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ANALYSIS
Flow-in~~i5n anaiysis (FIA) is an analytical tech- component analysis program to these spectra
nique of well-known potential, and its application to has allowed the resolution of mixtures of analytes
simultaneous determination of several analytes in the with strongly overlapped spectra and the cancellation
same sample is a subject of growing interest. One 5f of matrix effects.10-2 Derivative spectra have
the ways to increase its potential in the resolution recently been applied to the resolution of a
of complex mixtures is the use of image detectors. quatemary mixture by use of a combined FIA-DAD
Diode-array ~e~trophot5meters are image detectors system.3
capable ofcompletely recording an ultraviolet-visible The aim of this work was to expand the use of
spectrum in about 0.1 sec. Despite their increasing DADS in the simultaneous determination of several
use as HPLC detectors and their potential advan- species by FIA by exploiting all the information
tages,* they have only very recently begun to be used obtainable from the absarption and derivative
in FIA. For example, Vithanage and Dasgupta3 have spectra of each analyte.
applied diode-array detectors (DADS) to the study of Mul~~omp5nent analysis with an FIA-DAD
proton-iigand and metal-&and equilibria occurring system involves two essential aspects, namely (a) the
in the FIA-peak gradient zone. Valcarcel et al. have recording of the spectrum under conditions of maxi-
reported the simultaneous determination of Fe(U) mum sensitivity and repraducibility, and (b) the
and Cu(I1) with a mixture of specific chromogenic mathematical treatment of the information gathered,
reapnts,4 the metal concentrations being calculated to resolve the analyte mixture.
by means of a linear equation system. The use of This paper reports tw5 different procedures fur
DADs results in faster analyses thanks t5 the possi- spectrum recording, and three methods for treating
bility of making several simultaneous measurements the results, namely: (a) the resolution of a system
at various wavelengths. Although there are a number consisting of one equation per analyte; (b) multi-
of procedures available for optimization of the wave- component analysis (MA) by a computation program
lengths to be used, 5-8 the resolution of mixtures on supplied by Newlett-Packard; (c) multi-wavelength
the basis of a few linear equations is i~~~~~~ble to linear regression analysis (ML&X), a linear re-
systems having extensive spectral overlap. gression graphical method. All three procedures have
Derivative spectra are known to be more selective been applied to the simultaneous determination
than absorption spectra9 (zeroth-derivative). This in- of Fe(H) and Fe(II1) by use of a mixture of
creased selectivity has been exploited in the deter- 1, 10-phenanthroline and sulphosalicylic acid as
mination of a given component in a sample by chromogenic reagent. The results obtained by the
making measurements at a single wavelength. Diode- three methods are compared and discussed.
array detect5rs considerably facilitate the obtainment
of derivative spectra. The application of a mdti- FXPRRIMENEAL
Reagents
Stock iron solutions containing 1000 ppm Fe(H) and
*To whom all correspondence should he addressed. Fe(W) were prepared by dissolving Fe(NH4),(S04), . &I20
988 M. BLANCO et al.
and FeNH,(SO,),. lZH,O, respectively, in 0.05M sulphuric greater than 1% were achieved for integration times
acid and standardized both titrimetrically with a standard of 0.3 set or longer. Thus, a T, of 0.4 set, sym-
dichromate solution and p-diphenylaminesulphonate as in- metrically distributed about the residence time of the
dicator and gravimetrically by weighing as Fe,O, . Working
solutions were prepared by appropriate dilution with de- FIA peak, was chosen for further experiments.
gassed distilled water. The concentration of the Fe(H) Flow-injection analsysis is characterized by the fact
solution was checked daily by titration with standard di- that the dispersion of the sample in the manifold is
chromate solution. a reproducible function of time. Thus, the repro-
I, lo-Phenanthroline (Phen) solution 0.5%. Prepared from
ducibility of the sample image obtained at a given
1,lo-phenanthroline hydrochloride.
Suiphosalicylic acid solution (SSA), 2.5%. time will depend chiefly on the reproducibility of the
Acetate buffer, IM, pH 5.7. Adjusted with sodium hy- time elapsed between image recording and the refer-
droxide. ence time. Every FIA recording features two points
The stock reagent solutions were used to prepare those that are eligible as a reference time, namely sample
injected into the manifolds, as follows.
injection and sample arrival at the detector. A suit-
Manifold A: 1 mM Phen, 15 mM SSA, 0.2M acetate buffer. able computer program facilitates recording of spec-
Manifold B: 10 mM Phen, 25 mM SSA, 0.2M acetate buffer.
tra under preselected conditions (wavelength range,
All solid reagents used were of analytical grade. l time interval, integration time, etc.).
Apparatus and manifolds The simplest approach uses sample injection as
A Hewlett-Packard 8451A diode-array spectrophoto- the reference time. Obviously, the injection should
meter furnished with a 128-K RAM HP-82909A module, an be synchronized as accurately as possible with the
HP-9 121 floppy disk drive, an HP-98155A keyboard and an
triggering of the program handling the spectrum
HP-7440A plotter was used as the detection and data-
processing system. An HP-82940 GPI/O interface was used recording, in order to ensure reproducibility; this is
to control the automatic injection valve. Other equipment made possible by the use of an automatic valve, the
included a Gilson Minipuls-2 peristaltic pump and a Hellma control scheme of which is depicted in Fig. l(A).
178.712-QS flow-cell (inner volume 18 ~1, optical path A suitable alternative is to record the spectra once
length 10 mm).
An automatic Tecator V-200 and a manual Rheodyne
a preselected time has elapsed after arrival of the
5020 injection valve were used in manifolds A and B, sample at the detector. This needs no synchron-
respectively. All reaction coils and sample loops were made ization, so the analyst can inject the sample manually
from PFTE tubing of 0.5 mm bore. and then execute the recording program.
Normalized standard spectra A computer-controlled injection system (manifold
Three pure standard solutions of each component of the A) allows the recording of spectra in both ways,
mixture, with a concentration lying in the linear concen- whereas a manual system (manifold B) can only cope
tration range and giving absorbance readings lower than with the second of the two.
1.0, were prepared. The batch solutions were injected in Figure 2 shows the flow diagrams of the programs
triplicate and a spectrum for each standard was obtained
by averaging the three spectra obtained. A normalized used to control the recording of the spectra.
spectrum for unit concentration was obtained for each FIARECl uses the first few injections to calculate the
component by regression of the absorbance us. concen- residence time, TR; the spectrum is recorded from
tration. These normalized spectra were used as references further successive injections at TR - (T/2) set after
for resolution of mixtures.
each.
FIAREC2 employs the first few injections to calcu-
late the time elapsed between the start and the
RESULTS AND DISCUSSION maximum of the FIA peak, T, the spectrum being
subsequent19 recorded at T - ( T,/2) set after arrival
Recording of spectra
of the sample at the detector.
The HP-8415A spectrophotometer is capable of FIARECl and FIAREC2 were used in conjunc-
recording a whole spectrum (190-820 nm) in 0.1 set tion with manifolds A and B, respectively.
every 0.7 sec. Such a high recording speed allows a
vast amount of information to be gathered from an
Data treatment
FIA peak, yet a mixture can be resolved from a single
spectrum. In order to ensure the greatest possible The analytes were determined by processing the
sensitivity and reproducibility, the spectrum was information abstracted from the spectra of the mix-
recorded at the maximum of the FIA peak. The first tures, by three different procedures, as follows.
few attempts at recording spectra at high speed failed (a) By solving a system of two linear equations
because of the poor reproducibility of the results based on the absorbances of the two complexes,
obtained in successive injections. The reproducibility measured at their respective absorption maxima (510
was improved by increasing the integration time, T,, and 486 nm). The equations concerned were:
the period over which the spectrophotometer is con-
tinuously measuring the spectrum, the end of which Manifold A
is marked by delivery of the spectrum. Obviously, A,,, = O.O682[Fe*+]+ 0.0255[Fe3+]
since FIA signals are transient peaks, T, should be as
short as possible. Relative standard deviations not Ad86= O.O634[Fe*+]+ 0.0280[Fe3+]
Diode-array detectors
COMPUTER
Fig. 1. Schemes of the flow-injection manifolds. S = sample; I.V. = injection valve; L = reaction coil;
D = diode-array detector; W = waste. (A) V,, = 120 ~1; flow-rate 0.95 ml/min; L = 270 cm; TR = 39 set,
(B) V,, = 8 ~1; flow-rate 1.07 ml/min; L = 50 cm TR = 7 sec. In manifold A the sample is injected into
the reagent stream (normal FIA mode) while in manifold B the reagent is injected into the sample stream
(reverse FIA mode).
SELECTION L
.. I= l,...N 4 v J=I,...N
SELECTION L =+,...LI
J
SELECTION ~1, ~2
1
REFERENCE
t
REFERENCE INJECTION
1
=l....Y
1REFERENCE 1 1
INJECTION MEASURE ABSORBANCE
1 t
1 INJECTION 1 TIME=+
I
RECORDING FIA PEAK
YEXT J 4
TIME = +
1
MEASURE FOR Tl SELECTION Ll , L2
I
L WAIT TS-T1/2
1 I
AVERAGE SPECTRA FOR ALL INJECTIONS MEASURE FOR Tl
E*T K
Fig. 2. Flow chart of programs for recording of spectra. A: FIARECI; B: FIAREC2. See Table I for definition of variables
Diode-array detectors
0.6 - (A)
2
0.1 - -0.010
)&
I I I I I
0.0 -0.014
400 450 500 550 600 650 400 450 500 5x) 600 650
O.Ocm4
(u
P
E
1 A2
B o-
Z
l!s
-0.OOQ4 -
WOVelenQth ( nm )
Fig. 3. Spectra obtained for 20 ppm of Fe(III) (1) and 8 ppmFe(I1) (2) with a normal nFIA manifold.
A, absorption spectra; B, first-derivative spectra; C, second-derivative spectra; D, third-derivative spectra.
should he pointed out that the volume injected into were processed over the whole wavelength range
the manifold was kept rather low in order to avoid covered (40&650 nm).
the appearance of double peaks. The results obtained in the resolution of the
Fe(II)-Fe(II1) mixtures (Tables 2 and 3) are shown as
Mixture resolution the correlation between the concentrations taken and
A series of 12 mixed solutions containing various found, for the different calculation procedures used.
amounts of Fe(I1) and Fe(II1) in the ranges 0.25155 The real achievement of this work is not the
and 3-20 ppm, respectively, and [Fe(II)]/[Fe(III)] accurate calculation of the concentration of a given
ratios from 0.6 to 40, were prepared for manifold A. species in the presence of a higher concentration of
Similarly, another set of 14 solutions with Fe(I1) and another, but rather the possibility of simultaneously
Fe(II1) contents in the ranges 0.5-5.2 and l-12.4 determining both under all types of conditions.
ppm, respectively, in ratios from 0.25 to 16, were As would he expected from the degree of spectral
prepared for manifold B. The conventional and the overlap, the classical method of mixture resolution
first, second and third-derivative spectra (Fig. 3A-D) based on a linear equation system resulted in substan-
Table 2. Least-squares statistical data for fits of calculated vs. added concentrations (manifold A)
Fe+ Fez+
Correlation Correlation
Intercept is Slope zks coeff. (IP) Intercept * s Slope & s coeff. (RZ)
Linear equations
-0.33 f 0.97 1.06 f 0.09 0.937 O.f0 If:0.08 0.95 & 0.01 0.998
Multicomponent analysis
Absorption spectrum 0.32 f 0.37 0.97 f 0.03 0.988 -0.12 & 0.08 1.02 * 0.01 0.998
First derivative 0.45 f 0.50 0.97 k 0.05 0.978 -0.05 f 0.08 0.99 * 0.01 0.999
Second derivative 0.10 &OS6 1.OOf 0.05 0.976 -0.03 If:0.07 0.99 + 0.01 0.999
Third derivative 0.02 + 0.65 I .OI & 0.06 0.967 0.01 f 0.07 0.98 + 0.01 0.999
MLRA
400-600 nm 0.42 + 0.40 0.96 & 0.04 0.986 -0.15 kO.09 1.02 f 0.01 0.998
420-580 nm 0.24 + 0.40 0.97 f 0.04 0.987 -0.11 *Oo.08 1.01 + 0.01 0.999
tial errors and hence in poor precision, as cart be seen selection of the most suitable wavelength range for
from the deviation from linearity and the low cor- resolution of the mixture quite a simple task. The
relation coeEcient obtained. reliability of mixture resoln~on has been reported
The use of a calculation program (MA) operating to depend strongly on the wavelength range
throughout the visible spectrum provides better re- selected, but up till now the selection has been made
sults for the slope, intercept and precision: However, empirically.
deviations from linearity are still significant and the The MLRA program provides the user with the
slope is rather low for Fe(III) and high for Fe@), wavelength range that gives the best linearity of
The application of the MA program to the deriva- response. This is illustrated in Fig. 4 for a mixture
tive spectra results in considerably improved results, containing 0.52 ppm Fe(II) and 8.32 ppm Fe(III). In
particularly for Fe(II). The precision achieved is this case, deviations from linarity are observed only
virtually independent of the spectral mode and the near the ends of the wavelength range, where the
best slope and intercept are obtained for the first and absorbance values are significantly smaller. This is
second-derivative spectra. consistent with the findingi that one of the requisites
Although the use of derivative spectra also im- for accurate mixture resolution with the aid of the
proves the slope and intercept for FelfIIf, increasing MA program is that al1 species to be determined must
the derivative order results in poorer precision as a absorb significantly in the selected wavelength range.
result of the concomitantly decreasing spectral Narrowing of the spectral interval to 42G580 nm
signal. results in a better fit, as can be seen from Tables 2 and
It should be noted that manifold l3 yiefds more 3. This range narrowing also resulted in slight im-
precise results than manifold A, probably because of provements in the results obtained by MA from the
the greater sensiti~ty it produces. absorption spectra, though not from the derivative
The MLRA treatment, as applied to the whole spectra. No other wavelength range assayed yielded
wavelength range, yields similar results to those better results.
obtained by multicomponent analysis, but has
two significant advantages, namely the resolution CONCLUSIONS
procedure is straightforward and transparent to
the analyst-whereas that of MA is obscure This paper demonstrates the ~ssibili~y of accom-
and inaccessible-and (most important), it makes plishing simultaneous determinations by FIA with
Table 3. Least-squares statistical data for fits of calculated US.added concentrations (manifold B)
Fe3+ Fez+
-..-
Correkition Correlation
Linear equations
-0.09 zt 0.26 1.l2-tO.04 0.983 0.17 rf:0.09 0.88 + 0.03 0.986
Multicomponent analysis
Normal spectrum 0.03 _t 0.08 0.9s * O.OI 0.998 0.06 + 0.02 1.036 f 0.006 0.9996
First derivative 0.09 & 0.06 1.02 i_O.Of 0.999 0.06 * 0.03 0.994 + 0.009 0.999 t
Second derivative 0.08 f 0.08 I.os+o.or 0.999 0.05 + 0.03 0.99 * 0.01 0.999
Third derivative 0.02 kO.15 1.07& 0.02 0.994 0.06 & 0.03 0.99 f OxJl 0.999
MLRA
400-600 nm 0.02 f 0.08 0.9s * 0.01 0.998 0.05 * 0.02 1.04 f 0.006 0.9996
420-580 nm 0.04 & 0.07 0.96 * O.OI 0.999 0.05 + 0.02 1.029 & 0.006 0.9996
Diode-array detectors 993
0.30
1 E 0.20
P
\
i
f
^9; 0.10
0.00
Fig. 4. MLRA plot for a mixture of 8.32 ppm Fe(H) and 0.52 ppm Fe(B). Data obtained with manifold
B. (A) Wavelength range, 401X650 nm; Fe(II1) found, 8.22 ppm; Fe(I1) found, 0.60 ppm; regression
coefficient, r* = 0.997. (B) Wavelength range, 420-580 nm; Fe(II1) found, 8.26 ppm; Fe(I1) found, 0.58
ppm; regression coefficient, r2 = 0.9994. Absorbance measurements made at 4-nm intervals.