Ultraviolet (UV) detectors are the most common liquid chromatography (LC) detector, and perhaps the most reliable ones. But
they are not without problems.
I recently received an e-mail question from a reader complaining of a leaking detector cell in the ultraviolet (UV) detector
attached to his liquid chromatography (LC) system. This seems like a good opportunity to address that specific question, other
UV detector problems, and some of the more recent advances in detector design. Although the present discussion centres on
UV detectors, many aspects will apply to other optical detectors, such as fluorescence or refractive index detectors.
First, let's take a look at how the typical detector cell is constructed and we'll be able to see where leakage can occur.
After we have that information, correcting the problem should be straightforward.
For UV detectors designed to operate with conventional LC systems with upper pressure limits of 6000 psi (400 bar), the construction
shown in Figure 1 is common. The cell itself is made by drilling a 1-mm hole through a 10-mm-long block of stainless steel.
To contain the liquid, a quartz window is attached to each end of the cell and a seal is formed with a polymeric gasket. The
mobile phase needs to pass through the cell, so a provision is made for this by drilling small-diameter (for example, ≤0.125
mm i.d.) holes to connect the outside world with the cell cavity. Most commonly, the entrance and exit holes are on opposite
sides and opposite ends of the cell so that the flow path is Z-shaped. This allows efficient washout and clearance of bubbles,
should they enter the cell. UV light then passes through the cell from one end to the other. When sample peaks pass through
the cell, some of the UV light is absorbed, and a photodiode measures this absorbance by the change in the intensity of light
passing through the cell.
Figure 1: Schematic of a conventional UV detector cell. See text for details.
The amount of light passing through the mobile phase at steady state (no sample present) is affected by the refractive index
of the mobile phase. Any change in refractive index will result in more or less light making it through the cell, and the
baseline will drift or exhibit other disturbances. To help mitigate temperature-related refractive-index disturbances, a heat
exchanger is usually fitted to the inlet of the cell. Most commonly this is a stainless steel capillary wrapped around the
body of the cell and covered with a heat-conducting material so that the fluid entering the light path is thermally stabilized,
and temperature-induced background disturbances are minimized.
Another potential problem is the presence of air bubbles in the flow cell. Even though the mobile phase is usually degassed
before use, when the mobile phase leaves the column, it moves from a high-pressure region to a near-atmospheric pressure region.
When this happens, any residual air present tends to outgas from the mobile phase and form physical bubbles. When air bubbles
enter the flow cell, they disrupt the light path and result in a noise spike or false peak in the chromatogram. Usually, bubbles
continue through the flow cell and clear by themselves, but tiny microbubbles sometimes become lodged in the corners of the
cell that are less well swept. These bubbles can "bounce" in the flow stream and cause additional baseline problems. To help
avoid bubble problems in the cell, pressure can be applied to the cell outlet so that the internal pressure in the cell is
sufficient to keep the bubbles in solution. Restricting the flow at the outlet of the flow cell can be somewhat delicate —
we want to have enough pressure to keep any bubbles in solution until they leave the cell, but we don't want so much pressure
that the cell leaks. The most common practice to provide back pressure on the cell is to use a piece of capillary tubing as
the waste line. A narrow capillary will restrict the mobile-phase flow and create pressure upstream. The pressure thus created
is dependent on the mobile-phase viscosity, temperature, and flow rate. Because pressure will increase with flow rate, there
is a risk that increased pump flow, such as might be used to flush a column, will create sufficient pressure to exceed the
flow-cell pressure limits (cell pressure limits should be listed in the "specifications" section of the detector manual, but
typically are in the 150-psi or 10-bar region). A better solution to provide back pressure on the cell is to use a back-pressure
restrictor that generates a constant pressure. These restrictors are constructed like a spring-loaded check valve that opens
when the pressure exceeds a set value less than the detector cell maximum. Back-pressure restrictors can be purchased from
most suppliers of LC tubing and fittings.