A PROJECT REPORT ON ANALYSIS OF ORGANIC ACIDS IN COMMERCIAL

DRINKS

BY

OLAYIWOLA AYOMIDE GODWIN

MATRIC NO:

1808005158

SUBMITTED TO THE DEPARTMENT OF INDUSTRIAL CHEMISTRY,

FACULTY OF SCIENCE, EKITI STATE UNIVERSITY, ADO-EKITI,

NIGERIA

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF

BACHELOR OF SCIENCE (B.S.C HONS)

DEGREE IN INDUSTRIAL CHEMISTRY

SEPTEMBER,2023

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 CERTIFICATION
This is to certify that this work on Analysis of Organic Acids on Commercial Drinks was carried
out by OLAYIWOLA AYOMIDE GODWIN with matric no: 1808005158 of INDUSTRIAL
CHEMISTRY DEPARTMENT FACULTY OF SCIENCE, EKITI STATE UNIVERSITY, ADO
–EKITI.

------------------------------- ----------------------------------
Project Supervisor Head of Department
Dr. M.A. Azeez Prof. F.J. Faleye

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 DEDICATION
I dedicate this work to God who has been my fortress right from my childhood till this very day.

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 ACKNOWLEDGEMENT
I am indeed grateful to God for the wisdom he gave me to go this far with the project work and
study, my appreciation also goes immensely to my amiable supervisor Dr. M.A. Azeez for his
encouragement and insights throughout the course of this project work.

My gratitude goes to my parents as well Mr and Mrs Olayiwola, may God continue to uphold
you in all ramifications.

I appreciate all my friends, who in one way or the other assisted me during the course of writing
this report.

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 ABSTRACT
Organic acids are very vital compounds in food products and they occur naturally and in some
number of food products. Even though, they are not considered as nutrients, however they are
responsible for giving a characteristic taste to food and likewise preserve food products. In this
work, analytical method, High Performance Liquid Chromatography was employed in
determining the concentration of organic acid in commercial drinks, mainly Ceres 100% White
Grape juice and in comparison, to Caprisun Orange juice. These two samples show variation in
the concentration of organic acid present. The result of the analysis shows that Ceres 100%
White Grape juice has: Tartaric acid – 105.12, Citric acid – 27.59, Fumaric acid – 23.72 part per
million concentrations. While for Caprisun Orange juice, Ascorbic acid – 100.1, Citric acid –
27.8, Oxalic acid – 18.2 part per million concentrations. Ceres 100% White Grape juice has high
concentration of tartaric acid in abundance than Caprisun Orange juice, where tartaric acid is
undetected. On the other hand, Caprisun Orange juice shows high concentration of ascorbic acid
which is not detected for Ceres 100% White Grape juice.

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 TABLE OF CONTENT
CHAPTER ONE
1.0. Introduction to Organic Acids……………………………………………...
…………………1
1.1. Background of the Research…………………………………………….……….
…....2
1.2. Aim and Objectives of the Research………………………………….
………………3
1.3. Groups of organic acid …………………………………………………………….
…3
1.3.1. Monocarboxylic Acids……………………………………………………4
1.3.2. Dicarboxylic Acids……………………………………….……………….5
a. Citric Acid………………………………………………………….…6
b. Succinic Acid…………………………………………………………7
c. Oxalic Acid………………………………………………………..….9
d. Ascorbic Acid………………………………………………………..10
e. Fumaric Acid………………………………………………………...11
f. Tartaric Acid………………………………………………………....12
g. Benzoic Acid………………………………………………………....13

CHAPTER TWO
2.0. Literature Review on High Performance Liquid Chromatography…………………………15
2.1. Types of HPLC techniques………………………………………………………………….15
a. Based on mode of chromatography. ……………………………………………...……..16
b. Based on elution technique. ……………………………………………………………..16
c. Based on a scale of operation…………………………………………………………….16
d. Based on the type of analysis ………………………………………………………..…..17
2.2. Instrumentation…………………………………………………………………………..….17
A. Solvent Reservoir………………………………………………………………....18
B. Pump……………………………………………………………………………...18
i. Direct Gas Pressure System……………………………………………….…19
ii. Syringe Type pumps…………………………………………………………19
iii. Pneumatic Intensifier…………………………………………………….…..19
iv. Reciprocating pumps…………………………………………………..…….19
C. Sample Injector………………………………………………………………..…..20

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 D. Columns………………………………………………………………………...…20
i. Separation Column …………………………………………………………….……21
ii. Guard Columns……………………………………………………………..………..21
iii. Derivatizing Columns……………………………………………….……….………21
iv. Capillary Columns………………………….…………………………………..……22
v. Fast columns…………………………………………………………………………22
vi. Preparatory columns…………………………………………………………………22
E. Detector……………………………………………………………………….…..23
1.Molecular Spectroscopic Techniques………………………………...……23
a) UV Detector…………………………………………………..……..23
b) Refractive Index Detection (RID)………………………………..….24
c) Fluorometric Detection (FD) …………………………………….….24
2. Atomic Spectroscopic Techniques…………………………………….….24
2.3. Data Collection Devices………………………………………………………………….…24
2.4. Selection of Chromatographic Condition………………………………………….………..25
A. Buffer Selection and Concentration……………………………………………..25
B. Isocratic and Gradient Separations………………………………………………25
C. Internal Diameter…………………………………………………………….….26
D. Particle size and pore size……………………………………………………….26
E. Selection of Mobile Phase ………………………………………………………26
F. Selection of Detectors……………………………………………………………26
2.5. Different Types of HPLC Detector………………………………………………………….27

A. UV-Visible- Ultraviolet Visible Detector………………………………………….…….27

B. Refractive Index Detectors ………………………………………………………..…….27
C. Photodiode Array Detector (PAD), Diode Array detector……………………………….28
D. Fluorescence Detector……………………………………………………………………28
E. Electrical Conductivity Detector…………………………………………………………29

2.6. Developing The Approach for Analysis……………………………………………….……30

2.7. Sample Preparation…………………………………………………………………….……30

2.8. Method Optimization………………………………………………………………..………30

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2.9. Method Validation…………………………………………………………………..………30
2.10. Application of HPLC …………………………………………………………….………..31

CHAPTER THREE
3.0. RESEARCH METHODOLOGY
A. Chromatography conditions……………………………………………………….…..31
B. Standards Mixes and Sample Preparation……………………………………………..31
C. Buffer Preparation (25mM monobasic potassium phosphate) ………………………..31
D. Preparation of Mobile phase…………………………………………………….…….34
E. Instrumentation………………………………………………………………………..34
F. Solvents, Standards and Samples………………………………………………..……35

Chapter FOUR
4.0. Results…………………………………………………………………………………….37
4.1. Calibration Curve for each standard…………………………………………………..….40
4.2. Chromatogram Image for Ceres 100% White Grape Sample (Sample One)…………….43
4.3. Chromatogram Image for Caprisun Orange Sample (Sample Two)……………………..44
4.4. Conclusion ……………………………………………………………………...………..45
REFERENCE………………………………………………………………………………….46

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 CHAPTER ONE
1.0. INTRODUCTION TO ORGANIC ACIDS
Organic acids refer to organic compounds that are acidic and contain one or more carboxyl
groups. The most common organic acids are carboxylic acids (R-COOH), whose acidity
originates from the carboxyl group (-COOH), except for organic acids such as sulfonic acid (R-
SO₃H), sulfinic acid (R-SOOH), and sulfuric acid (R-SH) (Qui et al., 2021). Organic acids can
be classified according to the differences in the number of carboxyl groups, hydroxyl groups, and
carbon–carbon double bonds in their molecular structure: (1) aliphatic, alicyclic, aromatic, and
heterocyclic acids, such as benzoic acid; (2) saturated or unsaturated acids, such as acetic acid
and acrylic acid; (3) the number of carboxyl groups and whether the carboxyl groups are
substituted, e.g., acetic acid (one carboxyl group), malic acid (two carboxyl groups), and citric
acid (three carboxyl groups). Amino acids are organic compounds containing basic amino and
acidic carboxyl groups (R-CHNH₂-COOH), which are formed when the hydrogen atom on the
carbon atom of a carboxylic acid is replaced by an amino group (Qui et al., 2021). Fruit juice is
taken into account to be one in every of the healthiest foods in human diet, because of their well-
known according health edges (source of natural vitamins and antioxidants, anti-inflammatory
properties, bar of chronic diseases, etc.). during this sense, business ready juices claim to
preserve these nutritionary and healthy effects. Organic acids area unit chemicals found naturally
in fruits and vegetables. as a result of organic acids have such an outsized impact on the
organoleptic qualities and stability of fruit juices, the character and concentration of organic
acids in fruits area unit of nice interest. Organic acid identification and detection in fruit juices is
important for quality and method management. Organic acid concentrations in fruits area unit
essential as a result of they need an effect on the organoleptic qualities of fruit juices,
significantly in terms of flavour, colour, and scent. Organic acid level in fruit juices affects not
solely the organoleptic aspects of the liquids, however conjointly their stability, nutrition,
satisfactoriness, and quality. Natural and industrial fruit juices have varied levels of organic
acids, additions, and preservatives. many natural action ways are developed for distinguishing
and quantifying severally organic acids in several matrixes (Büyüktuncel et al., 2017)

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Organic acids play an important role in maintaining the nutritional value and sensory quality of
foods and are also an important class of food additives, including their use as preservatives,
acidity regulators, antioxidants, etc., with a wide range of applications. Organic acids are a group
of natural compounds known as weak acids used as food additives, but not all of them have
antimicrobial activity. The most effective antimicrobials are acetic acid, lactic acid, propionic
acid, sorbic acid and benzoic acid. The activity of organic acids is related to pH and to the
undissociated form of the acid. The use of organic acids is generally limited to foods with a pH
less than 5.5 (Doores, 1993). Organic acids are either naturally present in foods or chemically
synthesized and added, directly or indirectly, to food products, with some of them formed during
fermentation of carbohydrates in foods (Theron and Lues, 2011).

The aim of the current research was to determine the oxalic, tartaric, ascorbic, citric, fumaric,
and succinic acid content in fresh and pre-treated with steam vegetables and spices using high
performance liquid chromatography (HPLC) method.

1.1. BACKGROUND OF THE RESEARCH

Organic acids have long been used in their natural forms. These are low-molecular weight
compounds that contain one or more carboxyl groups and are observed in almost every
microorganism. They have numerous applications in industries related to bio commodities such
as food, cosmetics, surfactants, and textile industriies. Production of organic acids for
commercial exploitation is carried out either by chemical synthesis or fermentation, with
preference for fermentation because of its high yield in the microbiological processes. The initial
production practices of acetic acid date back to 1823 and citric acid to 1913. But commercial
fermentation of citric acid production via microbe-assisted processing started around 1920. Over
the years myriad organic acids of biosynthetic origin have been produced, most of which are
natural bioproducts of microbes, or may be expected as intermediary in important metabolic
pathways (Alonso et al., 2015). Because of their functional groups such as keto and hydroxyl
groups, organic acids are enormously valuable as initial substances for chemical industries.
Besides their traditional usage in food, feed, and pharmaceuticals of late, production of organic
acids as monomers with bifunctional characteristic has been triggered due to the resurgence of
bioplastics. Indulgence of recombinant DNA (Deoxyribonucleic acid) technology and metabolic

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engineering techniques have efficiently engineered organic acid-producing microorganisms to
enhance and speed up process developments for preferred bioproducts at high concentration,
quality, and productivity. Even novel microbial isolates demonstrating potential production
properties of interest are effectively engineered for the overproduction of organic acid on a
commercial scale (Li and Borodina 2015). This chapter provides an overview of the types of
organic acids, its chemistry and application in various section.

1.2 AIM AND OBJECTIVES OF THE RESEARCH

HPLC is widely used for the separation and detection of contaminants and additives in the
analysis of food or fruit drinks. Our fruit drinks contain quite a lot of extra ingredients such as
colorant which helps to increase acceptability and appeal of the food, food preservatives and
anti-oxidants to increase the life of the product, natural and artificial sweeteners and flavors to
improve the taste of fruit drinks. All these are required in small quantities and therefore
analytical methods have been developed for the quantitative and qualitative analysis of food
product and one of such method is the High-Performance Liquid Chromatography (HPLC). The
aims of the research according to this work can be summarized into two valid points:

1. Quantification and qualification of identified analyte

2. Identification of the analyte of interest.

1.3 GROUPS OF ORGANIC ACIDS.

Organic acids are grouped into the monocarboxylic, dicarboxylic, alpha hydroxyl and sugar
acids. Monocarboxylic acids include formic, acetic, propionic and sorbic acid. Acetic acid is
used as emulsifiers, stabilizers, preservatives, flavour enhancers and firming agents (Smith and
Hong-Shun, 2011). Formic acid which is the simplest carboxylic acid with one carbon atom, is
used as a preservative, acidifier in animal feed as well as a flavouring agent at low
concentrations (Burdock, 2004). Propionic acid can be used as a pH control agent, preservative

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and flavour enhancer while sorbic acid has found application as preservatives (Quitmann et al.,
2014).

Dicarboxylic acid includes adipic, fumaric and succinic acid and have found application in
beverage, feed and food preservation. Salts of adipic acid such as calcium and magnesium
adipate are used in food processes as sequestrants, acidity regulators, baking additives,
preservatives and flavour enhancers. Fumaric acid can be used as pH control agents, flavour
enhancers, firming agents and as emulsifiers and dough conditioner during esterification
process. Succinic acid is also used as flavour enhancers, preservatives, pH control agents and
in baking (Quitmann et al., 2014).

Alpha hydroxyl acids which include citric, lactic and malic acid have found application in
beverage, food and animal nutrition. Citric acid and its salts are used as sequestrants, pH
regulators, preservatives, flavour enhancers, and firming agents. Esters of citric acid can also
be used as emulsifiers and solvents. Lactic acid has been used for a long time as acidity
regulators, preservatives, baking additives, and flavour enhancers (Anyasi et al., 2015).
Lactic acid can also be used as a humectant due to its hygroscopic activity. Malic acid on its
part can be used as synergists, acidity regulators, preservatives, and flavour enhancers
(Quitmann et al., 2014).

Other organic acids are called the sugar acid and they include ascorbic, gluconic, lactobionic
and tartaric acid. Ascorbic acid and its isomer erythorbic acid are used as antioxidants,
synergist, sequestrants and reducing agents (Smith and Hong-Shun, 2011). Gluconic acid and
its salt are used as processing aids in the prevention of milk-stone in dairy industry and also
in animal nutrition. Lactobionic acid is used as gelling agent, flavour enhancer, antioxidant,
acidity regulators, baking additives and firming agents (Quitmann et al., 2014).

1.3.3. Monocarboxylic Groups

For example, unsaturated (cinnamic, sorbic), hydroxylic (citric, lactic), phenolic (benzoic,
cinnamic, salicylic) and multi carboxylic (azelaic, citric, succinic) acids (Cherrington, et al.,

4
1991). Organic acids are distinguished from other acids by the carboxylic functional group -
COOH to which an organic acids group or a hydrogen atom are attached.

Fig.1.1. Monocarboxylic acid

Common names used to describe this group of organic compounds include fatty, volatile fatty,
lipophilic, weak, or carboxylic acids (Cherrington, et al., 1991). The acetic acid of vinegar, the
formic acid of red ants, and the citric acid of fruits all belong to the same family of compounds -
carboxylic acids (Anon 2012).

1.1.2 Dicarboxylic Acids

Dicarboxylic acids (HO₂C-R-CO₂H) is an organic compound which contains two carboxyl
functional groups (-COOH). They have similar chemical properties to monocarboxylic acids.
However, they have two dissociation constants; one for the dissociation into a mono anion and
the second into a di anion (McMurry, 2008). The first ionization constant of dicarboxylic acids is
usually larger than their monocarboxylic analogues because there are two potential sites for
ionization making the effective concentration of the carboxyl group twice as large.

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 Fig.1.2. Dicaboxylic acid
Dicarboxylic acids are widely used in industries for the production of polymers (adipic acid), as
food preservatives (oxalic acid) and as amino acids in human body (glutamic acid). Dicarboxylic
acids can take various configurations depending on whether they are linear, branched chain,
unsaturated or aromatic. Dicarboxylic acids are found naturally in plants and animals sources but
in very little amount. Most dicarboxylic acids used industrially are synthetically produced from
the oxidations of fatty acids.

A. CITRIC ACID

Fig.1.3. Structure of Citric acid

The name citric acid was first derived from the latin word citrus, the citron tree that bears fruit
which resembles a lemon. Citric acid was first extracted from lemon juice in 1784 by a Swedish
chemist called Carl Scheele (Mattey and Kristiansen, 1999). It is an organic compound with the

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chemical formula HOC(CO₂H) (CH₂CO₂H)₂ . Citric acid has found application in food,
beverages, pharmaceuticals and industrial fields with its application dependent on acidity,
flavour and salt formation. Citric acid sold in a dry powdered form is commonly sold in markets
and groceries as "sour salt", due to its physical resemblance to table salt. It has use in culinary
applications, as an alternative to vinegar or lemon juice, where a pure acid is needed. Citric acid
can be used in food coloring to balance the pH level of a normally basic dye. Chemically, citric
acid is 2-hydroxy-1,2,3-propane tricarboxylic acid (CAS No. 77-92-9) composed of three pKa
values at pH 3.1, 4.7 and 6.4. Citric acid forms a wide range of metallic salts including
complexes with copper, iron, manganese, magnesium and calcium. Citric acid is a hydroxy
tricarboxylic acid produced naturally by various plants. Citric acid is water soluble, approved for
direct addition to multiple foods, is affirmed as generally regarded as safe (GRAS) and is
approved for use in the manufacture of fresh and processed meats and poultry at concentrations
specific to its purpose (USDA-FSIS, 2010). Citric acid and its salts have demonstrated efficacy
for pathogen control in both fresh and processed meat and poultry, but their usage is potentially
limited by possible negative sensory impact and the need for low pH maintenance for optimum
antimicrobial activity (Mani-Lopez et al., 2012).

B. SUCCINIC ACID

Fig.1.4. Structure of Succinic acid

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 Fig.1.5. Succinic acid
Succinic acid, which is also known as butanedioic acid, 1,2-ethanedicarboxylic acid and amber
acid, occurs in nature as such or in various forms of its esters. Succinic acid is a dicarboxylic
acid with the chemical formula (CH₂)₂ (CO₂H)₂ . Succinic acid is a white, odorless solid with a
highly acidic taste. In living organisms, succinic acid takes the form of an anion, succinate,
which has multiple biological roles as a metabolic intermediate being converted into fumarate by
the enzyme succinate dehydrogenase in complex 2 of the electron transport chain which is
involved in making ATP, and as a signalling molecule reflecting the cellular metabolic state.
Traditional applications of succinic acid include food additives, detergents, cosmetics, pigments,
toners, cement additives, soldering fluxes, and pharmaceutical intermediates. Succinic acid is
marketed as food additive E363. The name derives from Latin succinum, meaning amber.
(Tretter et al., 2016).

Succinic acid is a white, odorless solid with a highly acidic taste. Succinic acid is primarily used
as an acidity regulator in food and beverage industry (Chikhalia et al., 2006). It is also available
as a flavoring agent. contributing a somewhat sour and astringent component to umami taste.
(Thakker et al., 2017) As an excipient in pharmaceutical products, it is also used to control
acidity or as a counter ion. (Thakker et al., 2017)

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C. OXALIC ACID

Fig.1.6 Structure of Oxalic acid

Fig.1.7. Oxalic acid

Oxalic acid is an organic acid with the systematic name ethanedioic acid and formula
HO₂C−CO₂H, also written as (CO₂H)₂. It is the simplest dicarboxylic acid. It is a white
crystalline solid that forms a colourless solution in water. Oxalic acid has much greater acid
strength than acetic acid. It is a reducing agent and its conjugate base, known as oxalate (C₂O 2−
4), is a chelating agent for metal cations. Typically, oxalic acid occurs as the dihydrate with the
formula C₂H₂O₄ ·2H₂O. (Ullmann, 2005). Oxalic acid is mainly manufactured by the oxidation
of carbohydrates or glucose using nitric acid or air in the presence of vanadium pentoxide. A
variety of precursors can be used including glycolic acid and ethylene glycol (Eiichi et al., 1969).
Oxalic acid's main applications include cleaning or bleaching, especially for the removal of rust
(iron complexing agent). Its utility in rust removal agents is due to its forming a stable, water-
soluble salt with ferric iron, ferrioxalate ion. Oxalic acid is an ingredient in some tooth whitening
products. About 25% of produced oxalic acid will be used as a mordant in dyeing processes. It is

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also used in bleaches, especially for pulpwood, and for rust removal and other cleaning, in
baking powder, and as a third reagent in silica analysis instruments. (Eiichi et al., 1969).

D. ASCORBIC ACID

Fig.1.7. Structure of Ascorbic acid

Fig.1.8. Ascorbic acid

Vitamin C (also known as ascorbic acid and ascorbate) is a water-soluble vitamin found in citrus
and other fruits and vegetables, also sold as a dietary supplement and as a topical "serum"
ingredient to treat melasma (dark pigment spots) and wrinkles on the face. (Nathan and Patel,
2021) The term vitamin C encompasses several vitamers that have vitamin C activity in animals.
Ascorbate salts such as sodium ascorbate and calcium ascorbate are used in some dietary
supplements. These release ascorbate upon digestion. Ascorbate and ascorbic acid are both
naturally present in the body, since the forms interconvert according to pH. Oxidized forms of
the molecule such as dehydroascorbic acid are converted back to ascorbic acid by reducing
agents (Marriott, 2020). Vitamin C functions as a cofactor in many enzymatic reactions in
animals (including humans) that mediate a variety of essential biological functions, including
wound healing and collagen synthesis. In humans, vitamin C deficiency leads to impaired
collagen synthesis, contributing to the more severe symptoms of scurvy. Another biochemical

10
role of vitamin C is to act as an antioxidant (a reducing agent) by donating electrons to various
enzymatic and non-enzymatic reactions. (Meister, 1994). Doing so converts vitamin C to an
oxidized state - either as semi dehydroascorbic acid or dehydroascorbic acid. These compounds
can be restored to a reduced state by glutathione and NADPH-dependent enzymatic mechanisms.
(Michels and Frei, 2012).

Vitamin C has a definitive role in treating scurvy, which is a disease caused by vitamin C
deficiency. It is required for the functioning of several enzymes and is important for immune
system function (Nathan and Patel, 2021). It also functions as an antioxidant. Ascorbic acid and
some of its salts and esters are common additives added to various foods, such as canned fruits,
mostly to slow oxidation and enzymatic browning. It may be used as a flour treatment agent used
in breadmaking. (Washburn and Jensen, 2017).

E. FUMARIC ACID

Fig.1.9. Fumaric acid

Fig.1.10. Structure of Fumaric acid

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Fumaric acid (FA) originally derived its name from the plant, Fumaria officinalis (family:
Papaveraceae), from which this organic acid was isolated for the first time (Roa Engel et al.,
2008). It has a molecular formular of C₄H₄O₄, with an IUPAC identification as butenedioc acid,
E297. Some other common names of FA are allomaleic acid, boletic acid, lichenic acid, and
tumaric acid. The plant is an herb with pink-colored flowers that appears from April to October
in the northern hemisphere. The plant is well known for its medicinal uses and is considered to
be the major source of fumaric acid. Fumaric acid is a multifunctional chemical intermediate that
finds applications in nearly every field of industrial chemistry.

Fumaric acid is mostly used in the feed industry as an antibacterial agent. Food and beverages
account for 33% of the world consumption of fumaric acid, followed by rosin paper sizes
(20.0%), unsaturated polyester resins (18.6%), and alkyd resins (12.3%). Moreover, fumaric acid
and its ester derivatives (FAEs) have been explored with a number of newer applications in the
fields of medical science (such as neurology, immunology, and dermatology); veterinary science
(reduction of CH₄ emission up to 70% and improvement in feeding efficiency in the dairy and
poultry industries); and bio nanotechnology (drug delivery and tissue engineering) (Yang et al.,
2011). FA is the least expensive of the food-grade acids. It has been used as a nutritional additive
and acidulant in various forms in the food and farming industries without untoward effects since
1946. It is the strongest tasting food acidulant that can control the growth of microorganisms,
adjust pH, and enhance flavors (Yang et al., 2011).

F. TARTARIC ACID

Fig.1.11. Structure of Tartaric acid

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 Fig.1.12. Grape
Tartaric acid is a white, crystalline organic acid that occurs naturally in many fruits, most notably
in grapes, but also in bananas, tamarinds, and citrus (Duarte et al., 2012). Its salt, potassium
bitartrate, commonly known as cream of tartar, develops naturally in the process of fermentation.
It is commonly mixed with sodium bicarbonate and is sold as baking powder used as a leavening
agent in food preparation. The acid itself is added to foods as an antioxidant E334 and to impart
its distinctive sour taste. Naturally occurring tartaric acid is a useful raw material in organic
chemical synthesis. Tartaric acid, an alpha hydroxy-carboxylic acid, is diprotic and aldaric in
acid characteristics, and is a dihydroxyl derivative of succinic acid. Tartaric acid is a muscle
toxin, which works by inhibiting the production of malic acid, and in high doses causes paralysis
and death. As a food additive, tartaric acid is used as an antioxidant with E number E334;
tartrates are other additives serving as antioxidants or emulsifiers. Tartaric acid and its
derivatives have a plethora of uses in the field of pharmaceuticals. For example, it has been used
in the production of effervescent salts, in combination with citric acid, to improve the taste of
oral medications. The potassium antimonyl derivative of the acid known as tartar emetic is
included, in small doses, in cough syrup as an expectorant (Blair and DeFraties, 2000).

G. BENZOIC ACID

Fig.1.13. Structure of Benzoic acid

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 Fig.1.14. Benzoic acid
Benzoic acid (C₆H₅COOH) is one of the oldest and most commonly used food preservative
(Barbosa-Canovas et al., 2003). It is widely used as a preservative for food and beverages.
Benzoic acid occur naturally at a high level in many fruits such as cranberries, plums, cinnamon
and prunes. Indeed, some berries, such as cloudberries, contain so much benzoic acid that they
can be stored for long periods without bacterial or fungal spoilage (Piper, 2011). Benzoic acid, a
white crystalline solid is slightly soluble in water and this restricts its use as a preservative in
food industries. Sodium benzoate a salt of derivative of the acid (C₆H₅COONa) is more soluble
in water than the benzoic acid and it is commonly used as a preservative in food industries than
benzoic acid because of its greater solubility in aqueous solution. Benzoates are derived from a
neutralization reaction with benzoic acid and are more commonly used as food preservatives
than the acid. Its preservative effectiveness depends on the acidity of the food. It has been widely
used as preservatives in soft drinks, fruit drink and pickles (Clauden et al., 2012).

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 CHAPTER TWO
2.0. LITERATURE REVIEW ON HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Chromatography can be defined as an adsorption-based mass transfer method. Pumps transport a
pressured liquid and a sample mixture through a column loaded with adsorbent, allowing the
sample components to be separated. HPLC (high-Performance Liquid Chromatography),
formerly known as high-pressure liquid chromatography, is an analytical chemistry technique for
separating, identifying, and quantifying each component in a mixture (Karger and Barry,1997).
Pumps are used to move a pressured liquid solvent containing the sample combination through a
solid adsorbent material-filled column. Each component in the sample interacts with the
adsorbent material in a slightly different way, resulting in varying flow rates and separation of
the components as they flow out of the column. HPLC has been used for manufacturing, legal
(e.g., identifying performance enhancement drugs in urine), research (e.g., separating the
components of a complicated biological sample, or of comparable synthetic substances from
each other), and medicinal (e.g., detecting vitamin D levels in blood serum) applications (Karger
and Barry,1997).

The term chromatography is derived from the Greek words namely chroma (color) and graphein
(to write). It is defined as a set of techniques used for the separation of constituents in a mixture.
This involves 2 phases; they are stationary and mobile phases. The separation is based on the
difference between the partition coefficients of the two phases. The instrumentation includes a
solvent reservoir, HPLC pump, injector, HPLC column, detector, and data acquisition. It is used
for purposes like identification of compounds, chemical separation, purification of compounds,
etc. HPLC is mainly used in analytical chemistry (Liming, 2008).

2.1. TYPES OF HPLC TECHNIQUE

A. Based on mode of chromatography.
B. Based on elution technique.
C. Based on a scale of operation.
D. Based on the type of analysis.

15
 A. BASED ON THE MODE OF CHROMATOGRAPHY
HPLC is divided into normal phase and reverse phase based on the mode of chromatography. In
normal phase mode, the stationary phase is polar and the mobile phase is nonpolar. Here
nonpolar compounds travel faster and get eluted first. This is due to the less affinity between
solute and stationary phase. Polar compounds are retained for a longer time in the column due to
higher affinity towards the stationary phase and take more time to elute (Gupta et al., 2012).

B. BASED ON THE ELUTION TECHNIQUE

Based on the technique of elution they are divided as isocratic and gradient separation. In
isocratic separation, the combination of the mobile phase is used throughout the process of
separation. Same polarity or elution strength is maintained. The concentration of the mobile
phase is also the same. It influences the retention of the analyte. It is used in order to maximize
the loading capacity. In gradient separation, the mobile phase combination of lower polarity or
elution strength is used followed by gradually increasing polarity or elution strength. Their
selectivity depends on the dimensions of the column used. The advantages of gradient elution are
that it enhances the peak resolution, faster analysis time, and better detectability (Gupta et al.,
2012).

C. BASED ON THE SCALE OF OPERATION

In analytical HPLC only analysis of the sample is done. Recovery of sample is not done during
analytical high-performance liquid chromatography. Reusing is also not done for analytical
purposes as the sample quantity retained is very low after the process. For this technique
analytical columns are used. By using preparative HPLC individual fractions of pure compounds
can be collected using the fractional collector. In this HPLC the retained samples can be reused

16
hence wastage is low. Specialized preparative columns are used for this method. Preparation of
pure compounds can be done using HPLC (Gupta et al., 2012).

D. BASED ON THE TYPE OF ANALYSIS

Qualitative analysis helps in identifying the compounds from a mixture. This is done by
comparing the retention time of the sample compound with that of the standard
compound. Detection of impurities is also possible by the method of qualitative analysis.
Quantitative analysis is used in the determination of the quantity of individual or several
components, it is the main function of the quantitative analysis in HPLC. Here the analysis is
based on the comparison with the measurement of peak height from a sample with the
unknown concentration. This method should be subjected to validation before starting the
analysis (Gupta et al., 2012).

2.2. INSTRUMENTATION
The HPLC instrumentation involves a pump, injector, column, detector, integrator, and
display system. It is illustrated in figure below

Fig.2.1. Instrumentation Of HPLC

17
 A. SOLVENT RESERVOIR

Fig.2.2. Solvent Reservoir in HPLC

The contents of the mobile phase are present in a glass container called solvent reservoir
as shown in fig. above. In high solvent is a mixture of polar and non of the sample, the polar
and non-- performance liquid chromatography, the mobile phase or the polar liquid
components. Depending on the composition polar solvents will be varied. The solvent is used to
carry the sample through the system. From the solvent reservoir, the pump collects the
solvent then passes it to the injector.

B. PUMP

Fig.2.3. Pump System in HPLC

18
The pump suctions (Fig.2.3) the mobile phase from the solvent reservoir and forces it to column
and then passes to the detector. 42000 KPa is the operating pressure of the pump (Shinde Manjir,
2021). This operating pressure depends on column dimensions, particle size, flow rate, and
composition of the mobile phase. Some of the different types of pumps are discussed below

i. Direct gas pressure systems

This system consists of a cylinder gas pressure, which is applied directly to the eluent in a
holding coil. Advantages of this pump are that it is reliable and economical although solvent
changing is found to be tedious (Sanjay Kumar, 2010).

ii. Syringe type pumps

In these pumps, an electrically driven lead-screw moves a piston, which is able to pressurize a
finite volume of solvent, and thus delivers a pulseless constant flow of solvent to the system.
These pumps are found to be reliable although they are expensive, solvent changing is tedious
and they have a finite capacity (Sanjay Kumar, 2010).

iii. Pneumatic intensifier

Pneumatic intensifier pumps are operated via gas pressure. A large area piston drives a small
area piston when acted on by pressure from a gas line. The gas pressure is thus amplified in the
ratio of the areas of the forces of the pistons and a high-pressure liquid at constant pressure is
introduced into the system. If a partial blockage occurs in this system a drop-in flow rate occurs
but the pressure remains constant (Sanjay Kumar, 2010).

iv. Reciprocating pumps

A reciprocating pump is the most generally used, as it is economical and allows a wide range of
flow rates. With this pump, there is no limit on the reservoir size or operating time as is
commonly found with other pumps. This type of pump is electrically driven by a motor, which
moves back and forth within a hydraulic chamber (Shinde Manjiri, 2021).

19
 C. SAMPLE INJECTOR
The injector can be a solitary infusion or a computerized infusion framework. An injector for an
HPLC framework should give an infusion of the fluid specimen inside the scope of 0.1 mL to
100 mL of volume with high reproducibility and under high pressure (up to 4000 psi) in the load
position a sample loop is filled with the sample while the system is equilibrating. The flow
delivered by the pump flows through the loop and feeds the sample onto the column (Shinde
Manjiri, 2021).

D. COLUMNS

Fig.2.4. Columns used in HPLC

Columns as shown in figure 4 are typically made of cleaned stainless steel, are somewhere
around 50 mm and 300 mm long, and have an inward distance across somewhere around 2 and 5
mm. They are generally loaded with a stationary phase with a molecule size of 3μm to 10μm.
Columns with inner diameters of less than 2mm are regularly alluded to as microbore segments.
Preferably the temperature of the mobile phase and the column should be kept consistent during
the investigation (Shinde Manjiri, 2021).

20
 i. Separation Columns
There are various columns that are secondary to the separating column or stationary phase. They
are guard, derivatizing, capillary, fast, and preparatory columns (Dennis,1996).

ii. Guard Columns

They are placed anterior to the separating column. This serves as a protective factor that prolongs
the life and usefulness of the separation column. They are dependable columns designed to filter
or remove. Particles that clog the separation column, compounds and ions could ultimately cause
"baseline drift", decreased resolution, decreased sensitivity, and create false peaks, compounds
that may cause precipitation upon contact with the stationary or mobile phase, compounds that
might co-elute and cause extraneous peaks and interfere with detection and/or quantification.
These columns must be changed on a regular basis in order to optimize their protective function.
The size of the packing varies with the type of protection needed (Dennis,1996).

iii. Derivatizing Columns

Pre or post-primary column derivatization can be an important aspect of the sample analysis.
Reducing or altering the parent compound to a chemically related daughter molecule or fragment
elicits potentially tangible data which may complement other results or prior analysis. In a few
cases, the derivatization step can serve to cause data to become questionable, which is one reason
why HPLC was advantageous over gas chromatography, or GC. Because GC requires volatile,
thermally stable, or nonpolar analytes derivatization was usually required for those samples
which did not contain these properties. Acetylation silylation is concentrated acid hydrolysis are
few derivatization techniques (Dennis,1996).

21
 iv. Capillary columns
Advances in HPLC led to smaller analytical columns. Also known as microcolumns, capillary
columns have a diameter much less than a millimeter and there are three types:
1. open-tubular
2. partially packed
3. tightly packed
They allow the user to work with nanoliter sample volumes, decreased flow rate, and decreased
solvent volume usage which may lead to cost-effectiveness. However, most conditions and
instrumentation must be miniaturized, the flow rate can be difficult to reproduce, gradient elution
is not efficient, and care must be taken when loading minute sample volumes. Microbore and
small-bore columns are also used for analytical and small volumes assay (Dennis,1996).

v. Fast columns
One of the primary reasons for using these columns is to obtain improved sample throughput
(amount of compound per unit time). For many columns increasing the flow or migration rate
through the stationary phase will adversely affect the resolution and separation. Therefore, fast
columns are designed to decrease the time of the chromatographic analysis without forsaking
significant deviations in results. These columns have the same internal diameter but much shorter
length than most other columns, and they are packed with smaller particles that are typically 3
um in diameter (Dennis,1996).

vi. Preparatory columns

These columns are utilized when the objective is to prepare bulk (milligrams) of samples for
laboratory preparatory applications. A preparatory column usually has a large column diameter
which is designed to facilitate large volume injections into the HPLC system. Accessories
important to mention are the back-pressure regulator and the fraction collector. The back-
pressure regulator is placed immediately posterior to the HPLC detector. It is designed to apply
constant pressure to the detector outlet which prevents the formation of air bubbles within the
system. This, in turn, improves chromatographic baseline stability. It is usually devised to

22
operate regardless of flow rate, mobile phase, or viscosity. The fraction collector is an automated
device that collects uniform increments of the HPLC output (Yu Shao, 2004).

E. DETECTOR
The HPLC detector, situated toward the end of the column distinguishes the analytes as they
elute from the chromatographic column. Regularly utilized detectors are UV spectroscopy,
fluorescence, mass spectrometric, and electrochemical identifiers (Yu Shao, 2004).

1. Molecular Spectroscopic Techniques.

a. UV Detectors
Works by measuring the change in UV absorption by the sample. The sample under analysis
should have absorbance in the region of ultraviolet radiation. In UV transparent solvent, these
detectors are concentration sensitive. Direct detection has a flaw as not all inorganic ions have
appropriate chromophores but this can be compensated by using the method of derivatization
(Pooja Rejakumar et al., 2022). This is done by mixing the effluent with a chromogenic reagent
in a post column reactor. It is shown in figure

Fig.2.5. UV VIS Detector

23
 b. Refractive Index Detectors (RID)
The refractive index of a medium is the ratio of the speed of light in a vacuum to the speed of
light in the medium. These detectors measure the change in refractive indices in the eluent as the
solute passes through the sample cell. This method of detection is less sensitive than UV
detection, although nonchromatographic compounds can be measured directly without
derivatization (Karger,1980)
c. Fluorometric Detection (FD)
The solute is excited by UV radiation at a particular wavelength and the emission wavelength is
detected. Used with naturally fluorescent compounds but compounds can be reacted to produce
fluorescent derivatives (Molander Paal,2003).

2. Atomic Spectroscopic Techniques

Atomic spectroscopy includes atomic absorption spectroscopy as well as atomic emission
spectroscopy. The spectroscopic determination of atomic species can only be performed in the
gaseous medium in which the individual atoms are well separated from one another. Thus, the
first step in the atomic spectroscopic technique is atomization, a process in which the sample is
volatilized in such a manner so as to produce an atomic gas (Aksu Abdulla, 2018).

2.3. DATA COLLECTION DEVICES

Data collection devices are also called integrators. Signals from the detector might be gathered
on graph recorders or electronic integrators that fluctuate in many-sided quality and in their
capacity to process, store and reprocess chromatographic information. The graph so produced is
used to interpret the result of the experiment. The PC coordinates the reaction of the indicator to
every part and places it into a chromatograph that is anything but difficult to interpret (Aksu
Abdulla, 2018).

24
 2.4. SELECTION OF CHROMATOGRAPHIC CONDITION
Selection of the stationary phase/column is the first and the most important step. To avoid
problems from irreproducible sample retention during method development, it is important that
columns be stable and reproducible. A C8 or C18 column made from specially purified, less
acidic silica and designed specifically for the separation of basic compounds is generally suitable
for all samples and is strongly recommended. The use of silica-based packing is favoured in most
of the present HPLC columns due to several physical characteristics (Aksu Abdulla, 2018)

A. Buffer Selection and Concentration

Choice of the buffer is governed by the desired pH. The typical pH range for reversed-phase on
silica-based packing is pH 2.0 to 8.0. It is important that the buffer has a pKa close to the desired
pH since the buffer controls pH best at their pKa. A rule is to choose a buffer with a pKa value
less than 2 units of the desired mobile phase pH. Generally, a buffer concentration of 10-50 mM
is adequate for small molecules no more than 50% organic should be used with a buffer. This
will depend on the specific buffer as well as its concentration. Phosphoric acid and its sodium or
potassium salts are the most common buffer systems for reversed-phase HPLC. Sulfonate buffers
can replace phosphonate buffers when analysing organophosphate compounds (Walker
Valerie,2002)

B. Isocratic and Gradient Separations

Isocratic mode of separation includes constant eluent composition; which means equilibrium
conditions in the column and the actual velocity of compounds moving through the column are
constant. As mentioned earlier isocratic elution is normally successful in the partition of sample
components that are not altogether different in their proclivity for the stationary stage and in
gradient elution the organization of the mobile phase is fluctuated ordinarily from low to high
eluting quality (Molander Paal,2003).

25
 C. Internal Diameter
The range of internal diameter of columns in standard columns for reversed-phase and normal
phase is from 3.9 to 4.6 and length should be 15cm and 25cm. It is an important parameter that
influences the detection sensitivity and separation selectivity in gradient elution. It also
determines the quantity of analyte that can be loaded into a column (Marchetti et al., 2009).

D. Particle size and pore size

The smaller particles usually provide more surface area and better separations but the pressure
required for the optimum linear velocity increases by the inverse of the particle diameter
squared. Larger particles are used in preparative HPLC. The pore size of the column defines the
ability of the analyte molecules to penetrate inside the particle and interact with its inner surface
(Molander Paal,2003).

E. Selection of Mobile Phase

The mobile phase affects resolution, selectivity, and efficiency. Mobile phase composition (or
solvent strength) plays an important role in RP-HPLC separation. A mixture of acetonitrile and
water is the best initial choice for the mobile phase during method development (Marchetti et al.,
2009).

F. Selection of Detectors
The detector is a very important part of HPLC. Selection of detector depends on the chemical
nature of analyses, potential interference, the limit of detection required, availability, and/or cost
of detector. UV visible detector is a versatile, dual-wavelength absorbance detector for HPLC.
Photodiode Array (PDA). The detector offers advanced optical detection for waters analytical
HPLC, preparative HPLC, or LC/MS system solutions. Its integrated software and optics
innovations deliver high chromatographic and spectral sensitivity. Refractive index
chromatographic and spectral sensitivity, stability, and reproducibility make this detector the
ideal solution for the analysis of components with limited or no UV absorption. Multiwavelength

26
Fluorescence Detector offers high sensitivity and selectivity fluorescence detection for
quantitating low concentrations of target compounds (Marchetti et al., 2009).

2.5. DIFFERENT TYPES OF HPLC DETECTORS

Detectors in HPLC is placed at the end of the analytical column. The function of the detector is
to examine the solution which is eluting from the column. An electronic signal is proportional to
the concentration of individual components of the analyte. HPLC detector has particular
characteristics such as excellent linear response as a function of the good sensitivity works
approximately in the range of 0.01- 100 µg of the compound in elutes (Marchetti et al., 2009).

A. UV-Visible- Ultraviolet Visible Detector

It is the most commonly used detector in the HPLC. Most of the organic compounds
absorb light in the region of UV (190-400nm) and in the visible region (400-750nm). It is
based on Beer-Lambert law; deuterium and high-pressure xenon lamp are the sources of
the UV. It has various advantages and disadvantages (Kraiczek et al., 2014).
Advantages: It has high sensitivity, relative robust to temperature, compatible with
gradient elution.
Disadvantages: only compounds with UV or visible absorption could be detected.

B. Refractive index Detectors: Refractive index is a bulk property of column eluent. In this
detector, detection depends on solute modifying the overall refractive index of the mobile
phase. Bulk property detectors have an inherently limited sensitivity. For the detection of
the non-ionic compounds refractive index can be very useful and non-ionic compounds
are not absorbed in the UV region and in fluoresce (Ping et al., 2018). Various types of
RI detectors are the following (Kupina et al., 2014).
i. Christiansen effect detector
ii. Interferometer detector
iii. Thermal lens detector

27
 iv. Dielectric constant detector

Advantages:

1. Responds to nearly all solutes

2. Unaffected by the flow rate

Disadvantages:

1. Not as sensitive as most other types of detectors

2. Could not be used with a gradient elution (Kupina et al., 2014).

C. Photodiode Array Detector (PAD), Diode Array Detector: Photodiode arrays

(semiconductor devices) are used in the detection unit. A DAD detects the absorption in
UV to VIS region. While a UV-VIS detector has only one sample-side light receiving
section, a DAD has multiple (1024 for L-2455/2455U) photodiode arrays to obtain
information over a wide range of wavelengths at one time, which is a merit of the DAD
(Pragst et al., 2004).
Advantages:
1. PDA Detector could analyse a sample simultaneously at many different wavelengths
2. UV visible spectra are useful for compound identification, checking peak purity, as
well as finding the optimum absorbance for the compounds.
3. UV visible spectra of many compounds could be stored in the spectrum libraries,
which are used for compound identification.
4. Relatively robust to temperature and flow rate fluctuations
5. Compatible with gradient elution.
Disadvantages:
1. Slightly less sensitive than the UV-Visible detector (Pragst et al., 2004).

D. Fluorescence Detector: It is the most sensitive, specific detector among all existing
HPLC detector. It is possible to detect the presence of single analyte molecule in the flow

28
 cell. The sensitivity of this detector is 10-1000 times higher than UV detector (Raut and
Charde, 2014). Various types of Fluorescence detector are:
i. The single wavelength excitation Fluorescence detector
ii. Multi wavelength Fluorescence detector
iii. Laser-induced Fluorescence detector

Advantages:

1. Sensitivity is higher than UV-Vis detector

2. Selectivity is high because of relatively few compounds fluorescence.

3. Compatible with gradient elution

Disadvantages:

1. Difficult to predict fluorescence

2. Greatly affected by environment, solvent, pH, temperature, viscosity, ionic strength,
dissolved gas (Raut and Charde, 2014).

E. Electrical Conductivity Detector: It provides universal, reproducible, high-sensitivity

detection of all charged species such as anions, cations, metals, organic acids. These
detectors measure the conductivity of the total mobile phase hence categorized in bulk
density detectors. Electrodes of this detector are usually made up of platinum, stainless
steel or some other noble metal (Zhang, 2016).
Advantages:
1. Respond to ionic compounds and suitable for ion chromatography.
2. High sensitivity for low concentration range.

Disadvantages:

1. Sensitive to the fluctuations in the solvent flow and mobile phase composition

2. Not compatible with gradient elution.

29
 2.6. DEVELOPING THE APPROACH FOR ANALYSIS
While developing the analytical method on RP-HPLC the first step which is followed is the
selections of various chromatographic parameters like the selection of mobile phase, selection of
column, selection of flow rate of mobile phase, selection of pH of the mobile phase. Detection
wavelength is usually the isosbestic point in the case of simultaneous estimation of two
components (Sanjay Kumar, 2010).

2.7. SAMPLE PREPARATION

Sample preparation is an essential part of HPLC analysis, intended to provide a reproducible and
homogenous solution that is suitable for injection onto the column. The aim of sample
preparation is a sample aliquot that, is relatively free of interferences, will not damage the
column, and is compatible with the intended HPLC method that is, the sample solvent will
dissolve in the mobile phase without affecting sample retention or resolution. Sample preparation
begins at the point of collection, extends to sample injection onto the HPLC column (Shinde
Manjiri, 2021)

2.8. METHOD OPTIMIZATION

Here the identification of the “weaknesses” of the method is done and the same is optimized
through experimental design. Here one has to understand the method performance with different
conditions, different instrument setups, and different samples. This enables better reproducibility
of the obtained results (Gupta et al., 2012).

2.9. METHOD VALIDATION

Validation is the confirmation by examination and the provision of objective evidence that the
particular requirements for specific intended use are fulfilled. A process of evaluating method
performance and demonstrating that it meets a particular requirement. In essence, it knows what
the method is capable of delivering, particularly at low concentrations (Patwekar,2015)

30
 2.10. APPLICATIONS OF HPLC
i. FOOD AND FLAVOUR APPLICATION
Sugar analysis in fruit juices, detecting polycyclic compounds in vegetables, analysis of
preservatives. Even though the food fortification campaign was working, a more precise
recommendation is pursued to stop having such a huge variation as discovered in many studies
(Gracia, 2006).

ii. CLINICAL APPLICATION

Detecting endogenous neuropeptides, analysis of biological samples like blood and urine. A
sensitive HPLC–APCI–MS method for the determination of vitamin K1 (VK-1) in human
plasma was established in a study (Gracia, 2006).

iii. FORENSIC APPLICATION

Analysis of textile dyes, quantification of drugs and steroids in biological samples, and several
varieties of blue ballpoint pen inks were analysed by high-performance liquid chromatography in
different studies. Applications of HPLC to the analysis of cannabis, opium alkaloids,
amphetamine-related materials, LSD, and polynuclear hydrocarbons were also estimated in other
studies (Mihaly Maria and Aurelia Meghea, 2014)

iv. PHARMACEUTICAL APPLICATION

The pharmaceutical applications include controlling drug stability, dissolution studies, and
quality control. Although expected at first to be used as a complementary method to gas
chromatography, the pharmaceutical industry now almost exclusively uses HPLC as a
chromatographic technique. Other forms of HPLC that are used in the pharmaceutical industry
include reversed-phase, denaturing, and immobilized enzyme reactor HPLC. However, one of

31
the disadvantages of HPLC is that it must be preceded by calibration tests which can increase
costs (Levesen Karsten,2000)

v. ENVIRONMENTAL APPLICATION
Monitoring of pollutants and detecting components of drinking water. Coupling of HPLC to
NMR (Nuclear Magnetic Resonance) was applied for the first time to the analysis of
environmental samples, i.e., water samples from an ammunition hazardous waste site (Exarchou
Vassiliki, 2003). Using the continuous flow mode at very low flow rates (≤0.017 mL/min) and
large volume injection (400 μL), the confirmation of many nitroaromatic compounds could be
achieved down to the microgram-per-litre level after solid-phase extraction of a groundwater
sample from a former ammunition production site (Chenghong Li, 2003).

32
 CHAPTER THREE
3.0. RESEARCH METHODOLOGY
A. Chromatography Conditions
The protocol for the chromatography separation was developed based on isocratic simple
conditions. The injection volume was set at 5µL and the column temperature at 20 ◦C.
Double distilled water was used as a washing solvent between the samples. The mobile
phase used was 25 mM monobasic phosphate solution of pH 2.4, and the binary pump was
fixed at a flow rate of 0.5 mL/min. As an attempt to investigate the possibility of achieving a
shorter runtime, the runs were then repeated using the same conditions but with a flow rate
of 0.3 mL/min. All species were detected using VWD (Variable Wavelength Detector)
wavelengths at 210 nm.

B. Standards Mixes and Sample Preparation

Reagents used: Standard substances of fumaric acid, succinic acid, citric acid, ascorbic
acid, tartaric acid, oxalic acid were obtained from Sigma-Aldrich, double distilled water,
orthophosphoric acid.

Materials: magnetic stirrer, volumetric flask (1000mL, 25mL), beaker (3L), analytical
balance, pH meter, spatula, disk filter, measuring cylinder, stirring rod, micro pipette (1000
µL), micro pipette tip, sample bottles, 5mL plunger.

C. BUFFER PREPARATION (25mM monobasic potassium phosphate)

10.207g of monobasic potassium phosphate was weighed in an analytical balance, 3 liters
of double distilled water was measured using a measuring cylinder into the beaker. The
weighed phosphate salt was dissolved inside the measured solvent. For the solution to get
agitated, a magnetic stirrer was employed and its speed was adjusted to be 3 meter per
seconds (3m/s). The pH for the solution was checked for 3 consecutive times and the
results were: 3.68, 3.74, 3.67 respectively. The mean of the result was 3.69. For the
fourth time, the pH of the solution was observed to be 3.67. The solution was stirred and
1 drop of Orthophosphoric acid was added in order to control and reduce the pH level.

33
 And the pH value was observed four times, the values obtained were 3.07,3.17, 3.05, 3.05
respectively. Another drop of Orthophosphoric acid was added and stirred, and the pH
was checked thrice, showing the values 2.94, 2.93, 2.92 respectively. Then the third drop
of Orthophosphoric acid was added and also stirred in the solution, the pH value obtained
was 2.84. For the fourth time, 2 drops of Orthophosphoric acid were added and the pH
was checked thrice, which were 2.64, 2.64, 2.64. Another single drop of Orthophosphoric
acid for the fifth time, the pH was checked twice showing 2.56, 2.56. The step was
repeated once again and the pH value gotten was 2.50 and 2.50

Using a filter paper, on an analytical weighing balance, 3.402g potassium phosphate

(monobasic) was weighed and introduced into 1ltr volumetric flask. 750mL double
distilled water was added, the solution was stirred with magnetic stirrer until it dissolves to
give a homogenized solution. Then the pH of buffer solution was adjusted to 2.4 with
orthophosphoric acid. Lastly the flask was made up to mark with double distilled water,
while the pH of the buffer solution was reconfirmed.

D. Preparation of Mobile phase

Using a measuring cylinder, 1000mL of prepared buffer solution was measured and introduced
into 1L volumetric flask. Then the solution was transferred into the mobile phase reservoir bottle.

E. Instrumentation
The apparatus consists of a container of the mobile phase, a pump capable of pressures up to
4000psi or greater, a valve for injecting the sample (usually 10 to 500 µL volumes), the column,
a detector, electronics associated with the detector, and a recorder.

Preparation of HPLC for analysis

i. Ready the HPLC by making sure the solvent reservoirs are full and the waste bottles
have at least 1 liter of volume available to accommodate the waste solvent.
ii. Launch Open Lab by clicking HPLC1 (online)

34
 iii. Use the Instrument Control Tab, confirm that all components are green and indicate
“ready”
a. If this is not the case, press the “On” button.
b. The DAD will take a few minutes to warm up; if there’s a lightning bolt through the
purple lamp, wait until it has gone away, and the ready bar has turned green

Preparing a Sequence for the Sample mixture

1. Set up five methods, one for each of the isocratic runs and one for the gradient run
2. Select ‘sample’ in the “sequence” menu bar
3. Go to “sequence” and click “sequence template” and see the orders of the samples.
4. Each method should be different for each run and to check this, click the “method” menu
bar, click “method” in the selection menu, and select “edit the entire method”.
5. Ensure that all parameters are correct, and save the method.
6. Confirm that you have enough of the sample mixture in the sample vial and that the
sample vial is in vial slot 1
7. Click “run sequence”
8. Print the report after each run or after all runs are complete by clicking the “data
analysis” tab in the bottom.
9. Confirm you have peaks of your run.

F. Solvents, Standards and Samples

The water, used for both solvent and diluent, was HPLC-grade. For buffering the mobile phase
and adjusting the pH to 2.4, both monobasic potassium phosphate and phosphoric acid were
used.

The following organic acids were used for this application: oxalic acid, tartaric acid, ascorbic
acid, citric acid, succinic acid and fumaric acid.
Standard Preparation
The standards of fumaric, succinic, ascorbic, oxalic, tartaric, citric was all weighed on the
measuring scale, each acids having different milligrams per 25mL of volumetric flask, resulting
into different concentrations

35
Fumaric acid -15mg/25mL = 0.6mg/mL
Succinic acid- 34.8mg/25mL = 1.392mg/mL
Ascorbic acid- 14.6mg/25mL = 0.584mg/mL
Oxalic acid- 35.8mg/25mL= 1.432mg/mL
Tartaric acid- 21.1mg/25mL = 0.844mg/mL
Citric acid- 77.9mg/25mL = 3.116mg/mL
These are known as Stock Solutions

Furthermore,5 serial dillution was done in the ratio of stock to distilled, that is, stock: distilled
that generate a new concentration of a new standard which will be labelled C1,C2,C3,C4 and C5
respectively. The serial dilution is aided by the use of micro pipette, setting the volume to the
required microliter and the solution will be retained in a sample bottle

Serial 1 2 3 4 5
dilution
Vol. of stock 2.5ml 1.25ml 0.63ml 0.32ml 0.16ml
standard
solution
Vol. of 2.5ml 3.75ml 4.37ml 4.68ml 4.14ml
double
distilled
water

Serial Dilution Table

This was done to help determine the calibration curve for each standard. The diluted solution will
undergo filtration to segregate the solution from any impurities or contaminants using a disk
filter and the filtrate will be deposited in the vial. HPLC analysis was performed on all the
diluted solutions and the peaks was obtained

36
 CHAPTER FOUR
4.0. RESULTS

FIG.4.1a. Integrated calibration 1

FIG.4.1b. Variable Wavelength Detector for calibration 1

FIG.4.2a. Integrated calibration 2

37
FIG.4.2b. Variable Wavelength Detector for calibration 2

FIG.4.3a. Integrated calibration 3

Fig.4.3b. Variable Wavelength Detector for calibration 3

38
 FIG.4.4a. Integrated calibration 4

Fig.4.4b. Variable Wavelength Detector for calibration 4

FIG.4.5a. Integrated calibration 5

39
 Fig.4.5b. Variable Wavelength Detector for calibration 5

4.1. CALIBRATION CURVE FOR EACH STANDARD

Calibration curve for succinic acid

450
400
f(x) = 157.272888666881 x + 0.484638215792018
350 R² = 0.999877441101261
300
B
250 Linear (B)
200
150
100
50
0
0 0.5 1 1.5 2 2.5 3

Fig.4.6.

40
 Calibration curve of tartaric acid
1200

f(x) = 453.457207661313 x − 44.0608198467958

1000 R² = 0.998453889009211

800
Peak area

Series2
600
Linear (Series2)

400

200

0
0 0.5 1 1.5 2 2.5 3

Concentration

Fig.4.7

Calibration curve for fumaric acid

2500

2000 f(x) = 823.095954105184 x + 24.9075286097614

R² = 0.99591647112629
1500 B
Linear (B)

1000

500

0
0 0.5 1 1.5 2 2.5 3

Fig.4.8

41
 Calibration curve for Citric acid
1600
1400
f(x) = 546.085208685256 x + 21.4199591579309
1200 R² = 0.999891591351066
1000 B
Linear (B)
800
600
400
200
0
0 0.5 1 1.5 2 2.5 3

Fig.4.9

Calibration curve for Ascorbic acid

1000
900 f(x) = 362.255311217995 x + 42.1267394961091
800 R² = 0.999733166070018
700
600 B
Linear (B)
500
400
300
200
100
0
0 0.5 1 1.5 2 2.5 3

Fig.4.10

42
 Calibration curve for Oxalic acid
500
450
f(x) = 177.986827855696 x + 0.247261324263576
400 R² = 0.998440747845872
350
300 B
Linear (B)
250
200
150
100
50
0
0 0.5 1 1.5 2 2.5 3

Fig.4.11

4.2. CHROMATOGRAM IMAGE FOR CERES 100% WHITE GRAPE SAMPLE

(SAMPLE ONE)

FIG.4.12 Peaks of organic acid in the sample

43
DISCUSSION
For the sample Ceres 100% White Grape, the peak height of organic acids attainable were:
Oxalic acid – 3.466
Tartaric acid – 3.609
Fumaric acid – 5.378
Citric acid – 6.348

Table of concentration in ppm (part per million) for each organic acids in the sample
Oxalic acid 18.08
Tartaric acid 105.12
Fumaric acid 23.72
Citric acid 27.59
Ascorbic acid Not Detected
Succinic acid Not Detected

From the result,the concentration of tartaric acid in the sample is highly distinctive from the rest,
then citric acid. The sample has a low concentration of oxalic acid,and likewise some organic
acids are undectectable. As a result of this, it enhances the grape flavor present in the sample and
the principal acid in a grape fruit is tartaric acid followed by small quantites of citric acid and
others.

4.3. CHROMATOGRAM IMAGE FOR CAPRISUN ORANGE SAMPLE (SAMPLE

TWO)

44
DISCUSSION
For the sample Caprisun Orange, the peak height of organic acids attainable were:
Oxalic acid – 3.484
Ascorbic acid – 5.848
Citric acid – 6.251
Table of concentration in ppm for each organic acids in the sample
Oxalic acid 18.2
Ascorbic acid 100.1
Citric acid 27.8
Fumaric acid Not detected
Succinic acid Not detected
Tartaric acid Not detected

The result above clearly shows the six organic acids likely to be present in the sample (Caprisun
Orange). And from the result, ascorbic acid has the highest concentration, followed by citric
acid, oxalic acid and minute quantity of others. The orange flavor and taste of the sample is
influenced by dominant presence of ascorbic acid.

4.4. CONCLUSION

In Caprisun Orange juice sample, fumaric, succinic and tartaric acid was undetectable. On the
other hand, organic acids of ascorbic and succinic were not detected in Ceres 100% White Grape
sample. The dominant organic acids found in each sample were tartaric acid for the former, and
ascorbic acid for the latter. The two dominant organic acids in each sample function as
preservatives and also has an effect on the taste sensitivity. Some organic acids in these two
compared samples lack sufficient sensitivity for detection, while other components commonly
present in these types of samples have a high UV absorption, which can interfere with the
detection of target analytes. Also, the detection and peak shape of some organic acids can be
compromised by the interaction with surfaces of the analytical flow path, including the column.
pH can affect the detection of organic acids in fruit drinks, the pH of the sample can affect the

45
separation and detection of organic acids in fruit juices. The optimal pH for the separation of
organic acids in fruit juices is always found to be in between 2.5 and 3.5, depending on the type
of organic acid. Therefore, it is important to carefully control the pH of the sample during
analysis to ensure accurate detection and quantification of organic acids.

46
 REFERENCES

Aksu Abdulla; Preparation of analytical columns and HPLC silica-based C 18 packing material
synthesis from non-toxic silica source. Journal of pharmaceutical researches and
education; 201841(10),1-5.

Alonso, L., Fontecha, J., & Lozada, L. (2015). Omega-3 Long Chain Polyunsaturated Fatty Acid
Content and Oxidation State of Fish Oil Supplements Marketed in South America. PLOS
ONE, 10(12).

Anyasi, T. A., Jideani, A. I. O. and Mchau, G. R. A. (2015). Effect of organic acid pretreatment
on some physical, functional and antioxidant properties of flour obtained from three
unripe banana cultivars. Food Chemistry, 172, 515-522.

Blair, G. T. DeFraties, J. J. (2000). Hydroxy Dicarboxylic Acids. Kirk Othmer Encyclopedia of

Chemical Technology. pp. 1–19

Büyüktuncel, E., Kalkan, Ö., & Şahin, E. (2017). Determination of Organic Acids in Natural and
Commercial Orange Juices by HPLC/DAD. Hacettepe. Journal of Biology and
Chemistry, 45(3), 411–416
Clauden, J., Greeves, N. and Warran, S. (2012). Organic chemistry (2nd edition) New York.
USA: Oxford University Press

Chenghong Li. (2003). Optimal Packing Characteristics of Rolled, Continuous Stationary-Phase

Columns; Research gate, 18(2), 309-16

Cherrington, C. A., Hinton, M., Mead, G. C. and Chopra, I. (1991). Organic Acids: Chemistry,
antibacterial activity and practical applications. Advanced Microbiology and Physiology,
32, 87-108

Dennis R. (1996) chromatographic method of validation: A review of current practices and

procedures III. Journal of liquid chromatography and related technologies, 19(2),1-43

Doores, S. (1993). Organic acids. In: P. M. Davidson, and A. L. Branen (Edition),

Antimicrobials in foods (2nd edition, pp 95 - 136). New York: Marcel Dekker Incorporation

47
Duarte, A.M. Caixeirinho D., Miguel, M.G., Sustelo, V., Nunes, C., Fernandes, M.M.,
Marreiros,A. (2012). Organic acids concentration in citrus juice from conventional versus
organic farming. Acta Horticulturae, 933 (933), 601–606

Exarchou Vassiliki. (2003). LC-UV-Solid-Phase Extraction-NMR-MS Combined with a

Cryogenic Flow Probe and Its Application to the Identification of Compounds Present in
Greek Oregano. Research gate, 75(22), 6288- 94

Eiichi, Yonemitsu, Tomiya, Isshiki, Tsuyoshi, Suzuki and Yukio, Yashima. (1969). Process for
the production of oxalic acid.

Gupta, V., Jain, A. D. K. J., N.S, G., and Guptan, K. (2012) Development and validation of
HPLC method - a review; International Research Journal of Pharmaceutical and Applied
Sciences, 2(4), 17-25

Karger and Barry L. (1997) HPLC: Early and recent perspectives. Journal on Chemistry
Education, 74, 45.

Karger B. (1980). Distribution phenomena of mobile phase compound and determination of dead
volume in reversed phase liquid chromatography. ACS publication, 52(14), 2249-2257

Kraiczek, K.G. (2014). Highly flexible UV-vis radiation sources and novel detection schemes for
spectrophotometric HPLC detection. Analytical Chemistry, 86(2), p. 1146-52.

Kupina, S. and M. Roman. (2014). Determination of total carbohydrates in wine and wine-like
beverages by HPLC with a refractive index detector: First Action 2013.12. Journal of
Association of Official Agricultural Chemist International, 97(2), p. 498- 505.

Levesen Karsten. (2000). Application of high-performance liquid chromatography coupled to

nuclear magnetic resonance and high-performance liquid chromatography coupled to
mass spectrometry to complex environmental samples. International journal of chemical
and pharmaceutical sciences, 19(1),27-48.

Li, M., & Borodina, I. (2015). Application of synthetic biology for production of chemicals in
yeast S. cerevisiae. FEMS Yeast Research, 15(1)

48
Liming P. (2008). Analysis of basic compounds by reversed-phase liquid chromatography-
electrospray mass spectroscopy in high pH mobile phase. Journal of chromatography,
1179(2),131-144.

Mani-López, E., García H. S. and López-Malo, A. (2012). Organic acids as antimicrobials to

control Salmonella in meat and poultry products. Food Research International, 45, 713–721.

Marchetti N. (2009) Determination of adsorption isotherms by means of HPLC: adsorption

mechanism elucidation and separation optimization. Journal on Separation Technique
and Science. 32(5-6), p. 727-41.

Marriott M. P, Birt D. F, Stallings V. A, Yates A. A, edition. (2020). Vitamin C. Present

Knowledge in Nutrition, Eleventh Edition. London, United Kingdom, Academic Press
(Elsevier). pp. 155– 70. ISBN 978-0-323-66162-1.

Mattey, M. and Kristiansen, B. (1999). A Brief Introduction to Citric Acid Biotechnology. In B.

Kristiansen, M. Mattey, and J. Linden (Eds), Citric Acid Biotechnology, London: Taylor
and Francis Limited.

McMurry, J. (2008). Carboxylic acids: Organic Chemistry (7th Edition). Belmont, C. A.

Thomson Brooks/Cole, Thomson learning Incorporation

Meister A. (1994). Glutathione-ascorbic acid antioxidant system in animals. The Journal of

Biological Chemistry, 269 (13), 9397–400

Molander Paal. (2003). The impact of column inner diameter on chromatographic performance
in temperature gradient liquid chromatography; International journal of chemical and
pharmaceutical sciences, 128(11),1341-5.

Michels A. and Frei B. (2012). Vitamin C. Caudill M. A, Rogers M. (edition). Biochemical,

Physiological, and Molecular Aspects of Human Nutrition (3rd edition). Philadelphia:
Saunders. pp. 627–654.

Mihaly Maria and Aurelia Meghea. (2014). Chromatic analysis of blue ballpoint pen inks and
related dyes. Research gate, 40(2), 25-28

49
Nathan N. and Patel P. (2021). Why is topical vitamin C important for skin health. Harvard
Health Publishing, Harvard Medical School. Retrieved October 14, 2022.

Patwekar S. (2015). HPLC method development and validation– A general concept.

International journal of chemical and pharmaceutical sciences, 6(1), 77-102.

Piper, P.W. (2011). Resistance of Yeasts to Weak Organic Acid Food Preservatives. Advanced
Application of Microbiology, 77, 97-113

Pooja Rejakumar and. Ijppr.Human (2022). An overview on high performance liquid

chromatography. Vol. 23 (4), 506-527.

Pragst, F., M. Herzler, and B.T. Erxleben (2004). Systematic toxicological analysis by high
performance liquid chromatography with diode array detection (HPLC-DAD). Clinical
Chemistry and Laboratory Medicine, 42(11), p. 1325-40

Qui, D., Qiu, K., Zhang, H., Wu, S., Han, Y., Wu, Y., Qi, G., & Wang, J. (2021). Organic Acids
as Alternatives for Antibiotic Growth Promoters Alter the Intestinal Structure and
Microbiota and Improve the Growth Performance in Broilers. Frontiers in Microbiology,
11, 14- 29
Quitmann, H., Fan, R. and Czermak, P. (2014). Acidic Organic Compounds in Beverage, Food,
and Feed Production. Advanced Biochemical Engineering and Biotechnology, 143, 91–
141.

Raut, P.P. and S.Y. Charde. (2014). Simultaneous estimation of levodopa and carbidopa by
RPHPLC using a fluorescence detector: its application to a pharmaceutical dosage form.
Luminescence, 29(7), p. 762-71.

Roa Engel, C.A., Straathof, A.J.J., Zijlmans, T.W., van Gulik, W.M., van der Wielen, L.A.M.,
2008. Fumaric acid production by fermentation. Applied Microbiology and
Biotechnology, 78, 379- 389

Sanjay Kumar. (2010) Importance of Rp-HPLC In Analytical Method Development: A Review;

International Journal of Pharmaceutical Sciences and Research. 6(2), 27-39.

Shinde Manjiri. (2021) A Review on HPLC Method Development and Validation. International
Journal of Research and Development, 10(6), 94-95.

50
Smith, J. and Hong-Shum, L. (2011). Food additives data book. Oxford, Wiley

Thakker, J. N., & Desai, J. D. (2017). Organic acids in the rhizosphere of maize: Interactions
with phosphorus availability and uptake. Journal of Plant Nutrition and Soil Science,
179(2), 163–174.

Theron, M. M., & Lues, J. F. (2011). Organic Acids and Food Preservation. CRC Press1

Tretter, E. D., & Lantzsch, H. J. (2016). Organic acids in the rhizosphere of maize: Interactions
with phosphorus availability and uptake. Journal of Plant Nutrition and Soil Science,
179(2), 163–174.

Ullmann's Encyclopedia of Industrial Chemistry. Wiley. 2005. pp. 17624/28029

V. C. Gracia. (2006). Typification of Fruit Juices by HPLC Flavonoid Analysis. Research gate,
56(06), 575-575.

Walker Valerie. (2002). Solid-phase extraction in clinical biochemistry. SAGE journals, 12, 464-
477.

Washburn C. and Jensen C. (2017). Pretreatments to prevent darkening of fruits prior to canning
or dehydrating. Utah State University.

Yang, S.T., Zhang, K., Zhang, B., Huang, H., 2011. Biobased chemicals – fumaric acid. In:
Young, M.M. (Edition), Comprehensive Biotechnology. Elsevier, Netherland, pp. 163-
177

Yu Shao (2004). A stability-indicating HPLC method for the determination of glucosamine in

pharmaceutical formulations. Pharmaceutical and biomedical analysis, 2(1), 8-10

Zhang, M. (2016). Monitoring gradient profile online in micro- and nano-high performance
liquid chromatography using conductivity detection. Journal of Chromatography A,.
1460, 68-73.

51
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