UV-Vis Frequently Asked Questions - Instrument Design

UV-Vis FAQ Index Page  

How does a spectrophotometer function?

Spectrophotometer component breakdown

There are two basic designs for a typical dispersive type spectrophotometer, the single beam and the double beam type instrument.

The term dispersive indicates that the instrument “disperses” white light with either a prism or diffraction grating monochromator.

There are four basic components to a simple single beam UV/Vis spectrophotometer; a light source, a monochromator, a sample, and a detector.

The monochromator of the instrument is composed of an entrance slit (to narrow the beam to a usable size), a dispersion device (usually a diffraction grating or prism that separates polychromatic white light into bands of monochromatic light of a single wavelength), and an exit slit (to select the desired monochromatic wavelength).

The figure in this slide shows a simple single beam optical design for a UV/Vis instrument.

What are the light source requirements for a spectrophotometer?

 

The requirements for a spectrophotometer light source include:

  1. Bright across a wide wavelength range
  2. Stable over time
  3. Long service life
  4. Low cost
 

Many light sources meet some of the requirements on this slide, but no light source can meet them all. Many spectrophotometers switch between a halogen lamp for the visible range and a deuterium lamp for the ultraviolet range according to the wavelength setting. This is because of the difficulty in achieving both "a high degree of brightness" and “a uniform brightness distribution" across a wide wavelength range using a single light source. Switching between light sources with different emission wavelength ranges also offers the advantages of reducing the excess incident light into the monochromator and reducing the amount of stray light. Other instruments use a xenon lamp or xenon flash lamp suitable for the analysis target and aim of the analysis. A low- pressure mercury lamp that produces multiple emission spectra is effective for spectrophotometer wavelength calibration.

What types of light sources are used in a typical spectrophotometer?

 
 
 Tungsten-Halogen Lamp

Spectrophotometer Tungsten-Halogen Lamp

Deuterium Lamp 

Spectrophotometer Deuterium Lamp

You need a light source which gives the entire visible spectrum plus the near ultraviolet so that you are covering the range from about 175 nm all the way out to 3300 nm. You can’t get this range of wavelengths from a single lamp, and so a combination of two is used, a deuterium lamp for the UV part of the spectrum, and a tungsten/halogen lamp for the visible part. The tungsten lamp emits light from about 340 nm in the UV region up to over 3500 nm in the near infrared. These lamps are similar to the high intensity tungsten/halogen lamps you find in the home (Left).

The deuterium lamp (right) is a gas discharge light source that uses deuterium gas (an isotope of hydrogen that contains an additional neutron in its nucleus). A deuterium lamp emits light in the near ultraviolet and ultraviolet regions from 150 nm to 400 nm (Right). A deuterium lamp contains deuterium gas (an isotope of hydrogen) under low pressure subjected to a high voltage. It produces a continuous spectrum in the part of the UV spectrum we are interested in. The combined output of these two sources is focused on to the monochromator entrance slit.

What is the output of a halogen lamp?

 

Spectrophotometer Halogen Lamp output - wavelength vs relative intensity

Similar to a normal incandescent lamp, a halogen lamp filament heats up and emits light when current flows through it. The tungsten used as the filament material evaporates at high temperatures. Consequently, the bulb containing the filament of a normal incandescent lamp is filled with an inert gas to prevent evaporation of the tungsten.

A halogen lamp contains a halide as well as the inert gas to create the halogen cycle that returns evaporated tungsten to the filament, resulting in a long lamp life. It also restricts blackening of the tube wall, due to adhering evaporated tungsten, to create a light source that remains bright over long periods. Tungsten that evaporates at high temperatures binds with halogen near the cool tube wall to form tungsten halide. The suspended tungsten halide moves inside the tube due to convection and separates into halogen and tungsten near the hot filament. The separated tungsten adheres to the filament and the halogen bonds again with evaporated tungsten. This repeated reaction is known as the tungsten cycle.

The figure here shows the light intensity distribution at 3000 K color temperature. The usable wavelength range is 350 nm to 3500 nm, but this is affected by the color temperature.

Halogen lamps are stable over time, offer a long service life (approx. 2000 hours) and are relatively inexpensive. As such, they many of the conditions required for a spectrophotometer light source.

What is the energy distribution of a deuterium lamp?

energy distribution of a deuterium lamp

 

A deuterium lamp is a discharge light source with high pressure (several hundred Pa) of deuterium sealed in a bulb. As it uses a hot cathode to achieve stable and reliable arc discharge, approximately 10 seconds for preheating is required before starting the discharge. A deuterium lamp requires a large and complex power supply, making it more expensive than a halogen lamp. However, it is one of the few continuous spectrum light sources that is stable in the ultraviolet range.

The deuterium lamp has a short emission wavelength of 400 nm, or less. The window material limits its use at the short wavelength end. The figure here shows examples using synthetic quartz and UV glass.

The use at the long-wavelength end is limited to about 400 nm. However, the low degree of attenuation toward the long- wavelength end permits use of light above 400 nm. Multiple emission spectra also exist in the range at 400 nm and above. Of these, the spectra at 486.0 nm and 656.1 nm are particularly strong and can be used for wavelength calibration of the spectrophotometer.

What does a low-pressure mercury lamp do?

 

Spectral distribution of a low-pressure mercury lamp used in wavelength calibration

The low-pressure mercury lamp is a discharge lamp designed to have a low mercury vapor pressure (100 Pa max.) when lit to efficiently emit the mercury resonance lines (254 nm or 185 nm). The figure here shows the spectral distribution of a low-pressure mercury lamp. Low-pressure mercury lamps are available in versions that use the emitted ultraviolet lights directly, or as so-called fluorescent lamps that use a fluorescent material to convert the wavelength to a different wavelength.

A spectrophotometer uses the mercury emission lines to calibrate the displayed wavelength values. The 254 nm, 365 nm, 436 nm, or 546 nm emission lines can be used for the calibration, but care is required with the slit width (spectral bandwidth) used during measurements. For example, as the 365 nm emission line is a triple line (three emission lines in proximity), the spectral bandwidth must be 0.5 nm max. to accurately measure the respective emission lines.

What is “lamp switching” in a spectrophotometer?

 

light source energy distribution measured by the UV-1900 spectrophotometer

As stated above, halogen lamps and deuterium lamps are used in many spectrophotometers.

The graph at left shows their respective energy distributions measured by a UV-1800 UV-VIS Spectrophotometer. The light sources are switched near 300 nm to 350 nm, where the emission intensities of the halogen lamp and deuterium lamp are approximately equal. The light sources can be switched by moving the lamps themselves or by rotating a reflector.

The figure at right shows the switching method by rotating a reflector. By changing the tilt of the reflector positioned between the halogen lamp and the deuterium lamps, the light beam that enters the monochromator can be switched. In most instruments, the optimal tilt with respect to each light source is automatically adjusted during the initial setup operation after the power is turned on, which eliminates the need for positional adjustment by replacing lamps.

What component detects the light in a spectrophotometer?

 

various detectors and wavelength ranges in nm

The mechanism for sensing light and converting it to signals that we are most familiar with is the human optic nerve. The human eye senses light in a wavelength range of approximately 400 to 700 nm, and sends signals to the brain through nerve tissue. You could say that the eye is the optical detector of visible light that we are most familiar with. The human eye is sensitive to light in the visible region, and is most sensitive to green light with a wavelength of around 550 nm. In the same way, the detectors in spectrophotometers also have a wavelength range that they can be used for, and their sensitivity varies with the wavelength. Representative detectors with sensitivity in the ultraviolet and visible region include the photomultiplier tube and the silicon photodiode. Regarding near-infrared detectors, PbS photoconductive elements were used exclusively in the past, although nowadays there are instruments in which InGaAs photodiodes are used for part of the near-infrared region. The bottom figure shows the relationship between various detectors and wavelength ranges.

The detector converts the incoming light into an electrical current that can be quantitated. The higher the current, the greater the intensity of the light. For each wavelength of light passing through the spectrometer, the intensity of the light passing through the sample cell is measured. The most common type of light detector in UV/Vis spectrophotometers is the photomultiplier tube (PMT). The wavelength range for PMT’s is from 150 nm to 900 nm, although the region between 850 nm to 900 nm is marginal. PMT’s are one of the most sensitive light detectors made. In many cases a PMT can detect a single photon (above). Under the photon theory of light, a photon is a discrete bundle (quantum) of electromagnetic (light) energy. Photons are always in motion and, in a vacuum, have a constant speed of light to all observers, at the vacuum speed of light of c = 2.998 x 108 m/s.

The chart at the bottom displays the functional wavelength envelopes of the various detectors found in UV/Vis and UV/Vis/NIR spectrophotometers.

How does a photomultiplier tube work?

 

Photomultiplier diagram

PMT’s function via the photoelectric effect. In the photoelectric effect, electrons are emitted from certain types of metals as a consequence of their absorption of a photon of light energy, such as visible or ultraviolet light (at left). PMT’s function by a photon striking the first dynode, which releases electrons into a dynode chain with progressively higher voltages. This causes an electron cascade down the dynode chain until the electrons hit the anode which generates the electrical charge that can be measured. The net result is one photon releases many electrons onto the anode (see picture above). PMT detectors multiply the current produced by incident light by as much as 100 million times (i.e., 160 dB), in multiple dynode stages, enabling, for example, individual photons to be detected even when the incident light flux is very low.

A photomultiplier tube utilizes the external photoelectric effect, the phenomenon whereby photoelectrons are discharged when light strikes a photoelectric surface. This slide illustrates the operating principle of a photomultiplier tube. Photoelectrons discharged from a photoelectric surface (i.e., primary electrons) cause the successive emission of secondary electrons from dynodes (electron-multiplier electrodes) arranged in multiple stages, and this cascade ultimately reaches an anode. If one primary electron causes the emission of δ secondary electrons, and this process is repeated n times, then a multiplication factor of δn is obtained. Because photomultiplier tubes ultimately produce a large output for a low level of light intensity, their most important feature is that they offer an outstanding level of sensitivity, which cannot be obtained with other optical sensors. δ is referred to as the "secondary emission coefficient." A high voltage (-HV) is applied from outside the tube in order to accelerate the electrons.

The higher the value of this voltage, the larger the secondary emission coefficient. Another feature of a photomultiplier tube, then, is that the multiplication factor can be adjusted by controlling this high voltage. If there is sufficient light intensity, the voltage is decreased. If the light intensity decreases, the voltage is increased. If the slit is changed, or if accessories that cause significant decreases in light intensity, such as integrating spheres, are used, the advantages offered by this photomultiplier tube become particularly important. For this reason, photomultiplier tubes are used in high-grade instruments.

What is a detectors spectral sensitivity curve?

 

Spectral sensitivity characteristic of a photomultiplier tube

The relationship between the sensitivity of a photoelectric surface and the wavelength of incident light is referred to as the "spectral sensitivity characteristic." It is mainly determined by the material of the photoelectric surface. This slide shows the spectral sensitivity characteristic of a multi-alkali photoelectric surface that has sensitivity in the ultraviolet and visible region. There are other “types” of PMT’s that shift the detectors peak sensitivity into different wavelength regions. Picture the curve presented here “sliding” the sensitivity curve to higher or lower wavelengths. The curve presented here is a “red sensitive” R-928 PMT.

How does a silicone photodiode detector work?

Energy model of silicon photodiode and spectral sensitivity characteristic of silicon photodiode

An alternative type of light detector is the solid-state diode detector. Silicone diode detectors have a greater wavelength range than a PMT, usually from 180 nm to 1100 nm. Unlike PMTs, diodes do not require a high voltage power supply (expensive). And finally the are more robust in being able to deal with high light intensities without saturating (overloading). The NIR region of UV/Vis/NIR instruments uses two different types of solid-state detectors.

A silicon photodiode utilizes the internal photoelectric effect, the phenomenon whereby the electrical properties of the detector itself change when light strikes it. As the name suggests, a silicon photodiode is a semiconductor. When light strikes this semiconductor, if the energy of the light is larger than the band gap, electrons in the valence band are excited into the conduction band, and holes are left in the original valence band. As shown at left, these electron-hole pairs are created throughout the semiconductor, but in the depletion region, the electric field causes electrons to be accelerated toward the N- region and holes to be accelerated toward the P-region. As a result, electrons accumulate in the N-region and holes accumulate in the P-region, and the two regions become, respectively, negatively and positively charged. If this is connected to a circuit, current flows. The band gap of silicon is approximately 1.12 eV, so current flows only for wavelengths that have an optical energy greater than this. This works out to a wavelength upper limit of around 1,100 nm.

The lead sulphide NIR detector has been the industry standard for over 40 years. It functions through the entire near infra-red range of 860 nm to 3300 nm and is fairly inexpensive. The newer wide band indium gallium arsenide (InGaAs) detector is more expensive but has over two orders of magnitude more sensitivity with less noise than the older PbS detector. When a wide band InGaAs detector is cooled to -50 degrees C it has a usable wavelength range of 800 nm to 2500 nm. Narrow band InGaAs detectors cover the range of 800 nm to 1600 nm.

The graph on the right shows the spectral sensitivity characteristic of a silicon photodiode.

Silicon photodiodes have some advantages over photomultiplier tubes: they are less expensive; there is little unevenness of sensitivity over their light-receiving surfaces; and they do not require a dedicated power supply. Even with respect to sensitivity, if the light intensity is relatively high, they can provide photometric data that is by no means inferior to that obtained with photomultipliers. If the light intensity is relatively low, however, because signals are amplified in the electronic circuit that gives a current, increasing the amplification factor decreases the response speed.

What does an InGaAs detector sensitivity curve look like?

 

InGaAs detector sensitivity curve graph

Indium gallium arsenide (InGaAs) is a compound semiconductor. Like a silicon photodiode, an InGaAs photodiode is a photovoltaic element that has a P-N junction. The band gap energy of InGaAs, however, is smaller than that of silicon, so it absorbs light of longer wavelengths. This means that InGaAs photodiodes are sensitive to wavelengths that exceed the range of silicon photodiodes. The graph displayed in this slide shows the spectral sensitivity characteristic of an InGaAs photodiode.

How does a PbS photoconductive detector work?

 

Operating principle of photoconductive element and spectral sensitivity characteristic of PbS Photoconductive element

A photoconductive element is a photoelectric conversion element that utilizes the phenomenon of photoconduction, whereby the electrical conductivity (resistance) of a material changes when it is irradiated with light. The figure on the left illustrates the operating principle. When light of energy greater than the energy gap between the conduction band and the valence band strikes the element, electrons in the valence band are excited into the conduction band, and holes are created in the valence band. With a PbS photoconductive element, the resistance is reduced in accordance with the intensity of incident light, and this is obtained as a signal using an external circuit.

If the element is cooled, the spectral sensitivity characteristic shifts to the long-wavelength end; as a result, the element becomes more sensitive to longer wavelengths. At the same time, however, the response speed decreases. Although PbS photoconductive elements can, unlike some other near- infrared detection elements, be used at room temperature, they are still delicate elements for which the sensitivity, response speed, and dark resistance change according to the temperature. The graph on the right shows the spectral sensitivity characteristic of a PbS photoconductive element. Note the wavelength axis is in micrometers, so 1 micrometer is equal to 1000 nanometers.

What is the function of the monochromator?

Monochromator diagram

 

The earliest type of light dispersion (separating) device known was the raindrop. Light changes speed as it moves from one medium to another (for example, from air into the water of a raindrop). Rain disperses the white light from the sun in its component colors (wavelengths) as a rainbow. Next best is the prism. This speed change causes the light to be refracted and to enter the new medium (air to glass) at a different angle (Huygens principle). The degree of bending of the light’s path depends on the angle that the incident beam of light makes with the surface, and on the ratio between the refractive indexes of the two media types (Snell’s law). The refractive index of many materials (such as glass) varies with the wavelength or color of the light used, a phenomenon known as dispersion. This causes light of different colors to be refracted differently and to leave the prism at different angles, creating an effect similar to a rainbow. This can be used to separate a beam of white light into its constituent spectrum of colors (top).

You are probably familiar with the way that a prism splits light into its component colors. A diffraction grating does the same job, but more efficiently. Diffraction manifests itself in the apparent bending of light waves around small obstacles or the spreading out of waves past small openings. In optics, a diffraction grating is a reflecting optical component with a periodic structure of a tiny regular series of ruled lines, which splits and diffracts light into several beams traveling in different directions (above).

The angles of the incident and diffracted beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as the dispersive element. Because of this, gratings are commonly used in monochromators of spectrometers. When there is a need to separate light of different wavelengths with high resolution, then a diffraction grating is most often the tool of choice. This “super prism” aspect of the diffraction grating leads to applications in measuring light spectra in both laboratory instruments and telescopes.

You see an example of a diffraction grating almost every day. The tracks of a compact disk act as a diffraction grating, producing a separation of the colors of white light. The nominal track separation on a CD is 1.6 micrometers, corresponding to about 625 tracks per millimeter. This is in the range of ordinary light diffraction gratings. For red light of wavelength 600 nm, this would give a first order diffraction angle of about 22° (bottom).

Most of the shiny iridescent colors seen in insects (butterfly wings) is the result of diffraction rather than from chemical dyes or pigments. Insect diffraction is the result of tiny scales on the body or wings that function like a diffraction grating. An integral part of the monochromator is the exit slit, which only allows light of a very narrow range of wave- lengths through into the rest of the spectrometer. By gradually rotating the diffraction grating, you can allow light from a portion of the spectrum (a tiny part of the wavelength range at a time) through into the rest of the instrument.

The monochromator comprises a dispersive element consisting of an entrance slit and mirrors to create a parallel beam similar to sunlight, and an exit slit and mirrors to extract the monochromatic light.

Which is better: prism or diffraction grating?

 
  Prism Reflective Diffraction Grating
Dispersion Principle Exploits differences in the material refractive index according to the wavelength   Exploits diffraction from a reflective surface with a regular grating structure.  
Light Utilization Efficiency High (Generally has high efficiency despite light losses from boundary reflection and absorption during transmission through the material. A single prism covers the range from 185 to 2500 nm.) Low (Light with the same wavelength is dispersed in several directions as higher-order light. High efficiency near the blaze wavelength.)  
Wavelength Dependency of Dispersion Variable. High for UV; low fo visible to NIR light.   High and approximately constant.
Temperature Dependency of Dispersion High (Effects of temperature on refractive index.)   Low (Deformation due to temperature.)
Higher-Order Light None Yes (Requires higher-order light cutout filter.)  
Stray Light Low High (Dispersion due to higher-order light and surface roughness. Modern diffraction gratings achieve comparatively low stray light.)  
Polarization Low High  

The prism and diffraction grating are typical dispersive elements. The table here shows their respective features. Due to their superior dispersion properties, diffraction gratings are often used in modern spectrophotometers. The prism achieves dispersion due to the difference in the material refractive index according to the wavelength. However, the diffraction grating uses the difference in diffraction direction for each wavelength due to interference.

How do diffraction gratings work?

 

Diffraction grating with row of slits and reflective blazed diffraction grating diagram

The first diffraction gratings were often a row of slits which functioned as a transmission grating, as shown at left. Modern diffraction gratings are a reflective blazed grating type that has a sawtooth cross-section, as shown at right. As light that passed through an adequately fine slit is diffracted, so light reflected from an adequately fine sawtooth surface is also diffracted. There are 500 to 2000 serrations per millimeter.

In the past, the sawtooth face of a commercially produced diffraction grating is the replica of a master grating. A thin synthetic-resin replica is stuck onto a glass sheet and coated with aluminum. The master was traditionally produced using a machine tool, but now the surface is formed by an ion beam or using laser beam photolithography. This photolithographic process produces gratings with fewer imperfections. This smoother surface reduces stray light (light at unwanted wavelengths) by significant amounts.

What is a “blazed angled” diffraction grating?

Blaze wavelength and high-order light diagram

 

The various light orders of a diffraction grating result in dispersion of the energy and a reduction in light utilization efficiency. However, the diffracted light energy from a diffraction grating with a fine sawtooth profile is concentrated in the direction of the specular reflection, as shown at left. This wavelength is known as the "blaze wavelength." The diffraction grating in a spectrophotometer is normally used near the blaze wavelength. However, multiple diffraction gratings can be used separately to increase the efficiency over a wide range of wavelength.

A different way of viewing the phenomenon of higher-order light is to say that, if d, i, and θ are fixed, a different value of m results in a different λ. This indicates that light of multiple wavelengths θ diffracts in diffraction angles λ, as shown on right. Therefore, a higher-order light cutout filter (short-wavelength cutout filter) is positioned after the monochromator exit slit to extract light at a specific wavelength (normally ±1st-order light).

What is a diode array spectrophotometer?

 

Single beam diode array spectrophotometer and single beam dispersive spectrophotometer components

A diode array spectrophotometer is a different type of single beam optical design when compared to a dispersive design (above) Diode array instruments are optimized for rapid, simultaneous acquisition of a full UV/Vis spectrum. The design is somewhat like a dispersive single beam instrument, except the diffraction grating is after the sample to directly disperse the transmitted light from the sample onto a diode array detector. Unlike its dispersive slower scanning cousin, the grating in a diode array instrument does not move or scan. The transmitted light from the sample illuminates the array detector continuously, thereby allowing fast spectral data collection. An additional novelty is the use of both source lamps simultaneously to illuminate the sample with all light wavelengths from 190 nm to 1100 nm. A photo diode array detector (PDA) is a linear array of discrete photo diodes on a single integrated circuit (IC) chip. For spectrophotometers, it is placed at the image plane from the grating to allow a range of wavelengths to be detected simultaneously. In this regard it can be thought of as an electronic version of photographic digital camera detector array. The diode array detector is the secret to fast spectra collection. A diode array instrument can collect a full range UV/Vis spectrum in milliseconds to seconds depending on design. Although a single beam type instrument, subject to long term drift, this fact is rarely an issue since background corrections and sample data can be acquired in under a second.

Diode array instruments are ideal for collecting complete spectral data on rapidly changing samples in disciplines such as kinetics, dissolution, liquid chromatography, and multicomponent analysis.

What goes on under a spectrophotometer’s cover?

 

spectrophotomoter function diagram

As seen in the figure above, a spectrophotometer measures the light that passes through the sample, to then strike the detector, where it is measured. Let’s call the amount of light at wavelength λ incident on the sample cuvette Io and the amount of light exiting the sample to hit the detector I. If we now calculate the percent of the ratio these two values (I/Io)*100 we get a percent transmission (%T) value. If the sample transmits all the light at a given wavelength then %T = 100; however, if the sample absorbs light then we will have the case where I < Io and %T will be a number less than 100. If the beam is totally blocked or absorbed by the sample, then %T = 0.

One more important item. When a spectrophotometer is turned on it is literally as “dumb as a stump”. In other words, the instrument has not been calibrated for the values of 100 %T or 0 %T. This 100 %T calibration is acquired by a process called background correction for a spectrum. it is also sometimes called auto-zero for a single wavelength measurement. A background correction is performed by removing the sample from the instrument and measuring the amount of light that strikes the detector in the scan range of all wavelengths in the spectrum. These 100 %T values (called a background scan) are then stored in memory for use in calculating accurate sample %T values for a sample.

This brings us to the main disadvantage of single beam instruments, a phenomenon known as drift. There are numerous components in an instrument that are not stable over time (usually due to electrical fluctuations or heat buildup). The lamps and detector are subject to variations in electrical output while electronic resistance values on circuit boards can change due to heat buildup in the instrument. The net effect is that %T values can change over time after the background correction is performed. This means that frequent and timely background corrections must be performed in conjunction with sample measurements. A solution to the “time drift problem” is the double beam instrument.

How are single beam and double beam instruments different?

 

Single beam vs double beam spectrophotometer

In single-beam systems, monochromatic light from a monochromator (only a sample beam) enters the sample compartment and hits the detector directly. In a double-beam system, however, the monochromatic light from the monochromator is split into a sample beam (S) and reference beam (R) before entering the sample compartment and hitting the detector. Each of these designs is illustrated here. The single-beam configuration has a simpler design because it does not need a mechanism for splitting the beam into sample and reference beams. Therefore, single-beam designs tend to be used in lower priced systems.

How does optical design affect instrument time stability?

 

Optical design and instrument time stability

As an example of the difference in time stability between the configurations, the fluctuations in measurement values over time (drift) were compared using a single-beam instrument (blue line) and a double-beam instrument (red line). The top figure shows the results from placing the single beam and double beam instruments in the same room and using each to obtain time-course measurements for one hour at 5-second intervals. The double-beam instrument had less time variability than the single-beam instrument.

This means the double-beam system provides more stable measurement values than the single-beam system. The single-beam system requires waiting until the light source and detector stabilize, performing frequent blank corrections to minimize such time variability. A summary of single-beam and double-beam characteristics is shown in the table at bottom.

How do spectrophotometers use the blank?

 

single beam vs double beam transmittance calculation

Single-Beam Spectrophotometers

Single-beam spectrophotometers perform blank correction and sample measurements using only the sample beam. First the instrument records the intensity of the sample beam during blank correction (SBlank). Then, during sample measurement, the instrument records the intensity of the sample beam (SMeas). Measurement results are then calculated using SBlank and SMeas. For example, transmittance (%T) is calculated using the following formula. However, if the intensity of the light source varies between the blank correction and sample measurement, it can appear as variations in SBlank and SMeas values. In other words, if the light source intensity varies between the time the blank correction and sample measurement are performed, inaccurate data may be acquired. Thus, in single-beam systems, fluctuations in the light source intensity can have a major effect on measurement results. Therefore, single-beam systems require waiting until the system stabilizes before starting measurements. As a general rule, they require waiting about one to two hours after switching the power ON.

Double-Beam Spectrophotometers

Double-beam spectrophotometers perform blank correction and measure samples using both sample and reference beams. During blank correction, the instrument records the SBlank/RBlank ratio, based on the sample beam intensity (SBlank) and the reference beam intensity (RBlank). Next, during sample measurement, the instrument records the SMeas/RMeas ratio, based on the sample beam intensity (SMeas) and the reference beam intensity (RMeas). Measurement results are then calculated using SBlank/RBlank and SMeas/RMeas. Unlike single-beam systems, double-beam systems determine measurement values using the ratio of sample beam intensity to reference beam intensity during both sample measurement blank correction. As shown, fluctuations in the light intensity do not affect measurement results. Essentially, using the ratio of sample and reference beam intensities in double-beam systems cancels out any fluctuations in the intensity of the light source and reduces the potential of such fluctuations affecting measurement values.

To summarize, data is not affected by light source fluctuations because the reference beam continuously compensates for any fluctuations in the intensity of the light source in real time. In other words, double-beam spectrophotometers accurately measure samples over long time periods by cancelling out lamp variations via ratio of the sample and reference beams. This means double-beam systems offer superior time stability.

How is a double beam spectrophotometer different from a single beam instrument?

 

Double beam spectrophotometer components

As you can see in the picture above, a double beam instrument looks like its single beam counterpart until just before the sample. In this type of instrument, the beam coming out of the monochromator is either split (using a 50-50 beam splitter optic) or chopped (using a rotating segmented wheel) into two beams that enter the sample compartment. In the instrument design above, the beam is split in two via a half- silvered quartz (to allow UV as well as visible light to pass through it) mirror which acts as an optical beam splitter. These more complex double beam instruments now have various mirrors to direct the light beam through the instrument. These mirrors are special “front coated” aluminum mirrors that have a highly reflective coating of aluminum on their front side (unlike your bathroom mirror that has the aluminum coating on the back side of the glass).

In the chopper version of a double beam instrument the above beam splitter is replaced by a rotating segmented wheel. The chopper disk (above) is made up of a number of different segments. Those in the instrument we are describing have three different sections, other designs may have a different number. The light coming from the diffraction grating and slit assembly will strike the rotating disk and one of three things can happen.

How does a beam chopper work?

 

beam chopper diagram

  1. If it hits the transparent section, it will go straight through the chopper and then pass through the cell containing the sample. It is then bounced by a mirror onto a second rotating disk. This disk is rotating such that when the light arrives from the first disk, it meets the mirrored section of the second disk. That bounces it onto the detector. It is following the red path in the diagram at upper left.
  2. If the original beam of light from the slit hits the mirrored section of the first rotating disk, it is bounced down along the green path in the diagram at lower right. After the mirror, it passes through a reference cell. Finally, the light gets to the second disk which is rotating in such a way that it meets the transparent section. It goes straight through to the detector
  3. If the light meets the first disk at the black section, it is blocked, and for a very short while no light passes through the spectrometer. This allows the computer to make dark current measurement baseline, which is an allowance for any current generated by the detector in the absence of any light (a form of zero measurement).

The sample cell contains a solution of the substance you are testing, usually a dilute solution. The solvent is chosen so that it doesn’t absorb any significant amount of light in the wavelength range of interest. The reference cell contains only the pure solvent. Now even with a double beam instrument a background correction must still be performed before the measurement of any sample; however, this instrument design offers a number of advantages over its single beam counterpart. First, the chopper spins at 60 Hz so that a complete cycle of sample, reference, and dark current occurs every 16 milliseconds (that’s 60 times a second). This cycle permits the instrument to be stabilized every 16 milliseconds. Which means that any fluctuations due to lamp instability, detector sensitivity changes, or resistance value changes in the electronics are compensated for in real time via a comparison (ratio) of the reference and sample beam measurements. The net effect, very small to insignificant drift levels over time.

Modern double beam spectrophotometers may only need a background correction performed every six to eight hours. Since the solvent can be placed in the reference beam for real time measurement, a comparison, it’s actually a ratio, of the solvent “blank” and the sample can be performed. This allows for the direct measurement of the dissolved compound of interest in real time. This means that if your solvent has an absorbance it is easily calculated away. Another feature is that reference beam attenuation can be performed when high absorbance values (usually above 4) need to be measured.

What is a high performance (HP) spectrophotometer?

 

high performance spectrophotometer components diagram

High performance spectrophotometers are the premier instruments in both the UV/Vis and UV/Vis/NIR wavelength range. The difference between these two optical designs is the addition of a second diffraction grating and a second detector (PbS or InGaAs) for the NIR spectral range. This, however, is not what makes them high performance. There is a key component of HP instruments that makes them able to measure very low values of %T (or high absorbance). By adding a second diffraction grating (top right) for each wavelength region (UV/Vis and NIR), the stray light of the instrument is reduced by a considerable amount.

This is a total of four gratings in the instrument, two each of the UV/Vis and NIR diffraction grating. All high-performance instruments have two of the same type gratings working in tandem to reduce instrumental stray light. The reduce stray light for dual monochromator spectrophotometers is what defines them as high performance (see optical diagram above). HP spectrophotometers are able to measure between 6 to 10 absorbance units in the UV/Vis and up to 7 to 8 absorbance units in the NIR with an InGaAs detector.

For Research Use Only. Not for use in diagnostic procedures.

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