“Measure Your Gradient”: A new way to measure gradients in high performance liquid chromatography by mass spectrometric or absorbance detection

• The usual approach to measure HPLC gradients yielded misleading results.
• A simple new approach was developed that yields more accurate gradients.
• The retention times of 20 standards are used to back-calculate the gradient.
• The methodology was accurate under a range of conditions.
• Software and instructions were made available online.

The gradient produced by an HPLC is never the same as the one it is programmed to produce, but non-idealities in the gradient can be taken into account if they are measured. Such measurements are routine, yet only one general approach has been described to make them: both HPLC solvents are replaced with water, solvent B is spiked with 0.1% acetone, and the gradient is measured by UV absorbance. Despite the widespread use of this procedure, we found a number of problems and complications with it, mostly stemming from the fact that it measures the gradient under abnormal conditions (e.g. both solvents are water). It is also generally not amenable to MS detection, leaving those with only an MS detector no way to accurately measure their gradients. We describe a new approach called “Measure Your Gradient” that potentially solves these problems. One runs a test mixture containing 20 standards on a standard stationary phase and enters their gradient retention times into open-source software available at www.measureyourgradient.org. The software uses the retention times to back-calculate the gradient that was truly produced by the HPLC. Here we present a preliminary investigation of the new approach. We found that gradients measured this way are comparable to those measured by a more accurate, albeit impractical, version of the conventional approach. The new procedure worked with different gradients, flow rates, column lengths, inner diameters, on two different HPLCs, and with six different batches of the standard stationary phase.