A UV Visible Spectrophotometer is the instrument which is used to measure the intensity of light before and after passing through a sample - the ratio of one to the other is called transmittance and is expressed as a percentage. From transmittance, absorbance can be calculated.
Laboratories seeking a new UV/Vis spectrophotometer 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 spectrophotometer.
Application Considerations
UV/Vis spectrophotometry can be used for a wide range of applications, including biological analyses, clinical diagnostics, food, environmental, pharmaceuticals and materials analysis. The first thing you will need to consider is the application(s) for your UV/Vis spectrophotometer. Applications considerations have several components such as:
- Types of samples to be analyzed – this will enable you to choose the absorbance range.
- Volume(s) of samples to be analyzed – will you need a standard spectrophotometer or a microspectrophotometer?
- Current laboratory applications – this will help determine features, detection range and characteristics you require.
- Future applications – will you have future applications that will require a broader range?
Considerations for Instrument Components
The different components of a UV/Vis spectrophotometer contribute to the overall performance of the instrument. Any UV/Vis spectrophotometer will have the following different parts:
- Light source – provides radiation of appropriate wavelength.
- Sample compartment – the area where sample is introduced into the light beam.
- 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.
Light Source
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.
Buying Guide Tip: Some UV/Vis instruments can be equipped with a variety of sample holders to suit changing needs, such as a change in sample volume and sample type (liquid, solid).
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. Xenon flash lamps have become more common because it covers the entire UV and visible range, has very long lifetime, does not require warm-up time, and does not raise the temperature of the sample compartment. Light-emitting diode (LED) is used in some instruments.
Sample Format
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 spectrophotometer’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.
Monochromator
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.
Detector
Ideally, the detector should give a linear response over a wide range, with low noise and high sensitivity. Photomultplier tubes (PMT) and photodiode are single channel detectors, and the most commonly used in the instruments currently out in 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.
Considerations for Optical Configurations/Optical Design
There are several optical configurations for the UV/Vis spectrophotometers you will find in the market. 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 splitterinstead of a chopper, 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 spectrophotometer, 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, which disperses the light onto a diode array where each diode measures a narrow band of the spectrum. Figure 7 illustrates the difference in which the instrument components are set-up in conventional (single beam configuration is shown) and multi-channel (PDA system is shown) systems.
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| Single Beam UV Visible Spectrophotometer |
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| Split Beam UV/Vis Spectrophotometer |
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| Double Beam UV Visible Spectrophotometer with Touch Screen |
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| Large Screen Spectrophotometer Double Beam |
Considerations for Performance Criteria
here are many aspects to consider when buying a new UV/Vis spectrophotometer, but the three main criteria are wavelength accuracy, wavelength reproducibility and noise levels. The list below describes in detail important considerations for performance criteria.
Wavelength Accuracy
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.
Wavelength Reproducibility
The instrument’s reproducibility when making repeated readings of the same wavelength.
Noise
Noises in UV/Vis spectrophotometers 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 and reduces the instrument’s sensitivity.
Photometric Range (Working Absorbance Range)
For some applications, specifically those that have strongly absorbing species, it is important to consider photometric range. A spectrophotometer 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.
Photometric Reproducibility
The precision with which the UV/Vis instrument can make repeated measurement. It indicates how well the measured absorbance value can be reproduced.
Photometric Stability
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.
Stray Light
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 spectrophotometers 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).
Baseline Flatness
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.
Wavelength Range
Range in which the instrument is capable of measuring. UV/Vis instruments typically have 190–1100 nm wavelength range.
Useful Questions
In order to make the most out of your purchasing power you should ask yourself the following questions before speaking to a manufacturer:
1. What is your budget?
2. How will be using the instrument? Does it need to have a simple operation?
3. Is speed of analysis important to you? What is the scan rate/speed?
4. Do you need specific accessories for your applications? Are you analyzing extreme volumes (large or small)?
5. Future-proofing: How many samples will you be running per annum?
6. What service package do you require?
By asking yourself these questions, you equip yourself with the knowledge and understanding to enable you to make the right purchase for you and your experiments.
