Anal. Chem.

1996, 68, 169R-183R

Ultraviolet and Light Absorption Spectrometry

L. G. Hargis

University of New Orleans, New Orleans, Louisiana 70148

J. A. Howell*

Western Michigan University, Kalamazoo, Michigan 49008

R. E. Sutton

Kalamazoo Valley Community College, Kalamazoo, Michigan 49009

Review Contents have appeared (12-15). Other reviews have dealt with applica-
Chemistry 169R tions of ultraviolet and visible methods as applied to the analysis
Metals 170R of foods (16), water quality (17), and tropospheric substances
Nonmetals 170R (18). A review of the developments in the area of solid-phase
Organic Compounds 171R spectrophotometry focuses on batch and dynamic sorption,
Simultaneous and Multiwavelength 172R properties of reagents and complexes, and application of this
Determinations
technique to flow injection analysis and HPLC detection (19). A
Derivative Determinations 172R
discussion of highly sensitive chromogenic reagents such as
Reaction Rate Determinations 173R
porphyrins, hydrazine and its derivatives, oximes, sulfonates,
Flow Injection Determinations 173R
Photoacoustic and Thermal Lens Determinations 174R surfactants, and coordinate compounds is the subject of another
Physics 174R review (20). The application of ultraviolet and visible spectro-
Optimization and Calculation of Results 174R photometry to the identification of gems (21) and studies of
Precision, Accuracy, and Selectivity 174R inorganic particle formation (22), solid-phase structure (23), and
Standards and Calibration 174R protein folding and unfolding (24) have been reviewed. Reviews
Stoichiometry and Physical Constants 174R of mathematic methods of data treatment (25) and chemometric
Algorithms and Software 175R techniques (26) have also been reviewed. In the area of
Instrument Components 175R instrument performance, reviews dealing with validation (27) and
Spectrophotometers 175R
quality assurance (28) have appeared. Another recent review
Specialized Instruments or Components 178R
discusses the theory of Hadamard transform spectroscopy and
Literature Cited 179R
imaging (29).
This review reports the developments in ultraviolet and light During the past two years only a limited number of books
appeared that deal with the general subject of ultraviolet and
absorption spectrometry from January 1994 through December
visible absorption spectrosocpy. The Milton Roy Co. has pro-
1995, primarily as documented in the Ultraviolet & Visible
duced an educational manual for their Spectronic 20 and 20D
Spectroscopy section of CA Selects, and extends the series of
spectrophotometers which contains a discussion of the theory and
reviews on these topics sponsored by Analytical Chemistry starting
principles of the process, general operating instructions for the
with Light Absorption Spectrometry in 1945 (1-3), followed by
Spectronic 20 and 20D spectrophotometers, and also a well-
Ultraviolet Absorption Spectrometry in 1949 (4-9) and combined
detailed series of 10 experiments. The Encyclopedia of Analytical
Ultraviolet and Light Absorption Spectrometry in 1978 (10, 11).
Science (30) contains several sections on ultraviolet and visible
As in the previous reviews, the subject matter is divided into
spectrophotometry discussing theory and instrumentation, tech-
sections on chemistry, physics, and applications. The applications
niques, and applications. Other books treating specific spectro-
section is presented in the form of tables which summarize the
scopic subjects were Computer-Enhanced Analytical Spectroscopy,
routine determinations of inorganic and organic substances. The
Vol. 4 (31); Spectroscopic Techniques for Food Analysis (32); and
literature dealing with ultraviolet and light absorption spectrometry
Charge-Transfer Devices in Spectroscopy (33).
continues to be so extensive and varied in scope that the citations
in this review must be limited to those developments which we, CHEMISTRY
the authors, consider to be of the greatest interest to analytical This section is devoted to the discussion of papers dealing with
chemists and others engaged in the chemical analysis of materials. the development of reagents, analytically significant chromophoric
As a result of this necessary selectivity, the authors apologize in systems, and spectrophotometric methodology. Activity in the
advance for any errors of judgment made in the omission of area of spectrophotometric analysis of inorganic species seems
specific citations. to have remained relatively constant since the last review. In the
Review articles dealing with specific topics covered in the latter area of photometric methods for organic substances, activity has
sections of this review can be found in their appropriate categories. not significantly increased but continues to center around applica-
Reviews of a general nature and miscellaneous topics will be tions in the clinical, pharmaceutical, and biological areas. Com-
discussed in this section. A number of reviews of a general nature puters and microprocessors still seem to be stimulating a great
S0003-2700(96)00010-8 CCC: $25.00 © 1996 American Chemical Society Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 169R
deal of interest in the areas of multiple-wavelength, derivative, and
flow injection techniques. A new area of development is spec-
trophotometry with supercritical fluids with papers describing
methods for detecting the presence of contaminants in a flowing
stream of supercritical fluid (34), a system for temperature and
pressure control of an ultraviolet detector for supercritical fluid
chromatography (35), and a method of making acoustic and
photoacoustic measurements in supercritical fluids (36).
Metals. One review of spectrophotometric analysis for trace
metals discusses kinetic differentiation coupled with HPLC and
also capillary electrophoresis, film colorimetry, and reagents such
as porphyrins, and 2,2′-dihydroxyazobenzene (37). Gallium
reagents such as triphenylazo, pyridylazo, and trihydroxyfluorone
(38) and tin(II) chloride with HCl as a reagent for platinum group
metals (39) have been the subject of two reviews. Bis(arylazo)
derivatives of chromotropic acid for the determination of non-rare-
earth elements (40), 4,4′-bis(diethylamino)diphenylthione as a
reagent a number of transition metals (41), and 8-hydroxyquino-
line azo derivatives as a general reagent for metals (42) have been
reviewed.
A number of papers have dealt with the use of specific reagents
for various metals. Crown ethers were used to form complexes
of Li+, Na+, K+, and Pb2+ and their ion associates with xanthene
or sulfophothalein dyes, which were then extracted (43). A
synthesized bis(thiacrown) compound, containing two chromoge-
nic groups, was used as an extractive spectrophotometric reagent
for soft bivalent metal ions of cadmium(II) and copper(II), which
exhibited absorption maxima at wavelengths considerably shorter
than those of Cu(I), Ag(I), or Hg(II) (44). Another paper reported
the extraction of metal chelates with micelles and the subsequent
colorimetric determination of the dithizone metal chelates with
Triton X-100. This method is reported to be fast and allows for
the sequential determination of Cu, Hg, Zn, Pb, and Cd metal ions
in the aqueous phase with ppm detection limits (45). The
absorption spectra of octa(4-tert-butylphenyl)tetrapyrazinophor-
phyrazine complexes of copper and zinc were determined and
reported to have potential as photodynamic sensitizers for tumor
therapy (46). A method for the microdetermination of copper-
(II) using 3-(2-acetylphenylhydrazo)-β-diketones has been studied
and was found to obey Beer’s law at an optimum pH of 5.5 (47).
Iron(III) chloride reacts with 5,6-diamino-3,8-dimethyl-4,7-phenan-
throline and has been found to give a labile red-brown complex
which transforms into a stable blue-green iron(II) complex (48).
Complexation of metal ions of group IIIA with o-phenanthroline,
fluorescein, and their derivatives has been described and applied
to the analysis of indium in nickel alloys and zinc ores (49). The
spectrophotometric properties of the noble metal ion complexes
of synthetic amphoteric ion-type water-soluble porphines were
studied and their molar absorptivities determined (50).
Several significant studies have appeared in the area of
reagents for metals and families of metals. The optimum condi-
tions for Fe(III), Cr(III), Mn(II), Ni(II), Cu(II), and Zn(II) with
1-(2-pyridylazo)-2-napthol as an immobilized analytical reagent
were established and semiquantitative analysis effected by the
difference in the luminance and saturation of the color of the
sorbent (51). 4-(2-Thiazolylazo)resorcinol was another immobi-
lized analytical reagent which was used to determine Co, Pd, and
U at levels of 0.005-0.03 µg/mL (52). Schiff bases derived from
salicylaldehyde and sulfonamides were used for the spectropho-
tometric determination of traces of Fe(III) and Cu(II) ions (53).
170R Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
Reexamination of established systems continues to be an active
area of interest. The chemical equilibrium of the K2[PdCl4]
arsenazo(III) system was studied and three variable stoichiometric
M/L ratios were found (54). Attempts to develop an online
technique for the analysis of lithium using primary absorption
were reported as unsuccessful; however, additional testing did
indicate possible reagents which formed chelates with absorbance
signatures (55). In studying the stoichiometry of Fe(III) and
chromazurol S (CAS) and cetylpyridinuim (CP), the ratios were
found to be 1:3:3 and 1:6:6, respectively. Also U(VI)/CAS/CP
formed complexes with 1:3:6 and 1:6:12 ratios. The molar
absorptivities of Fe(III)/CAS/CP (1:6:6) and U(VI)/CAS/CP
(1:6:12) complexes were 1.18 × 105 (pH 5.4; 650 nm) and 9.5 ×
104 L mol-1 cm-1 (pH 5.6; 620 nm) (56). The sensitization of
color reactions of Co, Ni, or Cu with 5-Cl-PADAB, 3,5-diBr-PABAD,
5-Br-DEPAP, and PAR resulting from the addition of sodium
nitrate was studied in detail (57). Modified ligand complexes
containing eriochromecyanine R or chromazurol S ternary com-
plexes forming metal/ligand ratios of 1:6 have been applied to
pharmaceutical analysis (58).
One of the new methods reported in the determination of
cadmium employs a low-power argon ion laser as a 514.5-nm
source to measure cadmium dithizonate that has been extracted
into CCl4. A detection limit of 7 fg or 0.05 ng/mL for cadmium
corresponding to 1.89 × 10-6 AU was observed (59). Mixtures
of copper and silver present in special glasses have been analyzed
by employing the extraction of their dithizonates on polyurethane
foam. Beer’s law was obeyed for the concentration range of 0.05-
2.5 and 0.10-6.0 µg/mL for Cu (550 nm) and Ag (500 nm),
respectively (60). A group of new reagents based on 2-[2′-(6′-
substituted benzothiazoly)azo]-5-(dimethylamino)benzoic acids
has been proposed as reagents for the determination of iron(II),
Co(II), Ni(II), Cu(II), and Pd(II) (61). Titanium(IV) in mine
drainage was determined at the nanogram per milliliter level by
employing a preconcentration/precipitation procedure (62).
New synthesized reagents for specific metals and groups of
metals include chlorocarboxyl antipyrinylazo, which exhibited
good sensitivity for Re, Th, Bi, and Ca but only Ca had a
reasonable selectivity (63). 3-Amino-5,6-di(2-pyridyl)-1,2,,4-triazine
has been reported as a selective new reagent for Fe(II) (64).
Mo(VI), W(VI), Ti(VI), Zr(IV), Fe(III), and Fe(II) in the presence
of cationic surfactants such as CTMAB and cetylpyridinium
bromide have been shown to have sensitive chromogenic reac-
tions with 4-(2-benzothiazolylazo)pyrogallol (65). The synthesis
of 1-hydroxy-2-(6-bromo-2-benzothizolylazo)-8-aminonaphthalene-
3,6-disulfonic acid has been reported, and the acid has been used
as a color reagent for Cu(II), Co(II), Ni(II), and Pb(II) in ethanolic
media (66). The synthesis and a study of spectrophotometric
properties of new thiazolylazo derivatives of carboxylic acid has
been reported for 11 new thiazolylazo chromogenic reagents (67).
Nonmetals. Again, the number of papers focusing on
inorganic nonmetals was small. A review of instrumentation for
industrial gas analysis by absorption spectrometry has appeared
(68). The analysis of nitrite in meat was reported to have been
achieved by the formation of a color chelate with resorcinol and
the zirconyl ion (69). The iodine-18-crown-6 complex has been
monitored by UV and visible spectrophotometry and its stability
constant determined (70). Trace amounts of nitrogen in sewage
samples containing seawater were determined by reaction with
potassium peroxydisulfate followed by UV absorption spectrom-
etry and found to agree with values obtained by copper-cadmium
column methods (71). Spectroscopic determination of nitrate in
municipal waste water at levels of 0.5-13.7 mg/L was performed
without filtering (72). Phosphate concentrations in the range of
0.1-30 mM in bioprocesses have been determined by employing
the reduced molybdophosphoric acid method and subsequently
measured by sequential injection and monitoring with an online
spectrophotometer at a frequency of up to 25/h (73).
Organic Compounds. There were numerous citations of the
determination of organic constituents in a variety of sample types.
The reviews included the UV/visible spectra of carotenoids (74),
protein content in milk and other foods (75), picosecond and
femtosecond spectroscopy of photoreceptive proteins (76), de-
termination of glucuronic acid and glucuronides in biological
samples (77), the use of micelles to enhance the determination
of organic species (78), and photochemical and photophysical
properties of fullerenes (79) and higher fullerenes (80).
Reagents reported suitable for groups of related organic
substances included iron(III) ion as a catalyst for the dimerization
of micromolar amounts of 2-aminophenols in biological samples
(81). Iron(III) was also used for the detection and quantitative
determination of 5-50 µM levels of catechol derivatives (82).
Acetaladehyde, vanillin, and p-(dimethylamino)benzaldehyde have
been used for the determination of some thiophene-containing
cephalosporins (83). A study of the usefulness of o-phenylene-
diamine in determination of R-dicarbonyl compounds reported
quantitation at concentrations as low as 5 µM (84). A patent
discloses the use of oxo[5,10,15,20-tetra(4-pyridyl)-21H,23H-por-
phyrinate]titan to determine peroxides as well as the use of this
reagent in conjunction with an immobilized enzyme-containing
column to determine glucose, galactose, pyruvate, uric acid,
cholesterol, and others (85). Natural indoles were analyzed by
complexation with 1,3,5-trinitrobenezene and measurement of the
absorbance in the region of 460-529 nm (86). Another paper
reports the analysis of a number of quinones by their reaction
with an excess of piperidine in ethanol to obtain a colored (λ )
370-510 nm) product with an average recovery of 93.75% (87).
The determination of six sulfonamides in bulk sulfa drugs has
been performed with derivatives of alizarine, alizarine blue,
alizarine red, and quinalizarine (88). Thiocarbamates and Ni(II)
complexes were studied and the molar absorptivities and stability
constants evaluated (89). An assay for aspartate transcarbamylase
was reported using a chromophoric nucleotide analog, methyl-
thioguanosine, and compared to other methods (90). Electro-
generated buckministerfullerene anions were studied and their
UV and near-IR spectra were reported (91). Quantitation of target
DNA on blots was accomplished using a tetrazolium salt, 3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide as a substi-
tute for nitroblue tetrazolium (92). Concentrations of the phar-
maceuticals isocarboxazide and gliclazide (1-5 µg/mL), isoniazid
(0.25-2.5 µg/mL), iproniazid (0.5-5 µg/mL), tolazamide (1-7
µg/mL), captopril (2-15 µg/mL), and sulfathiourea (1-6 µg/
mL) were determined using 2,2-diphenyl-1-picrylhydrazyl (93).
Reagents applicable to the determination of families of organic
compounds have been rather limited during the reporting period
of this review. The detection of saccharides was reported by
studying the reaction of five reducing sugars (glucose, fructose,
xylose, maltose, lactose) with six diazotized compounds (sulfanilic
acid, 4-aminobenzoic acid, 3-aminobenzoic acid, 4-iodoaniline,
4-chloroanaline, 4-aminoacetophenone) to form colored products
(λ ) 530-540 nm,  ) 216-649) under alkaline conditions (94).
o-Toluidine in environmental samples was detected using Chlor-
amine B in an acetic acid medium or p-dimethylaminobenzalde-
hyde in HCl medium, with the latter being reported more reliable
and sensitive (95).
There have been a number of comparative studies of organic
reagents reported. The assay of chlorophyllin copper complex
at 405 and 630 nm found that measurements at 630 nm gave better
agreement with results from elemental analysis (96). The Kahn
and the Harboe methods for the determination of free hemoglobin
in blood plasma in the presence of bilirubin were studied and the
Kahn method found to be more suitable for the analysis in cases
of hemolysis with elevated bilirubin levels (97). When measuring
the ratio of absorbance of nucleic acids at 260 and 280 nm, an
additional reading at 240 nm is reported to be essential to
distinguishing phenol from pure nucleic acids (98). The poly-
chromatic correction matrix used in total protein measurements
by an automated biuret method was found not to compensate for
the interferences from sulfasalazine and fluorescein (99). A study
of sources of interference in protein determination with the Lowry
method was focused on interferences by magnesium and calcium
ions and found magnesium to be more significant at the same
concentrations (100). The Lowry method has been modified in
order to accelerate the color development and eliminate sensitivity
to certain carbohydrates (101). The Lowry method was also part
of a comparative study with the Bradford and bicinchoninic acid
methods considering the effects of polyelectrolytes on protein
assays (102). These same three methods of protein determination
were found subject to interference by cyclodextrins due to what
is probably a three-way interaction between dye, protein, and
cyclodextrin molecules (103). The use of SDS and Triton X-100
has been shown to enhance the performance of the Bradford
method of protein analysis in urine (104). An analytical procedure
for protein levels in cerebrospinal fluid has been evaluated, and
the turbidity measurements caused by benzethonium chloride
showed it to be a satisfactory alternative to the biuret method
(105). The reagent 3,4-dimethylphenol was used to determine
sugars on the basis of the assumption that glucose, fructose,
lactose, and sucrose had the same absorptivities and permitted
the determination of total sugar in honey (106).
Among the many new methods reported was the direct
ultraviolet measurement of a new nonsteroidal antiinflammatory
drug, IDPH-8261, using the quadratic polynomial coefficient which
exhibited linearity between concentration and absorbance over a
2-20 µg/mL range (107). A spectrophotometric cell with rotating
and stationary bioreactors has been used in the determination of
glucose in serum samples for both humans and bovines (108).
An indirect differential spectrophotometric method employing zinc
and zincon reagent has been used determine 15-45 µM concen-
trations of porphyrin (109). A direct method for measuring
enzyme activity in heterogenous systems was reported for both
insoluble and immobilized particles which remained stable during
the analysis time (110). Two sensitive methods for the determi-
nation of several fluoroquinolone derivatives was studied and
found to obeyed Beer’s law (111). The simultaneous determi-
nation of sulfathiazole and sulfamethazine using solid-phase
spectrophotometry was applied to synthetic and pharmaceutical
samples with detection limits of 0.07 and 0.08 µg/mL respectively
(112).
Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 171R
 Phosphorus-containing podands were studied in the determi-
nation of amines, and satisfactory correlations were observed
between the values of log Kex and the Hildebrand solubility (113).
A new reagent, 2,6-diphenyl-4-(4-dimethylamino)styrylpyrylium
chloride, was reported to be highly sensitive and selective for the
determination of anionic surfactants (114). The determination
of up to 3 ppm of formaldehyde by the formation of the triiodide
complex was reported; a stable complex lasting for at least 1 h
was produced (115). Glycated proteins have been determined
by measuring 2-keto-glucose released by the reaction of the
glycated proteins with hydrazine (116) and also using glucose
phenlosazone as the colorimetric reagent which exhibited a wide
linear concentration range of 25-1000 µM (117). Humic and
fulvic acids were extracted from water with XAD-2 resin and,
following complete elution with sodium dodecyl sulfate, were
analyzed spectrophotometrically (118). The reaction of sulfadi-
midine and aldehydes to form intensely colored Schiff bases was
successful for the determination of sulfadimidine in the presence
of other sulfa compounds (119). A protein assay using tetra-
phenylporphin tetrasulfonate as the chromogenic reagent pro-
duces an absorption band at 488 nm with no overlap of the free
dye (120).
Simultaneous and Multiwavelength Determinations. Nu-
merous papers describing methods of simultaneous analysis have
been written during this review period. Some have involved the
use of derivative techniques and kinetic measurements, and many
employed various mathematical algorithms for data treatment.
These particular references will be discussed in their appropriate
sections later in this review. Several reviews of simultaneous
determinations have appeared dealing with pharmaceutical analy-
ses (121), the application of trihydroxyfluorones (122), and the
progress of simultaneous determination methodology in China
(123).
A number of studies reported involved the simultaneous
determination of inorganic species. Methods for transition ele-
ments included papers dealing with methods for copper(II) using
ternary complexes of copper(II) and pyridine with p-dihydroxy-
benzene, resorcinol, pyrogallol, or p-nitrophenol (124); the diva-
lent ions of copper, zinc, cobalt, nickel, and cadmium using a dual
chromophoric system and the Kalman filtering algorithm (125);
chromium(VI) and chromium(III) using EDTA (126); chromium
and manganese in steel (127); copper, zinc, and cobalt using PAR
(128); cobalt, nickel, copper, zinc, and iron using 2-(5-bromo-2-
pyridylazo)-5-(diethylamino)phenol and PAR as dual parallel chro-
mogenic reagents (129-131); and copper, nickel, and zinc in
heavy oil using the method of equal absorption point, multiwave-
length linear regression, deviate spectrophotometry (132). Meth-
odologies for the simultaneous determination of noble metals such
as platinum, rhodium, palladium, and gold employing several
chromogens including tin(II) chloride and butylrhodamine B and
partial least-squares treatment gave molar absorptivities ranging
from 1.74 × 106 to 2.47 × 108 (133) and for mixtures of gold(III),
platinum(IV), and palladium(II) as bromide complexes (134) have
been reported. A comparison of six chemometric methods applied
to the simultaneous determination of aluminum, copper, manga-
nese, zinc, and cobalt in foods using 2,6,7-trihydroxy-9-styrylfluo-
rone-3 has been described (135). Tungsten, molybdenmum, and
germanium in steel has been analyzed with phenylfluorone and
cetyltrimethylammonium bromide by the equal absorptive wave-
length and linear plot method (136). Plan factor analysis has been
172R Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
employed in the simultaneous determination of boron, tellurium,
aluminum, gallium, indium, and lead using p-nitrophenylfluorone
(137).
A great deal of interest has been centered around the
simultaneous determination of various mixtures of organic com-
pounds. Among these were mixtures of aldehydes, ketones, and
NOx in exhaust gases using 2,4-dinitrophenlyhydrazine as the
chromogenic reagent (138). Other simultaneous methods which
have been reported have dealt with mixtures of solasonine and
solasodine (139); and total starch, amylose, and amylopectin
(140). The multicomponent determination of theophylline,
ephedrine, and amobarbital in pharmaceutical products using the
Kalman filtering algorithm has been described (141). Simulta-
neous analysis of organic compounds include papers dealing with
mixtures of 1,1,1-trichloroethane, trichloroethylene, and chloro-
form employing multivariate calibration (142); 1-naphthylamine,
1-naphthol, 2,7-dihydroxynaphthalene, 2,4-dimethoxybenzalde-
hyde, methyl salicylate, and dibutyl phthalate using target factor
analysis (143); and adenine, thymine, cytosine, and guanine using
Kalman filtering (144).
Derivative Determinations. Derivative techniques continue
to be an area of high activity with many applications to the
simultaneous determination of pharmaceutical products and other
mixtures. A number of general reviews have appeared (145-
148), as well as some devoted to the general applications of (149,
150) and specifically to the analysis of drugs, biochemicals, and
foods (151).
One investigation describes the loss of information resulting
from second derivatives taken from spectra that are linear in
wavelength, particularly at longer wavelengths, and suggests an
equation for restoring the lost information from the second
derivative in the long-wavelength region (152). Another paper
describes a technique employing high-order derivative absorption
spectroscopy to demonstrate resolution of nearly overlapping lines
of disparate oscillator strengths by employing tunable dye lasers
and a relatively inexpensive wavelength resolution instrument
(153). The ratios of derivative maxima and minima have been
used to test for the presence of nonspecific matrix interference
and to predict the validity of applications of derivative spectro-
photometry during the analysis of pharmaceutical formulation
containing a single drug (154). Simultaneous determination of
binary drug mixtures has been carried out using the first-derivative
absorption spectrum shift length method (155).
Derivative techniques continue to be a method of choice for
the determination of inorganic species. Simultaneous derivative
determinations using PAR as the chromogenic reagent have been
reported using first-order derivatives for mixtures of nickel,
copper, and zinc (156) and for cobalt, nickel, copper, zinc, and
iron (157); and for the second-order derivative for mixtures of
nickel(II) (0.2-1.25 ppm) and cobalt(II) (0.25-1.25 ppm) (158).
First-order-derivative spectrophotometry has been used for the
simultaneous determination of copper (0.0125-0.25 µg/mL),
mercury (0.025-0.25 µg/mL), and lead 0.025-0.25 µg/mL) using
dithizone reagent (159). Manganese, copper, and zinc in alumi-
num alloy react with 2-(5-bromo-2-pyridylazo)-5-(diethylamino)-
phenol and cetylpyridinium bromide to provide a three-component
color system which can be analyzed by multiwavelength linear
regression and derivative spectrophotometric measurements
(160).
 Derivative methods seem to be even more widely used in the
analysis of organic compounds. First- and second-derivative
methods have been described for the assay of the antiinflamma-
tory drugs fentiazac, flufenamic acid, tiaprofenic acid, and pro-
quazone (161). Other drugs reportedly determined by derivative
techniques are metronidazole (1-20 µg/mL) in tablets (162);
carboplatin (first; e150 µg/mL) (163); anthralin in creams
(second) (164); paracetamol in blood serum (first) (165); and
mixtures of cocaine, procaine, and lidocaine in powder samples
(sequential second) (166). A second-order-derivative spectro-
phometric method has been described for the determination of
dithiocarbamate fungicide residues (second; thiram 0.03-0.1 mg
of CS2/kg) (167, 168).
Considerable interest in the area of drugs has been seen with
methodology for the analysis of mixtures such as paracetamol
and phenoprobamate (first) (169); 10-40 µg/mL paracetamol and
1-3 µg/mL caffeine (first and second) in tablets (170); acetyl-
salicylic acid and free salicylic acid in sustained release tablets
(171); cetrimide and chlorhexidine gluconate (first; 221.3 nm,
268.9 nm) in antiseptic solutions (172); clopamide and pindolol
(fourth) in tablets (173); lidocaine hydrochloride and 5-nitrox
(fourth; 266 nm, 269 nm) in liquid formulations (174); phenobar-
bitone in mixtures with oxyphenonium bromide and meprobam-
ate, paracetamol, or acetylsalicylic acid (first and second) (175);
procaine hydrochloride with benzoic acid, pyridoxine hydrochlo-
ride, 4-aminobenzoic acid (176); 0.5-14 µg/mL sulfanilamide and
1-20 µg/mL sulfadiazine (third) (177); and sulfamethoxazole and
trimethoprim (second) (178). Derivative procedures for vitamin
mixtures reported are for pyridoxine hydrochloride and thiamine
hydrochloride in tablets (first and third) (179); vitamins B6 (307
nm, 0.2 µg/mL), B1 (282.7 nm, 0.46 µg/mL), and B12 (360.5-374
nm peak to peak, 0.22 µg/mL), uridine 5′-triphosphate (261 nm,
0.2 µg/mL) in injections (second) (180); and sodium salicylate,
thiamine hydrochloride, and ascorbic acid in visalicyl tablets (first
and second) (181). First-derivative measurements have been
used to determine benzimidazole and cinnamate and also ben-
zophenone derivatives in order to characterize sunscreens in
cosmetic formulations (182). A direct method (first and second)
for evaluating bilirubin, albumin, and oxyhemoglobin in amniotic
fluid has been described (183).
A variety of other organic compounds have been determined
by simultaneous derivative techniques including the pesticides
atrazine (1-15 µg/mL), diurion (1-10 µg/mL), and chlorpyrofos
(1-10 µg/mL) in groundwaters and soils (first) (184, 185);
sodium o-nitrophenolate, sodium p-nitrophenolate, and 2-methoxy-
5-nitrophenoate in plant and animal growth regulators (first)
(186); and tyrosine, tryptophan, and phenylalanine (first and
second) (187). Several papers dealt with the simultaneous
derivative determinations of colorant and dye mixtures including
carminic acid, riboflavin, and erythrosine in yoghurt samples (first)
(188); Amaranth, Carmoisine, and Ponceau 4R (first; calibration
graphs linear up to 32 mg/L) (189); and tartrazine, riboflavin,
curcumin, and erythrosine (190).
Reaction Rate Determinations. The basic principles and
instrumentation, data analysis, and applications of rapid-scanning
stopped-flow spectrophotometry of biological systems with em-
phasis on proteins, enzymes, and cofactors have been reviewed
(191). Another recent review discusses problems associated with
the accurate determination of enzyme activity using coupled
spectrophotometric assays (192). A patent disclosure proposes
a mathematic equation, i.e., y ) A + (B - A)/ekt (y, absorbance;
A, end point absorbance; B, starting point absorbance; k, reaction
rate constant; t, measuring time) for accurate concentration
calculation in a biochemical spectrophotometric analyte analysis
(193).
Noncatalytic kinetic methods reported during the past two
years include the determination of copper(II) in the range of 0.6-
1.7 µg/mL by monitoring its reaction with 2-methylindole-3-
carboxyaldehyde thiosemicarbazone (194); ephedrine and am-
phetamine in pharmaceutical samples by their reaction with 1,2-
naphthoquinone-4-sulfonate (195); dithiocarbamate pesticides
(polycarbazine, polychrom, eptam) in potatos by their inhibition
of potassium ferrocyanide oxidation (670 nm) catalyzed by
mercury(II) ion (196); and corticosteroids (190-1.9 µM) employ-
ing the Blue Tetrazolium method and the continuous addition of
reagent technique (197). A stopped-flow method for the deter-
mination of 0-2.2 µg/mL iron(II) in the presence of iron(III) is
based on the inductive effect of the iron(II)-chromium(VI)
reaction on the chromium(VI) iodide reaction (198).
In the area of catalytic reaction rate methods, the determination
of 0-3.5 µg/mL cobalt(II) by its catalysis of the oxidation of (m-
nitrophenyl)fuorone with hydrogen peroxide in basic medium has
been reported (199). Silver(I) has been determined in the range
of 0.8-0.01 µg/mL by the catalytic mechanism on the indicator
reaction of potassium peroxydisulfate and 3-3,4-dihydroxyphenyl-
alanine (200). Concentrations of ruthenium in the range of
approximately 20-2 ng/mL have been determined on the basis
of its catalytic effect on the reaction between potassium iodate
and various arsenazo reagents (201). The simultaneous deter-
mination of osmium and iodide can be effected by their catalytic
behavior on the indicator reaction between cerium(IV) and
arsenic(III) provided that the orders of the indicator reaction
catalyzed by the catalysts are different (202).
Flow Injection Determinations. The application of flow
injection analysis for precious metals (203) and to online control
of some industrial processes (204) has been reviewed. Several
investigators have developed a flow injection system incorporating
the generalized standard addition method (205). Investigations
directed toward improving flow injection determinations include
a comparison of mixing devices (206); the application of rank
annihilation factor analysis for predicting a single analyte concen-
tration (207); the use of generalized Fourier smoothing of data
(208); and an approach to compensate for both refractive index
and turbidity effects (209).
Simultaneous determination of metals by flow injection meth-
ods have been described for calcium and magnesium (210) and
for barium and strontium (211) with both systems using chloro-
phosphonazo III and the pH gradient technique; mixtures of
calcium and magnesium, aluminum and manganese, and alumi-
num and iron(III) using dual-wavelength detection (212); and
cadmium, cobalt, copper, and iron(III) in the range of 30-0.08
µM by preconcentration with online coated columns (213). A
stopped-flow injection kinetic method based on ligand-exchange
reactions for the analysis of iron(III), cobalt, and zinc has been
described (214). Other applications of flow injection analysis
include methods for determining sulfate ion involving molybdic
acid reagent and electrochemical reduction to form a heteropoly-
molybdate complex (215) and for the analysis of 0-12 mg/L
nitrate ion in natural waters using cadmium reduction and
subsequent diazotization (216).
Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 173R
 Flow injection methods seem to have gained wide popularity
for the analyses of various components of foods such as fructose
and glucose in syrups and molasses based on periodate oxidation
of reducing sugars and monitoring of the generated triiodide ion
(217); tannins in tea and beer based on the reduction of iron(III)
to iron(II) by tannins and subsequent formation of iron(II)-1,-
10-phenanthroline (218); peroxidase and lactoperoxidase in
vegetable and dairy products employing chemiluminescence of
peroxide formed in the presence of luminol (219); and total free
fatty acids in olive oil, milk fat, and cocoa butter based on
continuous extraction of soaps formed by the reaction with copper
acetate which gives a linear calibration plot over the range of 5-0.1
mM oleic acid (220). Anionic surfactants have been determined
by ion pairing with methylene blue and extraction into chloroform
by flow injection analysis (221). Cationic disinfectants such as
benzethonium and berberine have been analyzed by reaction with
bromochlorophenol and quinidine in 1,2-dichloroethane to form
a blue ternary ion (222). Flow injection analysis of 1-20 and
20-250 mg/L tetracyclines has been successfully carried out
based on their reaction with 4-aminophenazone and hexacyano-
ferrate(III) (223).
Photoacoustic and Thermal Lens Determinations. The
applic-ations of photoacoustic spectroscopy to biological systems
in general (224) and specifically to the study of percutaneous
absorption of drugs or cosmetics (225) have been reviewed.
Another review discusses photoacoustic and photothermal meth-
ods to the analysis of microparticles in condensed matter (226).
One paper describes the construction of an ultraviolet-visible
photoacoustic spectrophotometer using relatively inexpensive
individual optical components (227). Other papers have dealt with
the study of instrumental aspects of photoacoustic measurements
and include the description of a three-chamber cell (228) and a
new technique based on the measurement of the phase angle of
the photoacoustic signal when studying melanins in the dry state
in order to reduce signal sensitivity to light scattering (229).
Studies of thin films of C60 have also been reported (230).
Three reviews of note discussing thermal lens spectrometry
have been published (231-233). A model for thermal lens
spectrometry in partial optical saturation condition has been
developed (234). A multiwavelength thermal lens spectropho-
tometer based on an acoustooptic tunable filter as a polychromator
has been described (235). Photothermal microscopy with the
excitation and probe beams coaxial under a microscope has been
shown to provide high-sensitivity spectra for microobjects (236).
The effect of the nature of the solvent on the limit of detection in
photothermal spectroscopy was the subject of one paper (237).
Another paper describes a new technique in which absorption in
the infrared region is measured in the visible region by using a
visible probe laser to monitor the thermal lens effect that was
induced in a sample as a consequence of absorption in the infrared
region (238). A computerized optical parametric oscillator that
is capable of continuous tuning from about 420 nm to 2 µm for
measuring the entire thermal lens spectrum of nitric oxide with
a continuous scan has been described (239). Thermal lens
spectrometry has found application in the analysis of p-aminoben-
zoic acid and arylamine diuretics in urine (240); pesticides in
water with limits of detection of about 8 × 10-6 absorption units
at 363.8 nm (241); and tannins in white wines after oxidation with
the Folin-Ciocalteu reagent and extraction of the resulting
174R Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
phosphomolybdenum-phosphowolframate blue chromogen with
chloroform in the presence of tetralkylammonium salt (242).
PHYSICS
This portion of the review deals with topics related to the
measurement of radiant energy, treatment of data, and instru-
mentation used in light absorption and ultraviolet spectrometry.
Optimization and Calculation of Results. The progress
of chemometrics in simultaneous multicomponent analysis (243)
and deconvolution of mixtures using factor analysis has been
reviewed (244). A two-part series entitled “Chemometrics in
spectroscopy: Experimental designs” has appeared (245, 246).
Validation of ultraviolet and visible methods using computer
optimization techniques has been discussed (247). Two papers
address the subject of computer-optimized wavelength selection
for multicomponent determinations (248, 249). The solving of
ultraviolet spectra vectors via stepwise regression has permitted
the simultaneous determination of multiple-component systems
with indefinite components (250). Two new methods for calcula-
tion of wavelength displacement in triwavelength spectrophotom-
etry have been proposed and illustrated with the analysis of
molybdenum in a molybdenum(VI)-tungsten(VI)-phenylfluor-
one-cetylpyridinium chloride system (251). The H-point stan-
dard additions method has been used to eliminate the unknown
irrelevant matrix absorbance (252).
Precision, Accuracy, and Selectivity. A comparative study
of four methods of assessing precision in linear spectrophotomet-
ric assays has been reported (253). Modeling the response of
an instrument’s photonic detection subassembly has been found
to provide a quantitative estimate of the minimal relative uncer-
tainty in analyte concentrations (254). The method of dual
measuring has been proposed to enhance sensitivity of three-
wavelength spectrophotometry (255).
Error analysis in decoloration-type indirect spectrophotometric
analysis has been discussed (256). The relationship between slit
width effect errors and spectral bandwidths and shapes has been
described (257). Errors associated with determinations of mul-
ticomponent mixtures even with modern computerized data
analysis are the subject of several papers published in the past
two years (258, 259).
Standards and Calibration. Multivariate calibration stan-
dardization methods (260) and liquid reference materials for
ultraviolet and visible spectrophotometry (261) have been re-
viewed. Concentrations of 0.002-0.012 g/dm3 congo red have
been used to calibrated colorimeters, spectrophotometers, and
analyzers in the spectral range of 485-505 nm (262). A
spectrometer employing waveguides containing certain impurities
or dopants that produce absorption lines has been used for
calibration (263). A comparative study of precision spectropho-
tometric methods which addresses error sources and precision
has been presented (264).
Stoichiometry and Physical Constants. The effect of
simultaneous and competing reactions on the method of continu-
ous variations has been reviewed (265). The absorption coef-
ficients at 193 nm for water vapor from 300 to 1073 K have been
measured and reported (266). Basic spectrophotometric methods
for determining the composition of complex compounds has been
discussed (267); an improved mole ratio method employing dual-
wavelength measurements (268) has also been described. An
automated gradient flow injection spectrophotometric technique
Table 1. Spectrophotometric Methods for Inorganic Substances
method or reagent (wavelength, nm; molar absorptivity;
constituent material concentration range or detection limit, µg/mLa) ref
As 12-molybdoarsenic acid blue (8.28 × 103; 0-800) 354
Au ores catalytic decolorization Neutral Red; H2O2 (0-0.03) 355
Cu Al alloys 4,4′-bis(8-hydroxyquinolyl-5-azo)-3,3′-dimethylbiphenyl, Triton X-100 (535; 6.02 × 104; 0-1.0) 356
Fe(II) electroplating baths FIA, o-phenanthroline (500-520) 357
wastewater stopped-flow kinetics, simultaneous with Sb(III) and V(IV); Cr(VI), KI, starch (0-1.6) 358
Ga 12-molybdogallic acid (425; 1 × 10-3-1 × 10-2) 359
Gd(III) concrete 2-(3,5-dichloro-2-pyridylazo)-5-(dimethylamino)phenol, Triton X-100 (1.45 × 105) 360
Hg blood, urine Mo3S44+, H2SO4 (556; 0.05) 361
La(III) concrete 2-(5-bromo-2-pyridylazo)-5-(diethylamino)phenol, Triton X-100 (1.76 × 105) 360
Mo(VI) steel simultaneous with W(VI) and V(V), Tiron 362
Ni aluminum alloys 2-(2-thiazolylazo)-5-diethylaminobenzoic acid (615; 9.2 × 104; 0-0.52) 365
NO2- food Neutral Red (630; 0.1-1.80) 363
NO3- water direct UV (210; 0.1-25) 364
electroplating baths flow injection analysis, direct visible (630) 357
Pb hair indirect, Arsenazo I, H2O2 (0-0.40) 366
Pd CsGeCl3 in HCl (330; 5-30) 367
Pu liquids direct thermal lens (831; 1 × 10-6 M) 368
rare earths bones, teeth Chlorophosphorazo MA (7.5 × 105 - 8.5 × 105) 369
Rh(III) plating baths, water Chrompyrazol I, SnCl2, cetyltrimethylammonium bromide (630; 1.1 × 105) 370
SO2 wine continuous-flow sensor, pararosaniline, formaldehyde, ion-exchange support (0.16-6.0) 371
SO32- drinks (p-aminophenyl)azobenzene (0-1.80) 372
S2O82- flour 1-pentanol extn (414; 0.030) 373
Sb(III) wastewater stopped-flow kinetics, simultaneous with Fe(III) and V(IV); Cr(VI), KI (0.-2.0) 358
Sc tungsten ores 2-(5-bromo-2-pyridylazo)-5-(diethylamino)phenol, lactic acid, Triton X-100 (565; 6.1 × 104) 374
Se biological material 4-nitro-1,2-diaminobenzene (350) 375
Ta(V) ores, steel butylrhodamine B, poly(vinyl alcohol) (>2 × 106) 376
Te indium phosphide Bismuthol I, HCl, CHCl3 extn (447; 2.1 × 105; 6.5 µg/g) 377
V(IV) wastewater stopped-flow kinetics, simultaneous with Fe(III) and Sb(III); Cr(VI), KI (0-2.8) 358
V(V) 8-quinolinol, HCl, Na2SO3, MIBK extn (430) 378
steels simultaneous with Mo(VI) 362
and W(VI); Tiron
W(VI) steels simultaneous with Mo(VI) and V(V); Tiron 362
Zr 3-hydroxyflavone, HClO4, benzene extn (405; 0-1.0) 379

a Unless specified otherwise.

has been proposed for the determination of formation constants
of complexes of micromolecules with cyclodextrins (269). A
method has been developed for the determination of equilibrium
constants for binary mixtures even when none of the component
spectra are known and the components cannot be obtained in pure
form (270). A simple and rapid method for determining dissocia-
tion constants of organic polyprotic compounds even when pKa
values are nearly equal has been proposed (271).
Algorithms and Software. Interest in developing and refin-
ing chemometric approaches in order improve analytical meth-
odology continues to grow each year. Applications of Fourier
transform in the ultraviolet-visible region have been reviewed
(272), and a software platform for computer-based education in
Fourier transform spectroscopy has been developed (273).
Chemometrics have been widely applied in the area of
multicomponent determination and include a comparative study
involving a genetic algorithm, simulated annealing, and stepwise
elimination as methods for wavelength selection (274); the use
of apparent content curves for the analysis of mixtures such as
fluorescein-eosine-acridine and methyl red-methyl orange-
cresol red (275) and theophylline in antiasthmatic pharmaceutical
products (276); and the application of the H-point standard
additions method for the determination of mixtures of phenol,
o-cresol, 4-chlorophenol, and 3,4-dichlorophenol (277), and also
mixtures of phenol, 4-chloro-2-nitrophenol, 2,4-dichlorophenol, and
2,4,6-trichlorophenol (278). Other multicomponent analysis sys-
tems have employed zero-crossing and derivative quotient spectra
with standard divisor and numerical methods MULTI and PLS
for the resolution of binary and ternary mixtures of acetylsalicylic
acid, caffeine, and thiamine (279); polynomial approximation and
nonlinear transformation of the wavelength axis for the analysis
binary mixtures of dyes (280); Fourier to orthogonal function
coefficients to provide exact compensation of any component in
binary mixtures (281); modified multiwavelength K-factor spec-
trophotometry for the analysis of ternary mixtures of sodium
diclofenac, chloropheniramine, and paracetamol (282); and the
introduction of a damping factor into the p-coefficient matrix in
order to reduce the “abnormality” of normal simultaneous equa-
tions to effect the analysis of multiple vitamin B components in
pharmaceutical samples (283). Principal component analysis has
been reported for the determination of six amino acid mixtures
by the use of the scan algorithm method for the choice of several
principle components that have the best linear correlation with
concentration from many principal components (284). Another
principal component regression employing a simulated set of
binary mixtures with Gaussian bands in order to study the
influence of spectral overlap on the precision of quantitation was
developed and applied to the analysis of mixtures of 8-hydroxy-
quinoline complexes of titanium, aluminum, and iron (285). A
general framework for manipulating spectra as functions in
traditional multivariate methods such as principal component
analysis and partial least squares has been described; the report
concluded that there are two fundamentally different types of
representation, namely, by functions and by function coefficients,
with the latter being the most practical (286). A comparative
study of five multivariate calibration techniques (direct multicom-
ponent analysis, stepwise multiple linear regression, principal
component regression, two systems of partial least squares)
evaluated for the determination of transition metal ions in model
multicomponent systems (287). Adaptive filter techniques have

Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 175R
Table 2. Spectrophotometric Methods for Organic Substances
method or reagent (wavelength, nm; molar absorptivity;
constituent material concentration range or detection limit, µg/mLa) ref
acesulfame food Sevron Blue 5G, CHCl3 extn (655) 380
acetaminophen injections direct UV (248.6) 381
acetylsalicylic acid suppositories first-derivative UV, simultaneous with paracetamol and phenobarbital (255.8) 382
adrenaline pharmaceuticals simultaneous with isoprenaline, flow injection analysis with metaperiodate [(0-2) × 10-4 M] 383
albumin urine bromophenol blue (5.0) 384
aldehydes, aromatic diphenylamine, HCl (590-640; 5-75) 385
alkaloids tobacco bromination, KI, starch (580; 0.15-1.2) 386
amines, aromatic FeCl3, K3Fe(CN)6 (4.2 × 104-5.4 × 104; 2-20 µg) 387
amines, aromatic primary pharmaceuticals 5-nitrobarbituric acid in DMF (399-402; 0.73-1.48) 388
amines, primary and secondary 4-chloro-5,7-dinitrobenzofuran (2 × 10-6 M) 389
4-aminobenzoic acid m-hydroxybenzaldehyde (460-480; 0.1-5) 390
1-amino-2-propanol m-hydroxybenzaldehyde (460-480; 0.1-5) 390
amodiaquine pharmaceuticals bromophenol blue (415-420; 1-8) 391
pharmaceuticals 7,7,8,8-tetracyanoquinodimethane (0.4-4.0) 392
amphetamine urine sodium 1,2-naphthoquinone-4-sulfonate (1.4-50) 393
anthraquinones pharmaceuticals direct visible (527; 7.9-32) 394
antipyrine injections direct UV (248.6) 381
arabic gum electroplating solutions concentrated H2SO4 (275; 0-20) 395
ascorbic acid food indirect, Fe(III) (265; 0-12) 396
Rutinal C tablets simultaneous with rutin, first derivative in methanol (258.8) 397
atrazine pesticide formulations simultaneous with chlorpyrifos, first derivative (0-15) 398
benzocaine m-hydroxybenzaldehyde (460-480; 0.1-5) 390
bupivacaine HCl gentamicin sulfate direct UV (268; 80-400) 399
catechloamines periodic acid (485; 1.0-12.0) 400
ceftriaxone sulfate antibiotics simultaneous with streptomycin sodium, zero-crossing third derivative (227.8; 1-40) 401
chloramphenicol lotions simultaneous with salicylic acid, first derivative (258.1, 296.6) 402
tablets direct 1st derivative (255.6, 298.0) 403
chlordiazepoxide pharmaceuticals hydrolysis, 3-methylbenzothiazolin-2-one, Ce(IV) (565; 1.0-10) 404
chloroquine pharmaceuticals bromophenol blue (415-420; 2-12) 391
pharmaceuticals 7,7,8,8-tetracyanoquinodimethane (0.4-4.0) 392
chlorpromazine HCl pharmaceuticals ZnCl2, diazotized 1-aminoanthraquinone (2-20) 405
chlorpyrifos pesticide formulations simultaneous with atrazine, 1st derivative (0-15) 398
cilastatin Primaxin simultaneous with imipenem, 1st derivative (243; 14-42) 406
ciprofloxacin HCl eye drops direct UV in 0.1 M HCl (277) 407
clemastine hydrogen fumarate p-chloranilic acid in methylene chloride (530; 40-400) 408
cyclamate food Sevron Blue 5G, CHCl3 extn (655) 380
diazepam pharmaceuticals hydrolysis, 3-methylbenzothiazolin-2-one, Ce(IV) (580; 0.5-5.0) 404
dimethindene maleate p-chloranilic acid in methylene chloride (530; 40-420) 408
ethanolamine m-hydroxybenzaldehyde (460-480; 0.1-5) 390
flucythrinate grains, crops 2,4-dinitrophenylhydrazine (465; 1-8) 409
gibberellins reduction of 12-molybdophosphoric acid (820; 1.6-64) 410
ibuprofen copper acetate, CHCl3 extn (680) 411
liquid formulations iodide, iodate (352; 10-40) 412
imipenem Primaxin simultaneous with cilastatin, 1st derivative (318; 14-42) 406
indoles maleic anhydride (490-530) 413
isoprenaline pharmaceuticals diazotized 1-aminoanthraquinone (505; 5-80) 414
pharmaceuticals simultaneous with adrenaline, flow injection analysis with metaperiodate [(0-2) × 10-4 M] 383
isoxuprine pharmaceuticals Fe(III), 2,2′-bipyridine (520) 415
ketoprofen copper acetate, CHCl3 extn (680) 411
lipohydroperoxides iodide, nonionic or cationic surfactants (365; 0.5 nmol) 416
oils iodide, acetic acid, cetylpyridinium chloride (500; 5 × 10-7-2.5 × 10-6 M) 417
loratadine p-chloranilic acid in methylene chloride (530; 40-320) 408
memantine HCl pharmaceuticals sodium 1-(4-methoxyphenyl)cinnamonitrile-2-sulfonate (324.5; 5.0 × 10-6-6.5 × 10-5 M) 418
methotrimeprazine pharmaceuticals ZnCl2, diazotized 1-aminoanthraquinone (10-60) 405
metoprolol tartrate pharmaceuticals benzyl orange, CHCl3 extn (401; 7.4 × 103; 0-3.42) 419
pharmaceuticals bromophenol blue, Tropeolin OO, CHCl3 extn (603; 1-6) 420
miconazol liniments direct UV (272) 421
moroxydine direct UV (237; 2-12) 422
2-naphthol air direct UV in ethanol (225; 0.018 mg/m3) 423
naproxen copper acetate, CHCl3 extn (680) 411
nicotine tobacco bromination, KI, starch (580; 0.15-1.2) 386
nylidrine pharmaceuticals Fe(III), 2,2′-bipyridine (520) 415
ofloxacin eye drops direct UV (293; 1.0-10.5) 424
orciprenaline pharmaceuticals diazotized 1-aminoanthraquinone (412; 1-8) 414
organophosphorus pesticides acid hydrolysis, 12-molybdophosphate, malachite green, N,N-diphenylbenzamidine in toluene 425
oxazepam pharmaceuticals hydrolysis, 3-methylbenzothiazolin-2-one, Ce(IV) (565; 1.0-10) 404
paracetamol suppositories 1st derivative UV, simultaneous with acetylsalicylic acid and phenobarbital (263.2) 382
phenobarbital suppositories 1st derviative UV, simultaneous with acetlysalicylic acid and paracetamol (218.2) 382
phenol borax, disinfectants direct UV (287) 426
phenol glycerine direct UV (270) 427
water isopentyl acetate extn, H2O back extn, fourth derivative UV (250-270; 0-12) 428
phenothiazines pharmaceuticals Brilliant blue in HCl 429
pharmaceuticals 1,2-naphthaquinone-4-sulfonic acid 430
pholedrine pharmaceuticals Fe(III), 2,2′-bipyridine (520) 415
porphyrins, cationic absorbance difference, zinc, zincon (622; 15-45 µM) 431

176R Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
Table 2 (Continued)
method or reagent (wavelength, nm; molar absorptivity;
constituent material concentration range or detection limit, µg/mLa) ref
primaquine pharmaceuticals bromophenol blue (415-420; 2-10) 391
pharmaceuticals 7,7,8,8-tetracyanoquinodimethane (0.4-4.0) 392
procaine HCl pharmaceuticals simultaneous with pyrodoxine HCl, 1st derivative (309.33) 432
proteins erythrosin B (545; 2-14) 433
Ponceau S (525; 0-250 µg) 434
pyridoxine HCl pharmaceuticals simultaneous with procaine HCl, second derivative (265.81) 432
rutin Rutinal C tablets simultaneous with rutin, 1st derivative in methanol (337.4) 397
saccharin foods Sevron Blue 5G, CHCl3 extn (655) 380
salbutamol pharmaceuticals diazotized 1-aminoanthraquinone (425; 2-15) 414
salicylic acid lotions simultaneous with chloramphenicol, 1st derivative (241.6, 274.1) 402
streptomycin antibiotics simultaneous with ceftriaxone sulfate, zero-crossing third derivative (241.7; 0-35) 401
temazepam pharmaceuticals hydrolysis, 3-methylbenzothiazolin-2-one, Ce(IV) (580; 0.5-5.0) 404
tetracaine HCl pharmaceuticals direrct UV (301) 435
theophylline direct UV in NaOH (274) 436
thiacetazone 2,6-dichlorquinone-4-chlorimide (540-550; 2-14) 437
2-thiobarbituric acid 2,6-dichlorquinone-4-chlorimide (540-550; 2-4) 437
thiols, aliphatic 2,6-dichlorquinone-4-chlorimide (435) 437
thiols, aromatic 2,6-dichlorquinone-4-chlorimide (495-503) 437
thiomersal tinctures direct UV (310; 20-100) 438
thiophosphoryls induction effect on iodine-azide reaction (0.5-5 nmol) 439
thioridazine HCl pharmaceuticals ZnCl2, diazotized 1-aminoanthraquinone (10-80) 405
timolol maleate pharmaceuticals bromophenol blue, Tropeolin OO, CHCl3 extn (540; 2-8) 420
treinoin pharmaceuticals direct UV (358) 440

a Unless specified otherwise.

demonstrated the prediction of relative standard deviations of
amplitude estimates for overlapped target and interferant Gaussian
peaks that simulate overlapped spectral responses (288). These
techniques have also shown the utility of the Kalman filter as an
algorithm for calibration in a real system and were compared to
classical least squares and pure-component calibration for deter-
mination of binary, ternary, and quaternary mixtures of pollutant
phenols (289). They have shown the effectiveness of simulta-
neous kinetic determination of mixtures of 1.25-15 µM concentra-
tions of phenols employing the Kalman filter algorithm and a
reaction system based on the oxidative coupling of the phenols
to N,N-diethyl-p-phenylenediamine in the presence of hexacyano-
ferrate(III) with monitoring of the reaction at 660 nm (290).
Library searches are another area where the application of
chemometric techniques is growing. An artificial neural network
has been used to implement a spectral library search system for
ultraviolet spectra (291). Another spectral library search method
of ultraviolet and visible spectra has been developed and applied
to binary and ternary mixtures (292).
Other software applications include the development of SPECA,
a program for calculating thermodynamic equilibrium constants
from spectrophotometric data by applying the Debye-Hückel
theory, the specific interaction theory, and the modified Bromley
theory (293). An algorithm for the analysis of second-order
kinetics data by the method of standard additions employs a
generalized eigenproblem to mathematically separate instrument
response of the analyte from that of interfering species has been
applied to the analysis of trichloroethylene in samples that have
matrix effects caused by an interaction with chloroform (294).
Instrument Components. Reviews of instrument compo-
nents of ultraviolet and visible spectrophotometers include one
of a general nature discussing cells, light sources, and temperature
control with particular emphasis given to the analysis of amino
acids and proteins (295); another addressing improvements of
detectors (296); and two on charge coupled devices as detectors
(297, 298). The development of cell technology continues with
a patent disclosure of an off-axis cavity cell with astigmatic mirrors
in the design to provide a closed beam path (299); a report of
flow cells for flow injection analysis (300) and for aqueous
solutions at high pressure and temperature (301); the develop-
ment of a high-sensitivity cell for capillary zone electrophoresis
(302); and a description of a cryogenic intracavity photoacoustic
cell (303). In the area of detector technology, papers describing
a novel material for visible-blind ultraviolet detectors (304); a
polarized photometric detector for optically active compounds
(305); and a diode-laser based detector for doublet peak measure-
ment in flow injection analysis (306) have appeared.
Spectrophotometers. The commercial spectrophotometers
exhibited at Pittcon ‘94 (307-311) and Pittcon ‘95 (312-318) have
been reviewed. “Wavelength Dispersive SpectrometrysPast,
Present and Future” is the title of a review discussing alternative
spectrometer configurations, dispersive element types, and exten-
sion to analytical electron microscopy (319). ATI Unicam have
introduced their Models UV3 and UV4 to join their earlier UV2
spectrophotometers. These two new entries are both grating
instruments with wavelength ranges of 190-900 nm and band-
widths 1.5 nm (fixed) for the UV3 and six stepwise variable from
0.2 to 4 nm for the UV4. Stray light for both units (NaI solution
at 220 nm) is <0.02% T. Available from Cecil Instruments Limited
are their 1000, 2000, 3000, 8000, and 9000 series spectrophotom-
eters. The 1000 and 2000 series have 8- and 4-nm bandpass
options, and wavelength ranges to 1000 nm, lower wavelengths
of 325, 280, 200, and 190 nm options, and typical stray light of
0.01% at 220 and 340 nm. The 3000 series is a Littrow grating
monochromator with a bandpass of 2 nm, a wavelength range from
190 to 1000 nm, typical stray light levels of 0.01% at 220 and 340
nm, and a variety of hardware and software options. The 8000
and 9000 series are dual-beam grating instruments with 2-nm
bandwidths (four stepwise bandpasses variable from 0.5 to 4 nm
for Model 9050), stray light levels typically <0.01%, and a variety
of hardware and software options. Fisher Scientific has made four
low-cost spectrophotometers available with their analog Model
410 and their digital Models 415, 450, and 470. All of these
instruments have a wavelength range of 330-999 nm and stray

Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 177R
light characteristics of 0.1% typical at 340 nm. Models 410, 415,
and 450 have nominal bandpass characteristics of 8 nm while that
of the Model 470 is 5 nm. The Hach Co. has introduced its Model
DR/4000 UV-visible spectrophotometer with a wavelength range
of 190-1100 nm with programs for 130 common water quality
parameters and 110 preprogrammed calibration curves. Hewlett-
Packard has developed the HP 8453 UV-visible spectroscopy
system which employs a diode array detector, a wavelength range
of 190-1100 nm, a 1-nm bandwidth, and less than 0.07% stray
light at 220 nm (NaI, ASTM). In early 1994 LaMotte Co.
introduced the small portable SMART colorimeter featuring a
microprocessor-controlled filter wheel with 40 preprogrammed test
factors (analytes) and reagent systems. The Spectronic Genesys
2 spectrophotometer by Milton Roy now joins its predecessor the
Spectronic Genesys 5 on the market. Both are split-beam, dual-
detector systems with wavelength ranges of 200-1100 nm and
stray light characteristics of 0.05% T at 220 and 340 nm. However
the Spectronic Genesys 2 has an improved bandwidth of 2 nm,
four scan speed options, and a wider variety of software features.
The FullWave spectrophotometer is a recent product of Ocean
Optics, Inc. This low-cost instrument, designed for university level
students and novice spectroscopists, features a compact charge
coupled device array system, measures absorbance from 400 to
850 nm at 1024 wavelengths simultaneously, and uses Windows-
based software. Now available from Perkin Elmer is the Lambda
900 UV/VIS/near-IR spectrophotometer, a double-beam ratio
recording instrument with microcomputer electronics and con-
trolled by personal computer. The wavelength range of this
instrument is 185-3300 nm with incrementally variable bandpass
of 0.05-5.00 nm in the UV-visible region (0.2-20 nm in the near-
IR range) and stray light less than 0.00008% T at 220, 340, and
370 nm. The Lambda 12 and Lambda 14 UV/VIS spectropho-
tometers, double-beam instruments, are additions to Perkin-
Elmer’s line of Lambda spectrophotometers. Both have a wave-
length range of 190-1100 nm with bandwidths of 2 nm (fixed)
for the Lambda 12 and incrementally variable 0.5-4 nm for the
Lambda 14. Stray light is less than 0.02% T at 220, 340, and 370
nm for both, although the Lambda 14P has a the stray light
characteristic of less than 0.005% T. Also available from Perkin-
Elmer is the Lambda 18 UV/VIS spectrophotometer, a double-
beam, dual-monochromator instrument with microcomputer elec-
tronics and personal computer compatibility. The wavelength
range of this instrument is 185-900 nm with an incrementally
variable bandpass of 0.1-5.00 nm and stray light less than 0.0001%
T at 220, 340, and 370 nm. Polytec PI, Inc. have developed the
X-dap diode array fiber-optic spectrophotometer. Four models
covering different wavelength ranges (UV, UV-visible, UV-
visible-near-IR, near-IR) and lamp options consisting of tungsten-
halogen, deuterium, and xenon and also software, probe, and fiber
options are available. Shimadzu has introduced their dual-beam
UV-1601 (stand alone version) and UV-1601PC (PC version) UV-
visible spectrophotometers with a wavelength range of 190-1100
nm, a fixed 2-nm bandwidth with stray light less than 0.05% at
220 and 340 nm. They have also presented their single-mono-
chromator model UV-2401PC and a dual-monochromator Model
UV-2501PC UV-visible spectrophotometer. Both instruments
exhibit wavelength ranges of 190-900 nm and an incrementally
variable bandpass of 0.1-5 nm. The stray light levels at 220 and
340 nm for the single-monochromator system is less than 0.015%
T and less than 0.0003% T for the dual monochromator. Spectral
178R Analytical Chemistry, Vol. 68, No. 12, June 15, 1996
Instruments, Inc. have developed their 410 (350-930 nm) and
420 (200-930 nm) models of high precision fiber-optic charge
coupled device array UV-visible-near-IR spectrophotometers,
each having a 1.2-nm bandpass. A new and innovative spectro-
photometer is the SpectroPette by World Precision Instruments,
Inc. This instrument resembles a standard pipetter except that a
fiber-optic piston draws sample into the wave guide core region
where the sample becomes part of the fiber-optic wave guide
optical system. This type arrangement permits the measurement
of adjustable path lengths from 1 to 50 mm and volumes of 60
nL/mm light path. The wavelength range is 380-760 nm with a
spectral bandwidth of 12 nm (2.5 nm is an option) and stray light
as low as 0.15% T at 550 nm. A variation of this system as a small-
volume long-cell path flow cell instrument is also available.
Noncommercial spectrophotometers reported during this
reporting period include an instrument and method involving three
signal components and normalization (320), a single-beam spec-
trometer for fast spectral measurements of highly turbid materials
(321), spectrophotometric sensor devices working in a raster of
a few wavelengths (322), and an instrument employing parallel
complementary modulated cells (323). A unique system for
remote environmental measurements of absorbance incorporates
fluorescent dyes (Stilbene 420, 3-hydroxyflavone) placed before
and after the sample to provide an internal reference signal
proportional to transmission that is independent of the source light
intensity (324). A miniature charge coupled device array spec-
trometer which uses a compact tandem optical fiber DIP probe
has been shown to exhibit performance with respect to spectral
resolution, stray light, noise, and dynamic range comparable to
non-fiber-optic analytical instruments (325).
Specialized Instruments or Components. Diode array
detection in HPLC systems has been the subject of a number of
reviews (326-330). A linear photodiode array has been inte-
grated with a charge amplifier, an analog-to-digital converter, a
digital programmable polynomial solver for pixel level calibration,
a set of control registers, and a processor interface in order to
reduce noise effects by using large pixels and by closely coupling
the analog-to-digital converter to the photosensors (331). A
multicomponent process instrument with a photodiode array and
a stable and long-lived xenon flash lamp using fast transputer
technology combined with a nonlinear regression algorithm
permits differentiation of similar spectra online in real time (332).
Optical detection systems for capillary electrophoresis have
been reviewed (333). A number of detection systems for capillary
zone electrophoresis have been reported and include an extended
path length postcolumn flow cell (334); simultaneous detection
of sodium, potassium, and magnesium in ocular lenses using
imidazole at pH 6 (335); laser-based ultraviolet detection yielding
improved detection limits (336); and ultraviolet longitudinal cell
design with enhanced sensitivity for micellar electrokinetic
chromatographic determination of phenols (337). Tryptophan
and tyrosine residues in peptides separated by capillary electro-
phoresis have been detected by their second-derivative spectra
using diode array detection (338).
Fiber-optic systems continue to be an active area of interest.
Barium gallogermanate glasses have been found to exhibit
potential for both fiber and bulk optical applications with transmis-
sion beyond 5 µm (339). A fiber-optic interface has been
developed to convert a Cary Model 14H spectrophotometer into
a fiber-optic instrument (340). A remote system for laser-induced
photoacoustic spectroscopy of weakly absorbing species in solu-
tion using an optical fiber for laser light transmission to a cell
has been developed (341). Two drug dissolution monitoring
systems employing multiple fiber-optic probes and diode array
spectrometers have been reported (342, 343).
Light-emitting diodes, available in six different colors, have
been used to construct a multichannel flow injection cell allowing
simultaneous multiwavelength determination (344). A modified
sample compartment employing a computerized pipet has been
described which can produce repetitive optical measurements at
very high rates of 360-1800 samples/h with relative standard
deviations between 0.3 and 0.7% (345). A millisecond time scale
ultraviolet-visible diode array spectrophotometer has been coupled
to a stopped-flow instrument (346). The thermal unfolding of
RNase has been observed with a multidimensional spectropho-
tometer that measures nearly simultaneous CD, steady-state
fluorescence, and absorbance values (347). A monitor for
measuring NO emissions employing a NO resonance lamp with
multiple passes across a roadway has been described (348). In
other applications, developments that have been reported include
a liquid crystal shutter array spectrometer (349), a sample
chamber that deflects the measuring beam from its original axis
to pass through a long sample and returns the beam to its original
direction (350), and a dual-beam spectrometer designed for optical
studies of spread monolayers at the air-water interface (351).
Laser photothermal refraction from a cylindrical capillary has been
used to permit monitoring of the Fresnell fringe obtained by a
second pinhole diaphragm and permit a detection limit comparable
to 3 × 10-7 absorbance unit (352). The design and operation of
a wavelength-multiplexing mask for a computer-controlled ultra-
violet-visible Hadamard transform spectrometer has been de-
scribed (353).
Larry G. Hargis is a Professor of Chemistry and Associate Provost at
the University of New Orleans. He graduated from Wayne State University
with a B.S. in 1961, an M.S. in 1963, and a Ph.D. in 1964, spent a
year as a Postdoctoral Research Associate at Purdue University, and joined
the faculty at UNO in 1965. He has authored numerous papers on topics
in ultraviolet and light absorption spectrometry, reaction rate determina-
tions, fast kinetics of analytical reactions, and heteropolymolybdate
chemistry. He has also written two laboratory manuals, an introductory
analytical chemistry text, and chapters in several monographs dealing
with spectrophotometry. Dr. Hargis holds membership in the American
Chemical Society, National Science Teachers Association, Phi Lambda
Upsilon, and Sigma Xi.
Robert E. Sutton is an Adjunct Instructor of Chemistry at Kalamazoo
Valley Community College. He graduated from Wayne State University
with a B.A. in Chemistry in 1980 and was subsequently employed as a
pesticide analyst with the U.S. Food and Drug Adminsitration in Detroit,
MI until 1986. He received a M.A. in 1988 and a Ph.D. in 1995 in
Science Education from Western Michigan University. His fields of
interest are ultraviolet and visible molecular absorption spectroscopy, flame
absorption spectroscopy, and capillary electrophoresis. He is a member
of the American Chemical Society, Association of Analytical Chemists
and Alpha Kappa Mu.
James A. Howell is a professor of Chemistry at Western Michigan
University and also a science advisor for the Detroit District Laboratory
of the Food and Drug Administration. He received his B.A. from Southern
Illinois University in 1959, his M.S. from the University of Illinois in
1961, and his Ph.D. in analytical chemistry from Wayne State University
in 1964. His particular fields of interest are in ultraviolet and visible
absorption spectrometry, flame emission and atomic absorption spectros-
copy, and also computer applications to chemical instrumentation. He
is the author of a number of research papers and chapters in books. Dr.
Howell is a member of the ACS, SAS, and the Association of Analytical
Chemists.
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