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Talanta
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a r t i c l e i n f o a b s t r a c t
Article history: A portable, microcontrolled and low-cost spectrophotometer (MLCS) is proposed. The instrument com-
Received 17 April 2008 bines the use of a compact disc (CD) media as diffraction grid and white light-emitting diode (LED) as
Received in revised form 18 August 2008 radiation source. Moreover, it employs a phototransistor with spectral sensitivity in visible region as
Accepted 19 August 2008
phototransductor, as well as a programmable interrupt controller (PIC) microcontroller as control unit.
Available online 27 August 2008
The proposed instrument was successfully applied to determination of food colorants (tartrazine, sun-
set yellow, brilliant blue and allura red) in five synthetics samples and Fe2+ in six samples of restorative
Keywords:
oral solutions. For comparison purpose, two commercial spectrophotometers (HP and Micronal) were
White LED
CD media
employed. The application of the t-paired test at the 95% confidence level revealed that there are not sig-
Programmable interrupt controller (PIC) nificant differences between the concentration values estimated by the three instruments. Furthermore,
microcontroller a good precision in the analyte concentrations was obtained by using MLCS. The overall relative standard
Visible spectrophotometer deviation (R.S.D.) of each analyte was smaller than 1.0%. Therefore, the proposed instrument offers an eco-
Food colorants nomically viable alternative for spectrophotometric chemical analysis in small routine, research and/or
Iron (II) teaching laboratories, because its components are inexpensive and of easy acquisition.
Published by Elsevier B.V.
Fig. 2. Electronic layout of the MCLS detector. Legend: PT = phototransistor; C = polyethylene capacitors; R = resistors; U1 = operational amplifier; U2 = quadruple operational
amplifier; U3 = quadruple switches. Details in text.
G. Veras et al. / Talanta 77 (2009) 1155–1159 1157
mulations were diluted to fit in the linear range of the analytical optical–mechanical system. Since the slit for the light entrance
curve. on the probe and the sensitive area of the phototransistor have
Recently distilled water was used to prepare all solutions. the same dimensions (ca. 0.3 mm), the spectra registered by the
ocean optics spectrometer represent the profile of the radiation that
reaches the phototransistor. In this process, the spectrometer reg-
2.2. Description of the proposed instrument
isters the reflected spectrum resulting from the radiation diffracted
on the CD media. It was carried out five scans to obtain an average
Fig. 1 illustrates the optical components employed in the MLCS
spectrum for each step of the stepper motor. Average spectra were
and Fig. 2, the electronic layout of the detection unit. This instru-
used to build a calibration curve between the number of steps and
ment was assembled into a box with 30 cm front, 20 cm height and
the wavelength of maximum emission of the spectrum. In this case,
30 cm length. The optical–mechanical system is composed by white
an equation was estimated by using the linear least-squares and the
LED, focus lens to create a small light image (2 mm diameter) on
result is presented below.
the diffraction grid (CD) and stepper motor with gear to move the
diffraction grid and to promote the scan of the spectral region of Step = wavelength × 0.50246 − 214.7837
interest. To increase the spectral resolution, the position of the step-
per motor was adjusted so that the phototransistor can detect the The number of steps and peak wavelengths are linearly cor-
radiation from the second overtone. related in the spectral range 440–640 nm with a correlation
The instrument uses a PIC 16F877 programmable microcon- coefficient of 0.99968. Therefore, practically the entire visible
troller as control unit driven by a stabilized power supply. The spectral region can be exploited for spectrophotometric measure-
control software was elaborated with C language. The microcon- ments by the MLCS. The overall standard deviation at the peak
troller presents a 10-bit multi-channel analog to digital converter wavelengths was 0.30 nm, indicating a good repeatability asso-
(A/D), 8k × 14 words of flash program memory, 256 × 8 bytes of ciated to the wavelengths obtained starting from the number of
EEPROM data memory, 368 × 8 bytes of RAM, three timers and steps.
three ports with eight channels (multiplexed pins) each, for gen- The collected data were also employed to establish the effective
eral purpose, that can be configured for data acquisition. The bandwidths at maximum wavelength for each step and the value
microcontroller allows drastic reduction of the number of elec- 13 nm was estimated in the range 440–640 nm.
tronic components in the spectrophotometer and makes possible The dispersion of the maximum signals measured for each
to introduce or alter functionalities in the instrument without any step was also assessed and the overall relative standard deviation
hardware modification. (R.S.D.) was estimated as 2.6%. This result indicates a good repeata-
The white LED Control Module pulses the white LED radiation bility of the signals associated to the radiant power of the white
at 7 Hz. The module turns the white LED on for readings of the ana- LED.
lytical signals and off for determination of instrumental noise. The
signals are detected, filtered and amplified in the Detection Module
3.2. Determination of food colorants in synthetic samples
of the MLCS, showed in Fig. 2. In the layout, five operational ampli-
fiers are used for different purposes: to convert the photocurrent
The performance of the MLCS was initially evaluated by ana-
into voltage (U1), to filter the high frequency noise (U2:A) as a high-
lyzing food colorants in the synthetic samples. The absorbance
pass Butterworth filter; to amplify the analytical signal (U2:B), to
measurements were accomplished at the wavelengths of its
detect the peak signal as a sample-hold unit (U2:C) and to fit the
maximum absorption: 486 nm (sunset yellow), 500 nm (allura
analytical signal (U2:D) in the A/D scale (5 V) using CMOS switches
red) and 582 nm (brilliant blue). However, in the case of tar-
(U3:B–D) to change the gain depending on the value of the input sig-
trazine solutions, the measurements were performed at 440 nm
nal. C5 and R11 are used as low-pass filter and P1 is used for off-set.
due to the low sensibility of the MLCS in the wavelength of
CMOS switch U3:A is used to discharge the capacitor of the sample-
maximum absorption of this colorant (426 nm). For reference
hold circuit. The switches are controlled by the PIC. The processed
measurements, the food colorants were also analyzed by an HP
signal E is sent to the A/D converter of the PIC to be converted into
diode-array, model 8453, and a Micronal spectrophotometer, model
absorbance values. To calculate the absorbance values, the loga-
B34211.
rithm function used the measurement of blank (water) as reference.
In order to investigate the maximum concentration for linear
Both blank and sample measurements were registered turning the
response (the limit of linearity—LOL), ANOVA (analysis of variance)
LED on to consider maximum light power and off in order to con-
and F-test for lack of fit were applied to the models elaborated
sider the dark signal. After digitalization, the absorbance values are
with the concentration range 2.0–26.0 mg L−1 , for each colorant.
sent by the microcontroller to the liquid crystal display (LCD) mod-
The results showed no lack of fit up to the LOL as presented in
ule, a TECH2004D-FL-GBS-S character type LCD with two buses:
Table 1. An F-test for the regression significance revealed that the
eight bits for data and three bits for control. In this work, the com-
performed linear regressions are highly significant up to the LOL
munication between microcontroller and LCD was always carried
values.
out with six bits, four being used for data transmission and two
The values of limits of detection (LOD) and limits of quan-
for data control. The Stepper Motor Control Module controls the
tification (LOQ), which were estimated according to IUPAC
angular displacement of the CD media.
recommendations [7], are also shown in Table 1. It is worth not-
A clock elaborated with a 4-MHz piezoelectric crystal is used for
ing that LOL, LOD and LOQ present similar values for the three
synchronization of the microcontroller internal functions.
instruments and for the four food colorants.
After the study above, the analytical curves were constructed
3. Results and discussion employing the following concentration ranges: 2.0–12.0 mg L−1 for
allura red and 3.0–18.0 mg L−1 for the other colorants. In Table 2 are
3.1. Calibration of the MLCS optical–mechanical system presented the results for the colorants determinations in synthetic
samples.
An ocean optics spectrometer, model USB450, which substituted As can be seen, there is a good agreement between the results of
the phototransistor as detector, was used to calibrate the MLCS concentration determined by the proposed instrument and those
1158 G. Veras et al. / Talanta 77 (2009) 1155–1159
Table 1 Tabela 4
Values of LOD, LOQ and LOL obtained for the four colorants Recovery percentage obtained for Fe2+ in six samples of restorative oral solutions
Colorant Instrument LOD LOQ LOL Sample Added Fe2+ (mg L−1 ) Recovery (%)
(10−2 mg L−1 ) (10−2 mg L−1 ) (mg L−1 )
Micronal HP MLCS
Micronal 3.1 9.4 14.0
3.0 99.31 100.34 97.18
Allura red HP 0.7 2.2 14.0
01 5.0 98.86 100.19 100.56
MLCS 0.1 0.2 12.0
7.0 98.98 97.99 96.41
Micronal 3.2 9.7 18.0
3.0 99.65 100.00 97.18
Sunset yellow HP 0.7 2.1 18.0
02 5.0 98.67 99.24 98.05
MLCS 13.0 39.4 18.0
7.0 96.81 97.27 97.25
Micronal 2.6 7.8 18.0
3.0 96.19 94.56 93.85
Tartrazine HP 0.8 3.6 18.0
03 5.0 96.01 98.48 99.86
MLCS 8.1 24.7 18.0
7.0 98.55 99.28 99.05
Micronal 5.5 16.6 18.0
3.0 101.39 98.30 98.72
Brilliant blue HP 7.8 23.8 18.0
04 5.0 99.62 100.19 105.29
MLCS 3.1 9.3 18.0
7.0 99.27 98.28 98.10
01 4.9 ± 0.1 4.9 ± 0.0 4.9 ± 0.1 A portable, inexpensive and microcontrolled spectrophotome-
02 5.8 ± 0.1 5.7 ± 0.0 5.8 ± 0.1 ter was developed in this work. Such advantageous characteristics
03 4.2 ± 0.1 4.1 ± 0.1 4.3 ± 0.1 were achieved due to the incorporation of a CD media as diffraction
04 6.3 ± 0.1 6.3 ± 0.1 6.2 ± 0.1
grid and a white LED as radiation source. Other relevant char-
05 4.5 ± 0.0 4.4 ± 0.0 4.5 ± 0.0
06 9.0 ± 0.1 8.9 ± 0.1 8.9 ± 0.0
acteristic of the proposed instrument concerns to the use of a
phototransistor with spectral sensitivity in the visible region as
R.S.D. 0.8% 0.5% 0.7%
phototransductor.
G. Veras et al. / Talanta 77 (2009) 1155–1159 1159