” or non-polar embedded phase type) reversed-phase column is suspected of experiencing phase collapse (shorter retention time, inefficiency) due to the use of a 100% aqueous mobile phase, it should be reactivated at high flow rates Column 100 % acetonitrile at 60 °C (be careful not to precipitate any solid buffer from the remaining eluate in the column). In both cases, 50 to 100 column volumes may be required to properly reequilibrate or reactivate the phase.
1. Current content is 90% acetonitrile, 10% organics every 2 column volumes, and maintain for 10 column volumes (again be careful to avoid solid buffer precipitation by slowly increasing the acetonitrile concentration, as recommended here).
2. Add 10% acetonitrile to 50:50 acetonitrile-water every 2 column volumes and maintain for 10 column volumes.
3. Remove the end cap from the system and store it. Column endcapping is important to avoid phase transition to dryness.
One may use older HPLC systems as column “wash stations” which can save significant operating time on “real time” instruments.
If the sample is likely to contain particulates, choose a good quality in-line filter with appropriate mesh size; 0.45μm for conventional columns and 0.2μm for U HPLC columns are typical
If the sample matrix or diluent may damage the sorbent (e.g., due to pH) or the chemistry is particularly dirty or difficult to handle, a guard column may be used and matched to that of the analytical column. Great care should be taken when sizing the guard column and connecting it to the analytical column to ensure that separation efficiency is not compromised.
If column or frit contamination is suspected due to peak splitting or loss of efficiency, the direction of the column can be reversed to backwash, and the column wash procedure described above is a good “recipe” for this purpose
The column should be removed from the detector to avoid fouling, and be aware that backflushing should only be used as a last resort and may not achieve the original efficiency of the column.
Remember that the column volume can be estimated using π × r2 × L × 0.6 (the approximate interstitial porosity of silica used for HPLC column packing materials)
Therefore, for a 150×4.6mm column, this would be approximately:
3.142×(2.3)2×150×0.6=1496μL or 1.5mL
Extending the lifetime of your HPLC columns is crucial for maintaining accurate and reliable separations in analytical workflows. By implementing the strategies discussed in this blog post, these practices not only save costs associated with frequent column replacements but also ensure consistent and high-quality results. Remember, proper care and maintenance are key to achieving optimal performance and longevity from your HPLC columns.