Colorimetric Determination of Sulfadiazine in Tablets Through Derivatization with p-

Dimethylaminobenzaldeyhde

Samantha T. Radaza, Krizia Marie G. Queque

Pharmaceutical Chemistry Department, College of Pharmacy, University of the Philippines Manila

Abstract
This study aims to compute the amount of sulfadiazine in tablets by utilizing the Beer-Lambert law
as well as to determine the figures of merit given the data obtained. Single and multiple-standard calibration
methods were employed using the external calibration technique. The single-standard method made use
of a final working concentration of 0.0502 mg/mL while that of the multiple standard method were 0.0208,
0.0408, 0.0802, 0.1600, and 0.3216 mg/mL. An assay preparation was prepared and subjected to a UV/VIS
spectrophotometer in order to determine the amount of sulfadiazine present in the tablets. The spectral
absorbance curve was then generated obtaining an λ max of 319 nm. Percent label claim and percent active
ingredient were computed for both methods. Results show that the computed percentage labeled claim and
percent active ingredient for the single-standard method were 192.27% and 63.91% respectively while that
of the multiple-standard method were 171.10% and 56.88%. Both methods did not conform with USP
standards.

Keywords: sulfadiazine, Beer-Lambert’s Law, external standard calibration

1
Background
Spectroscopy is the study of interaction of matter and electromagnetic radiation. Molecular absorption
spectophotometry measures the amount of electromagnetic radiation absorbed by a molecular species
(Watson, 2005). When light asses through a solution, a fraction of that light is absorbed while the other
fraction is transmitted. Absorbance can be demonstrated by the Beer-Lambert’s Law:
A=εbC
where A is absorbance, ε is the molar absorptivity of the species, b is pathlength or thickness of
absorbing layer in centimeters, and C is molecular concentration.
Visible spectrophotometry or colorimetry measures absorbance of molecules in the visible region (350-
900nm) of the spectrum. Substances used for this test are either highly colored or can be reacted with a
chromogenic agent. Colorimetric methods can also be used to analyze compounds that have absorbance
in the uv regions. Since there are fewer compounds absorbed in the visible region, this technique is utilized
in the presence of substances that would react if the UV region were used. (Knevel and DiGangi, 1977)

Figure 1. Structure of Sulfadiazine

Sulfadiazine or 4-amino-N-pyrimidin-2-ylbenzenesulfonamide (Figure 1) belongs to the class of
antibiotics known as sulfonamides --used orally to treat mild-to-moderate infections. It acts by inhibiting the
folic acid synthesis that is essential for bacterial growth. A condensation reaction takes place when
sulfadiazine reacts with p-dimethylaminobenzaldehyde (p-DMAB) forming a yellow-colored Schiff base
whose maximum absorbance (𝜆𝑚𝑎𝑥 ) falls within the visible region, thus can be quantified via colorimetry.
The reason for the formation of the yellow-color is because of the increase of conjugations in the product.

Figure 2. Condensation reaction of Sulfadiazine and p-DMAB

The aim of this experiment is to compute the amount of sulfadiazine in tablets using external standard
calibration techniques. Figures of merit, limit of detection (LOD) and limit of quantification (LOQ), are used
to compare the single and multiple-standard calibrations.

2
Methods
In the standard preparation for the single standard method, 25 mg of dried sulfadiazine standard
was added with 10 mL 1N HCl. The resulting solution was then heated, cooled to room temperature,
transferred to a 25 mL volumetric flask and brought to volume with 1N HCl. This solution, with a
concentration of 1mg/mL, is the standard stock solution in which 1.25 mL was withdrawn and transferred
to another 25 mL volumetric flask. To this 1.25 mL solution, 5.0 mL of p-DMAB was added and then followed
by 1N HCl bringing the solution to volume. In the multiple standard method, the same procedure was done
as the single standard method but there were 5 concentrations of stock solution instead of only one. The
concentrations were 0.4 mg/mL, 0.8 mg/mL, 1.6 mg/mL, 3.2 mg/mL, and 6.4 mg/mL.
In the sample preparation, 75.50 mg of powdered sulfadiazine tablets was added with 10 mL 1N
HCl. The solution was gently heated, constantly stirred for 5 minutes, cooled to room temperature, and
filtered. The filtrate was then transferred to a 25 mL volumetric flask and was brought to volume using 1N
HCl. After mixing, 1.25 mL of the solution was transferred to a 25 mL volumetric flask to which 5.0 mL of p-
DMAB was added and 1N HCl was used to bring the solution to volume. A blank solution was prepared in
a 25 mL volumetric flask containing 5.0 mL of p-DMAB and a sufficient amount of 1N HCl to reach the
volume.
For the determination of wavelength and the absorption spectrum of sulfadiazine, the Genesys 10S
UV/Vis Spectrophotometer was used. First, the 0.0502 mg/mL standard solution was scanned from 230-
480 nm with 1 nm intervals. The λmax obtained was 319 nm. Then, the 1 mg/mL standard stock solution was
scanned in the same manner as the 0.0502 mg/mL standard solution and the λ max obtained was 309 nm.
The λmax of the 0.0502 mg/mL standard solution, which was 319 nm, was then used to measure the
absorbance of the standard and the assay preparations against the blank solution.

Results and Discussion

A spectral absorbance curve was generated from the data in the scanning of the 1mg/mL standard
stock solution and the 0.0502 mg/mL standard solution. This was done to determine the λmax which is the
wavelength at which the absorbance is highest. The λmax obtained would then be used to measure the
absorbance of the standard and the sample since it is the most sensitive to changes in concentration
(Knevel and DiGangi, 1977).
Theoretically, the superimposed absorption spectra should show the shift of λmax in the standard
stock and the standard solution. That shift of λmax from the UV region to the visible region is an indication
that the sulfadiazine was derivatized upon the addition of p-DMAB. Derivatization of sulfadiazine is done
so that there would be a chromophore in which analysis can be carried out at wavelengths beyond the non-
specific UV region. The properties and concentrations of the derivatized compound can now be related to
the original compound. (Adegoke, 2012)
However, the actual results in Figure 3 show that the derivatization of the sulfadiazine failed. The
λmax of the standard stock was 309 nm while that of the standard solution is 319 nm, a wavelength which is

3
still in the UV region. This was likely because of the very long standing time of sulfadiazine – 4 hours –
before it was read in the spectrophotometer. The solution itself was yellow which was mistakenly identified
as a Schiff base. Schiff bases are, in fact, chemically unstable and they degrade quickly which is likely the
reason that the λmax obtained was low and still in the UV region. (Ibrahim & Sharif, 2007) The wavelength
used for the measurement of absorbance was 319 nm since it was the higher of the two peaks obtained.

Figure 3. Superimposed Absorption Spectra of Sulfadiazine sample and standard.

To determine the amount of sulfadiazine in tablets, the single standard method and the multiple
standard method were employed. In the single-standard method with the sample, the final working
concentration was 0.0502 mg/mL with a mean absorbance of 0.525, a standard deviation of 0.0353 and a
relative standard deviation (RSD) of 0.0093. In the multiple standard method, different concentrations of
sulfadiazine were used to create a calibration curve. Table 1 below shows the absorbance of the derivatized
standard solutions obtained during the experiment.
Table 1.
Absorbance Readings of Derivatized Sulfadiazine Standard Solutions using Multiple-Standard Method
Solution Final Working SDZ Concentration Absorbance (A)
(mg/mL) Reading Reading Reading Mean RSD
1 2 3
1 0.0208 0.115 0.079 0.089 0.094 0.1969
2 0.0406 0.222 0.223 0.228 0.224 0.0143
3 0.0802 1.058 1.080 1.079 1.072 0.0116
4 0.1600 1.258 1.263 1.264 1.262 0.0025
5 0.3216 2.499 2.496 2.496 2.497 0.0007

From the scatter plot of the calibration data found in Figure 4 below, the relationship of the
concentration of sulfadiazine and the absorbance is shown. The 𝑟 2 value generated which is 0.9476 implies
imprecision in the experiment and shows that the relationship between the two variables is not that strong.
Molar absorptivity of sample was computed to be 1529.6508 L mol -1 cm-1 while that of the standard was

4
1361.0529 L mol-1 cm-1. The discrepancy is due to the higher concentration of sulfadiazine in the sample,
which was computed to be 0.0965 mg/mL, than the concentration of the standard which is 0.0502 mg/mL.
Following Beer-Lambert Law, the concentration is directly proportional with molar absorptivity.

Figure 4. Scatter Plot of Calibration data from Multiple-Standard Method using Standard Solutions of
Sulfadiazine
Comparing the two methods, the single-standard method is more precise given its smaller RSD
value. (Pharmaceutical Technology, 2010) However, the multiple-standard method is still preferred over
single-standard because the latter still carries over all experimental errors in the calculation for the method’s
sensitivity (k). From the equation:
k = Sstandard / Cs
where k is the method’s sensitivity, Sstandard is the measured signal of the standard, Cs is the concentration
of the analyte, errors affecting k would mean uncertainty in the analyte’s concentration. Furthermore, the
equation above is limited only to single concentration of analyte. Since concentrations of analytes can differ
from the concentration of the standard, the assumption that the value of k is constant can lead to errors.
On the other hand, errors from using single-standard can be avoided by using a series of standards with
different concentrations of the analyte. (Harvey, 2000).
According to the USP, the amount of sulfadiazine in tablets should not be less than 95% and not
more than 105%. Both the single-standard and the multiple-standard methods did not fall within the range
with 192.27% and 171.10%, respectively. The computed amount of sulfadiazine in the single-standard was
48.25 mg while that in the multiple-standard was 42.95 mg. Limits of detection and quantitation were not
calculated since the blank solution was read only once leading to insufficient data. (Theodorsson, n.d.)

Conclusion
The computed amount of sulfadiazine in tablets using the single-standard was 192.27% while that
of the multiple-standard method was 171.10%. These values do not comply with the USP’s official
requirement of sulfadiazine. The multiple-standard method is more preferred than the single-standard

5
because of the presence of multiple concentrations which reduces or eliminates error. LOD and LOQ were
not calculated due to insufficient data.

References
Adegoke, Olajire. (2012). Chemical derivatization methodologies for UV-visible spectrophotometric
determination of pharmaceuticals. International Journal of Pharmaceutical Sciences Review and
Research, 14 (2), 6-24. Retrieved from https://www.researchgate.net/publication/286322136_Che
mical_derivatization_methodologies_for_UV- visible_spectrophotometric_determination_of_phar
maceuticals
Harvey, D. (2000). Modern Analytical Chemistry. New York: NY, McGraw-Hill Companies, pp. 108, 109
Ibrahim, M.N. & Sharif, S.E.A. (2007). Synthesis, Characterization, and Use of Schiff Bases as Fluorimetric
Analytical Reagents. Retrieved from https://downloads.hindawi.com/journals/jchem/ 2007/191805.pdf
Knevel, A. M. & DiGangi, F. E. (1977). Jenkins’ Quantitative Pharmaceutical Chemistry (7 th ed.). USA:
McGraw-Hill, Inc. pp. 293-300, 307
Theodorsson, E. (n.d.). Limit of Detection, Limit of Quantification and Limit of Blank. Retrieved from
https://www.eflm.eu/files/efcc/Zagreb-Theodorsson_2.pdf
The United States Pharmacopeial Convention (2012). The United States Pharmacopeia 35th Revision and
the National Formulary 30th Edition. USPCI: Maryland, p. 4704
Torbeck, L.D. (2010). Statistical solutions: %RSD: Friend or foe?. Pharmaceutical Technology, 34 (1)
Retrieved from http://www.pharmtech.com/statistical-solutions-rsd-friend-or-foe
Watson, D.G. (1999). Pharmaceutical Analysis. UK: Harcourt Publishers Limited. pp. 75-79

6
Appendix
Sample Computations
Final working concentration
a) from Single standard b) from Sulfadiazine Sample
25.10 mg 1.25 mL wt. of sample = 75.5mg
= x
25 mL 25mL
label claim = 250mg/tablet
= 0.0502 mg/mL
ave. wt. of 20 tablet = 0.7521g or 752.1 mg
752.1mg 75.5mg
=
250mg x
X = 25.10 mg
(25.10 𝑚𝑔) 1.25 𝑚𝐿 49.86𝑢𝑔
𝑥 =
25 𝑚𝐿 25 𝑚𝐿 𝑚𝐿

= 0.0502 mg/mL

Amount of Sulfadiazine in Tablets

a) from Single Standard b) from Multiple Standard
Asample Based from the Equation of the Line
Csample = Cstd ×
Astd Given: y = 7.6862x + 0.0719
0.0502mg 0.525 Asample = 0.525
Csample = ×
mL 0.273
0.525 = 7.6862x + 0.0719
= 0.0965 mg/mL
0.525 − 0.0719
(0.0965) 25 𝑚𝐿 X=
AI = 𝑥 25 𝑚𝐿 𝑥 1.25 𝑚𝐿 7.6862
𝑚𝐿
x = 0.0859 mg/mL
= 48.2500 mg
(0.0859 𝑚𝑔) 25 𝑚𝐿
48.25 AI = 𝑥 25 𝑚𝐿 𝑥 1.25 𝑚𝐿
%AI = 75.5𝑚𝑔 𝑥100 = 63.9072% 𝑚𝐿

752.1 𝑚𝑔
= 42.95mg
%LC = 63.91% x = 192.2668% 42.95 𝑚𝑔
250 𝑚𝑔
%AI = 𝑥100 = 56.88%
75𝑚𝑔
752.1 𝑚𝑔
%LC = 56.88% x 250 𝑚𝑔
= 171.10%

Molar Absorptivity
MW Sulfadiazine = 250.28 g/mol
a) Sulfadiazine Standard b) Sulfadiazine Sample
1g 1g
(0.0502 mg x 1000mg) 1 mol (. 0859 mg x 1000mg ) 1 mol
x x
1L 250.28 g 1L 250.28 g
(1mL x 1000mL) (1mL x 1000mL)

= 2.0060 x 10−4 mol/L = 3.4322x 10−4 mol/L

A = εbc 0.525 = X(1 cm)( 3.4322 x 10−4 mol/L)
0.273 = X(1 cm)( 2.0060 x 10−4 mol/L) X = 1529.6312 L mol−1 cm−1
X = 1361.0529 L mol−1 cm−1