Liquid Chromatography Blog

Method Adjustment vs Change Part 5: Volume, Column Temperature and Detector Wavelength

In the last few articles we've focused on the guidelines for method adjustment listed in the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP). We looked at pH (Part 1), mobile-phase composition (Part 2), column dimensions (Part 3), and flow rate and particle size (Part 4). This week we’ll focus on the remaining variables listed in Table 1.

Injection Volume
The EP and USP agree that any reduction in injection volume can be made, as long as system suitability passes. The key factor to watch for is that the peak(s) is sufficiently large to meet the precision and accuracy requirements of the method. Special attention should be paid to the injection volume if the column diameter is reduced. When a smaller column is used, the amount of packing is decreased, and the original injection volume may create column overload conditions. The common signs of overload are reductions in retention time and increases in peak tailing. If these occur, reduce the injection volume in proportion to the change in cross-sectional area of the column (the same proportion as the change in flow rate discussed in Part 4).

 HPLC Solutions #60: Method Adjustment vs Change Part 5: Volume, Column Temperature and Detector Wavelength Table 1 

Column Temperature
The EP and USP have slightly different guidelines in terms of allowable temperature adjustments, as noted in Table 1. The EP allows a 10% change in temperature and a maximum temperature of 60 ºC. The 10% change would allow new conditions of 31.5-38.5 ºC for a method normally run at 35 ºC. This is a reasonable change if we consider the rule of thumb that a 1 ºC change in temperature will give ≈2% change in retention. The USP limit of ±10 ºC seems to be a little extreme, especially if we consider the potential for a change in the peak spacing when temperature is changed (see 'Temperature and Retention'). The upper limit of 60 ºC in the EP guidelines are a bit puzzling. I know that in our lab we commonly run at temperatures of 70 ºC with no ill effects. Perhaps this is a holdover from days when columns were not as stable as they are today.

   Another area related to temperature should be considered, especially if the method is temperature sensitive. That is the calibration of the oven and sufficient preheating of the mobile phase so that there is not a temperature gradient along the column. If you are in doubt, you can easily check these by using a thermocouple to check the temperature of the mobile phase entering and leaving the column.

The EP and USP agree that no change in wavelength of a UV detector is allowed, which makes sense. What is a bit puzzling is the USP ’s allowed ±3 nm variation in wavelength if the detector is changed. This probably is a holdover from the days when most detectors did not have internal calibration procedures. Most of today’s detectors automatically check the wavelength during the power-up cycle and either prevent you from continuing or at least warn you if the wavelength calibration is off. So this restriction is a bit of a moot point.

1. European Pharmacopoeia 6.0 (2010) Section
2. United States Pharmacopoeia 34 (2011) Section 621.

This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to


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