How to Buy UV/Vis Spectrophotometers



UV/Vis image

UV/Vis spectrophotometry is a common technique used for qualitative and quantitative chemical analyses, for a wide range of applications. Laboratories seeking a new UV/Vis spectrophotometer have many choices, from the simplest single-wavelength instruments to high-performance, multi-spectrum analyzers. This guide will highlight important considerations for purchasing a UV/Vis spectrophotometer.

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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 over many disciplines including life sciences, clinical diagnostics, drug discovery, food & beverage, environmental and forensics. The application performed using your UV/Vis spectrophotometer is a very important consideration.

  • 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?

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.


For example, METTLER TOLEDO’S range of UV/Vis Excellence instruments (Figure 1) are designed to optimize spectroscopic workflows. Using FastTrack™ technology, the compact UV7 is a sound investment for high quality analyses. The automated performance verification tool CertiRef™ facilitates installation qualification, instrument maintenance and compliance to the latest Pharmacopeia regulations. Watch this video to find out more about FastTrack™ technology and the reverse optic design of the UV7 spectrophotometer. The UV5Nano, using LockPath™ technology, is designed for fast, accurate micro-volume measurements over a wide concentration. Watch this video to find out more. The range of spectrometers can be easily integrated with other METTLER TOLEDO instruments using LabX software, to facilitate any laboratory workflow.

 METTLER TOLEDO UV/Vis Spectrophotometers

Figure 1: METTLER TOLEDO UV/Vis Spectrophotometers

Small samples of DNA, RNA and protein are routinely quantified using UV/Vis spectrophotometry for downstream applications. Watch the video (Figure 2) to find out more about the DeNovix DS-11 FX+, which is an all-in-one spectrophotometer/fluorometer for rapid and accurate 1 µL UV/Vis quantification.

DeNovix DS-11 FX+

Figure 2: DeNovix DS-11 FX+


The NanoDrop™ 2000/c Microvolume Spectrophotometer from Thermo Fisher Scientific (Figure 3), is also ideal for quantifying small volumes of DNA, RNA and protein and provides a purity ratio for additional sample quality determination. Watch the video to find out more. The recently released NanoDrop One/Onec is the next generation of this instrument, with many enhanced functions and features. Download this article to find out more.

UV/Vis Image 3

Figure 3: Thermo Fisher Scientific NanoDrop 2000/c Microvolume Spectrophotometer


The DropSense®96 Content Analysis Platform (Figure 4), from Trinean, is designed for high throughput quantification of nucleic acid or protein samples. Quantification is performed in 96 well microfluidic plates and additional sample quality data for compliance in regulatory environments is also provided. Find out more in this video.

Trinean DropSense96® Content Analysis Platform

Figure 4: Trinean DropSense96® Content Analysis Platform


The versatile Epoch™ 2, from BioTek Instruments Inc. (Figure 5) is a compact microplate spectrophotometer that performs UV-Vis measurements in 6- to 384-well microplates, cuvettes and in micro-volume samples, with additional features to accommodate temperature sensitive applications. Watch the video to find out more.

BioTek Instruments Inc. Epoch 2 Microplate Spectrophotometer

Figure 5: BioTek Instruments Inc. Epoch 2 Microplate Spectrophotometer


The SPECORD® PLUS from Analytik Jena (Figure 6), features quartz-coated optical components for higher quality, precise data. The monochromator with imaging holographic grating reduces stray light and provides exact measurements with improved signal-to-noise ratio. Watch the video to find out more.

Analytik Jena SPECORD® PLUS

Figure 6: Analytik Jena SPECORD® PLUS

The Aquarius CE 7400S, CE 7400/7500 spectrophotometers from Cecil Instruments Limited feature variable or fixed bandwidths, for the highest accuracy of measurement coupled with high stability over time and can be fully accessorized with a wide range of additional features, including pre-programmed methods and temperature control.

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).


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 or 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 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.

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. This eliminates the need for filling the 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


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. 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.

Table 4. Various detectors for UV/Vis spectrophotometers

Single Channel

Multi-Channel/Array

Photomultiplier (PMT)

  • Wide spectral range

  • Quick response

Photodiode

  • Most common

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

Photodiode array

Charge-coupled device (CCD)

  • Fast acquisition of entire spectrum

  • Fewer moving parts


Signal Handling/Data Management/Software
Most standalone spectrophotometers include their own software that drives the instrument and manipulates data. They may have pre-programmed methods to perform routine analyses and calculate common parameters. Higher-performance instruments are often designed for use with a personal computer and may require additional software from manufacturers. Sometimes users can pick and choose specific software modules and upgrades to match their analysis needs.

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 and 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 differences between the instrument component set-ups in conventional (single beam configuration is shown) and multi-channel (PDA system is shown) systems. UV/Vis Image 8

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, typically 190–1100 nm, which allows them to perform analyses far faster than conventional scanning instruments.

Considerations for Performance Criteria

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
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
Noise in UV/Vis spectrophotometers 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.

Photometric Range (Working Absorbance Range)
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.

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.

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.

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 becomes 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 and 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 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.

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 you be using the instrument, what functions do you need?
  • 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 will have the knowledge and understanding needed to make the right purchase for you and your experiments.

Future Trends

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, in the hardware and software components.

Miniaturization and portability will continue to be a trend, which enhances mobility and versatility. Improved flexibility and sample handling are areas that will increase efficiency and reduce the need for specialist training of operators. Advances in software and interfaces also improve efficiency for analysis and data handling.

The ability to rapidly and accurately quantify nucleic acids and proteins is a fundamental step in numerous life sciences and drug discovery applications, with an increased demand for smaller volume analysis and higher throughput capabilities.

Editor's picks

Dr Lois Manton-O'Byrne Dr Lois Manton-O'Byrne
Applied Science Editor

UV/VIS Spectrophotometers – UV5Nano
( METTLER TOLEDO)

Product image

4 out of 5

“This very compact instrument is ideal for accurate microvolume and cuvette measurements.”
Marcel Hollenstein, Institut Pasteur

Read more


DS-11 UV-Vis Spectrophotometer
( DeNovix Inc.)

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5 out of 5

"This is a wonderful product, compact, and easy-to-use."
Christopher Garrett, BMC Princeton





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SPECORD® Plus (Analytik Jena)

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4 out of 5

“A very good spectrophotometer with a very good rate speed - sensitivity.”
David G. Calatayud, University of Bath




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NanoDrop™ 2000/c Spectrophotometers (Thermo Fisher Scientific)

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5 out of 5

"The rapid turnaround time makes this system critical for our day-to-day needs.”
William Wittbold, Ajinomoto Althea Inc.


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DropSense96® Content Analysis Platform (Trinean)

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4 out of 5

"Superb and powerful QC tool! Combines the best features from nanodrop and picogreen and even adds more info on top!.”
Morten Flobakk, BioBank AS

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Epoch™ 2 Microplate Spectrophotometer (BioTek Instruments, Inc.)

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5 out of 5

“The BioTek systems are a favorite in our lab. The Epoch 2 is great quality for the price, reliable, and easy to use.”
Nichole Lobdell, Vanderbilt University

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