HPLC troubleshooting - part 2
Maintaining a high-performance liquid chromatography (HPLC) system in good condition provides users with consistent data quality and accuracy. A well-functioning system also minimizes downtime, helping to maintain lab efficiency.
As discussed in Part I, problems in HPLC systems might arise from several sources. When using a UV detector, the chromatogram can indicate the presence of a problem and provides clues about where to find the cause.
What problems can show up on a chromatogram?
A UV chromatogram plots absorbance at the selected wavelength over time. As the mobile phase carries the sample through the HPLC column, peaks indicate the relative abundance of compounds eluting from the column.
In the preferred scenario, sharp uniform peaks with clear separation and high signal to noise ratio will allow the identification of each analyte. Sometimes, however, the chromatogram might show distortions, including:
- Shouldered peaks
- Twin peaks or split peaks
- Tailed peaks
- Low signal to noise ratio
These distortions can add complexity to analysis, indicate upstream problems, and affect data accuracy and reliability.
What causes chromatogram distortions?
Physical contamination of the HPLC system and column can present as peak tailing, splitting, and shouldering.
On an uncontaminated column, sample application is immediate. Uniform application in a short timeframe allows efficient separation, supporting consistent and accurate results.
Contamination of the column or frit can increase the time taken to apply a sample in comparison with a clean column. This increased time can lead to poor resolution of the analytes.
Any chemicals or compounds that absorb at the same wavelength as the analyte can distort chromatograms and confuse analysis. If contaminants elute at a similar time as the analyte, the chromatogram might report inaccurate absorbance values or unexpected peaks. Any contaminants in the mobile phase can also cause background noise.
Possible sources of contamination include extractables from filter devices or other system components. Filter materials that release extractables or housings with low solvent resistance have the potential to interfere with the chromatogram.
However, extractables only become a problem if they are detectable and co-elute with the analyte of interest. Other sources of contamination might include residue on glassware from previous experiments.
High pressure in the HPLC system keeps gas dissolved. Generally, dissolved gas is only likely to cause problems if it comes out of solution to form bubbles. These bubbles are most likely to arise at the detector where the pressure drops.
Detectors vary in their sensitivity to gas bubbles, but the effect is often evident as baseline noise on the chromatogram, leading to a low signal-to-noise ratio.
A low signal to noise ratio is a common indicator of a high proportion of dissolved gas. This noise can affect the reliability of peak identification, making HPLC analysis particularly difficult when the analyte is limited and has a low absorbance level.
How can problems in HPLC analysis be minimized?
Reducing sources of contamination
Filtering the sample can reduce particle contamination by preventing undissolved particulates from entering the system.
Solvent compatibility and level of extractables are considerations in selecting an appropriate filter device. Running a comparative test with and without a standard in place of the sample can assess the effect of extractables on the chromatograph.
Reversing the solvent flow is also a common technique to clear particulates from the column and frit. However, note that this technique can disrupt packing and affect separation efficiency.
Sufficient HPLC column packing and column equilibration procedures can help make sure sample application is uniform. These actions can also reduce the likelihood of bubbles forming in the column.
It is common practice to degas the mobile phase before mixing it with the sample. Degassing with a cellulose filter minimizes bubble formation at the detector.
Some HPLC systems incorporate a degasser, but an alternative option is vacuum filtration of the mobile phase before use. Degassing before each HPLC run also reduces the likelihood that gases dissolved in the mobile phase reservoir between runs will affect results.
The quality and resolution of UV chromatogram data can provide users with feedback to help them achieve and maintain high-quality HPLC data.
Some HPLC problems can show up in the chromatogram, but others might be evident from the HPLC system itself. Examples of in-system problems are increasing back pressure, leakages and loss of pressure, and inconsistent flow rate. Find out more in part 1 of the HPLC troubleshooting guide.
Try our Whatman Filter Selector App to find out if you are using the most appropriate filtration solution for your samples. To discuss any challenges you are facing, please contact GE's Life Sciences Scientific Support.