Getting Started in HPLC

Section 2E. HPLC Detectors

The detector measures the concentration of sample bands as they leave the column and pass through the detector flow cell. When no band is passing through the detector, a constant signal is recorded -- called the Baseline of the chromatogram or detector. When a sample band reaches the detector, the detector responds to the difference in the mobile phase properties caused by the presence of the sample compound, giving rise to a change in detector signal, seen as a Peak.

How does the detector work? In most cases we will be using a photometric detector, commonly called a UV detector. In its simplest form, this consists of a light source, a flow cell (or "sample cell"), and a light sensor as shown at the right.

Simplified schematic diagram of a UV detector.

When no sample is flowing through the detector, light passes through the flow cell and generates a maximum signal at the light sensor. If a sample band enters the detector, the sample reduces the amount of light reaching the sensor and causes a change in the detector signal. This signal is electronically inverted by the data system resulting in the appearance of a positive peak in the chromatogram. The signal displayed increases in proportion to the concentration of sample in the flow cell. The detector will also respond to other changes in the contents of the flow cell. For example, if particulates or "dirt" are trapped within the flow cell, these will interrupt the the light passing through the flow cell and cause a change in the baseline. Or an air bubble can be trapped in the flow cell, causing a large change in the amount of light passing through. In this case, the signal might go completely off-scale, then return to the original baseline if the bubble passes through the cell.

Two types of UV detectors are commonly used today:
  • Variable-wavelength (sometimes called "spectrophotometric" detectors
  • Photodiode Array (sometimes simply called "diode array" detectors.

Variable-wavelength detectors are less expensive; they are the standard detector type for quantitative analysis and routine assays. Photodiode array detectors are more versatile, because they allow simultaneous acquisition of both chromatographic and spectral information; they are frequently used in method development.

The detector wavelength is an important characteristic of an HPLC separation. As a general rule, the wavelength is set to the absorbance maximum of the analyte. Using the wrong wavelength may result in decreased peak sizes, or even no peaks at all!

Because different compounds can have different absorbance spectra, a direct quantitative comparison of different peaks in the same chromatogram can be misleading. A small quantity of a compound which absorbs strongly at the detector wavelength can give a bigger peak than a large quantity of a weak absorber. For reliable quantitation, a calibration must be carried out with a known quantity of the exact compound to be analyzed.

The Variable Wavelength UV Detector uses a monochromator (slits and a grating) to select one wavelength of light to pass through the sample cell.

The Photodiode Array Detector passes all wavelengths of light through the sample cell, then focuses each wavelength on a single sensor element.

There are a number of other settings or controls for the detector; these may be built in to the detector itself, or may be part of the data system:

The size of all the peaks in the chromatogram can be changed using the attenuator or range settings. The attenuation scale usually indicates the size of a peak which will appear as a full-scale peak on the display. This scale is measured in absorbance units (which are proportional to sample concentration); it is often expressed as "Absorbance Units Full Scale" (AUFS). The figure at the right illustrates the effect of a change in attenuation. As the attenuation setting is increased (AUFS is decreased), the detector becomes more sensitive, and all peaks increase in size.

On many systems, the range or attenuation can be set either on the detector itself or on the data system. If the attenuation or range is set on the data system, it usually affects only   the display. It does not   change the way quantitation is carried out because it does not change the information presented to the data system. If the attenuation or range is set at the detector itself however, it is possible that the size of large peaks may exceed the capabilities of the data system.



Another detector setting is the Baseline Zero (sometimes called the "Zero Offset"). Moving this setting (shown at the right) simply shifts the entire chromatogram up or down on the display. As with the attenuation setting, if the zero is adjusted at the data system, it affects the display only; quantitation remains unchanged. If the baseline zero is changed on the detector itself, integration may be compromised if the baseline is offset sufficiently low to send a negative voltage signal to the data system (most data systems can only deal with positive voltage signals).




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Last revised: April 06, 2001.