Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, October 11, 2014

Appropriate Mixer Volume for HPLC and UHPLC Applications

For gradient analysis, most HPLC (UHPLC) systems incorporate a solvent mixer which is designed to balance the requirements of moderate dwell volume, low noise and mixing efficiency. Depending on the method run, the ideal mixer's volume may in fact be completely different than the one installed in your chromatography system. Here are some very general guidelines to determine the appropriate mixer volume for your HPLC system. Note: Since many types of mixer designs exist, these are guidelines only.

HPLC System Mixer Volume Choices; Which Size Is Best?

SMALL: Fast or ultrahigh speed separations using low volume, small particle columns. These types of applications depend on a low dwell volume mixer. To achieve this, your HPLC system should be plumbed with narrow bore capillary tubing (example: 0.005" ID; 0.12mm ID) and include a gradient mixer with a volume of less than 100 ul (example: ~35 ul is rather common size). *Don't forget to address the dwell volume of the autosampler, loop and flow cell too when optimizing your system. 

LARGE: High Sensitivity Analysis: Gradient analysis where sensitivity is key, benefit from larger volume mixers to minimize contributions of any UV absorbing additives (e.g. TFA) and turbulence in the flow. Traditional 300 to 750 ul mixers often work well in these applications, provided that the column volumes are also large. Smaller column volumes will require smaller mixer volumes to reduce the added dwell effect.

MEDIUM: Routine HPLC Analysis: Typical analytical separations using 3 to 5 mm ID columns (x 100mm or longer) usually benefit from modest sized mixers within a range of 200 to 400 ul volume. For these applications, I often start with a recommendation to use a mixer which has 10% of the columns volume as a starting point. For a typical 4.6 x 250mm, 5 micron porous support column, which has about 3mls of volume, a 300 ul volume mixer should provide enough volume for routine gradient analysis. As mentioned before, the type of mixer, column volume, flow rate and mobile phase characteristics will help suggest the most applicable size for your application. Remember, these are general guidelines only. Your exact application must be taken into account to determine which size is best.