# Liquid Chromatography Blog

This is the last in a six-part series on allowed adjustments to HPLC methods. In the previous instalments (Part 1, Part 2, Part 3, Part 4, Part 5), we’ve looked at each of the specific variables listed in Table 1.

Interactions
The allowed adjustment of the variables listed in Table 1 are each listed separately, but a change in some of these can have a cascading influence on other variables. Let’s consider a change in packing particle size as an example. Let’s say we start with a 150 x 4.6 mm, 5 µm column operated at 1.5 mL/min. This will generate a column plate number, N, of ≈10,000 in a real application. If we cannot pass system suitability because the plate number specification cannot be met, we might change to a 3 µm column packing. This would be expected to generate N ≈ 15,000, and may allow us to pass the system suitability. But what else will we observe?

Table 1

A decrease in particle size will cause an increase in column pressure – inversely proportional to the square of the particle size. This means the pressure will increase by 1/(5/3)2 ≈ 2.8 times. If the pressure originally was 150 bar (2200 psi), it would rise to >400 bar (>6000 psi). This would exceed the upper pressure limit of a standard HPLC system, so some other adjustment, such as a reduction in flow rate, would be required.

In another example, let’s change from our 150 x 4.6 mm, 5 µm column at 1.5 mL/min to a 100 x 2.1 mm, 3 µm column, still operated at 1.5 mL/min. The two columns should generate N ≈ 10,000, so they are equivalent from that standpoint. But the reduction in column diameter will cause the pressure to rise by the ratio of cross-sectional areas, approximately fivefold. The shorter column will reduce the pressure to 100/150 ≈ 65%. This is on top of the 2.8-fold increase in pressure resulting from the particle size change. So the net change would be ≈ 5 x 65% x 2.8, or a little over ninefold. Definitely this would be a problem with nearly every method. If we followed the instructions in (Part 3), we would reduce the flow rate fivefold to accommodate for the diameter change, so the pressure change would be a little less than twofold – much more tolerable.

The results of changes in physical dimensions and flow rate, such as those just mentioned, are easy to predict from simple calculations. Changes in variables that may change selectivity are less predictable, especially when interactions are possible. For example, a change in mobile phase composition will usually change retention, but also may change selectivity (peak spacing). Similarly, a change in the column temperature will change retention (approximately 2%/1 ºC temperature change), but may or may not change selectivity. So a simultaneous change in mobile phase and temperature can give unexpected results.

The bottom line here is that if you change more than one variable of Figure 1 to enable the method to pass system suitability, take special care to inspect the chromatogram for subtle (or not so subtle) changes in selectivity.

The adjustments from the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP) listed in Table 1 are intended for isocratic methods – those with a constant mobile-phase composition. If gradients are used, all bets may be off. For example, a change in column size or flow rate can have unexpected results that are much different than those predicted for isocratic methods. If you are using gradient methods and need to make adjustments, be extra careful to look for changes in selectivity if changes are made.

Am I Safe Now?
One question I get frequently is if these allowable adjustments can be made for any method with impunity. This gets an emphatic “NO” for an answer. These are guidelines for adjustments that can be made for many methods to improve them sufficiently to pass system suitability. For some methods, even a change of pH by 0.1 unit or temperature by 3 ºC will have disastrous results. Be deliberate with the changes, look carefully at the results, and proceed with caution. Remember, the system suitability test is the measure to use – if the change allows you to pass system suitability, you’re probably safe.

Documentation
And finally, Document, Document, Document! Be sure to include the who, what, why, when of a good newspaper article. And if you are cautious, I recommend getting your supervisor to sign off on the adjustment before putting it into routine use. Good luck!

References
1. European Pharmacopoeia 6.0 (2010) Section 2.2.4.6.
2. United States Pharmacopoeia, 34 (2011) Section 621.

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 TechTips@sepscience.com