Before beginning HPLC troubleshooting procedures related to retention time changes, compare the retention times of several runs to verify that the problem is reproducible and determine if there is a pattern to the variation.
may result from changes in mobile phase flow rate, mobile phase composition, column stationary phase, and column temperature. If the retention time for the column void volume, t0, changes and there have been no changes in the column configuration, the problem is related to flow rate. System leaks and air bubbles in the pump cause t0 to increase. To troubleshoot, follow the recommendations in the previous paragraph for low-flow problems. Increased flow rates cause t0 to decrease. Verify proper flow rate selection on the pump controller and check the pump flow rate accuracy as described in the “Low Pressure” section.
in one direction without a change in t0 represents a change in the mobile phase. Such changes are often caused by the reduction of at least one liquid mobile phase component due to evaporation or improper mixing procedures. When mobile phase is suspect, mix a new batch and determine if this corrects the problem. Degassing solvents using helium sparging may lead to the evaporation of volatile solvents, thus affecting the mobile phase concentration. Solvent degassing using an Inline HPLC Solvent Degasser (see listing in Instrumentation Section of this catalog) is the recommended alternative to helium sparging.
,the most common source of problems includes mobile phase pH and buffer concentration. Depending on the pKa, the retention time of ionizable compounds can shift up to 10% when the mobile phase pH changes by as little as 0.1 pH unit. Remember to measure the pH of the aqueous component only, as the addition of organic solvents will alter pH readings. Buffer concentrations should be typically 20-50 mM. Lower levels may provide irreproducible results and higher levels may reduce column life.
with the starting mobile phase before sample injection is essential for reproducible retention times. Determine adequate HPLC column equilibration by allowing longer equilibration times to see if the problem improves. Allow at least 10 to15 column volumes of solvent to pass through the column before injecting a sample. When using on-line solvent mixing instrumentation, compare isocratic results with a mobile phase that has been mixed manually. In gradient separations, exchange solvent inlet lines and make appropriate program adjustments. If the system is operating correctly, the results should remain the same in both configurations. Retention time drift may be observed during the first few samples of a given analysis. If retention times move and then stabilize after several sample injections, deactivation of the column may be taking place through sample loading onto the column. This may not be a problem if sample supply is not limited and the resultant chromatography is acceptable. As an alternative, a different type of column chemistry changes in sample preparation procedures, or mobile phase modifiers may help eliminate this phenomenon. If mobile phase modifiers such as ion-pairing reagents are being used, drifting retention times may also be the result of incomplete column equilibration.
such as contaminant buildup or general column deterioration can manifest as retention time drift. A reduction in HPLC column efficiency, or plate number (N), may result in broad peaks with poor retention. HPLC Column contamination that contributes to retention time drift is sometimes accompanied by changes in relative peak spacing and usually takes place over a large number of samples or over a long period of time. If a defective analytical column is suspected, replace or remove the guard column and run several more samples. If this does not correct the problem, or if a guard column is not being used, flush the analytical column with strong solvent to remove any contaminants. If the problem persists, replace the analytical column. Generally, a guard column should always be used to protect an analytical column. Periodic cleaning of the analytical column using strong solvents helps extend useful column lifetime and reduces long-term contamination.
may show up as shifts in retention times that cycle with changes in ambient laboratory temperature. Retention times can change as much as 2% with each 1°C change in ambient temperature, depending on the analysis. It is difficult and expensive to control the ambient air temperature through control of the laboratory air conditioning or heating system. As a cost effective alternative, column thermo stating can be used to either maintain the column at ambient temperature, or slightly elevate the column temperature above ambient. This helps to maintain reproducible retention times without affecting the HPLC chromatography. For a listing of thermo stated HPLC column heaters and HPLC chillers, see the HPLC Instrumentation Section of this catalog.
can also negatively impact retention times. If the sample is injected in a large volume of strong solvent, poor peak shape and irreproducible retention times may result for early eluting peaks. To avoid this problem, samples should be injected using the starting mobile phase solvent whenever possible.
can cause rapid contamination buildup on the guard and analytical columns, and this can reduce column life as well as affect retention times. Using an appropriate sample preparation technique such as solid phase extraction (SPE) may alleviate this problem.