In the Laboratory

UV–Visible First-Derivative Spectrophotometry

Applied to an Analysis of a Vitamin Mixture
F. Aberásturi, A. I. Jiménez, F. Jiménez, and J. J. Arias*
Departamento de Química Analítica, Nutrición y Bromatología, Universidad de La Laguna, Facultad de Química,
E-38071 La Laguna, Tenerife, Spain; *jjarias@ull.es

This article reports a method for the simultaneous For quantitative analysis, if the Beer–Lambert law (A
determination of a ternary mixture of vitamins using = ε c) is obeyed for the normal spectrum, the following
derivative spectrophotometry. The earliest application of equation can be obtained:
this technique was developed in 1954 by Stacy French et n
dA n
dε
al. (1), who used the wavelength modulation principle of
Hammond et al. (2) to examine the first-derivative spectra =
of photosynthetic systems.
dλ n dλ n c
where A is absorbance, ε is molar absorptivity, is cell path
Background length, and c is the concentration of the analyte. This forms
the basis for analytical determinations (4 ). Theoretically,
For a single-peak spectrum, the first derivative is a plot of dA/dλ is zero at λ max for the band in the normal spectrum.
2 2
the gradient dA/dλ of the absorption envelope versus wave- The second-derivative spectrum d A/dλ versus λ has two
length and features a maximum and a minimum. The vertical maxima with a minimum between them, at λ max of the
distance between these is the amplitude, which is proportional to normal absorption band (5). In principle, peak heights
the analyte concentration. Derivative spectra may be produced n n
(measured from d A/dλ = 0) are proportional to the
by processing the spectrophotometer output signal by electronic analyte concen-tration but the amplitude can also be
or numerical differentiation or by using spectrophotometers with measured by the so-called tangent method, in which a
specially designed optics (3). The differentiation dis-criminates tangent is drawn to the maxima and the amplitude is
against broad bands, emphasizing sharper features to an extent measured vertically from the tangent to the minimum.
that increases with increasing derivative order. For this reason, Measures thus obtained are called graphical measures.
the use of derivative spectra can increase the detection For a mixture (M) of non-interacting species (A and B)
sensitivity of minor spectral features (e.g. shoulders) and reduce absorbing in the UV–vis region, absorbance can be expressed as
the error caused by the overlap of the analyte spectral band by
AM = AA + AB = ε A cA + ε B cB
interfering bands of others species in the sample. The main
Hence,
inconvenience of the derivative technique is that the signal-to-
noise ratio becomes progressively worse for higher orders. dA dε dε
Current diode array spectrophotometers allow spectra of nth
M = Ac + B c
A
order to be obtained in an expeditious, cost-effective manner. dλ dλ dλ B

Derivative spectroscopy has been used to solve real-life

It is possible to measure the absolute value of the de-
problems in a variety of fields; of special in-terest in this respect
rivative of the sum curve at an abscissa value (wavelength)
are its pharmaceutical applications.
corresponding to a zero-crossing of one of the components in the
mixture. This is termed a zero-crossing measure and can be
applied to the first and second derivatives. The zero-crossing
measure would seem to be ideal in terms of systematic errors;
(a) (b) (c) but in fact, it suffers in comparison with the graphical measures,
A
with their relatively greater sensitivity to small changes in the
IA position of the interfering band.
Analyte A
Firstderivative
Firstderivative

A The “zero-crossing” derivative spectroscopic mode

Absorbance

IB
P
P allows the resolution of binary mixtures of analytes by
Q Q
B recording their derivative spectra at wavelengths at which
one of the components exhibits no signal. Zero-crossing
B
measurements for each component of the mixture are
Analyte B therefore the sole function of the concentration of the other.
Figure 1a shows the zero-order spectra of two analytes (A
and B). The first-derivative spectra are given in Figure 1b. The
first-derivative spectrum of analyte A is seen to cross zero
Wavelength / nm Wavelength / nm Wavelength / nm (dA/dλ = 0) at P wavelength, whereas the first-derivative spec-
trum of analyte B exhibits a zero-crossing at Q wavelength. The
Figure 1. (a) Absorption, (b) first-derivative spectra of two analytes
first-derivative spectrum for a mixture of A and B is given in
A and B (showing their zero-crossing), and (c) first-derivative
spectrum of their mixture showing the points where the signal Figure 1c. The amplitude measured at Q wavelength (I A) is
depends solely on one of the analytes. dependent only on the concentration of analyte A, and the
measure at P (IB) is dependent only on the concentration of

JChemEd.chem.wisc.edu • Vol. 78 No. 6 June 2001 • Journal of Chemical Education 793

 In the Laboratory

B; so, applying the same measures to the standard and to 1.5

(A)
the mixtures allows the determination of both analytes.
Mixtures of more than two analytes are resolved by
using successive zero-crossing measurements. Alternatively,
calibration curves can be constructed at wavelengths where Mixture
the overall signal is the sum or difference between the 1.0
individual signals of two or more analytes.

Absorbance
The “zero-crossing” method is convenient to implement;
it requires no sophisticated equipment and uses reagents
sparingly. Although it is usually applied to binary samples
(6– 9), in this work it was used to resolve a ternary mixture
0.5
of vitamins (folic acid–pyridoxine–thiamine). This particular
mixture was chosen for two main reasons: (i) its components FOLI
possess overlapped spectra and are thus difficult to resolve THIA PYRI
by conventional spectroscopic methods, and (ii) we were
interested in developing simple, reliable methods for
determining several analytes in a same sample without any 0
240 280 320 360
prior separation.
Wavelength / nm
Materials FOLI (B)
Apparatus PYRI

Spectra were recorded on an HP 8452A diode array

0
(Avondale, PA) detector interfaced to a Vectra ES computer,
both from Hewlett-Packard. Quartz cuvettes of 1-cm light path
were used throughout. First-derivative spectra were obtained
Firstderivative
THIA
using the software provided with the spectrophotometer.
Measurements of pH were made using a Radiometer PHM84
− 0.04
digital potentiometer (Copenhagen, Denmark) equipped with a
glass–saturated calomel dual electrode.
Reagents
1
Standard solutions containing 100  g mL of folic acid
(Sigma lot 30H02224, [59-30-3]), pyridoxine (Sigma lot − 0.08 Mixture
40H0321, [65-23-6]), or thiamine (Sigma lot 24H0450, [67-
03-8]) were prepared in volumetric flasks by direct weighing 200 240 280 320
of the commercially available pure products, dis-solution, Wavelength / nm
and diluting to the mark with Millipore Milli-Q deionized
water. An acetic acid–sodium acetate buffer of pH 5.5 and
Figure 2. (A) Absorption spectra and (B) first-derivative spectra:
CT = 0.20 M (Merck) was also used. 1
( ) folic acid (FOLI), 8  g mL ; ( ) pyridoxine (PYRI),
1 1
8  g mL ; ( ) thiamine (THIA), 8  g mL ; and (––––) their mixture;
Hazards 1
pH = 5.5, HAc/NaAc buffer, C T = 0.20 mol L .
There are no significant hazards related to the
procedures and reagents involved in this experiment.
at concentrations spanning the vitamin’s linear range, and
Procedure, Results, and Discussion deionized water to the mark. The spectra for these
solutions were recorded between 200 and 360 nm. First-
Preliminary tests revealed that the optimum pH for derivative spectra were obtained using the
resolving the ternary mixture studied was 5.5, adjusted with
spectrophotometer-bundled software and signals were
5 mL of acetic acid–sodium acetate buffer. The linear
measured at the 266, 282, and 334 nm for pyridoxine, 266
concentration ranges for the vitamins, established by testing
nm for folic acid, and 282 nm for thiamine.
across a broad span of concentrations, were 1.02–14.28
Figure 2A shows the absorption spectra for the individual
µg mL 1 for folic acid, 1.00–16.00  g mL 1 for pyridoxine, and 6.00–
vitamins and the mixture. The strong spectral overlap prevents
20.00  g mL 1 for thiamine. the individual determination of the vitamins in the mixture by
Construction of Calibration Curves conventional spectrophotometric methods. Figure 2B shows the
first-derivative spectrum for each vitamin and for the mixture.
To eight 25-mL volumetric flasks were added 5 mL of
The signal at 334 nm corresponds to pyridoxine, the 266-nm
HAc/NaAc (pH = 5.5, CT = 0.2 M), the required volume of signal to the sum of folic acid and pyridoxine, and the 282-nm
standard solutions of each vitamin to obtain as many solutions
signal to thiamine plus pyridoxine.

794 Journal of Chemical Education • Vol. 78 No. 6 June 2001 • JChemEd.chem.wisc.edu

 In the Laboratory

These wavelengths were then used to construct Table 1. Parameters of Regression Lines by Derivative
calibration curves. The equations for the calibration lines
Spectrophotometry
obtained at different wavelengths for the three vitamins,
and their statis-tical figures of merit, are given in Table 1. a DL/
λ /nm 1
Analyte Regression Equation r µ g mL
Simultaneous Determination of Vitamins in a Mixture Folic acid 266 0.000037 + 1.96E 3C 1 0.076
The first-derivative spectrum for a solution containing Pyridoxine 266 0.00002 – 6.55E 4C 1 0.138
the three vitamins was obtained as described and the signal at Pyridoxine 282 0.000039 + 4.89E 4C .999 0.145
334 nm was measured. The concentration of pyridoxine was Pyridoxine 334 0.00001 – 1.17E 3C 1 0.129
determined from this and the corresponding calibration
Thiamine 282 0.000112 – 1.02E 3C .999 0.115
equation and the signals at 266 and 282 nm corresponding to
NOTE: The number of standard solutions, n, is 24 (8 × 3); r is the correla-
that concentration were read (Fig. 3).
tion coefficient; DL is the detection limit. The variance, s 2, is 0 in all cases.
Next, the signal of the mixture at 266 nm was measured a 1
C denotes analyte concentration in µ g mL .
and the signal of pyridoxine at this wavelength was subtracted.
This value, in conjunction with the calibration equation for folic
acid, provided the concentration of folic acid (Fig. 3). Table 2. Simultaneous Determination of Folic Acid,
The thiamine concentration was determined by measur- Pyridoxine, and Thiamine by the Zero-Crossing Method
ing the signal at 282 nm for the mixture, subtracting the Mixture Founda/ g mL 1
1
signal for pyridoxine, and repeating the previous calculation Amount Added/ g mL Amount SD
using the calibration equation for thiamine. Foli Pyri Thia Foli Pyri Thia Foli Pyri Thia
Table 2 gives the results of applying this method to 122 832 140 120 824 144 4.63 7.11 6.44
seven mixtures of the vitamins. All errors and deviations 102 124 800 102 120 839 4.34 7.57 7.16
were very small. Consequently, the method allows the 816 145 100 825 140 102 4.18 8.20 6.66
accurate resolu-tion of the type of mixture studied. 612 104 180 629 100 181 4.22 7.20 7.37
408 124 160 423 122 160 4.46 7.63 6.73
Acknowledgment 204 145 120 208 143 118 4.89 8.31 6.41
816 124 800 820 120 832 4.18 7.57 7.19
We wish to acknowledge the financial support from a
the Ministerio de Educación y Cultura (DGICyT), Mean of three determinations.
research project No. PB98-0439.

Literature Cited 100

1. French, C. S.; Church, A. B.; Epply, R. W. In Carnegie

Insti-tution of Washington Year Book; Carnegie Institution 80

of Wash-ington: Washington, DC, 1954; pp 182–183. nm)

nm)
Signal increase

2. Hammond, V.; Price, W. C. J. Opt. Soc. Am. 1953, 43, 924. 266
60 282
3. Schmitt, A. Tech. Lab. 1978, 5, 1207–1214. λ
(
=

λ
=

4. Fell, A. F. Spectrom. Group Bull. 1980, 5–31. (

5. Green, G. L.; O’Haver, T. C. Anal. Chem. 1974, 46, 2191– 40

PYRI PYRI

2196.
6. Morelli, B. Analyst 1988, 113, 1077–1082. nm)
7. García Fraga, J. M.; Jiménez Abizanda, A. I.; Jiménez 20 λ = 266 =334nm)
( (λ
Moreno, F.; Arias León, J. J. J. Pharm. Biomed. Anal. 1991, FOLI PYRI nm)
(λ = 282
2, 109– 115. THIA
0
8. Bautista, R. D.; Jiménez, A. I.; Jiménez, F.; Arias, J. J. J. 0 5 10 15 20 25
Pharm. Biomed. Anal. 1996, 15, 183–192. 1
Concentration /  g mL
9. Toral, M. I.; Richter, P.; Tapia, A. E.; Hernández, J. Talanta
1999, 50, 183–191. Figure 3. Calibration graphs for pyridoxine (PYRI), folic acid
(FOLI), and thiamine (THIA) at the indicated values of λ .

JChemEd.chem.wisc.edu • Vol. 78 No. 6 June 2001 • Journal of Chemical Education 795