In this series on method adjustment vs method change, we first took an overview of the concept (Part 1) then at specific guidelines for mobile-phase adjustment (Part 2). In this article, we’ll take a closer look at some of the potential adjustments related to the column.
The guidelines for adjustments to the column length are the same for the European Pharmacopoeia (EP) and the United States Pharmacopoeia (USP), as summarized in Table 1 – a change of ±70% in column length is allowed. For example, let’s consider the most popular column size, 150 x 4.6 mm i.d. This guideline would allow 70% x 150 = 105 mm of length adjustment. Thus, any column in the 45 to 255 mm range would be allowed. Of course, only certain column lengths are available, but these limits allow for a change to most of the common column lengths: 50, 75, 100, 150, and 250 mm.
An increase in column length also will cause other expected changes in the chromatogram. The run time should increase in proportion to the column length, as should the column plate number, N, and system pressure, so each of these would increase by ≈65% for a change in column length from 150 to 250 mm. Resolution should improve as the square-root of the plate number, or about 30% for the same change in length. For most separations, the increases in run time and pressure may be inconvenient, but the other changes will benefit the separation. A decrease in column length may require some other changes to the system, such as a decrease in packing particle size. This is because a shorter column will reduce the plate number and resolution – if these were marginal with the original column, they likely will be unsatisfactory under the revised conditions if only the column length is changed.
The allowed changes to column diameter listed in Table 1 differ slightly between the EP and USP , but my interpretation is that the same result is allowed. The EP guidelines actually state that an adjustment of ±25% is allowed, but in the fine print is the statement, “When in a monograph the retention time of the principle [sic] peak is indicated, the flow rate has to be adjusted if the internal diameter is changed,” but no specifics are given. My interpretation is that this agrees with the recently revised USP recommendations.
Let’s first look at the EP (and old USP) ±25% guidelines with the same reference column as the one we used for column length, 150 x 4.6 mm i.d. A 25% change for a 4.6 mm column would be 1.1 mm, or an allowable range of 3.5 to 5.7 mm. If we consider the most common column diameters in use today – 1.0, 2.1, and 4.6 mm – this guideline does not allow us to change to the second most common diameter, 2.1 mm. This seems overly restrictive. I suspect that this guideline was introduced originally to allow for the use of the once-popular µBondapak columns from Waters that came in a 3.9 mm internal diameter. But for today’s applications, the 3.9 mm column is not so common.
The USP (and my interpretation of the EP) allows for any change in column diameter, provided that the mobile-phase flow rate is adjusted so that the linear velocity is constant. This may sound complicated at first, but in practice is pretty simple. Remember that the velocity of a fluid flowing through a tube will be related to the flow rate and the cross-sectional area of the tube. The cross-sectional area, of course, is proportional to the square of the diameter. So for a change from a 4.6 mm to a 2.1 mm column the adjustment is (4.6/2.1)2 = 4.8 ≈ 5-fold. Thus, if we were operating at 1 mL/min with the 4.6 mm column, we would run the 2.1 mm column at 1/5 = 0.2 mL/min. This should give the same linear velocity; we can verify that the correct adjustment was made, because the retention time and system pressure should be the same for both columns (assuming the same column length). The USP procedure makes sense, both from a scientific and practical standpoint, so perhaps the EP will clarify their guidelines at some point.
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