Across the Spectrum: Instrumentation for UV/Vis Spectrophotometry

Date: February 2, 1998 Chart 1 Spectrophotometry is a technique most scientists have used at some point in their careers. Whether for confirmation of a compound's identity or for quantitation of a protein, spectrophotometry in the ultraviolet and visible spectrum has rapidly found increasing applications in numerous fields within the last few years. To help meet this demand, manufacturers of instrumentation for UV/Vis spectrophotometry now provide machines with a wide spectrum of features and

By | February 2, 1998

Date: February 2, 1998 Chart 1

Spectrophotometry is a technique most scientists have used at some point in their careers. Whether for confirmation of a compound's identity or for quantitation of a protein, spectrophotometry in the ultraviolet and visible spectrum has rapidly found increasing applications in numerous fields within the last few years. To help meet this demand, manufacturers of instrumentation for UV/Vis spectrophotometry now provide machines with a wide spectrum of features and specifications. LabConsumer contacted 16 companies and received information on over 50 UV/Vis spectrophotometers to choose from. As you will see, spectrophotometry has come a long way since Dr. Arnold Beckman constructed the original device in the 1940's.

Adapted from Hewlett-Packard's "Fundamentals of Modern UV-Visible Spectroscopy" publication number 12-5965-512E, 1996.
What magic happens inside that UV/Vis spectrophotometer on the bench? Several things have to happen in order for you to jot down numbers in your notebook. First, a light source generates light at a specific wavelength or wavelengths. Commonly, UV/Vis spectrophotometers utilize two light sources: a deuterium arc lamp for consistent intensity in the UV range (190 to 380 nm) and a tungsten-halogen lamp for consistent intensity in the visible spectrum (380 to about 800 nm). Some spectrophotometers use xenon flash lamps, which offer decent intensity over the UV and visible regions.

The source light is then directed to a dispersion device that causes different wavelengths of light to be dispersed at different angles. Two common dispersion devices used in UV/Vis spectrophotometers are prisms and holographic gratings. The angle of dispersion with a prism, however, can be nonlinear and sensitive to changes in temperature. Holographic gratings are glass blanks with narrow ruled grooves. The grating itself is usually coated with aluminum to create a reflecting source. Holographic gratings eliminate nonlinear dispersion and are not temperature sensitive. They do require filters, though, since light is reflected in different orders with overlapping wavelengths.

Once the light has been passed through the dispersion device and the sample of interest it reaches a detector. Detectors in UV/Vis spectrophotometers come in a variety of shapes and sizes. Photomultiplier tubes are common; they provide good sensitivity throughout the UV/Visible spectral range and are highly sensitive at low light levels. Photodiodes have seen increasing use as detectors in spectrophotometers, bringing to the table a wider dynamic range. A photodiode is generally made up of a semiconductor and a capacitor to charge the semiconductor. As light hits the semiconductor, electrons flow through it, thereby lowering the charge on the capacitor. The intensity of light of the sample is proportional to the amount of charge needed to recharge the capacitor at predetermined intervals. As opposed to having single photodiodes, some spectrophotometers are composed of a photodiode array. Here, several photodiode detectors are arranged on a silicon crystal. The advantage of an array is the ability to do side-by-side readings, thus increasing speed.

The entrance slit, dispersion device, and exit slit are referred to as the monochromator. Light passing through the monochromator exits as a band. The width of this band of light at half the maximum intensity is the spectral bandwidth. Bandwidth comes in to play with regard to accuracy, since the accuracy of any absorbance measurement is dependent on the ratio of the spectral bandwidth to the natural bandwidth of the substance being measured. The natural bandwidth is the width of the absorption band of the sample at half the absorption maximum. As a rule, a ratio between spectral bandwidth and natural bandwidth of 0.1 or less will generate absorbance measurements 99.5 percent accurate or better. Above this, accuracy deteriorates.

In a conventional spectrophotometer, polychromatic light from the monochromator is transmitted through the sample, and the sample absorbance is determined by comparing the intensity of the light hitting the detector with just a sample blank with the intensity of light hitting the detector with the sample in place. With a diode array spectrophotometer, polychromatic light passes through the sample and is focused on the entrance slit of the polychromator (the entrance slit and dispersion device). The light is then dispersed onto the diode array with each diode measuring a portion of the spectrum.

Adapted from Hewlett-Packard's "Fundamentals of Modern UV-Visible Spectroscopy" publication number 12-5965-512E, 1996.

Single-beam spectrophotometers (which include conventional and diode array) are highly sensitive devices. Spectra can be acquired quickly in diode array spectrophotometers due to the single-beam design. In conventional spectrophotometers with a single beam, the blank and the sample are not measured simultaneously, and the interval between measurements-especially if measuring a broad spectrum-can subject results to lamp drift. Dual-beam spectrophotometers utilize a "chopper" that alternates the light path between the reference optical path and sample optical path to the detector at a speed that minimizes medium- or long-term effects of lamp drift. A variant of this is the split-beam design, which uses a beam splitter rather than a chopper. This allows light to be sent to the reference cell and sample cell simultaneously.

All said and done, which spectrophotometer will eventually end up on your lab bench? The specifications of the spectrophotometer you purchase should fit your applications. Specifications such as wavelength range, wavelength accuracy, and bandwidth should be considered. Perhaps you want to read your samples in a 96-well microplate as opposed to a normal cell or a sipper. Many spectrophotometers are PC-based, and if this is what you desire, there are plenty machines to choose from. Many of the simpler models have on-board software, while many of the PC-based models require-and some manufacturers include with the instrument-application-specific software. Machines range widely in specifications, capabilities, and features. The following are brief summations of what each company has to offer based on literature received from the companies.

Amersham Pharmacia Ultrospec 3000
The Ultrospec 1000 is Amersham Pharmacia's basic model, combining basic and enhanced software functionality. The Ultrospec 2000 features a Deuterium Lamp Saver Mode, Absorbance Ratio for quality control and purity checking, and printouts with GLP requirements. The spill-proof keypad and rugged construction highlight the features of this instrument. The Ultrospec 3000 comes with built-in software for a variety of applications, including wavelength scanning, standard curve, substrate concentration, and enzyme kinetics. The Ultrospec 4000 was designed to meet the demands of the Pharmacoepeia standard. The Ultrospec 4000 is supplied with SWIFT II METHOD (Windows® 95 format) and SWIFT METHOD (Windows® 3.1 format) software. Lastly, the GeneQuant II RNA/DNA Calculator measures absorbance at 230, 260, 280, and 320 nm. The GeneQuant II provides direct readings of DNA, RNA, and oligonucleotide concentration and A260/A280 ratio purity check.

AVIV Instruments 14DS-UV-VIS
AVIV offers three instruments in the UV/Vis range. The 14DS UV-VIS utilizes a double monochromator and is a digitally controlled, dual-beam spectrophotometer. The 17DS UV-VIS-NIR also uses a double monochromator and extends the wavelength into the near-infrared range (2,500 nm). Lastly, the 202 Circular Dichroism Spectrophotometer measures differential absorbance, a technique well suited for chiral molecules.

Beckman Instruments DU 7500
The DU Series 500 from Beckman features interchangeable sample modules and a complete set of application programs. Full-text prompts and soft keys guarantee simple interaction with the system via the graphical, backlit LCD. The DU Series 600 includes various configurations for different needs and applications and is available with or without a 14-inch VGA color monitor. The DU Series 7000 features diode array technology and is also available with or without a 14-inch color monitor. The DU Series 600 and 7000 units also come with programs for various measurements.

Bio-Tek PowerWave 200
The PowerWave 200 and PowerWave 340 are dual-beam microplate spectrophotometers, the 200 with an extended wavelength range. Both have an optional incubation feature (ambient +4°C to 50°C); offer onboard data reduction, curve-fitting, and 10-plate test results storage; and come with the KC4¦ for Windows® software package. Bio-Tek Instruments also offers bar code and robotics options for both spectrophotometers.

Hac DR/4000U
The DR/4000 U from Hach is a split-beam spectrophotometer that gives users several sample format options, including slide-in, multiposition carousels, pour-through cells, and a sipper system. Digital readouts of direct concentration units, absorbance, or percent transmittance are provided by the DR/4000 U. This system comes with over 130 preprogrammed calibration curves, numerous accessories, RS232 interface, and an illustrated instruction manual.

Hewlett-Packard HP#8453
The HP 8453 is a diode array spectrophotometer with a graphical user interface and built-in GLP capabilities. This instrument allows better than 2 nm resolution and less than 0.03 percent stray light. The Windows-based HP ChemStation software can be extended to advanced software for spectroscopy method development, biochemical analysis software for enzyme kinetics assays and DNA melts, and dissolution testing software for pharmaceutical applications. The HP 8453 also interfaces with a variety of accessories including a multicell transport, a Peltier-thermostatted cell holder, a sipper system, and an autosampler.

Hitachi Instruments U-3010
The U-2001 from Hitachi is a photodiode-based spectrophotometer featuring optional application-specific software. The built-in high- resolution LCD allows simple graph and data review. A GLP/GMP software card provides quality assurance validation. The U-2010 is a compact, double-beam spectrophotometer with Microsoft" Windows 95® software that allows quantitative analysis, wavelength and time scans, and wavelength programming. The U-3010 and 3310 are dual-beam spectrophotometers with Windows 95® or Windows NT® software packages. These units feature low stray light, variable bandpass, and linearity to greater than 3.0 absorbance (4.0 or higher with the 3310). Hitachi also recently introduced the new U-1500, a high-performance, low-cost spectrophotometer.

Cecil Sepctrophotometers from ISC BioExpress
ISC Bioexpress markets the Cecil (U.K.) line of spectrophotometers. The Cecil 1000 Series has a deuterium (UV) lamp that may be switched off when not required. The Cecil 2000 Series consists of single-beam models for routine analysis and features a high-resolution LCD screen, curve fitting, method storage, software extension modules, and immediate software upgrades. The Cecil 3000 Series consists of high-performance single-beam spectrophotometers with a wide range of quantitative methods made available with the embedded software. The Cecil 8000 and 9000 Series units are double-beam spectrophotometers with built-in software. These instruments can be controlled using a personal computer with GRAMD/UV software for Windows".

Jasco V-530
The V-Series from Jasco-all double-beam spectrophotometers-provides researchers with a wide range of spectrophotometric tools. The V-530 is a general-purpose spectrophotometer with silicon photodiode detectors. The V-550 utilizes a photomultiplier tube, and bandwidth can be set down to 0.1 nm for high-resolution work. The V-560 also uses a photomultiplier tube but incorporates two symmetrical Czerney-Turner mount monochromators for higher resolution and very low stray light. The V-570 employs two detectors: a photomultiplier tube detector is provided for the UV/Vis region and a Peltier-cooled PbS detector for the NIR region, covering the range of 190 to 2500 nm. The J-715 measures UV/Vis/NIR absorbance along Circular Dichroism for analysis of chiral molecules. The Model 6405 can handle numerous sample formats, including various cells and test tubes, and even an eight-position motorized multicell changer. Features of this unit include analog output and a bidirectional RS232 interface, long-life lamps, continuous real-time display, and graphical display of data. Users can choose among three scan speeds (250, 500, or 1,000 nm/min) and three scan intervals (0.25, 1, or 5 nm increments). Jenway also offers comprehensive option packs such as an in-built printer, a Peltier-controlled temperature cell block, and a sipper pump.

The SPECTRAmax PLUS from Molecular Devices
Molecular Devices offers three microplate spectrophotometers. The SPECTRAmax® PLUS can read cuvettes and 96-well microplates and can compare data between the two as well. With this unit, 96 samples can be read in as little as 10 seconds. The SPECTRAmax 190 and SPECTRAmax 340 both read 96-well microplates. Both the SPECTRAmax PLUS and SPECTRAmax 190 employ a new technology that determines the pathlength of light traveling through a sample in a microplate. Software for Windows® 95/NT or Mac/OS users is available.

Perkin-Elmer Lambda Bio 20
The Lambda Bio Systems from Perkin-Elmer all include ready-to-run methods for DNA, protein, and enzyme analysis. The Lambda Bio 10 is a single-beam spectrometer for all basic routine applications such as DNA quantification and colorimetric tests. The Lambda Bio 20 is a double-beam spectrometer offering enhanced stability and greater spectral resolution than the Lambda Bio 10. The stable optics of this system make it ideal for enzyme kinetic measurements. The Lamda Bio 40 is a double-beam spectrometer with variable slit widths. This high performance system is easy to use and flexible. All Lambda Bio spectrometers come standard with a disk of UV BioLab¦ methods for the PC.

Shimadzu Scientific's UV-1201
Shimadzu's UV-1201 features a large, adjustable LCD, and users can choose from 17 different application-oriented Program Packs to configure the system to their needs. The UV-1601 is a double-beam spectrophotometer with numerous standalone or personal computer operations (UV-1601PC). Users can rapidly change sample formats, from a standard cuvette to a capillary cell or to a powder or film holder. The UV-2401PC is a single monochromator system suited for a wide range of applications. From small sample volumes to highly absorbing samples, the software gives users the versatility of a sophisticated system. The UV-2501PC features a double-blazed grating double monochromator for low stray light levels. Shimadzu's UV-3101PC has two sets of three gratings to cover wavelengths from UV to near-infrared. The UV-3101 is designed for use with Shimadzu's Personal Spectroscopy Software for instrument control and data analysis.

Spectral Instruments 400 Series
The Model 400 spectrophotometers from Spectral Instruments combine fiber optic sampling technology with a highly sensitive charge coupled device (CCD) array detector. This feature makes these units well suited for spectrophotometry with low light levels, such as diffuse reflection measurements and colorimetry, and even spectroradiometry and many luminescence applications such spectrofluorimetry. The use of an array detector also enhances speed, allowing acquisition of five full-range measurements every second. Spectral Instruments offers four models in the 400 Series with varying spectral ranges.

SPECTRONIC Instruments
SPECTRONIC brings several UV/Vis models to the lab bench, beginning with the SPECTRONIC 21 DUV. The SPECTRONIC 21 DUV spectrophotometer is a digital-readout instrument that displays transmittance, absorbance, concentration, and absorbance-to-concentration conversion factor. The SPECTRONIC 401 is a microprocessor-controlled unit featuring a built-in RS232 interface, Auto Zero, and an easy-to-read digital readout of wavelength and data in absorbance, percent transmittance, and concentration modes. The SPECTRONIC 20 GENESYS¦ spectrophotometer is a compact, grating-based unit designed for rugged yet simple use. A two-line LCD readout displays data and gives understandable messages. The SPECTRONIC GENESYS spectrophotometers run tests which cover a wide range of applications and feature backlit, LCD screens which present test parameters, test results, and easy-to-read instrument status pictograms. The full-color display of the GENESYS 2 further enhances data presentation.

Varian Cary 500 Scan
The Cary 50 Series boasts a xenon flash lamp with a scan rate of up to 24,000 nm/minute. The Cary 100 and 300 are upgraded versions of the Cary 1 and Cary 3, respectively, utilizing Windows®-based software as opposed to DOS. These spectrophotometers incorporate a dual-beam design, but can be operated in single, dual, or dual-single beam modes. The Cary 100 and 300 are suitable for routine laboratory work and feature a patented optical system with a quartz overcoating that protects the optical system from damage and degradation. The Cary 300 has a premonochromator, extending the working range of the unit past 5 absorbance units by lowering levels of stray light. The Cary 400's working range extends beyond 6 absorbance units, allowing measurement in the low UV range. The Cary 500 is a research-grade reference UV-Vis-NIR instrument, incorporating a cooled PbS detector to reduce photometric noise. Both the Cary 400 and 500 use the Non-Measurement-Phase-Stepping wavelength drive system to ensure the measured photometric value at a particular wavelength will remain the same, regardless of scan speed. The Cary 400 and 500 are upgraded versions of the Cary 4 and 5.

Chart 1

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