A second factor that affects refractive index is pressure. For the quietest baselines, the pressure in the flow cell needs
to be constant. Most RI flow cells have an upper pressure limit of no more than approximately 100 psi (7 bar), and the use
of a back-pressure restrictor after the cell is common. A back-pressure restrictor can be thought of as a spring-loaded check
valve that maintains a fixed pressure, such as 75 psi (5 bar), at all times. This will keep the pressure constant and also
will keep the system from exceeding the maximum cell pressure. A piece of capillary tubing after the flow cell also can function
as a back-pressure restrictor, but the pressure will be related to the flow rate — if the flow rate is inadvertently set too
high, a capillary restrictor may cause the permissible cell pressure to be exceeded.
Because LC systems are operated in a constant-flow mode, the pressure should be constant. This usually is the case, but problems
with the pumps can cause the pressure to fluctuate sufficiently that the baseline is disturbed, even though other problems
such as retention-time shifts are not observed. Pressure problems because of pump malfunctions often will create cycling baselines.
To confirm this, you can change the flow rate and the frequency of the baseline cycle should change in accordance to the flow-rate
change. For example, a change from 1 mL/min to 2 mL/min should double the frequency of the baseline cycle. Common sources
of pressure fluctuations are faulty check valves, leaky pump seals, air bubbles in the pump, and more rarely a broken pump
piston. The easiest things to check are bubbles in the pump and degassing problems — make sure the degasser is working properly,
then purge the pump to release any trapped bubbles and resume operation. Check-valve sonication in methanol for a few minutes
often will clean a dirty or sticking check valve, or the check valve can be replaced. Pump seal replacement is a little more
work, but is something that can be done by following the instructions in the pump service manual.
Any change in the chemical composition of the mobile phase will change its refractive index, as will the presence of dissolved
air in the mobile phase. For these reasons, RI detectors are always operated only in the isocratic (not gradient) mode and
the mobile phase must be thoroughly degassed. If you have an in-line degasser, as is the case for most LC systems today, be
sure to use it. Otherwise, helium sparging is suggested to degas the mobile phase. Because of the extreme sensitivity of the
detector to very small changes in refractive index, on-line mixing of the mobile phase usually will create problems. As a
result, mobile phases must be hand-mixed so that no change in mobile phase composition occurs within the LC system. It is
best to use the mobile phase as the injection solvent so the refractive index change at the column dead-time is minimized.
Remember that the RI detector measures the difference in refractive index between the contents of the sample and reference
cells, so the reference cell needs to be purged with fresh mobile phase whenever the mobile phase is changed or replaced with
a fresh batch. It is a good idea to purge the reference cell daily to ensure its contents are matched with the mobile phase
exiting the column. When changing from one mobile phase to another or washing the mobile phase into a new column, complete
equilibration may take longer than you normally allow with UV detection. With UV and most other detectors, allowing 10 column
volumes of mobile phase (˜≈15 mL for a 150 mm × 4.6 mm column) to pass through the column is sufficient for equilibration.
It may take longer with the RI detector. Watch the baseline when changing the solvent; drift is common during solvent changeover,
so a nondrifting baseline is a good indicator of column equilibration with RI detection. As mentioned above, be sure to change
the mobile phase regularly to avoid problems with microbial growth, especially in highly aqueous mobile phases.