Incandescent, Arc, and Cathode Lamps: The light source for measurements in the visible portion of the spectrum is usually a tungsten light bulb. The lifetime of a tungsten filament is greatly increased by the presence of a low pressure of iodine or bromine vapor within the lamp. An example is the quartz-halogen lamp, which has a fused-silica envelope and which provides high-intensity light over a wide spectrum for extended operating periods (2000 to 5000 hours) before replacement is necessary.

However, a tungsten light source does not supply sufficient radiant energy for measurements in the UV region (below 320 nm). In the UV region of the spectrum, hydrogen and deuterium lamps, as well as high-pressure mercury and xenon arc lamps, provide sources of continuous spectra in the UV region with some sharp emission lines. These sources are more commonly used in UV absorption measurements. Low-pressure mercury vapor lamps also provide spectra in the UV region and are useful for calibration purposes, but they are not very practical for absorbance measurements, because of their limited wavelengths.

Mercury arc lamps emit an intense 254-nm resonance line and are widely used as detectors in high-performance liquid chromatography (HPLC) (see Chapter 13). Alternatively, some HPLCs employ a miniature hollow cathode lamp as a very-narrow-wavelength intense source. For example, a zinc hollow cathode lamp emits a line at 214 nm that is close to the maximum wavelength of peptide bond absorption (206 nm); this emission property permits the usage of such lamps to measure peptides and proteins. Details on the hollow cathode lamp are found in the section on “Atomic Absorption Spectrophotometry.” The hollow cathode lamp also has a long, useful lifetime if a lower-current, nonpulsed power supply is used.

Laser Sources: A laser (light amplification by stimulated emission of radiation) is a device that controls the way that energized atoms release photons; lasers are used as light sources in spectrophotometers because they provide intense light of a narrow wavelength. Through selection of different materials, different wavelength(s) of light are emitted by different types of lasers (Table 10-3).

Three properties of laser sources distinguish them from “conventional” sources: (1) spatial coherence is a property of lasers that allows beam diameters in the range of several microns; (2) lasers produce monochromatic light; and (3) lasers have pulse widths that vary from microseconds (flash lamp–pulsed lasers) to nanoseconds (nitrogen lasers) to picoseconds or less (mode-locked lasers) in duration. Air-cooled argon ion lasers produce about 25 mW of energy output at 488 nm and have plasma tube lifetimes of 6000 hours or longer. Continuous-wave dye lasers typically use an argon ion laser with an output of 1 W or less as an energy pump and use different fluorescent dyes to achieve excitation wavelength ranges of 400 to 800 nm. Helium-neon and helium-cadmium lasers are useful because of their low cost and ease of operation, and because they emit a number of excitation wavelengths; however, the power output of helium-neon lasers has been limited to about 2 mW at 594 nm.

Infrared diode lasers are used in compact disc players and laser printers, and in bar code readers (see Chapter 19). They are solid-state devices, typically constructed of gallium arsenide, and energy is pumped into them at a low potential of −1.5 V. Depending on its construction, the wavelength output of the laser ranges from 550 to 1810 nm. Development of inexpensive near-IR lasers has led to interest in using reflective techniques in the near-IR region of the spectrum (0.8 to 2.5 µm wavelength). Reflectance spectrophotometry is now used clinically for the transcutaneous measurement of bilirubin in neonates.¹⁴ Another application of reflectance spectrophotometry is measurement of blood oxygen saturation in near-IR and IR regions.

Filters: The simplest type of filter is a thin layer of colored glass. Certain metal complexes or salts, dissolved or suspended in glass, produce colors corresponding to the predominant wavelengths transmitted. The spectral purity of a filter or other monochromator is usually described in terms of its spectral bandwidth. This is defined as the width, in nanometers, of the spectral transmittance curve at a point equal to one half the peak transmittance. Because glass filters have spectral bandwidths of approximately 50 nm, they are referred to as wide-bandpass filters.

Other glass filters include narrow-bandpass and sharp-cutoff types. Operationally, a cutoff filter typically shows a sharp rise in transmittance over a narrow portion of the spectrum and is used to eliminate light below a given wavelength. Historically, narrow-bandpass filters were constructed by combining two or more sharp-cutoff filters or regular filters. Currently, however, the availability of high-intensity light sources now favors the use of narrow-bandpass interference filters.

A narrow-bandpass interference or dichroic filter uses a dielectric material of controlled thickness sandwiched between two thinly silvered pieces of glass. The thickness of the layer determines the wavelength of energy transmitted after constructive and destructive wavelength interference caused by reflections between the glass surfaces separated by the dielectric spacing. These filters have narrow spectral bandwidths, usually from 5 to 15 nm. Because they also transmit harmonics, or multiples, of the desired wavelength, accessory glass filters are required to eliminate undesired wavelengths. Thus an interference filter designed for 620 nm will also transmit some radiation at 310 and 1240 nm unless accessory cutoff filters are provided to absorb this undesired stray light.

Prisms and Gratings: Prisms and diffraction gratings are widely used as monochromators. A prism separates white light into a continuous spectrum because shorter wavelengths are bent, or refracted, more than longer wavelengths as they pass through the prism. A diffraction grating is prepared by depositing a thin layer of aluminum-copper alloy on the surface of a flat glass plate, then ruling many small parallel grooves into the metal coating. Better gratings contain 1000 to 2000 lines/mm and must be made with great care. These are then used as molds to prepare less expensive replicas for general use in instruments.

Modern holographic gratings are made using a laser in a “high-precision machining” mode. The focused beam of the laser is accurately scanned over a photosensitive material termed a photoresist. After multiple lines have been scribed on the photoresist, chemicals are used to dissolve and elute the exposed photoresist to create channels that become the lines of the grating. A layer of a highly reflective material is then sputtered onto the surface of the laser-etched channels, and the grating is ready for use. A flat photoresistive surface or a concave surface can be used to make this type of grating. These types of gratings are extremely accurate, have low light scatter, and are widely used in the spectrophotometers found in clinical chemistry instruments. For example, most UV-visible spectrophotometers and virtually all IR spectrophotometers use reflective gratings. In addition, HPLC detectors frequently use a concave holographic reflective grating in their optical system.

Each line ruled on the grating, when illuminated, reflects light and gives rise to a tiny spectrum. An array of parallel wave fronts are formed that reinforce those wavelengths in phase and cancel those not in phase. The net result is a uniform linear spectrum. Some instruments contain diffraction gratings that produce spectral bandwidths of 20 nm or more; higher-priced instruments may have a resolution of 0.5 nm or less.

The flat surface grating discussed previously is called a plane transmission grating. Lines are engraved on the surface of a mirror, which may be a polished metal slab or a glass plate on which a thin, metallic film has been deposited. A grating may also be ruled at a specified angle, so that a maximum fraction of the radiant energy is directed into wavelengths diffracted at a selected angle. This type of grating is called an echelette and is said to have been given a blaze at a particular angle or to have been blazed at a certain wavelength (e.g., 250 nm).

Selection of a Wavelength Isolation Device: The type of monochromator chosen depends on the analytical purpose for which it is to be used. For example, narrow spectral bandwidths are required in spectrophotometers for resolving and identifying sharp absorption peaks that are closely adjacent. Lack of agreement with Beer’s law will occur when a part of the spectral energy transmitted by the monochromator is not absorbed by the substance being measured. This is more commonly observed with wide-bandpass instruments. In practice, an increase in absorbance and improved linearity with concentration are usually observed with instruments that operate at narrower bandwidths of light. This is especially true for substances that exhibit a sharp peak of absorption.

The natural bandwidth of an absorbing substance is defined as “the bandwidth of the spectral absorbance curve at a point equal to one half of the maximum absorbance.” As a general rule, for peak absorbance readings to be within 99.5% of true values, the spectral bandwidth should not exceed 10% of the natural bandwidth. For example, many chemistry procedures used in the clinical laboratory produce an absorbing species for which the natural bandwidth ranges from 40 to over 200 nm. The natural bandwidth of NADH is 58 nm (λmax = 339 nm). Therefore, for accurate measurements of this compound, a spectral bandwidth of 6 nm or less should be used.

In practice, the wavelength selected is usually at the peak of maximum absorbance to attain the maximum measurement; however, it may be desirable to choose another wavelength to minimize interfering substances. For example, turbidity readings on a spectrophotometer are greater in the blue region than in the red region of the spectrum, but the latter region is chosen for turbidity measurements to avoid absorption of light by bilirubin (460 nm) or hemoglobin (417 and 575 nm). The absorbing species developed in the alkaline picrate procedure for creatinine produces a relatively flat peak in the visible region of the spectrum at approximately 480 nm, but the reagent blank itself absorbs light strongly below 500 nm. A compromise is made by selecting a wavelength at 520 nm to minimize the contribution of the blank. Blank readings should, of course, be kept to a minimum. A small difference between two large numbers is subject to greater uncertainty; hence minimizing absorbance of the blank improves precision and accuracy. The linear working range of a method can be expanded by not measuring at the peak absorbance. However, measurements should not be taken on the steep slope of an absorption curve, because a slight error in wavelength adjustment would introduce a significant error in absorbance readings.

Photomultiplier Tubes: A photomultiplier tube (PMT) contains (1) a cathode, (2) a light-sensitive metal, and (3) a series of dynodes, all of which are enclosed in an evacuated glass enclosure. As many as 10 to 15 stages or dynodes are present in common photomultipliers. Photons that strike the photoemissive cathode emit electrons that are accelerated toward the dynodes. Additional electrons are generated at each dynode. Depending on the number of dynodes and the accelerating voltage, the cascading effect creates 10⁵ to 10⁷ electrons for each photon hitting the first cathode. This amplified signal is finally collected at the anode, where it can be measured.

When such a tube is operated, voltage is applied between the photocathode and each successive stage. The normal incremental increase in voltage at each photomultiplier stage is from 50 to 100 V larger than that of the previous stage (Figure 10-5). Typically, a conventional PMT tube has approximately 1500 V applied to it.

PMTs have (1) extremely rapid response times, (2) are very sensitive, and (3) are slow to fatigue. Because these tubes are very sensitive and have a rapid response, they must be carefully shielded from all stray light. A PMT with the voltage applied should never be exposed to room light because it will burn out. The rapid response times of PMTs are needed when a spectrophotometer is being used to determine an absorption spectrum of a compound. Also, PMTs are sensitive over a wide range of wavelengths.

When voltage is applied to a PMT in the absence of any incident light, some current is usually produced. This current is called dark current. It is desirable to have the dark current of a PMT at its lowest level because this current appears as background noise.

Photodiodes: Photodiodes are solid-state photodetectors that are fabricated from photosensitive semiconductor materials such as (1) silicon, (2) gallium arsenide, (3) indium antimonide, (4) indium arsenide, (5) lead selenide, and (6) lead sulfide. These materials absorb light over a characteristic wavelength range (e.g., 250 nm to 1100 nm for silicon). Their development and use as detectors in spectrophotometers have resulted in instruments capable of measuring light at a multitude of wavelengths. When a photodetector consists of two-dimensional arrays of diodes, it is known as a photodiode array. Each photodetector within the array responds to a specific wavelength. For example, photodiode arrays have been designed to have a 2-nm resolution per diode from 200 to 340 nm, and a 1-nm resolution per diode from 340 to 800 nm.

In practice, all diodes are initially charged to 5 V, and they discharge when they are struck by light. Each diode then is sequentially scanned and recharged to 5 V. The amount of energy required for recharging is proportional to the quantity of light striking that diode. Because scan time for all diodes is in the millisecond range, many scans are typically taken. The resultant data is processed using a variety of algorithms, including signal averaging, background subtraction, and correction for scattered light. Consequently, an optical spectrum of an ongoing chemical reaction can be monitored as a function of time with a high degree of resolution and accuracy.