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_____________________________________________________________________________________________________
1
Department of Chemistry, St. Xavier’s College,
Maharashtra, India.
*Corresponding author: E-mail: pralhad1806@gmail.com;
Chapter 3
Print ISBN: 978-93-90206-65-0, eBook ISBN: 978-93-90206-66-7
Simultaneous Estimation of Ofloxacin and
Ornidazole from Pharmaceutical Dosage
Form by Spectrophotometric Methods
Pralhad Rege
1*
, Kyle Meyers
1
and Neha Kapadia
1
DOI:
10.9734/bpi/tprd/v4
ABSTRACT
Two simple, sensitive, accurate, precise, rapid and economical methods were developed for the
simultaneous estimation of Ofloxacin and Ornidazole from combined tablet dosage form. First method
is based simultaneous equations and second method is based on Q-analysis (absorbance ratio
method). Ofloxacin and Ornidazole show absorbance maxima at 293nm and 275nm in 0.1N HCI
respectively. The linearity was obtained in the concentration ranges of 2-14 μg/ml for Ofloxacin and 5-
35 μg/ml for Ornidazole with Regression Coefficient (R
2
) greater than 0.999. In the first method
concentration and subsequently amount of drug determined by using simultaneous equations and in
second method concentration and amount of drugs determined by using ratio of absorbance at iso-
absorptive point(which was found to be 284 nm) and at λmax of one of the drug. The results of
analysis have been validated statistically and by assay and recovery studies. Therefore the proposed
validated method can be successfully applied for routine quality control analysis and simultaneous
determination of Ofloxacin and Ornidazole in combined drug formulations.
Keywords: Ofloxacin; Ornidazole; Absorbance ratio; iso-absorptive point; regression coefficient.
ABBREVIATIONS
OF : Ofloxacin
OZ/ ONZ : Ornidazole
SEM : Simultaneous equation method
ARM : Absorbance ratio method
1. INTRODUCTION
In the topical countries like India, the major problems of health arise due to improper lifestyle,
unhealthy environmental conditions, unhygienic and substandard food. Infections caused by the
microorganisms like, fungi, protozoa, are most common. Drugs with antifungal and antiprotozoal
activity have been used in the treatment of the same.
Ofloxacin, C
18
H
20
FN
3
O
4
with molecular weight 361.368 g/mol is used in the treatment of bacterial
infections, while Ornidazole, C
7
H
10
ClN
3
O
3
with molecular weight 219.63 g/mol is used as an
antiprotozoal agent. Ofloxacin and Ornidazole in combined dosage form is available in the market,
has gained great acceptance in diarrhea, bacterial and protozoal infections. In many cases, drugs with
two active ingredients are prescribed to the patients to have an added advantage. Many of these
antibacterial drugs are found in combination with antifungal and antiprotozoal drugs which are highly
effective against fungal and protozoal infections.
A literature surveys reveals few Chromatographic methods [1] i.e. HPLC HPTLC, Derivative and
Extractive spectrophotometric methods for the simultaneous determination of ofloxacin and
ornidazole. Very little attention has been paid to the use of electroanalytical method.
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidazole from Pharmaceutical Dosage Form by Spectrophotometric Methods
24
In the present study a successful attempt has been made to estimate both these drugs i.e. Ofloxacin
and Ornidazole simultaneously in combined pharmaceutical formulation by two simple
spectrophotometric methods. First method is based simultaneous equations and second method is
based on Q-analysis (absorbance ratio method). The proposed methods have been validated as per
ICH guidelines
1.1 Structure
Ofloxacin Ornidazole
2. MATERIALS AND METHODS (EXPERIMENTAL)
2.1 Instruments
Systronics UV – VIS spectrophotometer type 118, single beam spectrophotometer with spectral width
of 2.0 nm. Wavelength accuracy is + 1.0 nm and a pair of 10 mm matched quartz cells was used to
measure the absorbance. The wavelength ranges from 200 – 1000 nm obtained by a 1200
grooves/mm grating with Czerny – Turner Mount. Fused Silica deuterium lamp and tungsten iodide
sources are used to cover the wavelength range. High sensitivity, wide range, reliable silicon photocell
is used as the detector. The photometric readings of %T (Percent Transmittance), ABS (Absorbance)
and CONC (Concentration) are displayed on an electronic display. Stray light corrections are made
using a built-in filter wheel containing four filters. There is a built-in 4-position sample changer
provided for 10mm cuvettes. The instrument runs on 230V, 50Hz.
2.2 Materials
Pure standard of Ofloxacin and Ornidazole was obtained from Cipla pharmaceutical Pvt. Ltd. The
tablet formulations of the
said combination were purchased from a local pharmacy (The label claim
contained 200 mg of Ofloxacin and 500 mg of Ornidazole.) All the solutions were prepared in double
distilled water. All the reagents use were of AR grade.
2.2.1 Preparation of standard solutions
10 mg of standard Ofloxacin and 25 mg of standard Ornidazole was accurately weighed and
dissolved in 0.1N HCI and made up to a volume of 50 ml in standard flask to give stock solution (200
μg/ml of Ofloxacin and 500 μg/ml of Ornidazole respectively). Further all the standard solutions
containing the mixture of Ofloxacin and Ornidazole were prepared by using this stock solution.
2.2.2 Preparation of the sample solution
Twenty tablets of the combined drug formulation O2 (Medley Pharma Limited) were weighed and
powdered. 51 mg the powdered sample equivalent to (10 mg of OF and 25 mg of OZ) was weighed
and transferred to a 50 mL standard flask. To it 25 mL of 0.1N HCI was added to dissolve the sample.
The mixture was sonicated for 10mins with intermittent shaking. The sample was cooled to room
temperature and diluted to 50 mL with 0.1 N HCI and mixed well. The above solution was filtered
through Whatman filter paper no. 41 and Further 1.0 mL of this filtered solution was diluted to 10 mL
using 0.1 N HCI. The solution so obtained is supposed to contain 20µg/mL of OF and 50µg/mL of OZ.
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidazole from Pharmaceutical Dosage Form by Spectrophotometric Methods
25
Absorbances of these solutions were measured at appropriate wavelengths, i.e. at 293 nm (λ
1
) and
275 nm (λ
2
).
2.3 Method-1: Simultaneous Equation Method [2,3,4,5,6]
Working standard solutions were scanned in the entire range of 200-400 nm to determine λmax of
both the drugs. The λmax of Ofloxacin and Ornidazole were found at 293 nm and 275 nm
respectively. A series of solutions were prepared having concentration ranges 1-40 μg/ml by using
0.1N HCI from working standard solutions. Absorbances of resulting solutions were measured at 293
nm and 275 nm and calibration curves plotted at these wavelengths. The linearity was obtained in the
concentration ranges of 2-14 μg/ml for Ofloxacin and 5-35 μg/ml for Ornidazole. The absorptivity
coefficients of these two drugs were determined by using calibration curve equation. Two
simultaneous equations were formed using these absorptivity coefficients values.
A
1
= 60.1 C
X
+ 15.0 C
Y
(1)
A
2
= 35.7 C
X
+ 23.4 C
Y
(2)
Where, A
1
and A
2
are the absorbances
of sample at 293 nm and 275 nm respectively. C
X
and C
Y
are
the concentration (μg/ml) in of Ofloxacin and Ornidazole in sample respectively. From the resulting
concentration obtained after solving above equations, then amount of Ofloxacin and Ornidazole
present in the given sample was found out.
2.3.1 Analytical method validation [7,8]
2.3.1.1 System suitability
System suitability tests are used to ensure reproducibility of the equipment. The test was carried out
by recording absorbance at working concentrations for Ofloxacin (4 μg/ml,8 μg/ml, 12 μg/ml) and for
Ornidazole (10 μg/ml, 20 μg/ml, and 30 μg/ml) with five replicates and the mean was used for the
whole calculations.
2.3.1.2 Specificity
The specificity of method was confirmed by recording the spectra of both the standard solution
and the drug sample solutions. The spectra obtained from the drugs sample solution were
found to be identical to those obtained for standard solution.
The addition of the standard solution to the drug sample solution for recovery analysis did not
change the characteristics of spectra confirms specificity.
2.3.1.3 Linearity and range
A good linearity was achieved for Ofloxacin and Ornidazole in the concentration ranges of 2-14 μg/ml
for Ofloxacin and 5-35 μg/ml Ornidazole. The calibration curves were constructed with concentration
(C) against absorbance of both drugs.
2.3.1.4 Limit of detection and limits of quantitation
The signal-to-noise ratio of 3:1 and 10:1 was used to establish LOD and LOQ, respectively. For LOD
and LOQ analysis twenty readings for blank recorded then their standard deviation calculated i.e. for
LOD= (SD×3+ Mean absorbance of Blank) and for LOQ= (SD×10 + Mean absorbance of Blank.)
2.3.1.5 Intra-day and inter-day precision/ ruggedness
The intra-day and inter-day precision was used to study the variability of the method. According to
USP, ruggedness is the degree of reproducibility of the results obtained under variety of test
conditions. It is expressed as percent RSD. It is also called as reproducibility or intermediate
precision. It is the analysis of same sample under variety of normal test condition such as different
laboratories, different analysts, different instruments, different lots of reagents, different days etc.
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidazole from Pharmaceutical Dosage Form by Spectrophotometric Methods
26
It was checked by recording the absorbance as well as spectra of standard solutions of Ofloxacin and
Ornidazole i.e. working concentrations for Ofloxacin (4 μg/ml, 8 μg/ml, 12 μg/ml) and for Ornidazole
(10 μg/ml, 20 μg/ml, and 30 μg/ml) with five replicates (both at intra-day (five times within 24 hour)
and inter-day (two times each. during 3 days intervals) to check the precision.
2.3.1.6 Assay
The validated spectrophotometric method was used for the simultaneous quantitative determination of
OF and OZ from the formulation. Quantification has been done by simultaneous equation method
(SEM). Two samples of different brands were used for the determination.
The absorptivity coefficients of these two drugs were determined by using calibration curve equation
(1) and (2) mentioned earlier.
C
X
= (λ
2
€2
× A λ
1
) - (λ
1
€2
× A λ
2
) (3)
(λ
1
€1
× λ
2
€2
) - (λ
1
€2
× λ
2
€1
)
C
Y
= (λ
1
€1
× A λ
2
) - (λ
2
€1
× A λ
1
) (4)
(λ
1
€1
× λ
2
€2
) - (λ
1
€2
× λ
2
€1
)
C
X
and C
Y
was calculated
Where C
X
= is Concentration of Ofloxacin, C
Y
= is Concentration of Ornidazole
From their concentration finally, the amount of OF and OZ in the tablet formulation is calculated.
Where,
λ
1
€1
= 60.1 × 10
- 3
-(OF at 293 nm), λ
2
€1
= 35.7 × 10
- 3
-(OF at 275nm)
λ
1
€2
= 15.0 × 10
- 3
-(OZ at 293 nm), λ
2
€2
= 23.4 × 10
- 3
-(OZ at 275 nm)
A λ
1
= Absorbance of sample mixture at 293 nm
A λ
2
= Absorbance of sample mixture at 275 nm
2.3.1.7 Accuracy (recovery)
The recovery was used to evaluate the accuracy of the method. Accuracy of the method was
determined using the method of standard addition. A fixed volume of standard Ornidazole solution
was mixed with different concentrations of preanalyzed sample solutions and mixtures were analyzed
by proposed method. The percent recovery was determined at different levels i.e. from 50% to 150%
level.
2.4 Method-2: Absorbance Ratio Method (Q-Analysis) [9,4,5,6]
Absorbance ratio method uses the ratio of absorbance at two selected wavelengths one at iso-
absorptive point and other being the λmax of one of the components. From the overlay spectra of two
drugs, it is evident that Ofloxacin and Ornidazole show iso-absorptive point at 284 nm and the second
which is the λmax of Ofloxacin (293nm). The quantification of both drugs was carried out at the
selected wavelengths. i.e. 284nm and 293nm. The absorptivity coefficients of these two drugs were
determined by using calibration curve equation at 293 nm and 308 nm.
2.4.1 Linearity and range
A series of solutions having concentration ranges 1-40 μg/ml were prepared by using 0.1N HCI
Absorbance of resulting solution was measured at 293 nm and 284 nm. Calibration curves were
plotted at these wavelengths. The linearity was obtained in the concentration ranges of 2-14 μg/ml for
Ofloxacin and 5-35 μg/ml for Ornidazole.
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidazole from Pharmaceutical Dosage Form by Spectrophotometric Methods
27
2.4.2 Assay
The validated spectrophotometric method was used for the quantitative determination of OF and OZ
simultaneously from the formulation. Quantification has been done by Absorbance Ratio method
(ARM). Two samples of different brands were used for the determination.
The Absorptivity coefficients of these two drugs were determined at 293 nm and 284 nm. The
concentration of OF and OZ was found out using following equations
(5)
(6)
Where,
C
x
= Concentration of X i.e. Ofloxacin
C
y
= Concentration of Y i.e. Ornidazole
Q
m
= Ratio of absorbance of mixture at λ
max
(293nm) and λ
iso
(284 nm)
Q
x
= Ratio of absorptivity of X at λ
max
(293nm) and λ
iso
(284 nm)
Q
y
= Ratio of absorptivity of Y at λ
max
(293nm) and λ
iso
(284 nm)
A = Absorbance of mixture at iso-absorptive point i.e. 284 nm.
Ax
1
= E (1%, 1cm) of X at λ
iso
(absorptivity of x drug at isoabsorptive point)
Ay
1
= E (1%, 1cm) of Y at λ
iso
(absorptivity of y drug at isoabsorptive point)
2.4.3 Sensitivity
Sensitivity refers to the smallest quantity that can be accurately measured. It also indicates the
capacity of the method to record or measure small variations in concentrations. In the case of
Spectrophotometric methods, a parameter known as “Sandell’s Sensitivity” is used to evaluate the
sensitivity of the method. It is the amount required to give an absorbance of 0.001 units in one square
centimeter path.
3. RESULTS AND DISCUSSION
1. The proposed methods were found to be simple, accurate, sensitive, precise and economical.
2. Both the methods have been validated as per as ICH guidelines. For system suitability, the mean
% RSD was found to be less than 1.for both OF and OZ, Regression analysis (Linearity and
Range) for both the drugs was > 0.999 indicates the precision of the validated method. Lower
value of LOD and LOQ confirm the sensitivity of the specified method.
3. Lower values of Sandell’s Sensitivity and higher values for Molar Absorptivity signifies the
effectiveness of the methods for routine use.
4. Repeatability and also inter and intra-day precision was studied where % RSD was found to be
less than 1) for both drugs (Table 1).
5. Accuracy was determined for the given methods by calculating the percent recovery at different
levels i.e. from 80% to 120% level. The percentage recovery at three different was found to be
from 98.00% to 102.00% for both the drugs (Table 3).
6. Quantification of drugs from the formulation was done by assay analysis Assay studies
were carried out at three different levels i.e. 80%, 100%, 120% level. The percentage
assay at three different levels for OF and OZ was found to be from 98.00% to 102.00% (Table
2).
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidazole from Pharmaceutical Dosage Form by Spectrophotometric Methods
28
Table 1. Method validation parameters
Parameters
Method 1 (SEM)
Method 2 (ARM)
Ofloxacin
Ornidazole
Ofloxacin
Ornidazole
System suitability
(n=5) %RSD
0.69% 0.46% 0.24% 0.51%
Linearity range
(μg mL
-1
)
2 to 14 µg/ml 5 to 35 µg/ml 2 to 14 µg/ml 5 to 35 µg/ml
Correlation
coefficient (R
2
)
0.9998 0.9999 0.9998 0.9998
LOD (μg mL
-1
) 0.3 µg/ml 0.8 µg/ml 0.3 µg/ml 0.8 µg/ml
LOQ (μg mL
-1
) 1 µg/ml 2.5 µg/ml 1 µg/ml 2.5 µg/ml
Intraday precision (n=5)
%RSD
0.75% 0.65% 0.35% 0.71%
Interday precision (n=5)
%RSD
0.60% 0.45% 0.29% 0.54%
Assay 98% to102% 98% to 102% 98% to 102% 98% to102%
Recovery 98% to102% 98% to 102% 98% to 102% 98% to102%
Sandell's sensitivity (µg
/cm
2
)
0.01034 0.02858 0.01184 0.02969
Molar Absorptivity
(mole/lit/cm)
21718.22 5139.23 18502.041 4128.95
Sample Used:
Brand Name
O2 (Medley Pharma Limited)
Batch No.
E00701
A.P.I.
Ornidazole- 500 mg
Ofloxacin – 200 mg
Excipients
q.s.
Colours
Sunset yellow FCF, & titanium dioxide IP
Table 2. Result of assay studies
Method 1 (SEM)
Method 2 (ARM)
Ofloxacin
Ornidazole
Ofloxacin
Ornidazole
Labeled claim
(mg)
200 mg 500 mg 200 mg 500 mg
Drug found in mg 198.8 mg 499.1 mg 201.2 mg 504.0 mg
% Assay 99.7% 99.85% 100.3% 100.6%
SD 0.366 0.498 0.524 0.467
% RSD (n=5) 0.311 0.472 0.515 0.461
Table 3. Results of recovery studies
Method
Name
Level of %
Recovery
% Recovery Found
Standard
Deviation (SD)
Relative Standard
Deviation % (RSD)(N=5)
OF
OZ
OF
OZ
OF
OZ
SEM
0 100.6% 100.2% 0.037 0.032 0.57 0.73
50 % 99.8% 99.4% 0.041 0.04 0.35 0.51
100 % 101.01% 101% 0.051 0.045 00.39 0.50
150% 100.2% 99.7% 0.01 0.07 0.69 0.79
ARM 0 99.6% 100.0% 0.02 0.056 0.33 0.62
50 % 99.5% 99.9% 0.025 0.015 0.53 0.22
100 % 100.3% 100.5% 0.032 0.05 0.52 0.55
150% 101.7% 100.7% 0.033 0.076 0.44 0.69
Simultaneous Estimation of Ofloxacin and Ornidaz
Ofloxacin standard (293nm)
Fig. 1. Linearity graph for :
Ofloxacin standard (284nm) Ornidazole standard (284nm)
Fig. 2.
(a)
Fig. 3.
Overlay spectra of the working range for (a) OF (293 nm) and (b) OZ
(275 nm), for a set
-0.1
1.1
0
0.5
1
200 250 300
350
Abs
Wavele ngth[nm]
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidaz
ole from Pharmaceutical Dosage
Form by Spectrophotometric Methods
29
Ofloxacin standard (293nm)
Ornidazole standard (275nm)
Fig. 1. Linearity graph for :
- (SEM)
Iso-absorptive point
Ofloxacin standard (284nm) Ornidazole standard (284nm)
Fig. 2.
Linearity graph for: - (ARM)
(b)
Overlay spectra of the working range for (a) OF (293 nm) and (b) OZ
(275 nm), for a set
of Linear working range
400
350
-0.623802
1.2513 1
-0.5
0
0.5
1
193.267 3 00
Abs
Wa velength[nm]
Trends in Pharmaceutical Research and Development Vol.4
Form by Spectrophotometric Methods
Ofloxacin standard (284nm) Ornidazole standard (284nm)
Overlay spectra of the working range for (a) OF (293 nm) and (b) OZ
406.733
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidazole from Pharmaceutical Dosage Form by Spectrophotometric Methods
30
Fig. 4. Overlain spectra of Ofloxacin (293nm), Ornidazole (275nm) and Iso-absorptive point
(284nm)
4. CONCLUSION
All above mentioned factors lead to the conclusion that, both the methods described in this paper for
simultaneous estimation of Ofloxacin and Ornidazole are found to be simple, accurate, precise,
accurate, economical, and rapid, therefore presented methods can be recommended for routine
quality control analysis of Ofloxacin and Ornidazole in their combined dosage form.
ACKNOWLEDGEMENT
We thank Cipla Pharmaceutical Pvt. Ltd (Vikroli) and our Department of Chemistry, St. Xavier’s
College, Mumbai for providing us all the Necessary instrumentation facilities and their technical
assistance.
COMPETING INTERESTS
Authors have declared that no competing interests exist.
REFERENCES
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585-589.
2. Gupta-R, Singhai-AK, Agrawal-RK. Derivative UV spectrophotometric determination of
norfloxacin and tinidazole combination in tablets. Indian-Drugs. Jul. 2000;37(7):348-349.
3. ICH Q2B, Validation of Analytical Procedure: Methodology, In. Proc. Int. Con. Harmonization,
Geneva; 1996.
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Nebivolol in pharmaceutical dosage forms by absorption ratio method International journal of
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5. Patel SA, Patel NM, Patel MM. Simultaneous spectrophotometric estimation of ciprofloxacin
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6. Sohan S. Chitlange*, Soni Ranjana, Sagar B. Wankhede, et al. Spectrophotometric Methods for
Simultaneous Estimation of Nimesulide and Drotaverine. International Journal of ChemTech
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9. Pralhad Rege
*
, Kyle Meyers, Neha Kapadia. “Simultaneous Determination of Norfloxacin and
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Biography of author(s)
Dr. Pralhad Rege
Department of Chemistry, St. Xavier’s College, Maharashtra, India.
He is working in capacity of Assistant Professor in the Department of Chemistry, St. Xavier’s College Mumbai. He completed
his Ph.D. in Analytical Chemistry. He also hold PGDHRM from Symbiosis, Pune and PG Diploma in Clinico-Regulatory and
Patents from Ruia College. His total teaching experience is 13 years and research experience is 12 years. He is a Mumbai
University recognized guide for M.Sc by research and Ph.D. Currently two students for M.Sc by research and three students for
PhD are working under his supervision. His area of research interest is Analytical chemistry, Pharmaceutical chemistry,
synthetic chemistry and Herbal science. He has published 21 research papers in various national and international, actively
participated in more than 70 conferences / workshops/ Seminars/ online FDP’s. Till date he has Presented 15 research papers
in the form of Oral / poster presentations. He has been part of various Departmental and College level committees. He is an
Advisory and Editorial member of various National and International journals. He has received Minor Research Grant from
University of Mumbai for the Academic year 2016-17. He has Worked as Reviewer for the book titled “Concise Graduate
Chemistry –II (ISBN: 978-81-942249-1-4) published by University of Mumbai. He has Written a book (As one of the Authors)
titled “Concise Graduate Chemistry –III (ISBN: 978-81-942249-5-2) currently in Press and will be published by University of
Mumbai. He is also a visiting faculty in Ruia, K.J.Somaiya, Mithibai, Guru Nanak, Vivekanand and Konkan Gyanpeeth, Karjat
College. He has invited as a Recourse Person in Chemistry in the International Junior Science Olympiad-Cum-Selection Camp
(OCC) at K.J.Somayia College. He worked as a Presiding Officer in various Assembly Parliamentary and Legislative Assembly
Elections. His prime objective is to make a significant contribution in the field of Education and Research.
Mr. Kyle Meyers
Department of Chemistry, St. Xavier’s College, Maharashtra, India.
He is an aspiring researcher with experience in education and chemistry. He is also a detail-oriented bench researcher with a
substantial background in various fields of chemistry some of which include Nanoparticles and analytical instrumentation. He
has been a Research Scholar with Dr. Pralhad Rege since his student life and he has shared many research and educational
endeavors with him.
Trends in Pharmaceutical Research and Development Vol.4
Simultaneous Estimation of Ofloxacin and Ornidazole from Pharmaceutical Dosage Form by Spectrophotometric Methods
32
Ms. Neha Kapadia
Department of Chemistry, St. Xavier’s College, Maharashtra, India.
She is an aspiring researcher with experience and a highly refined skill set, including strategic planning, confidentiality,
organization, and teamwork. She also worked as a research assistant for a year in the field of Nano-sciences and
Spectroscopy. She has been known and awarded for my optimistic nature which helps her maintain a positive vibe in her work
ethics. Social help has always been a part of her nature.
_________________________________________________________________________________
© Copyright (2020): Author(s). The licensee is the publisher (Book Publisher International).
DISCLAIMER
This chapter is an extended version of the article published by the same author(s) in the following journal.
Analytical Chemistry: An Indian Journal, 10(9): 570-575, 2011.
