While the relative cost of HPLC columns has been reduced over time, extending column lifetime remains an important consideration for most laboratories. The following tips should help to protect your columns and significantly extend the useful lifetime of most phases.
Always read the manufacturer's literature with respect to the recommended pressure, eluent pH and temperature operating ranges for the column, and stick 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 typically loss of efficiency (peak broadening) and at high pH, peak tailing and an increase in column back pressure.
Avoid mechanical shock of the column bed, such as dropping the column, and ramp the pressure or flow slowly (1 mL/min/min is ideal) each time eluent flow is initiated.
Most modern HPLC equipment is capable of achieving this flow or pressure ramp automatically through secondary instrument settings. Bed voiding due to pressure shock often manifests itself via split or very badly tailing peaks. Columns may be reversed for analysis if a replacement is not readily available; however, the efficiency of the column is likely to reduce much more quickly as the bed will ultimately slump in the opposite direction, leading to the same chromatographic symptoms.
If columns have dried out, initiate the flow very slowly (0.1 mL/min/min) using an eluent containing at least 50% acetonitrile.
If a "standard" (non "aq" or non-polar embedded phase type) reversed-phase column is suspected of phase collapse (shortening retention times, poor efficiency) due to use with 100% aqueous mobile phases, the column should be reactivated at high flow with 100% acetonitrile at 60 °C (take care to not precipitate any solid buffers from the eluent remaining within the column). In both of these cases, between 50 and 100 column volumes may be required to properly re-equilibrate or re-activate the phase.
Columns should be properly washed after each use.
A recommended washing routine may be:
One might use an older HPLC system as a column "wash station," which can save significant amounts of operating time on "live" instruments.
If samples are likely to contain particulate matter, choose a good quality inline filter with the appropriate mesh size; 0.45 µm for traditional columns and 0.2 µm for UHPLC columns is typical.
If the sample matrix or diluent is likely to harm the sorbent (due to pH, for example) or is particularly chemically dirty or intractable, a guard column may be used, and the phase should be matched with that of the analytical column. Take great care when selecting the dimensions of the guard column and connecting to the analytical column to ensure that the efficiency of the separation is not compromised.
If column or frit contamination is suspected due to peak splitting or loss of efficiency, it is possible to reverse the direction of the column for back flushing purposes, and the column washing procedure mentioned above is a good "recipe" for this purpose.
One should uncouple the column from the detector to avoid fouling, and note that reversal for flushing should only be used as a matter of last resort, and that the original efficiency of the column may not be achieved.
Remember that column volume may be estimated using π × r2 × L × 0.6 (the approximate interstitial porosity of silica used for HPLC column packing materials).
So, for a 150 × 4.6 mm column, this would approximate to:
3.142 × (2.3)2 × 150 × 0.6 = 1.496 µL or ~1.5 mL
These tips will help extend your column lifetimes.
Detangling the Complex Web of GC × GC Method Development to Support New Users
November 1st 2024In this article, the authors describe a simple workflow to develop a GC×GC method for a specific sample upon initial use, with the aim of decreasing the time to accomplish functional workflows for new users.
AI and GenAI Applications to Help Optimize Purification and Yield of Antibodies From Plasma
October 31st 2024Deriving antibodies from plasma products involves several steps, typically starting from the collection of plasma and ending with the purification of the desired antibodies. These are: plasma collection; plasma pooling; fractionation; antibody purification; concentration and formulation; quality control; and packaging and storage. This process results in a purified antibody product that can be used for therapeutic purposes, diagnostic tests, or research. Each step is critical to ensure the safety, efficacy, and quality of the final product. Applications of AI/GenAI in many of these steps can significantly help in the optimization of purification and yield of the desired antibodies. Some specific use-cases are: selecting and optimizing plasma units for optimized plasma pooling; GenAI solution for enterprise search on internal knowledge portal; analysing and optimizing production batch profitability, inventory, yields; monitoring production batch key performance indicators for outlier identification; monitoring production equipment to predict maintenance events; and reducing quality control laboratory testing turnaround time.
Green Chemistry: What is it (and What Is It Not)? And How Does It Apply to Gas Chromatography?
October 31st 2024Everyone is talking about sustainability, and organizations are creating sustainability programs. But what does green chemistry really mean, and how does it apply to gas chromatography?
AOAC International Awarded NIST Grant for Developing Drug Testing Standards
October 31st 2024The grant will be part of a new collaborative scientific initiative to address the need for standards that define the desired performance of lateral flow immunoassay test strips to detect illicit drugs in tablets and powders.