Questions from the e-mail bag are considered this month.
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
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
R is the retention time and t
0 is the column dead time, usually determined by the first rise in the baseline at the "solvent front." Resolution is a function
of k/(1 + k), so if we plot retention as the retention factor on the x-axis and resolution as k/(1 + k) on the y-axis, we see a plot like that of Figure 1. You can see that the resolution line starts out at a very low value and rises
to a plateau as k increases. This relationship is the basis of the recommendations for k-ranges for isocratic methods. When 2 < k < 10, you can see that the plot begins to flatten out, but run times aren't excessive. In this region small changes in k will result in very small changes in resolution. Or another way of looking at this is that the method is robust to small
changes in variables that might change retention, such as the percentage of organic solvent in the mobile phase, temperature
or pH. On the other hand, if we extend the acceptable k-range to 1 < k < 20, the early peaks lie on a much steeper portion of the curve. This means that the same change in k that caused little concern with longer retention times will cause larger changes in resolution. Thus, methods with k < 2 tend to be less stable. Another problem with peaks with k < 2, and certainly k < 1, is that there is more likelihood of interferences from unretained material at t0. I've also plotted the run time and peak height in Figure 1. As k increases, run time increases and peaks broaden and are shorter; both of these are undesirable, so smaller k-values for the last peak are desirable.
Figure 1: A plot of resolution, expressed as k/(1 + k) vs. retention (k). The effect of retention on peak height and run time
are also shown.
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.