LC Method Scaling, Part I: Isocratic Separations - - Chromatography Online
LC Method Scaling, Part I: Isocratic Separations

LCGC North America
Volume 32, Issue 2, pp. 98-102

What kind of adjustments need to be made when scaling an isocratic method?

Today we are often confronted with many different types of liquid chromatography (LC) methods. These may use conventional 250 or 150 mm 4.6 mm, 150 mm 2.1 mm, 50 mm 2.1 mm, and many other column configurations packed with particles generally ranging from <2 μm to 5 μm, and sometimes even 10 μm in diameter. One of the challenges this variety presents is transferring a method from one column configuration to another and still obtaining the same resulting separation. For example, you may use an ultrahigh-pressure LC (UHPLC) system to develop methods quickly in your research and development (R&D) laboratory, but want to transfer it to a conventional LC system for routine use. Or your conventional method with ultraviolet detection (LC–UV) may need to be transferred to an LC system with mass spectrometry detection (LC–MS). Alternatively, you may want to adjust a pharmacopeial method to use a different column configuration. In many of these cases, the method must be moved from one column size to another, yet maintain the same separation. The conversion is not difficult, but you do have to be careful to make the appropriate adjustments. Isocratic separations, in which the mobile-phase concentration is constant, are simpler to convert than gradient methods, where special care has to be taken to avoid inadvertent chromatographic changes. This month's "LC Troubleshooting" discussion focuses on isocratic separations, and next month we'll look at gradients.

Resolution Is the Key

Equivalent separations require that the resolution stays the same when conditions are changed. In the method development classes we teach, we use what is often referred to as the "fundamental resolution equation" as a guide for the method development process. We can use this same equation to guide us in method conversion:

where R s is resolution, N is the column plate number, α is the separation factor, and k is the retention factor. The first caveat is that the chemistry of the system cannot change when a change in the column or other conditions changes. This means that the mobile phase must remain the same, as well as the column temperature and column chemistry. With today's columns, it usually is valid to assume that the same brand name description of a column (for example, ACE C18 [Advanced Chromatography Technologies Ltd.) will have the same column chemistry, no matter what the particle size is (2, 3, 5 μm, and so forth). You'll recall that the retention factor is defined as:

where t R is the retention time of a solute and t 0 is the column dead time (retention time of an unretained peak). If we keep the chemistry of the system constant, the retention time relative to the dead time should stay constant, so k will remain unchanged. Any change in retention because of a change in column length, diameter, or flow rate will have a proportional change for t R and t 0, so k will stay constant in this case as well. For example, doubling the flow rate will halve t R and t 0, and k will be unchanged.

The separation factor α is simply the ratio of k values for two adjacent peaks, k 1 and k 2:

So if we keep k constant, as discussed above, α will be unchanged. If k and α are kept constant, to keep R s constant (equation 1), all that remains is to make sure that the column plate number stays constant.


blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:



Column Watch: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments. LATEST: When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: HPLC for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

MS — The Practical Art: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis

LC Troubleshooting: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: How Much Can I Inject? Part I: Injecting in Mobile Phase

More LCGC Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>

Source: LCGC North America,
Click here