UV/Vis Spectroscopy is a very popular technique because of its relative ease of use and the speed of analysis. It has been called the workhorse of the modern laboratory, used in the qualitative and quantitative analyses of organic and inorganic compounds in a wide range of products and processes.
Instrument designs of UV/Vis spectrometers have evolved since the first system became commercially available in 1947. Current instruments can handle a breadth of research, industrial and academic applications. Laboratories seeking a new UV/Vis spectrometer face no shortage of choices, from the simplest single-wavelength measurements to high-performance, multi-spectrum analyses. This guide will help you by discussing the important considerations for your next UV/Vis spectrometer.
UV/Vis refers to the ultraviolet (UV) and visible (vis) parts of the electromagnetic spectrum. The Joint Committee on Nomenclature in Applied Spectroscopy sets the far UV region at 10-200 nm, near UV at 200-380 nm, and visible at 380-780 nm. Modern UV/Vis spectrometers typically have a wavelength range from 190 nm to 1100 nm.
When a beam of electromagnetic radiation strikes an object, it can be absorbed, transmitted, scattered or reflected; or it can cause fluorescence. The processes that are involved in UV/Vis spectroscopy are absorption and transmission. When ultraviolet and visible radiation interacts with matter, electronic transitions take place; that is, electrons in the ground state are promoted to a high energy state. In particular, π to π* and n to π* transitions occur in the UV/Vis region.Transmittance and Absorbance
When a beam of light passes through a sample, the amount of light absorbed is the difference between the incident radiation (Io) and the transmitted radiation (I). Transmittance and absorbance are terms used to express the amount of light absorbed by the sample. Absorbance in older literature is also called ‘extinction’ or ‘optical density’ (OD).The Beer-Lambert Law
The Beer-Lambert Law (sometimes simply called Beer’s Law) states that the concentration of an analyte in solution is directly proportional to the absorbance (A) of the solution.
The first thing you will need to consider is the applications for your UV/Vis spectrometer. Application considerations have several components, as shown in Figure 1.
Figure 1. Application considerations for your next UV/Vis spectrometer
Start with your laboratory’s current and future applications
This will help you to determine the instrument features/characteristics you require. For example, in your current application, what is the detection range? Will you have future applications that may require a broader range?
Consider the samples you will be analyzing
The type of samples you will be working with will help you to determine what absorbance range you will need in your UV/Vis spectrometer. For example, if it is a turbid or concentrated liquid or a solid sample that is optically thick, you may require a working absorbance range between 5 A and 8 A or higher. Also, is your sample volume limited? There are systems that can measure a few microliters of sample, such as the system shown in this video.
A special type of UV/Vis spectrometer allows the user to measure spectra of microscopic samples or microscopic areas on samples. These UV/Vis microspectrometers consist of a UV-visible microscope integrated with a UV-visible spectrometer. Here is an example of a UV/Vis/NIR microspectrometer.
The different components of a UV/Vis spectrometer contribute to the overall performance of the instrument. Any UV/Vis spectrometer will have the following different parts:
The light source should be stable during the measurement period. That is, the intensity of emitted radiation should not fluctuate, and there should be adequate intensity over as large a wavelength region as possible. The ideal light source would yield a constant intensity over all wavelengths with low noise and long-term stability. Figure 2 lists the light sources for UV/Vis spectrometers. The different sources are not equivalent; they provide light intensities and noise at different parts of the spectrum.
Figure 2. Light sources for UV/Vis spectrometers
Many UV/Vis spectrometers use both deuterium and tungsten-halogen lamps to cover the entire UV (deuterium lamp) and visible (tungsten-halogen lamp) spectrum. Either a source selector is used to switch between the lamps as appropriate, or the light from the two sources is mixed to yield a single broadband source. Xenon flash lamps have become more common because they cover the entire UV and visible range, have a very long lifetime, do not require warm up time, and do not raise the temperature of the sample compartment. Light-emitting diode (LED) is used in some instruments. LED is a low cost solution for simple applications; the lamp life is almost infinite.
Most samples analyzed by UV/Vis are liquid. Traditional sample formats take sample cells, cuvettes, sippers (for automated sampling), and microtiter plates, as well as combinations of these. Some instruments feature fiber optic probes for measuring samples outside the UV-Vis spectrometer’s sample compartment. It eliminates the need for filling a sample cell, which is especially useful for quantitative analysis in quality control labs, where large numbers of samples need to be analyzed quickly.
The ideal monochromator should produce monochromatic light. In practice, however, the output is always a band, optimally symmetrical in shape. The dispersing device in monochromators can be a prism or diffraction grating. Most modern spectrometers contain holographic gratings instead of prisms.
Figure 3. The dispersing devices used in UV/Vis spectrometers
Ideally, the detector should give a linear response over a wide range, with low noise and high sensitivity. Figure 4 shows the different types of detectors used in UV/Vis spectrometers. Photomultiplier tubes (PMT) and photodiode are single channel detectors, and the most commonly used in the instruments currently on the market. Photodiode is usually found in low-end instruments, while PMTs are used in higher end instruments (research grade). Photodiode array (PDA) and charge-coupled device (CCD) are multi-channel detectors. They allow for fast acquisition of the entire spectrum, and since they have less moving parts, are more robust. However, they are not as sensitive as PMTs.
Figure 4. Various detectors for UV/Vis spectrometers
Signal handling/Data management/Software
Most standalone spectrometers include their own onboard software that drives the instrument and manipulates data. They can have pre-programmed methods to perform routine analyses, calculate common parameters, etc. Higher-performance instruments are often designed for use with a personal computer, requiring additional software from manufacturers. Sometimes users can pick and choose specific software modules and upgrades to match their analysis needs.
There are several optical configurations for the UV/Vis spectrometers you will find in the market, shown in Figure 5. The single beam configuration was the earliest design and is still in common use, especially among low-end instruments. Double beam and dual beam spectrometers measure the ratio of light intensities and, therefore, are not as sensitive to fluctuations in the light source or detector. Split beam resembles the dual-beam spectrometer but uses a beam splitter instead of a chopper, and uses two separate but identical detectors. Check out an example of a split beam UV/Vis spectrometer in this video.
Figure 5. Optical configurations of UV/Vis spectrometers
Single beam, double beam, and split beam are conventional UV/Vis spectrometers. In conventional systems, polychromatic light from the source is focused on the entrance slit of a monochromator, which selectively transmits a narrow band of light. This light then passes through the sample area to the detector. In a multi-channel UV/Vis spectrometer, such as those that use photodiode array (PDA) and charge couple device (CCD) detectors, polychromatic light from a source passes through the sample area and is focused on the entrance slit of the polychromator. This disperses the light on to a diode array, where each diode measures a narrow band of the spectrum. Figure 6 illustrates the difference in which the instrument components are set-up in conventional (single beam configuration) and multi-channel (PDA system) systems.
Figure 6. Schematic showing the difference between a conventional and PDA spectrometer.
Multi-channel/array-based instruments are mechanically simpler, and therefore more robust. They are able to simultaneously analyze a full spectrum, which is typically 190-1100, allowing them to perform analyses far faster than conventional scanning instruments.
Figure 7 lists the common performance criteria that are typically given by manufacturers of UV/Vis instruments. Gordon Bain, Product Manager for UV/Vis Spectroscopy at ThermoScientific, advises that potential buyers of UV/Vis spectrometers should pay close attention to these three criteria: wavelength accuracy, wavelength reproducibility, and noise.
The deviation of the wavelength reading at an absorption band from the known wavelength of the band. The wavelength deviation can cause errors in the qualitative and quantitative results of the UV/Vis measurement.
The instrument’s reproducibility when making repeated readings of the same wavelength.
Noises in UV/Vis spectrometers originate mainly from the light source and electronic components. Noise affects the accuracy at both low- and high-ends of the absorbance scale: photon noise from the light source affects the accuracy of the measurements at low absorbance, electronic noise from electronic components affects the accuracy of the measurements at high absorbance. High noise level will reduce the limit of detection as well as the instrument’s sensitivity.
Figure 7. Common performance attributes/criteria for UV/Vis spectrometers
Photometric range (working absorbance range)
For some applications, specifically those that have strongly absorbing species, it is important to consider photometric range. A spectrometer that can detect transmission of 10% has a photometric range of 1A, 1% is 2A, 0.1% is 3A, and so on. A photometric range of 3.5A to 4A means it can handle samples that absorb as much 99.99% of incident light.
Linear dynamic range
The concentration range over which absorbance and concentration remain directly proportional to each other. A wide linear dynamic range permits the analysis of a wide range of sample concentrations (optical densities), and reduces sample preparation (dilution) requirements.
Photometric (absorbance) accuracy
Photometric accuracy is defined as how accurately an instrument measures absorbance and is determined by the difference between the measured absorbance and the established standard value.
Variations in lamp intensity and electronic outputs between the measurements of the incident radiation (Io) and transmitted radiation (I) result in instrument drifts. These changes can lead to error in the value of the measurements, especially over a long period of time. Photometric stability is the ability of the instrument to maintain a steady state over time so that the effect of the drift on the accuracy of the measurements is insignificant.
This is the unwanted radiation or wavelength of light, other than the desired wavelength, that reaches the detector. Stray light causes a decrease in absorbance and reduces the linearity range of the instrument. High absorbance measurements are more severely impacted by stray light.
Spectral bandwidth, resolution
Spectral bandwidth and resolution are related: the smaller the spectral bandwidth, the finer the resolution. In general, poor resolution leads to a decrease in extinction coefficient across the spectrum and therefore inaccurate quantitation. The sensitivity of the measurement is also compromised. Most UV/Vis spectrometers in the market today provide adequate resolution for the most common applications. If your application requires detailed spectral information, you need an instrument with very small bandwidth (better resolution).
For UV/Vis spectrometers that have dual light sources (a deuterium lamp for the UV range and a tungsten lamp for the visible range), the intensity of the radiation coming from the light sources is not constant over the whole UV-Visible range. The response of the detector also varies over the spectral range. A flat baseline demonstrates the ability of the instrument to normalize the output of the lamp and detector responses.
Range in which the instrument is capable of measuring. UV/Vis instruments typically have 190–1100 nm wavelength range.
There are other practical considerations that you need to think about before purchasing your next UV/Vis spectrometer. These are shown in Figure 8.
Figure 8. Other considerations when purchasing a UV/Vis spectrometer
Ease of use, simple operation
The new UV/Vis system needs to be fairly easy to use. In many labs today, often there is no longer a UV/Vis expert, so personnel are expected to perform a wider variety of functions. With fewer trained UV/Vis analysts, systems that are easy to use, even for the most sophisticated applications, will result in greater productivity and efficiency.
Speed of analysis, overall productivity
Look at the specification for scan rate or scan speed. This tells you how fast the instrument can scan the entire wavelength range. What level of throughput do you need? Modern sophisticated UV/Vis instruments can yield remarkable gains in operator efficiency because they can perform automated tasks. For example, some instruments can self-align newly installed accessories which, if done manually, is time consuming and if not done properly compromises results and reduces the accuracy and reproducibility.
Maintenance and service
Quality maintenance and service should not be overlooked. Ideally, the UV/Vis instrument is designed for convenient ongoing maintenance and to minimize unexpected service. A simple maintenance task, such as replacing a light source, should be a task any operator can handle in a minute. Consider a service plan that fits the volume of work done by the instrument. Make sure the instrument manufacturer has deep expertise in UV/Vis application support, so that problems you may have in method development, operation and troubleshooting will be addressed. Other services that your instrument vendor should ideally provide include validation, education and training, and consulting that can increase your uptime and productivity.
Flexibility, accessories available
Your flexibility consideration will depend on your intended application(s) for the instrument. Will you be analyzing extreme volumes of samples? (2 microliters for some samples, milliliters of another set of samples) Will you be using the instrument for both liquid and solid samples? If this is the case, invest in an instrument that offers sampling flexibility.
Some manufacturers offer accessories that easily snap in and align themselves. The accessories are “smart” and communicate with the system software and other hardware. Ideally, accessories and system components should be easily inserted or swapped with other UV/Vis systems from the same manufacturer.
The instrument should be able to perform difficult analyses while maintaining quality results.
UV/Vis spectrometers can cost anywhere from $2,000 to $120,000. The low-priced systems are designed for repetitive tasks in specific application areas. It’s a cost-effective choice for laboratories that deal with easier samples and a small set of simple measurements. However, some labs that perform repetitive tasks also require high performance UV/Vis spectrometers. Mid-range to upper-end systems are commonly used in research laboratories including academic, industrial and government research facilities.
When considering your budget, do not forget to factor in the cost of maintenance and service along with the cost of acquisition.
Do you need to comply with regulatory guidelines? Many manufacturers offer programs/packages to facilitate system qualification validation procedures. Many systems have features that will ensure full regulatory compliance.
UV/Vis spectroscopy is a mature technique, but it continues to evolve. Since the first UV/Vis spectrometer in 1947, rapid advances in electronics, optics and computer/software controls have paved the way for instruments that are easy to use, more compact and flexible. Manufacturers will continue to find innovative ways to meet specific end-user needs and requirements, both in the hardware and software components. They want to enhance the users’ experience as they use the instrument, helping them to get the answers they need in the easiest, most efficient manner.
Miniaturization/portability will continue to be a trend, allowing for increased mobility, and paving the way for more applications. Flexibility and ease of sample handling are also areas that manufacturers will continue to work on. As instruments are increasingly operated by lab personnel that do not have formal spectroscopy backgrounds, software/interfaces that are very easy and simple to use will continue to be the trend.
With its relative ease of use, wide range of applicability, and low cost compared to other lab instruments, UV/Vis spectrometers will continue to serve as a workhorse in many laboratories.
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