Liquid Chromatography Blog

Method Adjustment vs Change Part 2: Aqueous-to-Organic Ratio

In the previous article (Part 1), we took an overview of the concept of method adjustment vs method change in reversed-phase HPLC, in light of the guidelines of the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP). We also looked at an example of the allowed changes in mobile phase pH and buffer strength. This week, we’ll move down the chart and look at the variable listed in Table 1.

A/B Ratio
The A/B ratio refers to the ratio of aqueous (A) to organic (B) components of a reversed-phase mobile phase. The summary statement in Table 1 is a bit confusing, but becomes clear with an example. A minor component of a mobile phase is one that is present at 50% or less of the total.

   First, let’s look at the ±30% relative guideline, with a binary mobile phase of 50/50 methanol (MeOH)/buffer. As the concentration of the A- and B-solvents is the same, both are “minor” components. We can calculate that 30% of 50% is 15%, but only ±10% absolute is allowed (USP ), so this would mean that any mobile phase from 40/60 MeOH/buffer to 60/40 MeOH/buffer could be used… provided that system suitability passes. It is highly unlikely that a method will tolerate such large changes in mobile phase concentration and still pass system suitability. However, the guideline gives us considerable flexibility in adjusting the mobile phase to meet system suitability.

HPLC Solutions #57: Method Adjustment vs Change Part 2: Aqueous-to-Organic Ratio Table 1

   The EP ’s recommendations are more conservative, with a ±2% maximum change. So, for the example above, the allowable change would be 48/52 MeOH/buffer to 52/48 MeOH/buffer. This is much more realistic, in terms of an “adjustment” to the mobile phase. With this limit in place, it is not clear to me when the ±10% limit would apply.

   For the allowed change in a mobile-phase component at a small concentration, we would apply the same rules. If the mobile phase were 97/3 MeOH/buffer, we could change the buffer by 30% of 3%, or 0.9%, or 97.9/2.1 to 96.1/3.9 (USP ). The EP would allow up to ±2% in this case, or 99/1 to 95/5. Both of these guidelines seem reasonable.

   When a ternary (three-component) mobile phase is used, the application of the guidelines gets a bit more complicated. The example from the USP is for a mixture of 60/35/5. In this case, 30% of 35% is 10.5%, which exceeds the 10% limit, so the second component can be adjusted from 25 to 45%. Then 30% of the 5% component is 1.5%, so it can be adjusted from 3.5% to 6.5%. The amount of the major component (the 60% one) is then adjusted to give 100%. In this case, 50/45/5, 70/25/5, 58.5/35/6.5, and 61.5/35/3.5 would all meet the requirements. Again, provided system suitability passes.

A Word of Caution
The guidelines and examples discussed above are fairly straightforward, but the impact of such adjustments may not be so simple. With many methods, particularly ones with just a few components, the effect of adjusting the concentration of a major mobile-phase component often will primarily affect retention. The minor components of a mobile phase, such as those present at ≤5%, often are added to provide changes in selectivity necessary to separate certain analytes. This is especially true with methods that have many analytes or large concentration differences, such as stability-indicating or purity methods. Caution and common sense should always be the guidelines whenever adjustments are made to the mobile-phase composition. Be sure system suitability still passes and that the changes make sense, both from a scientific and method-performance standpoint.

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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