Does This Make Sense?
Now, the final test has to do with whether or not all these estimates line up with what is observed in reality. First, let's
consider the data of Figure 1. Resolution is directly related to peak width, so a 1% or 10% loss in resolution would correspond
to a 1% or 10% increase in peak width, respectively. Also, any increase in peak width should give a corresponding decrease
in peak height, so we can indirectly measure any loss of resolution by a reduction in peak height. The 30-μL injections of
Figure 1 are approximately the same as the injection volume estimated above for a 10% loss in resolution. You can see that
the peak heights for injection of 30 μL of sample in mobile phase (Figure 1b) are 60–70% of those when 30 μL of water is injected
(Figure 1d). So some broadening is occurring, as expected — partly because of the injection in mobile phase rather than a
weaker solvent. Note that in all cases where the mobile phase or weaker injection solvent was used (Figures 1b–1d), the retention
times were unchanged (within normally expected sample-to-sample variability).
Contrast the results of Figures 1b–1d with those of Figure 1a. In the case of Figure 1a, too strong an injection solvent was
used. This resulted in two problems. First, the peaks broadened unacceptably, especially for the first peak, which is severely
distorted. Second, some of the sample molecules are swept down the column during the injection-solvent dilution process, giving
shorter retention times. These problems could be solved by injecting a much smaller sample volume or using a more dilute injection
The data of Table I show the same pattern, but less severely. This is because of at least two factors. First, the injected
volumes (20 μL) are less than those of the <15% rule, and the difference between the injection solvent strength and the mobile
phase is less. For Figure 1a, 30 μL of acetonitrile is injected into an 18% acetonitrile mobile phase, whereas in Table I,
20 μL of methanol is injected in a 60% or 70% methanol mobile phase.
We have seen that the combination of injection solvent strength and injection solvent volume must be chosen carefully to avoid
unwanted band broadening and the resulting loss of resolution. In the examples of Table I and Figures 1b–1d, the injection
conditions were responsible for some loss in resolution (measured indirectly from loss in peak height), but no change in retention
was observed. The peaks of Figure 1a, however, were distorted and lost retention because too much of too strong a solvent
The <15% and <40% rules discussed above are guidelines that seem to work fairly well. But, as with all guidelines, it is a
good idea to give the actual results a reality test. One way was discussed here, where estimates of peak volumes and injection
conditions were compared with actual experimental conditions. Another technique is simply empirical — try increasing or decreasing
the proposed injection volume by twofold. In the same manner, injection solvent concentrations that are closer to the mobile
phase than the 100% strong or weak solvent conditions used in Table I and Figure 1a would be expected to have a less detrimental
result. For example, injection in 70% methanol into a 60% methanol mobile phase would be expected to be less of a problem
than an injection solvent of 100% methanol. If significant differences in the chromatograms are observed, you should adjust
the conditions so that you are not operating too close to the reliability limits of the method.
(1) T.-L. Ng and S. Ng, J. Chromatogr. A
329, 13–24 (1985).
(2) L.R. Snyder, J.J. Kirkland, and J.W. Dolan, Introduction to Modern Liquid Chromatography, 3rd Ed. (Wiley, Hoboken, New Jersey, 2010), section 2.6.1.
is a PhD student in the laboratory of Dr. Hugh Flowers at Glasgow University in Scotland.
John W. Dolan
"LC Troubleshooting" Editor John Dolan has been writing "LC Troubleshooting" for LCGC for more than 25 years. One of the industry's most respected professionals, John is currently the Vice President of and a
principal instructor for LC Resources, Walnut Creek, California. He is also a member of LCGC's editorial advisory board. Direct correspondence about this column via e-mail to
John W. Dolan