UV/Vis Spectrophotometer Buying Guide

UV/Vis image

UV/Vis spectrophotometry 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.

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.

Basic Concepts

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 spectrophotometers 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(Table 1). Absorbance in older literature is also called ‘extinction’ or ‘optical density’ (OD).

Table 1: Table to show transmittance and absorbance calculations

Transmittance (T)

Absorbance (A)

T=I/I0

A = -logT

%T=I/I0 x 100

Unit: theoretically, none, but 'A' or 'AU' are used to report absorbance measurements ('OD' is also sometimes used)


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.

Beer-Lambert Law:

A = constant x concentration x cell length
A = εbc
ε is the molar absorption or extinction coefficient
b is the path length
c is concentration

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?

Buying Guide Tip: There are dedicated UV/Vis spectrophotometers for specific applications. For example, labs that need to quantify only nucleic acids and protein have several dedicated instruments to choose from. These instruments have built-in assay methods, which make analyses fast and simple. They also usually require very small volumes since biological samples are often restricted by the volume available for analyses.


Another UV/Vis spectrophotometer, the Xpose from Trinean, enables high speed, micro-volume nucleic acid and protein quantification (Figure 1).

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Figure 1: Trinean Xpose UV/Vis Spectrophotometer


During next generation sequencing (NGS) library preparation, it is important to extract, quantify and qualify DNA and RNA. Typically, DNA is quantified using a UV/Vis spectrophotometer and its purity assessed by visualization on an agarose gel. The Denovix DS-11 FX+ (Figure 2) is an all-in-one spectrophotometer/fluorometer for rapid and accurate 1µL UV/Vis quantification.

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Figure 2: Denovix DS-11 FX+


UV/Vis spectrophotometers from Cecil Instruments (Figure 3), respectively double beam and single beam instruments, are currently being used in hospitals in the UK. These spectrophotometers have a variety of uses, for example in the detection and quantification of xanthochromia in cerebrospinal fluid. These instruments from Cecil are ideal for diagnostic evaluation as they enable high performance wavelength analyses; multiple wavelength analyses; many routine analyses; such as creatinine, bilirubin and urea; kinetic analyses and spectral derivative.

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Figure 3: Cecil Aurius Series CE 2021UV/Vis Spectrophotometer


The ability to analyze samples in 6- to 384-well plates enables high-throughput and reduction in errors. The BioTek Epoch 2 UV/Vis spectrophotometer can accommodate cuvettes, microspots, and microplates (Figure 4). The system includes fully functional Gen5 software and is easily controlled with a touchscreen with a variety of data output options including WiFi, Bluetooth and a USB flash drive.

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Figure 4: BioTek Epoch 2 Microplate Spectrophotometer

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. 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.

Table 2. List of UV/Vis light sources

Deuterium (190 – 380 nm)

Tungsten-halogen (320 – 1100 nm)

Xenon (190 – 1100 nm)

Most common UV source; good intensity continuum in the UV region; typical life approximately 1,000 hrs.

Most common Vis radiation source; typical life approximately 2,000 hrs; relatively inexpensive.

Cover the UV and visible range but higher instrumental stray light and less energy at the far visible end; ideal for general measurements, long lifetime (typically seven years).


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, such as Nanodrop Lite. LED is a low cost solution for simple applications; the lamp life is almost infinite (Figure 5).

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Figure 5: Thermo Scientific Nanodrop Lite

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.

Table 3. The dispersing devices used in UV/Vis spectrophotometers

Prism

Grating

  • Simple and inexpensive

  • Drawbacks include non-linear dispersion and the temperature related characteristics of the commonly used prism materials.

  • Used in most modern UV/Vis spectrophotometers

  • Advantages over prism include better resolution, linear dispersion, constant bandwidth, simpler mechanical design for wavelength selection


The SPECORD PLUS series from Analytik Jena (Figure 6) has a monochromator with imaging holographic grating that enables stray light reduction and absolutely precise measuring results. The minimized number of movable components ensures best reliability, notably improved signal-to-noise ratio and energy throughput.

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Figure 6: Analytik Jena SPECORD PLUS

Detector

Ideally, the detector should give a linear response over a wide range, with low noise and high sensitivity. Table 4 shows the different types of detectors used in UV/Vis spectrophotometers. 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.

Table 4. Various detectors for UV/Vis spectrophotometers

Single Channel

Multi-Channel/Array

    Photomultiplier (PMT)

  • High sensitivity

  • Wide spectral range

  • Quick response


    Photodiode

  • Most common

  • Compared to PMT: less expensive, less sensitive, more robust

  • Fast acquisition of entire spectrum

  • Less moving parts



    Photodiode array


    Charge-coupled device (CCD)


Signal Handling/Data Management/Software

Most standalone spectrophotometers include their own on-board 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.

Buying Guide Tip: Ideally, the software should be easy to use, allowing ease of operation, design of experiment, and data analysis to increase productivity. Also, make sure it has adequate security options and tools for compliance, if you work in a regulated environment.

Considerations for Optical Configurations/Optical Design

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, uses two separate but identical detectors.

Table 5. Optical configurations of UV/Vis spectrophotometers

Single Beam

  • One beam of light to make measurements

  • Simple configuration (less complicated)

Double Beam

  • Light split into two different paths by a chopper, one detector

  • More complex optics: higher cost, good stability, lower sensitivity

Split Beam

  • Light is split into two different path by a splitter, one passes through the sample; the other is used as the reference

  • More complex optics: higher cost, good stability, lower sensitivity

Multi-Channel/ Array Based

  • All wavelengths from light source pass through the sample; light passing through the sample is dispersed by a diffraction grating; separated wavelengths fall on different pixels of the array detector

  • Fast acquisition of spectrum; simultaneous detection of all wavelengths


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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Figure 7. Schematic showing the difference between a conventional and PDA spectrophotometer.

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.

Considerations for Performance Criteria

There 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.

Buying Guide 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, ask.


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.

Buying Guide Tip: Most quantitation applications using UV/Vis involve the measurement of the standards and samples of comparable concentrations in rapid succession on the same instrument. As long as the photometric measurements are reproducible and the response is linear over a defined range, the absolute photometric accuracy may not be critical. However, photometric accuracy is critical when comparing the results of a sample measured on different instruments.


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:

  • What is your budget?
  • How will be using the instrument? Does it need to have a simple operation?
  • Is speed of analysis important to you? What is the scan rate/speed?
  • Do you need specific accessories for your applications? Are you analyzing extreme volumes (large or small)?
  • Future-proofing: How many samples will you be running per annum?
  • 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.

Future Trends

UV/Vis spectroscopy is a mature technique, but it continues to evolve. Since the first UV/Vis spectrophotometer 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 operated more by lab personnel who do not have a formal spectroscopy background, software/interfaces that are very easy and simple to use will still be the trend.

Light-emitting diode (LED) is being utilized more as they offer a low cost solution for simple applications and the lamp life is almost infinite. Additionally, the use of UV/Vis spectroscopy in the field of next generation sequencing is very exciting. The ability to quantify DNA in this way enables fast and reliable results in the area of life sciences. UV/Vis spectrophotometers are relatively easy to use, enable a wide range of applicability, have a low cost compared to other lab instruments and will continue to serve as a workhorse in many laboratories.

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