edited by FRANK A. SETTLE, JR. Vi~iniaMiiitavMilute Lexington. vA 24450
ch&miccrl injtrument~tion Evolution of Instrumentation for UV/Vis Spectrophotometry Part II lnes R. Alternose, Lesley E. DeLong, and Laurence E. Locke Milton Roy Company, Analytical Products Division, 820 Linden Avenue, Rochester, NY 14625
Evolutlon of Data Output The meter represents one of the earliest and simplest data displays on a photoelectric instrument. Termed a galvanometer or balancing potentiometer, it translates the electrical signal from the photodetector t o a needle deflection on the meter display. Values are read from a scale calibrated in percent transmittance andlor absorbance. An anti-parallax mirror along the face of the meter aids in making reproducible readings. The largest evolutionary step in data output was the introduction of the digital display. Direct readout of data values became possible, eliminating the need t o assess alignment of the meter needle visually with calibration marks. Dieital disolavs are thus
because of their expense. Today one commonly finds LED (light-emitting diode), LCD (liquid-crystal display), vacuum fluorescent display, and CRT's (cathode ray tube) huilt into spectrophotometers. Many simple general-purpose spectrophotometers now have upgraded models with a digital LED display. Alphanumeric readout requires an LCD, LED, or vacuum fluorescent display. A graphics display, such as a wavelength scan or time scan, requires a CRT or LCD. Some instruments have a built-in CRT, others use the video monitor on a computer. The advantages of a built-in CRT are that scans can he viewed during data acquisition without any peripheral equipment, such as a recorder, and they can be
able. As mentioned in Part I,' data on meter instruments can be in the absorbance and/
A log converter permits absorbance output from the dc signal. With additional electronic circuitry (such as a variable-gain amplifier), a factor can be introduced to convert absorbance data to concentration results. The Spectronic 20D is an example of
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the uoeradine" of a " eeneral-ouroose low-cost spectrophotometer to include direct concentration readout. Transmittance, absorbance, and coneentration data output are all analog. An analog output allows communication with an X-Y or Y-T recorder for scans, enzyme kinetic studies, or histograms. The recorder represents the oldest type of nonmanual hardcopy output. With theaddition ofan analogta-digital converter, digital output became possible. At first, digital signals were used for digital displays and BCD (Binary Code Decimal) output. BCD is the earliest digital output that enabled communication with a printer. More recent technology enables speetrophatumeters to send the digital signal via a serial or parallel port to communicate with a printer or computer. The computer in turn can communicate with recording devices, such as printers and recorders, by use of the appropriate interfaces. Hard copy of data from a printer is generally in a tabular format. From a recorded plotter or printerlplotter, output is in graph format, either as a eurve(s) or as a histog r a m ( ~ )With . the increasing "intelligence" of recorders, plotters, and spectrophotometers, graphical output has evolved from a simple line corresponding to voltage output to the ability t o control and label axes, plot curves and offset axes. Examples of hard c o w. from various data outout devices intert a t 4 o l t l >a rpeitn,phutumewr can lhe i w n in 1 1 1 ~liyurcs dcumpenylng the nrkr srrl i w u,n dala mdn~pulutiun. The i,hvwur sdvantage to the user of hard copy dataoutput is the elimination of tedious manual copying and plotting of data values, savina time and reducing the chances for errors. -
1960's, consisted primarily of clinical analyzers. These spectrophotometers with early microprocessors could be used for either endpoint or kinetic tests. An endpoint test allows for a short period of temperature equilihratian and then presents results automatically in percent transmittance, abosrbance, or concentration units. For kinetic te>r., the change in abwrbance over timr ~ A . l , a n drheenzymeart~viry(in Intern3lim*l L'nitsi are m l c ~ l a t e dand ~ r i n t r d . Figure 1 is a printout from the Spectronic 2100 Clinical Analyzer, a typical instrument from the 1960's generation, in the Kinetic1 Repeat Mode.
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SAMPLE IOENTIFICATION NO.
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INITIAL ABSORBANCE 'in ABSORBANCE CHANGE i,? (DELTA A)
ABSORBANCE CHANGE TIMES KINETIC FACTOR
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NEW INITIAL ABSORBANCE
i.Wi i i -. tur thia type uf test I t is used in the quahty control uf culurrd solutiuns such as beverages
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Kinetics Test This type of test, as previously mentioned, measures the change in absorbance during aprescribed period of time. The data printout, as shown in Figure 2, gives more information than those from earlier instruments leompnrp with Fig. I ). In addition rc, the ~ n ~ t i aahwrhnnre. l the ahsorhance change, and the absorbance change multiplied by the kinetic factor (IU-International Units), this printout contains the test type and name, analytical wavelength, time required to run the test, time(s) of measurement, total change, and average change. All data values as well as final answers appear on the printout. Most instruments also list test parameters.
A,
A,
A=ABSORBANCE = BASE WV,
A,
h =PEAKWV
A, = BASE WV,
Linear and Nonlinear Curve Fit
BASELINE CORRECTEDABS r
3 PNT NET NAME IEIITDLY CYCTIME CYCLES SIGAU FACTOR LOWLIMIT HI LIMIT EWE1 W U PEAK WU EASE2 WU
Three-Point Net Absorbance (Allen "Base-Line" Technique)
To obtain concentration data on unknown samples, a standard curve must first be established. In many modern spectrophotometers, the microprocessor can calculate a linear or nonlinear curve when provided with data from two or more standard solutions. For a linear curve fit, the standardsareused in thelinear equation,^ = rnx b, which is determined by the leastsquares linear regression method. If the relationship of absorbance versus cancentration is nonlinear, a standard curve can he established from two or more standards with the use of a segmented curve fit or a higher-order equation.
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3 2500 0000 100.0 5 7 0 . MNM 622.5NM 7 2 0 . 0HM
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The instrument will automatically search a given wavelength range for the major peak. This type of data is useful in research for identifying a material or for determining the optimum wavelength t o use for an endpoint analysis.
A,
WAVELENGTH (w)
The instrument measures the absorbance of a sample a t two different wavelengths (A, and hd and computes the ratio or difference ofthe two (AArlAA2or AXl - AX2).The data printout usually lists all of the datavalues as well as the final answer: the ratio or difference multiplied by a factor. Additionally, the spectrophotometer can be programmed to run tests for a number ofcycles a t specific time intervals. This kind of data manipulation is important for the assessment of puritv of samoles: . . for instance. the ratio of the abiorlumrpr at 261)nm a n d 2 ~ 0nm gives a. indirat~onc,t'I)N,\ p ~ r i t yin a preparation.
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Automatic Peak Location
Absorbance Ratio and Difference
This type of analysis is used to correct for the effects of a broad sloping baseline on a peak height determination. T h e absorb a n c e ~of a sample a t three wavelengths are measured, two "base-line" wavelengths and a peak wavelength. The spectrophotometer then calculates the peak height relative t o a calculated baseline. An example of this type of curve is shown in Figure 3, along with a printout of parameter values and sample test values. This technique can he used for the determination of benzoic acid., a commonly used preservative, in juices, jams, jellies, and tomato products.
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ID# 1 3 PNT NET RUN TIME
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Statistics
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3PTNET 0.8MIN
570.0 622.5 720.0 EASE1 PEAK EASE2 TIME ORMIN 3 9 6 1.501 ,255 NET 3 29.29 TIME ,287 NET 3
0.5tlIN 1.502 .225 30.98
The microprocessor in a spectrophatameter frequently can be used for further test data analysis by statistical methods. The mean and standard deviation are calculated for a series of data values obtained in a particular test, such as endpoint, kinetics, ahsorbance ratio, and absorbance difference.
Test Storage Although not data manipulation in the strictest sense, it is important to mention that the microprocessor in many spectrophotometers can be used to store user-programmed test programs. A user can "call up" the test every time i t will be performed,
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Figure 3. Three-point Net Absorbance Test, Curve showing the broad sloping baseline and calculations, test parameter listing, and data printout from a sample test.
TYPICAL PRIMOUT TEST NAME SAMPLE IDENTIFICATION NUMBER W ~17 TEST TYPETIME OF MEASUREMENT
KINETICS RUN TlME TIME 3 IS 13
A& ,190 372
555
U T EH a N a L ~~O~NM-WAVELENGTH 63 SEC -TIME DELTA
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IQ/CHANGEIN 184
u n c n L REQUIRED TO RUN TEST ABSORBANCE DURING KINETIC INTERVAI
I85 ~
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,165 TOTAL CHANGE 715~/ 715 85R51U ....... .. \FINAL
A V E U G E CHANGE
RESULT = AVERAGE CHANGE X FACTOR
Figure 2. Data printout for the Kinetics Mode from a modern micropracessor-controlledspectraphotometer.
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ANALYTICAL WAVELENGTHS IN nm Figure 4. Histogram data output for a Multiple Wavelengths Test.
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saving time and ensuring that test parameters remain constant from experiment to experiment.
Signal Averaging/Signal Smoothing On some microprocessor-controlled spectrophutometers signal averaging is available. This feature enables the microproeessor to take signal outputs and average them to produce a less noisy output. With a selection of signal average values, the operator can optimize t h e signal-to-noise ratio, thereby filtering out noise with minimal loss in resolution.
High-Low Limits In some spectrophotometers, a programrnahle "high-low" limit feature flags levels outside a user-specified range for easy interpretation, even by the inexperienced user.
WAVELENGTH Figrue 5. A wavelength scan plus repeat scans with both axes offset.
Summary of Flxed Wavelength Analyses Data Manlpulatlon It is readily apparent that the enhanced data manipulation capabilities of microprocessor-controlled instruments result in tests that are easier and quicker to set up and run and in relatively error-free analyses and data reporting. These features are especially useful in laboratories performing numerous routine analyses daily. The benefits to such laboratories include the use of relatively unskilled labor, fewer recording errors, and with enough automation, continuous operation. These increased capabilities are used to advantage in many applications, particularly clinical laboratories and those concerned with quality control.
Scanning Analyses Scanning and Baseline Storage In most spectrophotometers, to scan a sample over a range of wavelengths, the dispersing device (grating or prism) must he moved to obtain all the wavelengths desired. The most primitive scanning instruments relied upon manual adjustment of wavelength. More modern instruments have a wavelength drive, and automatic measurements can be made of absorbance or transmittance over a specified wavelength range. These measurements also can be made after an initial delay and a t fixed time intervals for a prescribed number of cycles. To scan, a baseline must be stored over the wavelength range of interest to compensate for differences in energy output of the lamp(s) over that wavelength range. Scanning has traditionally been accomplished with a double-beam optical design where the reference is scanned simultaneously with the sample. Since modern microprocessors allow the storage of a reference scan, scans are now possible with single-beam instruments. In the latter the reference and sample scans are separated in time; the reference dataobtained a t a n earlier time is subtracted from the sample data during the scan. In double-beam instruments the scans are separated in space; the reference and sample data are obtained simultaneausly. The split-beam optical configuration, as discussed in Part I, scans by
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WAVELENOTH
OF A8SORBANCE PEAK
0
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3. S.cond derirrtlrc
r a n ol I (llrst dnlratlre 012)
DECREASINGL" NEGATIVE ABSORBANCE CHANGE
0 WAVELENGTH OF
PEAK
Figure 6. Simulated derivative scan of a simple absorbance band.
diverting a portion of the light and uses it for a reference through air and by storing a baseline scan. In this way the split-beam design provides the high energy level of the single-beam design and the stability of double-beam design.
Repeat Scans A repeat scan program can he used to observe changes of a scan with time. The microprocessor can control the plot axes on the recorder so that changes are graphically emphasized either by superimposing the spectra;or by combining "stacking", using theyaxis offset, and "shifting", using the x-axis offset, to produce a three-dimensional effect. Figure 5 is a n example of repeat scans with both axes offset.
Derivative Spectroscopy Derivative spectroscopy measures the change in slope occurring in a spectrum as the wavelength changes. The calculation of
derivatives of a scan results in increased spectral resolution. Figure 6 shows a simulated absorbance scan with an absorbance maximum and its relationship to the first and second derivatives of this scan. First and second derivatives are important when overlapping bands on an absorption spectrum severely limit the ability to perform reliable qualitative and quantitative measurements. These features are also important for quantifying a shoulder peak. Derivative scanning can enhance resolution and discriminate in favor of sharp spectral features for the purposes of identification and quantification. Derivative spectroscopy is important in production quality assurance testing of organic solvents where slight impurities will alter the characteristics of a scan. It frequently is used in pharmaceutical and biomedical research. T o increase sensitivity, same scanning spectropbotopeters have a programmable
Ah. the interval (in nilnametersl~,used in the ~~~derivative calcul'ation. With a low Ah value, smaller changes can be detected. Although this results in greater sensitivity, it will em. phasize short-term phenomena, in particular, noise. The fact that it is programmable gives the operator more flexibility, and if signal averaging is available, the signal-tonoise ratio can be optimized. ~
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Log Absorbance Spectra Taking the log of an absorbance spectrum accentuates small details in the spectrum, providing a "fingerprint" for a particular material. It enables comparisons of spectra for the same material regardless of concentration and is used in forensic research.
Computer lnterfaclng t o Increase Data Manloulatlon C a ~ a b l l l t l e s ~ ~ ~ Today many spectrophotometers have computers built-in for complex data handlingor have the capability to interface with an external computer. Built-in computers have the advantage of providing data-bandling features in the same unit. The main disadvantage is that if the computer breaks down, the entire instrument becomes inoperable. With an external computer, in the event of a breakdown. the instrument can still be used for spectrophotometric determinations. The user also has the option of interfacing with another computer for enhanced data handling. Additionally, an external computer can be used for other operatims such as word prncessing and spreadsheets. Computers (either built-in or external) can provide data enhancement and manipulation features such as: spectrum smoothing scale and peak expansion eraohieal data disolav " ;se' of peripheral printers t o obtain hard copy data high-order derivatives (on wavelength o r time scans) comparison of spectra addition or subtraction of spectra location of peaks and valleys in normal and derivative spectra programming of complex procedure tests, such as kinetics, repeat scans, or multiple wavelength analyses
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A computer can be used t o store data. The main advantage of this is that data may he stored in a tabular form which can be easily changed to fit the user's needs. This storage can be in the computer, on tapeor disks, in a data logger, or in a peripheral computer. Once stored, the data can be formatted by order and labelled to produce a usable hard copy on a printer, in the form of ready-tocirculate reports; or as easy-to-interpret graphical hard copy via a plotter. Advances in software design now enable the merging ofstoreddata with software such as Lotus 12-:3 to format data into graphs and tables. Applications of these capabilities can include the following: tristimulus color measurements (important in foods, textiles, paints, colored glass, and plastics) repeat scan (quality control) batch processing of samples (quality control) rate measurements (clinical chemistry)
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calculation of absorbance derivative (trace analysis) matrix and component suhtraction (forensic) difference spectra (forensic)
Program Cartridges A recent development in data handling is the cartridge accessory. Cartridges contain a totally permanent memory (ROM) that cannot be erased and electrically erasable memory (EEPROM) that stores parameters for test setups. Many of the same data manipulation capabilities built into a larger instrument, such as the Spectronic 1001 or 1201, can he contained on these cartridges. Cartridges plug into the instrument and are generally programmed for a limited number of tests. This provides specifieity. A user need buy only those cartridges containing tests required for a particular application. tlecause the cartridges are separate units, they can be removed from the instrument for storage or be sent, already programmed, to another laboratory. An R&D or a QA laboratory thus can reproduce a test exactly as it was done in another laboratory. The individual cartridge design concept also allows development of additional data manipulation capabilities long after the main instrument is introduced. To summarize, the advantages of cartridges aver built-in test modes are: flexibility, specificity, easy repeatability of test parameters between laboratories, and unlimited test storage because additional units can be purchased once the memoryofa unit has been exhausted.
Evolution of Sample Handling The first generation of speetrophotometers had limited sample handling capabilities. They were designed primarily to handle manually inserted test-tube cuvettes. The main flexibility in sample handling was the diameter of the test tube. The introduction of the cuvette adapter permitted the use of standard 10-mm-square cuvettes. Greater accuracy can he achieved with reetangular cuvettes having parallel faces since cylindrical cuvettes present problems by acting as astigmatic lenses, causing the light to go out of focus. Cuvettes originally were made of glass. With the advent of quartz optics, use of the ultraviolet region of the spectrum became possible, requiring the use of silica or quartz cuvettes (since glass absorhs in the UV). Today, plastic disposable cuvettes for visible spectroscopy are available. The most commonly used euvette has a light pathlength of 10 mm, but longer or shorter pathlength cuvettes are available for samples with concentrations or absorbances outside normal ranges. For dilute solutions, the sensitivity of a speetrophotometric procedure can be increased by the use of a cuvette with a longer pathlength (common ones are 50 mm and 100 mm). Conversely, a concentrated solution must be measured in a shorter-pathlength cuvette. When sample volumes are restricted, microcells are particularly useful. Many spectraphotometers are equipped with a sample compartment that will accept one or more cuvettes of various pathlengths and sizes (with the appropriate adapters). Cell holders with multiple positions (typically four, five, and six) load and measure Volume 63
more than one sample a t a time, thereby saving time. Also, with multiple positions, the reference always can be kept inone position for a rapid reference cheek. To extend the usefulness and convenience afspectrophotometric teehniques,cells with flow-throughcapability have been designed. They enable both in-linesampling as wellas rapid discrete sampling, purging, and rinsing of cells when performing procedures that require many replicates. From the flow cell, cell contents can be purged automatically or manually into a waste receptacle or returned to the source container, thereby saving scarce samples. For high-pressure applications such as HPLC, flowcells cspable of withstanding high pressures are used. Because the temperature of the euvette is important for some applicati~ns(e.g., enzyme studies), special cells have been designed to regulate temperature. The simplest contain a water jacket and must be connected to a circulating water bath. A more modern, electronic means of regulating temperature is with a Peltier device, which commonly adjusts temperature in the range of25-37% in 0.1 OC increments but is capable of attaining higher and lower temperatures. Some spectraphotometers are equipped with a temperature programmer to raise or lower temperature with time and record absorbance changes. This accessory is used to perform a "DNA melt" analysis which can identify the type of DNA in a sample. Some spectrophotometers have speeialized accessories to handle specialty applications. Common ones are gel scanning attachments for quantitation of stained materials on a gel; dissolution apparatus for dissolution studies of tablets in the pharmaceutical industry (as a measure of bioavailability); and reflectance attachments t o measure reflected color of opaque objects. Recent trends in sample handling are toward increased automation. Many spectrophotometers can be interfaced with a frontloader, also called a shuttle, for automatic sample handling. This piece of equipment will handle many samples (typically 100200) contained in test tubes and bring them into position for sampling by the spectrophotometer (via the flow-through cell). For further automation of sample handling, the automatic sample handler can he used in conjunction with a diluter-pipettor, t o dispense reagents and dilute samples. The ultimate in automation is the robot.5 A robotics system for spectrophotometry consists of a spectrophotometer, a computer, and a robot(s). The computer controls the operation of the robot for performing various preparative steps such as weighing, mixing, and dispensing reagents and diluting samples. The computer then instructs the spectrophotometer t o run the test and the robot to remove and dispose of the samples. The most obvious advantage of a robotics system is the freeing up of technician time for other tasks. Additional advantages are the ability to run an operation continuously and unattended, and to improve precision due t o the elimination of operator error. The computer in a robotics system acts as an intelligent interface for all parts of the
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Future - Trends ~ ~
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~~~h of the systems we have discussed in thisoverview willcontinue toevolve as technology develops. In the optical system we will probably see the growth of fiber optics for remote sampling, use of lasers as light system. It not only controls the robot but sources. imoravements in eratines. also saves all of the samole oreoaration .. . . . and indara. Bpraure there is tno-way z ~ m n ~ u n i c a - crcnsed use u i diude arrays. The wntinued de\elupment c,f rol,~mciwrll result in more rim, the iumputer, U Y L ~rtcrivinj annlytipwerful ay*temr t
