How to Buy UV/Vis Spectrophotometers

The different components of a UV/Vis spectrophotometer contribute to the overall performance of the instrument. The UV/Vis spectrophotometer consists of five basic components:

• Light source – provides radiation of appropriate wavelength.
• Sample compartment – Those working with low volumes of precious samples, such as DNA or protein, will benefit from investment in a micro-volume spectrophotometer, which can analyze as little as 0.5µL of sample. On the other end of the scale, some can accommodate large cuvettes – to provide lower detection limits of trace analytes - or even solid samples.
• Monochromator – produces a beam of monochromatic light; in the conventional UV/Vis configuration, it consists of an entrance slit, collimating device, dispersing device, focusing lens or mirror, and an exit slit.
• Detector – detects and measures the intensity of radiation.
• Signal handling and measuring system – processes data and controls the instrument.

Figure 11 shows the components and setup of a typical array UV/Vis spectrophotometer.

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. Table 2 lists the light sources for UV/Vis spectrophotometers. The different sources are not equivalent; they provide light intensities and noise at different parts of the spectrum.

Many UV/Vis spectrophotometers 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. This type of setup is known as scanning. Xenon flash lamps have become more common because they cover the entire UV and visible range, have extended lifetimes, do not require warm-up time, and do not raise the temperature of the sample compartment. Light-emitting diodes (LED) are used in some instruments as low-cost solutions for simple applications as the lamp life is almost infinite. In this case, the setup is known as array.

Download this array versus scanning white paper for a comparison of these two well-established spectrophotometer setups and find out more about their individual performance.

Most samples analyzed by UV/Vis are in liquid or solution state. Traditional sample cells include cuvettes, sippers (an accessory which automatically fills the cells with sample solution), and microtiter plates, such as the Epoch™ 2. Micro-volume spectrophotometers, such as the Mettler-Toledo UV5Nano, use a micro-volume platform onto which the sample solution can be directly pipetted. Some instruments feature fiber-optic probes for measuring samples outside the UV/Vis spectrophotometer’s sample compartment. This allows analysis of the sample in situ, which is especially useful when it is not possible to physically remove samples, for example when monitoring industrial production lines, blood or the environment.

Another consideration is pathlength. Micro-volume spectrophotometers have a very short pathlength, normally between 0.1 to 1mm, which allows highly concentrated samples to be analyzed without the need for dilution. Figure 12 shows a diagram of the LockPath system from Mettler-Toledo, which allows micro-volume droplets of sample to be analyzed accurately.

At the opposite end of the spectrum, some instruments accommodate a very long pathlength. For example, the Spectroquant^® Prove 600 can accommodate cuvettes up to 100mm, maximising sensitivity and allowing detection of analytes at trace levels.

Figure 12: LockPath system from Mettler-Toledo ensures that the available pathlengths at 0.1 or 1mm are accurately defined. The pathlengths can be automatically and manually defined according to the user’s needs. Source: Mettler-Toledo International.

TIP: If using cuvettes as sample cells, it is important to consider the cuvette material. Common materials include optical glass, quartz and sapphire. The transmission range of the material will need to match the wavelength range of your analytes; materials which have a broader transmission range, such as sapphire, come at a higher cost.

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 spectrophotometers contain holographic gratings instead of prisms.

Ideally, the detector should give a linear response over a wide range, with low noise and high sensitivity. Table 3 shows the different types of detectors used in UV/Vis spectrophotometers. Photomultiplier tubes (PMT) and photodiodes are single-channel detectors, and the most commonly used in the instruments currently out in the market. Photodiodes are usually found in low-end instruments, while PMTs are used in higher-end instruments (research grade). Photodiode arrays (PDA) and charge-coupled devices (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.

UV/Vis software is an area which is constantly developing and expanding. Most spectrophotometers now include their own software which controls the instrument and collects, analyzes and manipulates data.

Many software packages now come with pre-programmed methods which allow users to perform routine analyses and calculate common parameters with just a few clicks. A combination of an intuitive user interface and software can make the instrument more accessible to occasional, non-expert users as well as speeding up routine analysis. For example, many instruments suited to life sciences include pre-programmed applications for nucleic acid quantification, protein and peptide quantification, kinetic assays and optical density and cell/mL calculations.

Examples include:

Many standalone instruments now come with a touchscreen user interface, making operation simple and efficient. For example, Mettler-Toledo UV/Vis Excellence spectrophotometers feature a One Click™ Operation system, which allows fast selection of pre-defined methods though a colour-screen touchscreen.

Higher-performance instruments are often designed for use with a personal computer, for example the DataStream Software from Cecil Instruments Limited.

Ease of data export is important for standalone instruments. Many models now come with USB and WiFi, giving you more freedom when organizing your laboratory space and making it easier to access your data. Another feature you might find useful is the ability to directly print results.

There are several optical configurations for the UV/Vis spectrophotometers you will find in the market, shown in Table 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 spectrophotometers 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 spectrophotometer but uses a beam splitter instead of a chopper and uses two separate but identical detectors.

Single beam, double beam and split beam are conventional UV/Vis spectrophotometers. 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 multi-channel UV/Vis spectrophotometers, polychromatic light from a source passes through the sample area and is focused on the entrance slit of the polychromator, which disperses the light onto a diode array where each diode measures a narrow band of the spectrum. This diode array, known as a photodiode array detector (PDA), is typically made up of a linear array of 1024 phototubes for each different wavelength.

Advantages of multi-channel/array-based instruments include:

• Greater robustness – require less moving parts, so require less maintenance
• Faster-full spectrum scan speeds – some instruments can provide full-spectrum scans in as little as 1 second.

Figure 13 illustrates the differences between the instrument component set-ups in conventional (single beam configuration is shown) and multi-channel (PDA system is shown) systems.

There are several criteria to consider when buying a new UV/Vis spectrophotometer but wavelength accuracy, wavelength reproducibility and noise levels are particularly important.

Wavelength accuracy is most important when you want to compare results between different instruments. Deviation in the wavelength can cause errors in both quantitative and qualitative results. Wavelength accuracy is most commonly checked by using certified reference standards.

TIP: The level of wavelength accuracy required is dependent on your application. A typical level of wavelength accuracy for UV/Vis instruments is around ±1nm, however, some instruments achieve accuracies of ±0.5nm.

Wavelength reproducibility is the ability of the instrument to produce the same wavelength over repeat readings. This is important as it allows for accurate comparison of one reading to another, for example a sample to a blank, or one sample to another.

Noise in UV/Vis spectrophotometry mainly originates from the light source and electronic components. This can affect 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, while electronic noise from the components affects high-absorbance measurement accuracy. High noise levels will reduce the limit of detection and the instrument’s sensitivity.

For some applications, specifically those that have strongly absorbing species, it is important to consider the photometric range. A spectrophotometer that can detect transmission of 10% has a photometric range of 1A, where 1% is 2A, 0.1% is 3A. A photometric range of 3.5A to 4A means it can handle samples that absorb as much as 99.99% of incident light.

TIP: The photometric range specified for a UV/Vis spectrophotometer does not mean that it is linear over that range. Not all instruments give their specifications for linear dynamic range, though they always give the photometric range. So, if linear dynamic range is important to you, be sure to ask about it.

Figure 12: LockPath system from Mettler-Toledo ensures that the available pathlengths at 0.1 or 1mm are accurately defined. The pathlengths can be automatically and manually defined according to the user’s needs. Source: Mettler-Toledo International.

This refers to 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 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. It can be considered the most important parameter for applications which involve comparison of extinction coefficients between instruments, spectral identification, and sample purity analysis.

TIP: Absolute photometric accuracy may not be critical to your application. In most quantitation applications, as long as the measurements are reproducible and linear over the wavelength range, the photometric accuracy is not critical. However, it does become a critical parameter when comparing the results over multiple instruments.

The precision with which the UV/Vis instrument can make repeated measurements. It indicates how well the measured absorbance value can be reproduced.

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 becomes 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 and resolution are related; the smaller the spectral bandwidth, the finer the resolution. In general, poor resolution leads to a decrease in the extinction coefficient across the spectrum and, therefore, inaccurate quantitation. The sensitivity of the measurement is also compromised. Most UV/Vis spectrophotometers provide adequate resolution for common applications. If your application requires detailed spectral information, you will require an instrument with very small bandwidth, to gain better resolution.

For UV/Vis spectrophotometers 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 190–1100nm wavelength range. However, some life sciences-specific instruments provide a narrower range, as common analytes such as dsDNA and protein all fall within the UV range (Table 6).

Resolution, or resolving power, is the main factor which determines the performance of a spectrophotometer. A low resolution will make it impossible to differentiate between absorbance peaks which are close together in wavelength, making spectral identification challenging.

In order to make the most out of your purchasing power, you should ask yourself and your colleagues the following questions before speaking to a manufacturer:

» What is your budget?

It is important to understand and work within a realistic budget.

» What applications will you use the instrument for?

Consider the hardware and software which will be best suited to your needs. Don’t forget to consider any future applications you may require the instrument for, or ways in which others in your laboratory may wish to use the instrument.

» Are you replacing an instrument?

Review with others what the advantages and limitations of the old instrument were. This will help you decide which features are essential or desirable in your next purchase.

» How important is speed and efficiency?

Which features or technologies would streamline your workflow and save time for users? How will the system integrate into your current workflow? Remember to take into account your future goals, such as the introduction of liquid handling robots or an increase in sample volume.

» What servicing and maintenance and support do you need?

Find out what type of maintenance the instrument requires and how often. Decide which type of servicing package you will require.

Although UV/Vis spectroscopy is a well-established technique, the technology continues to evolve. Since the first UV/Vis spectrophotometer in 1947, rapid advances in electronics, optics and software have paved the way for easier-to-use, more compact and flexible instruments. Manufacturers are devising innovative ways to meet specific end-user requirements, both in the hardware and software components.

• An exciting technology which is becoming more commonplace in UV/Vis spectrophotometers is photodiode array technology, which is helping to increase the speed and robustness of the instruments, ultimately leading to enhanced throughput.
• Miniaturization is a continuing trend. Microvolume spectrophotometers are becoming more commonplace, especially in life sciences applications, and allow for accurate measurements to be performed on as little as 0.5µL of sample.
• User interface and connectivity: Touchscreens and other user-friendly features are being seen increasingly on compact, standalone instruments, enabling easy user interaction and increased portability. Features such as WiFi and printer connectivity are allowing faster transfer of results.

• Pre-programmed and customizable methods enable non-expert users to carry out complex analyses with a single click
• Features such as sample contaminant identification are improving the quality of data produced and helping researchers identify problems in their workflows
• Data analysis software is continually improving and becoming more user-friendly, enabling fast and accurate interpretation of results