What causes peaks to appear where they don't belong?

This is the third installment in a series focusing on some of the basic principles of troubleshooting liquid chromatography (LC) methods. First, we looked at some general practices for troubleshooting any LC problem (1). Then we looked at problems whose symptoms are related to pressure changes (2). This month, we'll concentrate on problems exhibited as abnormal retention times. As a means to organize the discussion, let's look at situations where retention times are too long, too short, or inconsistent.

What Controls Retention?

The mobile phase can change because of an error in formulating it, such as a mistake in volumetric measurement or adjustment of the pH. If an error in formulating the mobile phase is suspected, it is best to make a new batch to see if it fixes the problem. Some mobile phases can change over time because of chemical degradation, selective evaporation of one component, or microbial growth in highly aqueous mobile phases. Again, reformulation is the best way to verify this problem source. Most of us use on-line mixing to prepare isocratic mobile phases. An error in instrument settings or hardware failure can be the cause of errors in on-line mixing. Check for proper degassing and pump operation, as well as the correct control-program settings. Sometimes hand-mixed mobile phases can be used as a check for on-line mixing, or alternate mixing channels can be used for both isocratic and gradient methods (for example, use the C and D solvent reservoirs instead of A and B in a four-solvent LC system).

The stationary phase in the column has a finite lifetime, generally in the 500–2000 sample range (or more), depending on the nature of the sample. Every column will die eventually, and some methods are harder on columns than others. For example, mobile phases outside the pH 2–8 region accelerate the degradation of silica-based columns. Some column types have shorter lifetimes than others. For example, cyano and amino columns are unlikely to last as long as C8 or C18 columns, which tend to be quite robust. In addition to changes in retention, column failure usually is accompanied by a rise in system pressure and an increase in peak tailing. Replacement of a suspect column with a new one is the easiest way to check for column-related problems.

System hardware problems that generate symptoms of changed retention most commonly are associated with pump malfunctions or leaks. Pump problems can be checked with a simple flow-rate measurement with a stop watch and volumetric flask. A secondary symptom of pump problems may be high, low, or fluctuating pressure. Low flow may be associated with faulty check valves, worn pump seals, air bubbles in the pump, or errors in pump settings. Cleaning, component replacement, or degassing should correct such problems. High flow rates usually are a result of improper settings.

The most common environmental cause of retention changes is a change in column temperature. This effect is common if the column oven is not used or is not working properly. Methods that operate under ambient conditions are highly susceptible to failure, especially if the laboratory temperature is not well controlled. In my travels, I have encountered laboratory temperatures ranging from 10 °C (central China in January) to 35 °C (Tel Aviv in June). If we use the rule of thumb that retention can change by 2% with each 1 °C change in temperature, you can imagine the result if the same method were run in both of those laboratories under ambient conditions! Use the column oven and make sure that it is operating properly.