Sometimes RI detectors are used for different applications with either aqueous and nonaqueous solvents. When this is the practice,
be sure to flush the entire system (reservoirs, degasser, pump, autosampler, and detector) with a series of solvents that
are mutually miscible. For example, go from aqueous solvents to 100% acetonitrile or methanol, then to organic solvents. If
you are not sure of the history of the system, remove the column and replace it with a piece of capillary tubing. Then flush
the entire system with 20–30 mL of isopropanol, which is miscible with both aqueous and organic solvents. Then flush to the
desired mobile phase.
Baseline noise can be a critical factor with RI detection. Because RI inherently has poor sensitivity when compared to UV
or other detectors, signal-to-noise can be a limiting factor. For this reason, you may want to take advantage of larger detector
time constants (noise filters) with RI than with other detectors. A good rule of thumb is to set the detector time constant
at 10% of the peak width at baseline or 20% of the half-height width. For example, if the peak is 10 s wide at the baseline,
you can use a 1 s time constant. A higher time constant value smoothes the baseline, but too high a value will "smooth" off
the top of the peaks, making them broader and shorter.
If you are having a hard time distinguishing the source of a baseline problem between the pump and mobile phase as opposed
to a temperature-related problem, turn off the pump (or set the flow to 0 mL/min). This will eliminate the pump or mobile-phase
problem. If the baseline problem persists, it is because of changing temperature.
Because of its extreme sensitivity to temperature, a byword for RI detection is patience. It will take longer to equilibrate
the mobile phase, to warm up the detector, or settle down from any system change. For this reason, if time is critical, it
is prudent to leave the detector turned on and in a mobile-phase recycle mode. You can reduce the flow rate under these conditions,
if you desire, but this will leave the system in a standby mode that will return rapidly to normal operation.
If you are looking for alternatives to the RI detector for universal detection, consider evaporative light scattering detection
(ELSD) or charged-aerosol detection (CAD). Both of these detectors rely on evaporation of the mobile phase and then detection
of the "dust" that is left behind. Both ELSD and CAD can be operated with gradients, which is an additional advantage, but
they are restricted to mobile phases that are volatile — so no phosphate buffer is allowed.
And if all else fails . . . read the directions! If you are a normal user of other detectors, such as UV, fluorescence, or
MS, troubleshooting RI problems may not be second nature. Consult the operation and service manual for your specific detector
for troubleshooting and preventive maintenance instructions. If you'd like advice from other users regarding specific problems,
consult one of the on-line discussion groups, such as Chromatography Forum (
http://www.chromforum.org/) or the HPLC Users Group at LinkedIn (
Equation 2 of the October 2012 installment (B. Alsehli and J.W. Dolan, LCGC North Amer.
10, 898–902 ) contained an error and should have read as follows:
With this change, all the calculated values of peak widths and recommended injection volumes should be increased fourfold.
The discussion and conclusions are still valid. For a fully corrected version, see
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
John W. Dolan