How It Works

To understand how the Measure Your Gradient methodology works, you first have to understand how gradient retention times are calculated from isocratic retention measurements.

Suppose you measured the isocratic retention of a compound over a range of mobile phase compositions...

...and you want to calculate its retention time in the following gradient.

The easiest way to do it is to think of the gradient as a series of very short isocratic steps, each with a certain mobile phase composition.

For each isocratic step, you could determine the compound's retention factor (from the isocratic retention vs. % B relationship above) and from that, calculate how far it will travel through the column during the step. After a number of isocratic steps, the compound will eventually have traveled the entire length of the column.

The number of isocratic steps it took to elute the compound multiplied by the length of each step gives its gradient retention time.

The Measure Your Gradient methodology uses the same basic approach, but in reverse. Instead of calculating gradient retention times from a measured gradient profile, it back-calculates what the gradient profile must have been to give a set of measured gradient retention times.

It uses an iterative process to back-calculate the gradient profile. It begins with the ideal (programmed) gradient profile, calculates retention times for each of 20 standards (using their known isocratic retention behavior; see below), and compares them with their measured retention times. Then it makes a small adjustment to the gradient profile, recalculates retention times for all 20 compounds, and checks to see if the accuracy of the calculated retention times improved. If so, it keeps the change, otherwise it discards it. It continues making small adjustments to the gradient profile until the difference between the measured and calculated retention times is minimized.

Advantages and Limitations

This approach offers a number of advantages over conventional approaches to measuring HPLC gradient profiles:

1) It is easy to use and fast. You simply run a test mixture containing 20 standards and report their retention times to the open-source Measure Your Gradient application.

2) Since the gradient profiles are measured from the retention times of the standards, it doesn't matter what type of detector you use - a mass spectrometer works great.

3) There's no need to reconfigure your HPLC to measure the gradient profiles it produces. All you have to do is run the standard mixture in a gradient.

4) The gradient profiles are measured under precisely the same experimental conditions as your regular reversed-phase LC-MS runs. Therefore, the back-calculated gradient profiles more accurately reflect the one that analytes experience in your real HPLC runs.

The limitations of the methodology are:

1) At this time, you must use an Eclipse Plus C18 (3.5 µm particle size) column, though any column length or inner diameter is okay.

2) The column oven must be set to 35 °C.

3) The mobile phases must be 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B).

Isocratic Retention Measurements

The Measure Your Gradient methodology uses a test mixture containing a set of 20 standards that are visible by both electrospray ionization (ESI) MS and by UV absorption. The 20 standards in the test mix are listed below along with their measured isocratic retention behavior. This data is coded into the Measure Your Gradient application where it is used to calculate gradient retention times.

Their isocratic retention was measured on an Eclipse Plus C18 column (3.5 µm particle size) using carefully prepared, pre-mixed water/acetonitrile mixtures containing 0.100 ± 0.005% (by volume) formic acid. The column temperature was held at 35.0 ± 0.1 °C. Uracil was used as the dead time marker.

You can also download this data in spreadsheet form: isocratic_data.csv

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