One aspect I enjoy about being the "LC Troubleshooting" editor is getting to interact with readers through a wide variety of liquid chromatography (LC) questions that I get via e-mail. This month I'll share some of the more interesting ones I've received recently. If you have a question for me, feel free to contact me at the e-mail address listed at the end of this article.
Reader: I've heard you say that the retention factor, k, should not be less than 2 for an isocratic method. Is this a hard-and-fast rule? I'm having trouble getting the first peak retained and would like to have a fast run.JWD: As a general rule, a retention factor in the range 2 < k < 10 will give you the "best" chromatography, but this is no guarantee of the best separation. Also, some samples have such a wide polarity range that you can't fit them in this retention window. In such cases, 1 < k < 20 certainly is acceptable. When even this extended range of k-values is not possible, you should seriously consider gradient elution instead of an isocratic method. Let's review why we set these k-value guidelines. First, recall that the retention factor is calculated as
However, it must be acknowledged that the recommendations of k-ranges shown in Figure 1 are just that, recommendations, not hard-and-fast rules; there will always be exceptions. For example, sometimes it is not possible to get sufficient retention of a very polar peak so that k > 1 can be obtained. Or for very clean samples, the baseline disturbance at t 0 may be small enough that k = 0.5 provides acceptable separation for adequate quantification. But when we make a decision to develop and validate a method with such small retention, we should go into it with our eyes open and recognize the potential problems.
What are some alternatives? If run time is your major concern, it may be possible to increase k-values so that the first peak has k > 2, then to increase the flow rate and reduce the retention time, because k is not affected by flow rate. Or if retention on a conventional C18 column is too short for a polar compound, maybe an embedded polar phase column will provide an acceptable alternative. Another alternative might be to use hydrophobic interaction chromatography (HILIC), which is a form of normal-phase chromatography. With HILIC, retention orders typically are the opposite of those obtained using reversed-phase chromatography, so polar compounds are well retained and nonpolar ones come out early.