National Ribat University

Faculty Of Graduate Studies & Scientific Research

Simultaneous Determination of Pseudoephedrine Hydrochloride and

Triprolidine Hydrochloride in Tablets by Multi-Linear regression

Spectrophotometry

A Thesis Submitted in Partial Fulfillment of the Requirements for Master Degree in

Drug Quality Control

By :Tagwa Rabie Ali Mansoor

Supervisor :Dr. Imad Osman Abu Reid

2017
 Dedication

This work is dedicated to my

Loving parents, brothers and sisters.

 Chapter 1

Introduction and Literature review

1.1 Introduction
Presently various combinations in dosage forms are present in enormous amount and are
increasing rapidly. These multicomponent formulations provide the increased therapeutic
index, multiple actions, less side effects and quicker relief. The analytical process deal
with two parts of chemical characterization either it is quantitative or qualitative. The
quantitative analytical analysis provides the amount of chemical identities present in the
formulation. The main objective behind the analytical estimation is to provide the
assurance that particular formulation contains the equal amount of active pharmaceutical
ingredient as mentioned in the label [1].

For the estimation of multicomponent in formulation the various instrumental techniques

like spectrophotometric and chromatographic techniques are used due to their advantages
of being less time consuming, cheap, specific and accurate [2].

Multicomponent UV Spectrophotometric methods are based on recording and

mathematically processing absorption spectra .They offer the following advantages
[3]:Avoiding prior separation techniques e.g. extraction, concentration of constituents
and cleanup steps that may required ,spectral data are readily acquired with ease, the
process is fast, accurate and simple, wide applicability to both organic and inorganic
systems, typical detection limits of 10-4 to 10-5 M and moderate to high selectivity.

Such methods include [4]:

• Simultaneous equation method: applied when a sample contains two absorbing drugs
(x and y) each of which absorbs at the λmax of the other.
• Difference spectrophotometry: the essential feature of difference spectrophotometry
is that the measure the absorbance difference (ΔA) between two equimolar solutions
 of the analyte in different chemical forms which exhibit different spectral
characteristics. The selectivity and accuracy of spectrophotometric analysis of
samples containing absorbing interferences may be markedly improved by this
technique [4].
• Derivative spectrophotometry (DS): the theory behind DS is the conversion of a
normal spectrum (zero-order spectrum) to its first, second or higher derivative spectra
by differentiating absorbance of the sample with respect to wavelength [5] .The
differentiation of zero-order spectrum can lead to separate of overlapped signals,
elimination of background caused by presence of other compounds in a sample [6],
improvement of resolution of mixtures, and enhancement of sensitivity and
specificity [7].
• Derivative ratio spectral method: based on the derivation of the ratio spectral for
resolving binary mixtures. It permit use of the wavelength of highest value of
analytical signals with several maxima and minima, which give an opportunity for the
determination of active compounds in the presence of other compounds and
excipients which could possibly interfere in the assay; by conversion of zero-order
spectra to its first, second and higher derivatives spectra [8,9].
• Double divisor ratio spectra derivative method: this method is based on the use of the
derivative of the ratio spectrum obtained by dividing the absorption spectrum of the
ternary mixture by a standard spectrum of a mixture of two of the three compounds in
the mixture, and the measuring at either the maximum or minimum wavelengths. It
can only be used for the mixtures that the ratio of the concentrations of two
interfering compounds (used as double divisor) is known [10].
• Successive ratio - derivative spectra method: this method is used for simultaneous
determination of the three compounds in ternary mixtures without need to know the
ratio of concentration of species. It is based on the successive derivative of ratio
spectra in two successive steps [11].
• Q-absorbance ratio method: is a modification of the simultaneous equations method.
According to this method, the ratio of absorbance at any two wavelengths for
substance, which obeys Beer’s law, is constant value independent to concentration
and path length. This constant is term “Hufner’s Quotient” or Q-value. The method
 involves the measurement of absorbance at two wavelengths, one being the λ max of
one of the components (λ 2) and the other being a wavelength of equal absorptivity of
the two components (λ 1) called iso-absorptive point [5,12].
• Isosbestic “isoabsorptive" point method: this technique can be used only if the spectra
of the same concentration of the two studied drugs cross at a point called isosbestic or
iso-absorptive point. At the isosbestic point both drugs have equal absorptivities and
their mixture acts as a single component and gives the same absorbance as pure drug.
• Absorpitivity factor method: the absorptivity factor is applied for the analysis of
binary mixture if only there is a large difference in the absorptivity between both
drugs, so there is no occurrence of an isoabsorptive point [13].
• Dual wavelength method: dual wavelength method facilitates analyzing a component
in presence of an interfering component by measuring the absorbance difference (ΔA)
between two points in the mixture, one of the drugs consider as a component of the
interest and other drug as an interfering component. The method based on selection of
two wavelengths where the interfering component shows the same absorbance (ΔA =
0) where as the component of interest shows significant difference in absorbance with
concentration. Δ A between two points on the mixture spectra is directly proportional
to the concentration of the component of interest independent of interfering
component [14].
• Ratio subtraction method (RSM): this method apply when we have two drugs with
overlapping spectrum (x and y), and the spectrum of x extended more than y. The
determination of x can be done by dividing the spectrum of the mixture by a certain
concentration of y as a divisor (y0) [15].
• Absorption factor method (AFM): this method describes the analysis of a binary
mixture where the two components x and y have overlapped spectra.y shows
interference at λ max of x, while x shows no interference with y at another wavelength
(λ 2).
• Multivariate Chemometric methods: Chemometrics recognizes that it is often better to
measure many nonselective signals and then combine them in multivariate model
(multivariate analysis), whereby multiple variables are considered simultaneously. A
multivariate measurement is defined as one in which multiple measurements are
 made on a sample of interest. So, more than one variable or response are measured
for each sample [16, 17].

1.2 Objectives
Rationale:

The individual spectra of TRI and PSH show extensive overlapping over the wavelengths
of 220 – 270 nm, hence their determination when present in combination is not possible
with classical spectrophotometric techniques.

The objectives of this research were:

• To develop a new spectrophotometric method based on multi-wavelength linear

regression to overcome the problem of spectral overlap.
• To apply this developed method for the estimation of PSH and TRI combined in
tablets.
1.3 literature review
Triprolidine, 2-[(1E)-1-(4-methylphenyl)-3-(pyrrolidin-1-yl)prop-1-en- 1-yl] pyridine
(Figure 1),act as anti allergic , histamine H1 Antagonist that blocks the action of
endogenous histamine, which subsequently leads to temporary relief of the negative
symptoms brought on by histamine[18].

Pseudoephedrine Hydrochloride, (1S, 2S)-2-(methylamino)-1-phenylpropan-1-ol (Figure

2), it acts as vasoconstrictor, adrenergic agents, sympathomimetic and bronchodilator
agents [18].
 Figure 1: chemical structure of Triprolidine.

Figure 2: chemical structure of pseudoephedrine.

The combination of triprolidine and pseudoephedrine is used for treatment of allergic

rhinitis, symptoms of the common cold nasal decongestion.

The combination of triprolidine (TRI) and pseudoephedrine hydrochloride (PSH) in

tablets and syrup dosage form is official in United State Pharmacopeia (USP), normal
phase HPLC method on silica column using mixture of alcohol and 0.4 % ammonium
acetate solution (17:3 V/V) as mobile phase , with ultraviolet detection at 254 nm was
employed for the determination of the two analytes [19].
Literature review revealed that only few methods have been developed for the
determination of PSH and TRI in combination.

First derivative spectrophotometric chromatography and high performance liquid

chromatography were developed for the determination of PSH and TRI in tablets. In the
UV-spectrophotometric method the determination was carried out at the wavelengths 240.2
and 263.5 nm; selected for the first order derivative method. The chromatographic method
was performed on a C18 column using methanol: water mixture (80:20 v\v) adjusted to pH
3.0 with phosphoric acid. The flow rate was 1ml/min and the analytes were monitored at
246.2 nm. [18]

Partial least-squares multivariate calibration over the range of 235 – 315 nm was used for
the determination of the two analytes. [20]

Second order derivative spectrophotometric method was developed and used for the
determination of the two analytes from dissolution study at 271 nm and 321 nm [21].

Proportional isoabsorptive point spectrophotometric method using 290 nm and 266 nm (the
iso absorptive point) was employed for the determination of two actives [22].

Comparison of the ratio spectra derivative spectrophotometry, derivative

spectrophotometry and vierordt’s method was used for quantitative analysis of two
analytes over the range of 264nm and -291.8nm. [23]
1.4 Theoretical back ground
A number of methods have been developed to determine the composition of binary
mixture spectrophotometrically. Most of these are directed at mixtures where one
component can be isolated from the other or they require a Beer’s law experiment to
measure the molar absorptivity of each of the substance in the mixture. However, multi-
wavelength linear regression analysis (MLRA) was described [24] and proved to be
capable of resolving mixture with overlapping spectra without determining molar
absorptivities or complicated mathematics. Using this method, the composition of a
binary mixture with overlapping spectra can be resolved with only three measurements,
the absorbance of a standard solution for each component, and the unknown mixture
itself.

Assuming additivity, the absorbance of a mixture is the sum of the absorbencies of its
components. If we have a mixture consisting of two components, 1 and 2 with an
unknown concentration of C1 and C2, then absorbance of the unknown mixture,

A mixture = A1 + A2 but applying Beer’s law: A1 = €1bC1 and A2 = €2bC2

Substituting: A mixture = €1bC1 + €2bC2

However, the absorbencies of standard solutions of the same substances will follow the
same Beer’s law relationship and have the same molar absorbance, €, and one centimeter
path length, b, as the unknown solutions under the same conditions.

Therefore, we can write:

Astandard1 = €1bC standard1 and Astandard2 = €2bCstandard2

A standard1 A standard2
Rearranging these relation: €1b = and €2 b =
C standard1 C standard2
Substituting:

A standard1 A standard2
AMIXTURE = C standard1 C1 + C2
C standard2

C1 C2
AMIXTURE = C standard1 A standard1 + A standard2
C standadr2

Divided by A standard 1 and simplifying we obtain:

A mixture C1 C2 A standard2
= C standard1 + X
C standard1 C standard2 A standard1

Therefore, a plot of:

A mixture A standard2
C standard against A standard1
1

Will give:

C2 C1
Slope = and intercept =
C standard2 C standard1

That is, the concentration of unknown component 2 (C2) in the mixture, equals the slope
time the concentration of the standard solution for component 2. Likewise, the
concentration of the unknown component 1 (C1) in the mixture equals the product of the
intercept times the concentration of the standard solution for component 1.
Or simply

C1 = intercept x C standard 1 and

C2 = slope x C standard2

Chapter 2

Materials and Methods

2.1 Materials

• Triprolidine hydrochloride and pseudoephedrine hydrochloride working

standards were obtained from (Blue Nile Pharmaceutical Company, Khartoum
North- Sudan).
• Trifed tablets (AL-Hikma Pharmaceutical Industry- Jordon): labeled to contain
60 mg of pseudoephedrine hydrochloride and 2.5 mg of triprolidine
hydrochlorides were purchased from local market.
• Hydrochloric acid analytical grade reagent (HCl).
• Laboratory produced distilled water was used throughout the analysis.

2.2 Instruments
 • UV-VIS spectrophotometer single beam (model UV min1240 Shimadzu -
Japan).
• Ultrasonic bath – Germany.

2.3 Reagents

• Hydrochloric acid (0.1 N ) diluent:

The reagent was prepared by transferring 8.1 ml of concentrated HCl into 1000 ml
volumetric flask, and diluted to volume with distilled water.

2.4 Samples and standard solutions preparation

2.4 .1 Stock standard solutions

Standard stock solutions of TRI (160 μg/ml) and PSH (3800 μg/mL) were prepared by
accurately weighing and diluting about 8 mg TRI and 190 mg PSH into two separate
50ml volumetric flask with 0.1N HCl.

2.4.2 Linearity standards

Separate linearity standards of two analytes were prepared by proper dilution of suitable
aliquots from their corresponding stock standard solutions with 0.1 HCl to give
concentration in the range of (6.4-32 μg/ml) for TRI and (152-760 μg/ml) for PSH.

2.4.3 Preparation of working standards

Working standards were prepared by quantitative dilution with 0.1 N HCl of suitable
volumes from the stock standard solutions and used in different parts of analytical work.

2.4.4 Laboratory synthetic mixtures :

Five synthetic mixture containing different amounts of TRI and PSH were prepared by
accurate dilution of aliquots from their corresponding stock standard solutions.

2.4.5 Preparation of the experimental sample:

A total of ten tablets were accurately weighed, powdered and mixed well. A quantity of
the resulted powder equivalent to one tablet was weighed and transferred into a 100 ml
volumetric flask , the final volume was adjusted with 0.1 N HCl. This process was
replicated for six different weights equivalent to one tablet to produce six experimental
sample. The solutions were sonicated for 15 min then filtered using 0.45 μ filter.

2.5 General procedure:

The absorbances of the working standards, mixtures and sample were read at 5 nm
intervals within the wavelength range of 240-265 nm.

The concentration of TRI and PSH in synthetic mixtures and the samples were calculated
according to the MLRA principle from the slope and intercept of the straight line, using
Microsoft Excel Spreadsheet.

Chapter 3

Results and Discussion

The individual spectra of TRI and PSH (Fig. 3), showed considerable overlapping over
the wavelength range of 220-320 nm, accordingly application of the classical
spectrophotometric techniques for their determination in combined dosage form is not
possible.

A= Triprolidine

B= Pseudoephedrine

Figure 3: UV spectra of TRI (160 μg/ml) and PSH (3800 μg/mL) in 0.1 N HCl.

Multi-wavelength linear regression analysis is one of the approaches that can be used
when the overlapping between the spectra of the two analytes is very extensive .The
application of the technique requires existence of a linear relation between the
concentration of the analytes and their absorbances over the wavelength range selected
and additivity of their absorbances at each wavelength.

3.1 Linearity

Both TRI and PSH showed good correlation between the concentration absorbance at
each of the selected wavelength over the range of 220-320 nm (r > 0.99) and small
intercept, the regression data of the two analytes is presented in Table 1 and 2. The
presented data suggests possible application of the proposed method for determination of
TRI and PSH in combined dosage forms using MLRA.

Table 1: Triprolidine linearity data at selected wavelengths

μg/ ml Absorbances
240nm 245nm 250nm 255nm 260nm 265nm
6.4 0.329 0.29 0.244 0.208 0.198 0.206

12.8 0.657 0.576 0.482 0.407 0.384 0.4

19.2 1.027 0.903 0.755 0.637 0.601 0.626

25.6 1.131 0.993 0.831 0.701 0.661 0.689

32 1.427 1.254 1.048 0.883 0.833 0.868

Slope 37500 32935.39 27485.955 23089.89 21727.53 22654.49

Intercept 0.1132 0.0997 0.0849 0.074 0.0713 0.0739
r2 0.9873 0.9872 0.9873 0.9874 0.9875 0.9875

Table 2: pseudoephedrine hydrochloride linearity data at selected wavelengths

μg/ ml Absorbances
240nm 245nm 250nm 255nm 260nm 265nm
152 0.063 0.107 0.155 0.178 0.156 0.109

304 0.128 0.197 0.285 0.326 0.283 0.194

456 0.193 0.292 0.422 0.482 0.419 0.287

608 0.217 0.338 0.497 0.572 0.497 0.337

760 0.285 0.434 0.627 0.716 0.624 0.426

Slope 0.0003 0.0005 0.0007 0.0008 0.0007 0.0005
Intercept 0.0173 0.0351 0.0504 0.0582 0.0508 0.0375
2
r 0.9911 0.9950 0.9962 0.9967 0.9968 0.9964
r2= correlation coefficient

3.2 Determination of synthetic mixture (Accuracy)

The method accuracy was tested by analyzing five laboratory prepared synthetic mixtures
containing different amount from TRI and PSH ,the results obtained showed good
agreement between actual and theoretical amounts of the two analytes. For TRI the
recovery from the synthetic mixtures was (99.66 - 107.18 %), and the recovery of PSH
was (98.04 - 99.87 %).

The absorbance data at selected wavelengths, the absorbances ratio and the accuracy
calculation are shown in tables 3, 4 and 5.

Table3: The absorbance data at selected wavelengths

ƛ(nm) TRI PSH m1 m2 m3 m4 m5

240 1.108 0.237 0.766 1.026 0.858 0.585 1.341

245 0.967 0.365 0.763 0.992 0.88 0.61 1.332
250 0.812 0.516 0.767 0.958 0.903 0.633 1.322
255 0.694 0.619 0.769 0.932 0.915 0.648 1.309
260 0.654 0.584 0.724 0.872 0.86 0.608 1.231
265 0.679 0.402 0.629 0.785 0.734 0.516 1.086
m= synthetic mixture

Table4: The absorbance ratio data at selected wavelengths

 ƛ(nm) TRI/PSH m1/PSH m2/PSH m3/PSH m4/PSH m5/PSH

240 4.6751 3.2320 4.3291 3.6202 2.5107 5.7553

245 2.6493 2.0904 2.7178 2.4109 1.6897 3.6897
250 1.5736 1.4864 1.8565 1.750 1.2363 2.5820
255 1.1211 1.2423 1.5056 1.4781 1.0536 2.1284
260 1.1198 1.2397 1.4931 1.4726 1.0482 2.1224
265 1.6890 1.5646 1.9527 1.8258 1.2709 2.6748
Slope 0.5600 0.7963 0.6036 0.4039 1.0033
Intercept 0.6119 0.6066 0.8022 0.5994 1.0009
Correlation coefficient 0.9999 0.9999 0.9999 0.9999 0.9999

Table 5: The accuracy results of the synthetic mixtures

sample Triprolidine (μg/mL) Content Pseudoephedrine (μg/mL) Content

Theoretical actual % Theoretical actual %
1 0.0181 0.0194 107.18 0.4699 0.4608 98.06
2 0.0258 0.0259 100.38 0.4658 0.4608 98.92
3 0.0195 0.0194 99.48 0.6161 0.6144 99.72
4 0.0132 0.0131 99.24 0.4580 0.4584 100.08
5 0.0329 0.0328 99.69 0.7649 0.764 99.88
mean 101.19 mean 99.33
Standard deviation 3.37 Standard deviation 0.836
RSD% 3.33 RSD% 0.0084

3.3 Analysis of commercial Sample

The proposed method was applied to analysis of five samples taken from commercial
tablets dosage.

The results obtained were in good agreement with the labeled amounts 104.20% and
92.55% with relative standard deviations of 0.77 % and 1.07% for TRI and PSH
respectively. This support the suitability of the proposed method for determination of TRI
and PSH in tablets dosage formulation .The tablets analysis data is presented in tables 6-
9.

Table 6: Samples weight taken:

Sample no. Weigh taken μg Active

Triprolidine Pseudoephedrine
S1 0.1339 24.96 598.92
S2 0.1341 24.99 599.82
S3 0.1340 24.97 599.37
S4 0.1339 24.96 598.92
S5 0.1341 24.99 599.82
S6 0.1340 24.97 599.37
S= sample

Table 7: The absorbance data at selected wavelengths (samples)

ƛ(nm) TRI PSH S1 S2 S3 S4 S5 S6

240 1.105 0.233 1.081 1.096 1.101 1.088 1.093 1.104

245 0.964 0.361 1.047 1.063 1.074 1.047 1.06 1.079
250 0.809 0.512 1.024 1.047 1.045 1.036 1.03 1.05
255 0.691 0.615 1.015 1.033 1.036 1.013 1.025 1.04
260 0.651 0.58 0.963 0.972 0.98 0.96 0.978 0.986
265 0.676 0.406 0.854 0.854 0.867 0.866 0.859 0.866
Table8: The absorbance ratio data at selected wavelengths (Samples)

ƛ(nm) TRI/PSH S1/PSH S2/PSH S3/PSH S4/PSH S5/PSH S6/PSH

240
4.7424 4.6394 4.7038 4.7253 4.6695 4.6909 4.7381
245 2.6703 2.9002 2.9445 2.9750 2.9002 2.9362 2.9889
250 1.5800 2 2.0449 2.0410 2.0234 2.0117 2.0507
255 1.1235 1.6504 1.6796 1.6845 1.6471 1.6666 1.6910
260 1.1224 1.6603 1.6758 1.6896 1.6551 1.6862 1.7
265 2.1330
1.6650 2.1034 2.1034 2.1354 2.1330 2.1157
Slope
0.8246 0.8368 0.84090 0.8307 0.8345 0.8428
Intercept
0.7188 0.7255 0.7333 0.7181 0.7230 0.7376
Correlation confident
0.99978 0.99986 0.999901 0.99961 0.99970 0.99989

Table 9: The assay results of samples

sample Triprolidine (μg/mL) Content Pseudoephedrine (μg/mL) Content
Theoretical found % Theoretical found %
S1 24.96 25.70 102.97 598.92 549.17 91.69
S2 24.99 26.08 104.34 599.82 554.33 92.42
S3 24.97 26.20 104.92 599.37 560.27 93.48
S4 24.96 25.89 103.73 598.92 548.67 91.61
S5 24.99 26.01 104.06 599.82 552.37 92.09
S6 24.97 26.26 105.16 599.37 563.54 94.02
Average Average
104.20 92.55
Standard deviation 0.80 Standard deviation 0.99
RSD% 0.77 RSD% 1.07
 Chapter 4

Conclusion and References

4.1 Conclusion

• The MLRA is straight forward procedure allowing the accurate resolution of

binary mixtures of compounds with overlapped spectra.
• The cost effectiveness and simplicity of method render it as suitable alternative
to other expensive methods e.g. chromatographic methods for the analysis of
binary mixtures of compounds with overlapped spectra in laboratories and
countries where such sophisticated equipments are not affordable.
• The accuracy and simplicity of the method suggested it suitability in case
where quick results are demanded e.g. as in-process analysis procedure during
blend analysis in industrial setups.
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 Table of Contents

Acknowledgements I

abbreviations II

Abstract ( English) III

List of figures IV

List of tables V

Chapter 1: Introduction and Literature Review

1.1 Introduction 1
1.2 Objectives 4
1.3 Literature review 5
1.4 Theoretical background 7
Chapter 2: materials and methods
2.1 Chemicals and Standards 9
2.2 Instruments 9
2.3 Reagents 9
2.4 Samples and Standard solutions preparation 9
2.4.1 Stock Standard solutions 9
2.4.2 Linearity standards 9
2.4.3 Working standards 10
2.4.4 Laboratory synthetic mixtures 10
2.4.5 Sample preparation 10
2.5 General procedure 10

Chapter 3 : Results and Discussion

3.1 Linearity over the selected wavelengths range 11

3.2 Determination of synthetic mixtures ( accuracy) 13
3.3 Analysis of commercial sample 14

Chapter 4 :
4.1 Conclusion 17
4.2 References 18
 Acknowledgements

I’m cordially indebted to my supervisor Dr. Imad for the efforts and supports he exerted
during my research work, gratitudes can’t pass without falling on all the staff of the
faculty of pharmacy at Al Ribat National University.

I’m also thankful to all my colleagues in batch one for the memorable time we spent
together.
 Abbreviations

AFM Absorption Factor Method

C18 Octadecylsilane
DS Derivative Spectrophotometry
HCl Hydrochloric acid
HPLC High Performance Liquid Chromatography
min Minute
mL Milliliter
MLRA Multi-Wavelength Linear Regression Analysis
nm Nanometer
PSH Pseudoephedrine Hydrochloride
RSM Ratio Subtraction Method
TRI Triprolidine hydrochloride
USP United State Pharmacopeia
UV Ultraviolet
μg Microgram
 Abstract

A simple, accurate and inexpensive method has been developed for the determination of
triprolidine hydrochloride and pseudoephedrine hydrochloride in tablets,Multi-
wavelength Linear Regression Analysis.The method utilized the slope and intercept of
the straight line obtained by plotting the ratio of the sample absorbances divided by the
pseudoephedrine standard of known concentration against the absorbances ratio of
triprolidine standard and pseudoephedrine standard.

The recovery the synthetic mixture was 99.33% and 101.19% withthe relative standard
deviations of 0.0084% and 3.33% for triprolidine and pseudoephedrine respectively

The results obtained by applying the method to the analysis of two analytes in tablets
were in good agreement with label claim, 104.20% and 92.55% with relative standard
deviations of 0.77% and 1.07% for triprolidine and pseudoephedrine respectively.
 List of figures
Figure 1 Chemical structure of Triprolidine 5
Figure 2 Chemical structure of Pseudoephedrine. 5
Figure 3 UV spectra of TRI (160 μg/ml) and PSH (3800 μg/mL) in 0.1 N HCl 11

List of tables
Table 1 Triprolidine linearity data at selected wavelengths 12
Table 2 pseudoephedrine hydrochloride linearity data at selected 12
wavelengths
Table 3 The absorbance data at selected wavelengths 13
Table 4 The absorbance ratio data at selected wavelengths 13
Table 5 The accuracy results of the synthetic mixtures 14
Table 6 Samples weight taken: 14
Table 7 The absorbance data at selected wavelengths (samples) 15
Table 8 The absorbance ratio data at selected wavelengths (Samples) 15
Table 9 The assay results of samples 16
 Chapter one

Introduction and Literature Review

 Chapter Two

Materials and methods

 Chapter Three

Results and Discussion

Chapter Four

Conclusion