7 UV-Visible Spectrophotometers
UV-visible spectrophotometers are instruments designed to quantitatively measure the amount of light absorbed by an analytical sample, which is then related to the identity and/or quantity of an analyte. Spectrophotometers come in single-beam and double-beam designs, and may also differ in the details of how absorption spectra are recorded. In most designs, the light source (Ch. 3) is either a combination of deuterium (UV emitting) and tungsten-halogen (visible emitting) lamps, or a xenon lamp (UV and visible emitting). The photodetector(s) (Ch. 5) can be a PMT, photodiode, photodiode array, or CCD. Instruments are characterized by their wavelength resolution, wavelength range, measurement speed, dynamic range, size, and cost, among other considerations.
Cuvettes
UV-visible absorption measurements are most commonly made on solution-phase samples in a transparent cell called a cuvette. Standard cuvettes have a square prism shape and are made from either plastic, glass, or quartz (i.e. silica). The principal difference between these materials is their transmissive ranges for UV and infrared light, with quartz having the broadest range and plastic having the least broad range. Plastic cuvettes are the most robust and lowest cost, whereas silica cuvettes are the most fragile and the most expensive.
Single-Beam Design
A single-beam design has a light source, a monochromator, a sample cell with known path length, and a photodetector along a common light path. A monochromator (Ch. 4), which can be placed either before or after the sample cell, is necessary because the Beer-Lambert Law is valid only for (approximately) monochromatic light. A blank sample and an analytical sample are measured sequentially. Absorbance spectra are measured by scanning the monochromator and calculating the transmittance at each wavelength step.
Double-Beam Design
A double-beam design has a light source and an adjacent monochromator. The light output from the monochromator is split into two pathways, each with its own sample cell. A blank sample and an analytical sample are measured concurrently, and absorbance spectra are measured by scanning the monochromator. There may be two photodetectors (i.e. one for each optical pathway) or a single photodetector that, via an optical chopper, measures the intensity of both beams in rapid succession. Fluctuation (i.e. noise) in incident light intensity is a significant limitation in measuring large and very small absorbance values. The double-beam design compensates for these fluctuations, greatly increasing precision and the reliable range of absorbance measurements. The trade-off for this increased performance is generally higher cost and larger size.
Array Detector Design
An array detector design has a light source and, after the sample, an entrance slit and grating (but no exit slit). The grating disperses the light across the array detector, which is usually a photodiode array (PDA) or a CCD. An exit slit is not required because each diode element in the PDA or pixel in the CCD is small enough to act as its own slit and detect only a narrow range of wavelengths. The array detector design is advantageous in that it is robust (no moving parts), acquires full spectra rapidly (no scanning required), and supports compact designs.
Connections
- Spectrophotometer designs use combinations of the light sources (Ch. 3), wavelength selectors (Ch. 4), and photodetectors (Ch. 5) learned about in previous chapters.
- The design of a spectrophotometer enables practical application of the Beer-Lambert Law (Ch. 6).
- Since light absorption is the first step in generating fluorescence (Ch. 8), some aspects of the design of a spectrophotometer will reappear in the design of spectrofluorometers (Ch. 9).
- UV-visible spectrophotometers have similar designs and components as those used for atomic spectroscopy (Ch. 11).
Post-Reading Questions
- Explain why the material from which a cuvette is made of matters.
- Explain why a tungsten-halogen lamp needs to be combined with a deuterium lamp for UV-visible spectrophotometry but a xenon lamp does not. (Hint: You may wish to revisit Ch. 3.)
- What is an advantage of a double-beam spectrophotometer design?
- What is an advantage of a detector-array spectrophotometer design?
- Explain the role of a monochromator or polychromator in a spectrophotometer design.
Topic Learning Objectives
The chapter is a primer for the following learning objectives, which will be covered in lecture and/or with additional assigned reading:
- Explain the role for a blank measurement and apply best practices in selecting a blank.
- Be able to select a suitable cuvette material with a particular wavelength range or chromophore of interest.
- Explain why absorbance measurements are best made at the peak of a molecular absorption band.
- Draw diagrams for single-beam and double-beam spectrophotometer designs.
- Draw diagrams for scanning and array detector spectrophotometer designs.
- Explain the operating principles of how different instrument designs record a UV-visible absorption spectrum.
- Relate instrument parameters with spectral resolution.
- List common sources of error and interference in spectrophotometric measurements.
- Explain when and how a double-beam spectrophotometer design improves analytical figures of merit.