The remaining items on my list fall in the extracolumn effects category. We've already looked at the column connections, but
extracolumn peak broadening can occur because of poor tubing connections wherever the sample contacts, so connections at the
autosampler and detector should be checked as well, especially if PEEK tubing and fittings are used. The detector flow cell,
time constant (noise filter), and data system data rate also can be sources of peak broadening. I'm assuming that the detector
was not changed, so the flow cell is not likely the source. The detector time constant is an electronic filter that helps
to reduce the noise through signal averaging, but if it is set to too large a value, it can result in broadened peaks. The
rule of thumb is that it should be set to ≈0.1 times the width of the narrowest peak of interest. Similarly, the data system
sampling rate should be set so that at least 15–20 data points are collected across the peak. Although neither of these settings
is likely to change without intervention by the operator, a power outage or other factor might cause these to be reset to
default values that are not appropriate for the present samples. It is easy to check both the time constant and data rate
to be sure they aren't the source of the problem.
The final item on the extracolumn effects list is the possibility of the use of too strong of an injection solvent, too large
of an injection, or both. My rule of thumb is that you can inject ≈15% of the volume of the first peak of interest if you
inject in mobile phase. So, for example, if the first peak is 0.1-min wide and the flow rate is 1 mL/min, the peak volume
is 100 μL. This translates to ≈15 μL for an injection volume if mobile phase is used as the injection solvent. If a stronger
solvent is used for injection, smaller volumes should be used; if a weaker solvent is injected, larger volumes may be possible.
The simplest way to check for injection problems is to inject a very small volume (for example, ≤5 μL) to see if the problem
is corrected. I've seen a case where a method worked well for years, then one day started giving broad or split peaks for
no apparent reason. In examining the conditions, it was discovered that too much of too strong a solvent was injected. By
diluting the injection solvent and increasing the injection volume to maintain the same injection mass, the problem was solved.
Unfortunately, too many methods are in use where the operating conditions are "right on the edge" of reliability — it takes
only a minor, and often unidentified, change in conditions to cause them to fail.
The preceding discussion followed a specific series of steps to solve the problem. In any particular case, some of these steps
could be eliminated by mental experiments or it may be more logical in your case to perform the test steps in a different
order. Additional questions might also be appropriate, such as asking if the method ever worked, or if it was being transferred
from another lab or a literature method. Do your best to eliminate possible causes by a bit of mental work before you start
doing physical experiments to correct the problem.
(1) J.W. Dolan, LCGC North Am.
29(11), 982–986 (2011).
(2) L.R. Snyder, J.J. Kirkland, and J.W. Dolan, Introduction to Modern Liquid Chromatography, 3rd ed. (Wiley, Hoboken, New Jersey, 2010).
John W. Dolan
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.Dolan@LCResources.com