Instrumentation

I
Dlama 0. Jones
Tracor Northern
2551 West Beltiine Highway
Middleton, Wis. 53562-2697

Photodiode Rrray Detectors in

UU-VIS Spectroscopy: Part 1
I
I n Part I, which appeared in last
months issue, the theoretical aspects
of diode array UV-VIS spectroscopy
were discussed. The second article in
this two-part series describes the applications of diode arrays in analytical chemistry.

Applications of the linear photodiode array (LPDA) spectrometer in

the UV-VIS spectral region can be
considered under four headings: (1)
molecular spectroscopy, (2) rapidscanning detection for time-dependent phenomena, (3)rapid-scanning
detection in HPLC, and (4) other ap-

plications, which include low-lightlevel applications and atomic spectroscopy.

w-mw
The combination of electrochemistry and spectroscopy was first reported in 1964 (I). Since that time spectroelectrochemistry has been used to
study the redox chemistry of inorganic, organic, and complex biological
molecules. The redox reactions in
these systems occur on the second to
millisecond time scale. Consequently,
the ability of the LPDA spectrometer
to measure spectra on a millisecond

time scale is important. Although Raman, fluorescence, and internal- and

specular-reflectance spectroscopy
have been used in spectroelectrochemistry, the majority of applications
involve light absorption or transmission in the UV to IR spectral region.
Transmission and absorption spectroelectrochemistry require that the
light first pass through an optically
transparent electrode and its diffusional neighborhood, then through the
solution, after which the transmitted
intensity can be detected (Figure 1).
Conventional electrochemical cells
with transparent electrodes have path

Figure 1. Typical spectroelectrochemical cells

Adapted Iran Referen-

0002-2700/85/A357- 1207$01 50/0

@ 1985 American Chemical Socieh

ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985

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lengths on the order of 1 cm. Thin-layer electrodes (TLE) with smaller path
lengths (< 0.02 mm) allow rapid electrolysis (20-120 s) of electroactive species (2).
Optically transparent electrodes
(OTE) are subdivided into three
classes (3).The first electrode class
consists of a very thin film of conductive material, such as platinum or
gold, deposited (100-5000 A) on a
transparent substrate such as quartz
(UV-VIS) or glass (visible). The second is the minigrid class, which consists of a minigrid or micromesh of
metal (Pt, Au, Ag, Cu, or Ni). The
third class is a mercury electrode in
which a thin film of mercury is deposited onto a Au or Ni minigrid or onto
Pt or carbon film electrodes.
OTEs have advanced to the stage
that routine spectrochemical measurements are possible, and the field is expanding to include OTE cells for
HPLC ( 4 ) .The small cell volume typically found in the TLE and resulting
small absorbance values for dilute
samples have resulted in a number of
studies to maximize cell geometry, to
use fiber-optic coupling, and to assemble averaged spectra from multiple experiments to improve S/N ratios.
In a basic cyclic voltammetry experiment, the spectra are recorded for
a series of applied potentials. Potentials are maintained until the solution
reaches equilibrium and electrolysis
ceases. The Nernstian equation and
Beers law can then be used to calculate the formal potential, the number
of electrons involved in the reaction,
and the ratio of the oxidized to the reduced form of electroactive species
(5).This information is used to hypothesize the redox reaction pathway.
The optically transparent thin-layer
electrode can also be used in the study
of reaction mechanisms. The oxidized
state of the compound is converted in
the redox reaction by the application
of a potential for a specified time. In a
manner analogous to stopped-flow
and kinetic techniques, the spectral
properties of the reduced state are
monitored and treated mathematically with conventional kinetic equations.
Unlike spectroelectrochemical measurements using scanning wavelength
spectrophotometers with photomultiplier detection, which suffer from slow
or inadequate detector and scanningsystem response time, the LPDA spectrophotometer can provide complete
spectral characterization of redox intermediates. Actual spectral data can
replace hypothesized reaction mechanisms and mathematical modeling.
The LPDA spectrophotometer has
been used in the spectroelectrochemical analysis of the biologically important class of compounds, metalloporphyrins (6, 7). With the complete
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spectral information provided by an

LPDA spectrophotometer system for
mitochondrial b-cytochromes, the
field of spectroelectrochemistryhas
been combined with mathematical
data manipulations, such as singularvalue decomposition, to identify different b-cytochromes in beef heart mitochondria (8).The ability of current
LPDA systems to skip diodes (thereby
drastically reducing detector response
time to less than 5 ms for the monitoring of predefined spectral regions of
interest) is an area yet to be explored.

HPLC
During the past 15 years, HPLC
(high-pressure liquid chromatography) has become one of the major analytical techniques for the analysis of
complex mixtures of chemical compounds, such as pharmaceutical formulations, plant extracts, and drugs in
human body fluids. With the advent
of integrated circuits and inexpensive,
powerful microprocessors, HPLC detection systems have become so refined that they are now commonly the
most sophisticated equipment in an
HPLC system. Yet even with the major advances in detector technology,
the technique of HPLC has suffered
from the lack of a universal detector
that provides optimum sensitivity and
flexibility.
The UV detector is one of the most
commonly used detection systems in
HPLC. The UV-VIS detector can be
used not only to detect that a component is eluting from the chromatographic column (qualitative analysis)
but also to determine the concentration of a compound provided that a
calibration curve from known concentrations of the compound of interest
exists (quantitative analysis).
The first HPLC photometric detectors were fixed-wavelength UV detectors using the strong 254-nm emission
line of low-pressure mercury lamps.
The low cost and the relative universality of this detector have made it the
most common type on the market today; however, these detectors are severely limited in that they analyze
only one wavelength. Chemical determinations that have depended on single-channel detection have often been
limited in specificity because of the
broadband absorption character of the
chromophores. When compounds
show little or no absorbance a t 254 nm
or when interfering substances exist,
accurately quantifying a single compound at a single wavelength is impossible.
These limitations led to the development of selective filter photometers
that monitor a limited number of
wavelengths and to variable-wavelength detectors offering a wide selection of UV and visible wavelengths.

ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985

With conventional variable-wavelength detectors the eluent flow must

be stopped to trap a peak in the flow
cell while a UV-VIS spectrum is obtained. This approach presents the analyst with many wavelengths but does
not provide spectrophotometric analysis at more than one of these wavelengths a t any given time.
The advent of the LPDA detector
has eliminated the necessity of the
single-wavelength approach to HPLC
detection. Although an LPDA detector is still limited to compounds that
absorb UV and visible light, this detector allows simultaneous acquisition
of spectral information, without stopping the eluent flow.
The LPDA HPLC detector has several advantages beyond simple component identification. These advantages
occur both in the temporal and spectral domains. In the temporal domain,
analysis time can be shortened because the wavelength dimension allows the analyst to observe all UVVIS-absorbing chromophores during a
single elution. In the spectral domain,
an improvement in detection level is
gained by virtue of the availability of
the total UV-VIS spectrum of the
chromophore. The analyst can sum
the output intensity over several discrete diodes or wavelengths (broadband integration);this provides a significant advantage over single-wavelength detectability. In most cases this
can account for increases of 5-500 X in
detectability (9).
The analyst can obtain multiwavelength chromatograms from a single
analysis of one sample. In situations
where the solutes are not resolved by
the column but where spectral overlap
is minimal, all the components comprising a single elution profile can be
determined simultaneously by monitoring each component at a wavelength that is free of interference. Simultaneous acquisition at different
wavelengths also ensures the detection
of all components of a complex mixture (universal detection).
Additionally, when using an individual narrow-wavelength band, wavelengths can be selected that optimize
sensitivity while minimizing interferences, The chromatographer can
choose the wavelength of maximum
absorbance to gain sensitivity or the
wavelength providing the optimum
signal relative to possible interfering
substances to gain selectivity. Additional specificity can be obtained from
information about peak position in
both the wavelength and time domains. Identification of unknowns can
be carried out concurrently with quantitative analysis of known species in
the same chromatogram. Increases in
sensitivity are obtained by adaptability in both wavelength and time aver-

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valve to connect a sample

mn directly to an analytical

a valve to exchange one column for another when

for manual operation. And each is also available with

a pneumatic actuator for automatic operation.
The valves are described in our catalog, which contains
diagrams showing how to connect them to columns,
pumps, and injectors.
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and refractive index changes found in

HPLC are similar to those found in
stopped-flow kinetic experiments. Although both types of experiments involve flowing systems, the spectral
data requirements of the two techniques place different demands on the
LPDA spectrometer and associated
computer. In the stopped-flow experiment, speed of acquisition is critical
for the characterization of reactive intermediates. In the HPLC experiment, memory capacity of the data acquisition system is usually more important than spectral acquisition on a
millisecond time scale.

I
Flgure 2. Reactants A, B, C. and D; mixers 1, 2. 3, and 4

%ppecl llow
Fast-flow kinetic studies of chemical reactions have traditionally been
conducted with single-wavelength
spectrometers or, if the reaction time
permitted, with conventional scanning
spectrophotometers. Recently, UVVIS spectrometers using LPDAs combined with a commercially available
stopped-flow device have been successfully used to study the oxidation
of cytochrome c oxidase in the
450-920-nm region (10) and to study
the catalytic cycle of molybdenumcontaining enzymes (11).
The speed of spectral acquisition
found in LPDA spectrophotometers
has made possible the spectral detection and characterization of chemically reactive intermediates directly
through spectral analysis rather than
through the classical reaction rate
techniques. Wavelength-dependent
anomalies, Rayleigh scattering, and
refractive index changes, which occur
in stopped-flow experiments, are
readily detected and corrected with
the information contained in the total
spectrum acquired with an LPDA
spectrophotometer. The overall information contained in a nonabsorbing
spectral region or a spectral region of
minimal absorption can he effectively
used, just as in HPLC, to correct for
system artifacts. Rayleigh scattering is
attributed to the presence of huhhles
or macrocontaminants in the sample
and appears as temporal random noise
at the detector. This noise limits the
dynamic range of the system. Refractive index changes are attributed to
insufficient temperature control in
some cases and the subsequent thermal lensing effects (12).
One commercially available
stopped-flow system (with a digitally
controlled, positive-displacement, motor-driven ram) incubates both the optical cell and reactants in the same
isothermal bath to minimize these refractive index anomalies (13). The
ram drives the syringes in a programmable fashion and also responds to a
stop command from the receiver syringe (14).

~ d a p t e dwiul pamission of Update Instruments

aging. Theoretical analyses show that

significant increases in sensitivity are
obtained hy adapting the spectral
bandwidth to the chromonhores natural bandwidth.
With an HPLC-LPDA svstem the
analyst can use data in thecomputer
memory from other wavelengths rather than repeating analyses a t different
wavelengths. Simultaneous detection
of a sample absorption hand and an
unabsorbed wavelength provides a
means of signal correction. Ratioing
the intensity of the absorbing wavelength to that of a minimally ahsorbing wavelength effectively removes
system fluctuations and temperaturedependent refractive index changes.
Typically, the error introduced by using a wavelength of minimal ahsorption is less than other sources of experimental error. Optical compensation with a separate photodiode
measuring source intensity allows a
dynamic reference and true spectral
ratio measurement.
HPLC data pmcessing
The commercial availability of
HPLC-LPDA systems and the advent
of the 16-and 32-hit microcomputer
in the analytical laboratory have made
it possible to use on a routine basis a
number of data-processing techniques
previously unavailable to the chromatographer.
Peak purity has long been a validation problem for the analyst. It often
requires a t least two analyses, if not
more, under various chromatographic
regimes. Multiple analyses of a peak
at best yield only single wavelengths
per analysis. Under those conditions
the analyst must rely on wavelength
ratios as an indication of purity. The
absorbance ratio of two carefully chosen wavelengths, when plotted as a
function of time, changes through the
peak only if the eluent is not a pure
compound (8).
Alternatively, with an LPDA system
121OA

the spectra obtained a t the up slope,

apex, and down slope can be used to
determine coelution. If the eluting
compound is pure, spectra taken anywhere along the envelope of the peak
should exhibit changes only in the amplitude of the response. Shifts in the
maxima or minima are an immediate
indication of the presence of an impurity in the chromatographic peak. Superposition of these three spectra
after normalization should show a net
difference of zero if the chromatographic peak is pure. Automatic acquisition of spectra during elution can
he achieved manually or automatically
by computer software that can detect
when the signal leaves or returns to
the baseline or passes through up- and
down-slope inflection points or peak
apexes. Periodic acquisition of spectra
at constant time intervals throughout
an analysis can also he of value.
Accurate and precise representation
of the spectral information in HPLC is
critical. Spectral quality can he severely affected by the choice of the detector integration time. The integration time must be sufficiently short to
allow sufficient data points to be taken from the spectra to define the chromatographic peak clearly. An integration time that is too long will distort
the location of the peak maxima. The
integration time must be short enough
so that no noticeable concentration
change will occur during spectral acquisition. Short integration times result in a decreased S/N ratio; however,
significant improvement can be obtained by time averaging a number of
spectra or scans. On the basis of the
Ramsey criterion, five data points
must be taken across a peak to define
the peak envelope minimally (with a
cubic-spline fit algorithm). Some commercial instruments require that a
minimum of 25 data points be taken
across a chromatographic peak so that
simpler fitting algorithms can be used.
The effects of temperature control

ANALYTICAL CHEMISTRY. VOL. 57. NO. 11. SEPTEMBER 1985

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weivei
rringe

Figure 3. Block diagram of

a stopped-flow system using a diode array spectrophotometer

Adapted with permission from Updale lnslrumenb company literature

In a typical stopped-flow experiment (Figure 2). two svrinees are loaded with reactants. Thisyrrnges are actuated, causing the reactants to flow
a t a constant velocity. Mixing occurs
in the cuvette, which is located in the
thermostated bath. In one mode of operation, the LPDA spectrophotometer
is triggered to take data during a period of continuous flow (Figure 3). After
a certain interval of continuous flow
required to purge the cuvette, the flow
is stopped. Spectra continue to be acquired after the flow is stopped. As
many as four reactant syringes and

0.102

three mixers can be used. Spectral information is acauired on a millisecond

time scale (15).
In the past, because of the lack of
LPDA systems, the study of reaction
rates has led to speculation of reaction
intermediates based on kinetic data.
In the past the chemical dead time
(16) has been used to characterize the
performance of a stopped-flow instrument, based on the assumption that
the reactants were homogeneously
mixed in a very small volume. The
question of homogeneity can now he
tested by the LPDA stopped-flow sys-

tern. If a reaction is very fast with resDect to the Dhvsical dead time, then
the homogeneity of the samplecan be
detected by the LPDA spectrophotometer during steady flow. If the reaction is strictly second order and the
mixing is complete, in light of the
speed of the reaction, there should be
no spectral evidence of reactants in
the flow chamber or cuvette, and the
products should be fully developed
(Figure 4).
General applicatkns
Previously the use of the LPDA
spectrophotometer in atomic spectroscopy was limited because the requirements for high resolution and large
spectral coverage could not be readily
achieved. The availability of longer
(2048- and 4096-element) arrays
makes the application of the LPDA
more feasible for general atomic spectroscopy. Currently, the LPDA spectrometer is used as a diagnostic tool in
the semiconductor industry for plasma etching of semiconductor wafers.
Atomic lines of various plasma gases
are monitored and used as a diagnostic parameter to determine the extent
of wafer etching.
Some work has begun in the application of the LPDA spectrometer to
optical rotatory dispersion or circular
dichroism (17). The study of lowlight-level phenomena in the UV-VIS
region, such as chemiluminescence
and fluorescence, requires an intensified LPDA. The same detector arrays
are used with the addition of a microchannel plate image intensifier to the
face of the detector. The sensitivity of
the intensified LPDA compares favorably with that of a photomultiplier.
The speed of the LPDA and intensified LPDA compared with conventional scanning spectrophotometers,
which use photomultiplier tubes, al-

s
t

.....

.: b ..,

......

452

515

577

40

Wavelength (nm)
Fbure 4. Rapid-flow, raplbscan spectra of an acid-base reaction with a dye, Bromcresol purple
(a) Specbum of remainder of 188t expsrlment being cleared from the cuvene: e l m lime. 12.5 ms
(b) Acid and base forms of the dye give a sleedy specbum; elapsed time. 82.5 ms; flow slopped.
(c) Decay 01 base form of me dye; elawed time. 75 ma. (d) Base fOrm of the dye decaying; slapped
lime. 100 ms. Adapted with pe(mi98ion of Update lnsbumenls

1212A

ANALYTICAL CHEMISTRY, VOL. 57. NO. 11, SEPTEMBER 1985

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More than ten applications
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ANALYTICAL CHEMISTRY. VOL. 57, NO. 11. SEPTEMBER 1985

1213A

lows the characterization of transient

luminescent intermediates and the
study of reaction decay lifetimes. The
LPDA spectrophotometer is becoming
the detector of choice in many areas of
UV-VIS spectroscopy (HPLC, kinetics, molecular and atomic spectroscopy, luminescence studies, and spectroelectrochemistry) in which the
speed of acquisition and completeness
of spectral information are important.
Future trends
On the technological front the next
horizon in array spectrophotometers
will be the two-dimensional array. Future trends in research and industry
for LPDA spectrophotometers can be
expected to include improved software
and computer systems to take advantage of the speed of the LPDA and the
wealth of information available from
the array, longer arrays, and dedicated
application-oriented LPDA system
packages. The most important trend
of all is the impact that the LPDA
spectrophotometer has had and will
continue to have on all areas of chemistry-the ability to acquire complete
spectral information on short-lived reaction intermediates. The advancements in LPDA technology now place
the analyst in a position to interpret
structure in terms of the resulting
spectroscopy.
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References
(1) Kuwana, T.; Darlington, R.; Leedy, D.
Anal. Chem. 1964,36,2023-26.
(2) Heineman, W. R. Anal. Chem. 1978,
50,390-400 A.
(3) Heineman. W. R. J . Chem. Ed. 1983.
60,305-8.
(4) Pinkerton, T. C.; Hajizadeh, K.;
Deutsch, E.; Heineman, W. R. Anal.
Chern. 1980,52,1542-44.
(5) Fritz, M. L.; Durst, R. A. Talanta
1983,30,933-39.
(6) Rhodes, R. K.; Kadish, K. M. Anal.
Chem. 1981,53,1539-41.
(7) Kadish, K. M.; Rhodes, R. K. Inorg.
Chern. 1981,20,2961-66.
( 8 ) Reddy, K.V.S.; Hendler, R. W. J . Biol.
Chem. 1983,258,8568-81.
(9) Dessy, R. E.; Reynolds, W. D.; Nunn,
W. G.; Titus, C. A.; Moler, G. F. J. Chromatogr. 1976,126,347-68.
(10) Armstrong, F.; Shaw, R. W.; Beinert,
H. Biochim. Biophys. Acta 1983,722,
61-71.
(11) Barber, M. J.; Salerno, J. C. In Mo_

lybdenum and Molybdenum-Containing

Enzymes; Coughlar, M. P., Ed.; Pergamon Press: New York, N.Y., 1980; Chapter 18.
(12) Chattopadhyay, K.; Coetzee, J. F.
Anal. Chem. 1972,44, 2117-18.
(13) Miller, M. L.; Gordon, G. Anal. Chem.
1976,48, 778-79.
(14) Hansen, R. E., Update Instruments,
persona1 communication, 1985.
(15) Hansen, R. E.; Beinert, H. Anal.
Chem. 1966,38,484-87.
(16) Sturtevant, J. M. In Rapid Mixing

and Sampling Technique in Biochemistry; Chance, B., Ed.; Academic Press:

New York, N.Y., 1964; p. 89.
(17) Aiello, P. J.; Enke, C. G. In Multichannel Image Detectors, Vol. 2; Talmi,
Y., Ed.; ACS: Washington, D.C., 1983;
Symposium Series 236, pp. 57-73.

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ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985