How to Obtain a Flat Baseline in Gradient Mode

You developed a nice gradient method for your pharmaceutical impurities analysis. The peaks are sharp and symmetrical. The separation is good. The only problem is the baseline, which slopes and makes accurate quantitation of your peaks difficult. There are a couple of ways you can avoid this problem and you might find one easier than the other depending on your situation.

                 The first way is to run a blank. Typically this will be your sample diluent with nothing else in it. You only need to do this once for the method and then you can use it for each sample. Most modern chromatography software has a feature called “blank subtraction.” Here, you would load your sample chromatogram with the sloping baseline and then apply the blank subtraction operation. Every chromatographic feature in the blank will be subtracted from your sample data, resulting in a completely flat baseline. Some analysts may find this cumbersome though because it requires you to run an additional sample. Still, many protocols may require the analyst to run a blank anyway to demonstrate that they don’t have any “ghost peaks” in their sample chromatograms.         

                The second method doesn’t require an additional run but it can be more tricky to initially develop. This technique is called “absorbance matching.” The reason the gradient slope occurs is because of a difference in UV absorbance between the A and B solvents. You don’t notice it in isocratic methods because the solvent composition never changes. Generally, the B solvent will absorb more because of the organic solvent (e.g. acetonitrile, methanol, etc.). In this approach, the goal is to achieve identical absorbance between the two solvents. The two don’t have to have the same absorbance across the whole UV spectrum, just the wavelength you are operating at. To achieve this, add a UV-absorbing species to the A solvent until you don’t see any change in the baseline. Once you know the correct amount, the process should be easy to write into your SOP. Generally though, determining the proper amount is accomplished by trial and error. The additive should be unretained and should not interact with or affect the sample. Examples include nitrate, nitrite, azide compounds, etc.

An example of blank subtraction