BAB I

INTRODUCTION

1.1 Background

Polycyclic aromatic hydrocarbons which are often called PAHs (polycyclic

aromatic hydrocarbons) are a group of organic compounds that have two or more

aromatic rings, usually produced from incomplete combustion such as fossil fuels,

wood, or during food processing such as combustion and fumigation. Polycyclic

aromatic hydrocarbons (PAH) or also known as polycyclic organic matter (POM)

are aromatic molecules consisting of two or more aromatic ring molecules arranged

by carbon and hydrogen atoms (Scott, 2015).

There are approximately 10,000 polycyclic aromatic hydrocarbon (PAHs)

compounds, and the most wellknown of those are acenaphthene (ACE),

acenaphthylene (ACY), anthracene (ANTH), benzo[a]anthracene (BaA),

benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF), fluoranthene (FLT),

naphthalene (NAPH), and phenanthrene (PHEN). PAHs are colorless, white or pale

yellow-green solid organic compounds that contain at least two fused six-sided

benzene rings that include only carbon and hydrogen. Many PAHs are insoluble in

water, but a few of them vaporize easily. Although about 10,000 PAHs exist in the

environment, the US Environmental Protection Agency (US EPA) recognizes only

16 of them as priority pollutants owing to their carcinogenic properties. These 16

PAHs are NAPH, ACY, ACE, fluorene (FLU), ANTH, FLT, pyrene (PYR), BaA,

chrysene (CHR), benzo[b]fluoranthene (BbF), BkF, BaP, dibenzo[a,h]anthracene,

benzo[g,h,i]perylene (BghiP), and indeno[1,2,3-cd]pyrene (INPY). PAHs are

derived from the incomplete combustion of oil, coal, petrol, wood, garbage,

tobacco, meats, and other carboncontaining organic materials.

Few PAHs are known for their utilities, for example, NAPH is known for

its use as mothballs, and ANTH is used for making dyes, explosives, plastics,

lubricants, and moth repellents. The products that contain coal tars contain PAHs

at low concentrations as well. The primary sources of PAH emissions are smoke,

automobile emissions, and industrial exhausts of petroleum refineries, fossils fuel

power plants, and coking plants, paper mills. In addition, naturally occurring fires,

such as bush or forest fires, and active volcanoes release PAHs (Kurnia, etc., 2018).

Methods of determining PAH compounds usually employ chromatographic

techniques, such as gas chromatography and liquid chromatography. Research on

the analysis of PAHs has been conducted, including the development of extraction

methods for different types of samples and assembly of various types of stationary

phases. Research on analytical methods to detect PAHs is growing, including

research to increase sensitivity and find the separation factor of chromatography to

detect these compounds. Researchers continue to develop PAH separation methods

that are easier and cheaper in practice ((Kurnia, et al., 2018).

Technology chromatographic analysis is widely used for analyze organic

combinations, one of which is considerations with High Performance Liquid

Chromatography or High Performance Liquid Chromatography (HPLC). The

working principle of HPLC is a different molecule held at different times in the

column chromatography corresponds to the polarity of the structure the molecule.

Analysis by HPLC has several advantages such as easy to operate and possess High
resolution method. Therefore, the above needs to be shared with this paper which

contains the analysis of PAH use of HPLC (Hirjani, et al., 2018).

1.2 Formulation Of The Problem

The formulation of the problem of this paper are:

1. what is meant by polycyclic aromatic hydrocarbon compounds?

2. what does it mean with high performance liquid chromatography instrument ?

3. how to analyze polycyclic aromatic hydrocarbons using HPLC?

1.3 Purpose

The purpose of this paper are:

1. to find out what is meant by polycyclic aromatic hydrocarbon compounds.

2. to find out what a high performance liquid chromatography instrument.

3. to find out how to analyze polycyclic aromatic hydrocarbons using HPLC.

 BAB II

DISCUSSION

2.1 Polycyclic Aromatic Hydrocarbons

Polycyclic Aromatic Hydrocarbons (PAHs) are one of the main groups of

organic pollutants that are persistent for the environment. PAH is also known as

polycyclic aromatic hydrocarbons which are widely distributed and detected in

various media, including air, water, food, soil, sediment, industrial waste, animal or

human tissue. PAHs consist of two or more benzene rings in a linear or cluster

arrangement, with complex chemicals including carbon and hydrogen. Generally,

PAH exists as a yellow-green, white or pale solid with a characteristic odor. PAH

is formed due to a complex mixture due to natural and synthesized products. Based

on its formation mechanism, PAHs are divided into two different categories. The

categories are pyrolitic and petrogenic forms. Petrogenic PAH is their natural form

while pyrolitic PAHs come from incomplete combustion or pyrolysis of organic

matter, crude oil and its products (Arowojolu et al., 2018).

Polycyclic aromatic hydrocarbons (PAHs) or polynuclear aromatic

hydrocarbons are organic compounds that are mostly present as colorless, white, or

pale yellow solids. Polycyclic aromatic hydrocarbons consist of two or more single

fused aromatic rings with a pair of carbon atoms shared between rings in their

molecules. PAHs with six fused aromatic rings are often known as “small” PAHs,

and those with more than six aromatic rings are called “large” PAH. The general

characteristics of PAHs are high melting points, boiling points, occurrence as

solids, low vapor pressure and low aqueous solubility that further decrease with
increasing molecular weight. PAHs have good solubility in organic solvents

because they are highly lipophilic (Singh, et al., 2019).

Generally, PAHs can be formed during incomplete combustion of organic

matter from both natural combustion sources (forest and brush fres, volcanic

eruption) or man-made combustion sources (automobile emissions, cigarette

smoke, and aircraft). The food processing include (such as drying and smoking) and

cooking of food at high temperatures (grilling, roasting, frying) are major sources

of PAH production. Contamination of vegetables oils (including olive residue oils)

with PAH usually occur during technological processes like direct fire drying.

Polycyclic aromatic hydrocarbons (PAHs) can be formed in processes of

production/distillation of high-boiling petroleum products as a result of thermal

cracking (Singh, et al., 2019).

Polycyclic aromatic hydrocarbons (PAHs) omnipresent in environment and

formed of group of several chemical compounds, they are persistent in environment

with diferent structures and varied toxicity. PAHs toxic efects and cause inhibition

in the function of cellular membranes as well as with enzyme systems in organism

and they also afect environment (air, water and soil). PAHs can cause carcinogenic,

mutagenic, teratogenic efects and are potent immunesuppressants. The

International Agency for Research on Cancer classifes some PAHs as carcinogenic

to humans (Group 1, 2A or 2B). It is because of harmful efect of some of PAHs,

that diferent regulatory bodies have put restrictions on their content in various

industries. In this context, it becomes imperative to have methods that may help in

precise detection and quantitative estimation of PAHs in diferent matrices

(Singh, et al., 2019).

2.2 High Perfomance Liquid Chomatography (HPLC)

Chromatography is defined as a set of techniques which is used for the

separation of constituents in a mixture. This technique involves 2 phases stationary

and mobile phases. The separation of constituents is based on the difference

between partition coefficients of the two phases. The chromatography term is

derived from the greek words namely chroma (colour) and graphein (to write). The

chromatography is very popular technique and it is mostly used analytically. There

are different types of chromatographic techniques namely Paper Chromatography,

Gas Chromatography, Liquid Chromatography, Thin Layer Chromatography

(TLC), Ion exchange Chromatography and lastly High Performance Liquid

Chromatography (HPLC) (Thammana, 2016).

High Performance Liquid Chromatography which is also known as High

Pressure Liquid Chromatography. It is a popular analytical technique used for the

separation, identification and quantification of each constituent of mixture. HPLC

is an advanced technique of column liquid chromatography. The solvent usually

flows through column with the help of gravity but in HPLC technique the solvent

will be forced under high pressures upto 400 atmospheres so that sample can be

separated into different constituents with the help of difference in relative affinities.

In HPLC, pumps will be used to pass pressurized liquid solvent including the

sample mixture which is allowed to enter into a column filled with solid adsorbent

material. The interaction of each sample component will be varies and this causes

difference in flow rates of each component and finally leads to separation of

components of column (Thammana, 2016).

 Chromatography can be depicted as a mass exchange process including

adsorption. HPLC depends on pumps to pass a pressurized fluid and an example

blend through a section loaded with adsorbent, prompting the partition of the

specimen segments. The dynamic segment of the section, the adsorbent, is regularly

a granular material made of solid particles (e.g. silica, polymers, etc.) 2 μm to 50

μm in size. The segments of the example mixture/blend are isolated from each other

because of their distinctive degrees of connection with the retentive particles. The

pressurized fluid is commonly a blend of solvents (e.g. water, acetonitrile and/or

methanol) and is known as ‘mobile phase’. Its organization and temperature plays

an important part in the partition procedure by affecting the connections occurring

between sample segments and adsorbent (Thammana, 2016).

2.2.1 History

Preceding HPLC researchers utilized standard liquid chromatographic

methods. Liquid chromatographic systems were to an inefficient because of the

flow rate of solvents being reliant on gravity. Separations took numerous hours, and

some of the time days to finish. Gas chromatography (GC) at the time was more

effective than liquid chromatography (LC), in any case, it was trusted that gas stage

partition and investigation of extremely polar high atomic weight biopolymers was

impossible. GC was ineffectual for some organic chemists due to the thermal

instability of the solutes. Accordingly, alternative techniques were hypothesized

which would soon bring about the advancement of HPLC.

Taking after on the original work of Martin and Synge in 1941, it was

anticipated by Cal Giddings, Josef Huber, and others in the 1960s that LC could be

worked in the high-proficiency mode by decreasing the pressing molecule

measurement generously beneath the run of the mill LC (and GC) level of 150 μm

and utilizing pressure to expand the versatile stage velocity. These expectations

experienced broad experimentation and refinement all through the 60s into the 70s.

Early developmental exploration started to enhance LC particles, and the innovation

of Zipax, an externally permeable molecule, was promising for HPLC technology.

The 1970s achieved numerous advancements in equipment and instrumentation.

Specialists started utilizing pumps and injectors to make a simple configuration of

a HPLC system. Gas amplifier pumps were perfect since they worked at consistent

pressure and did not require release free seals or check valves for steady flow and

great quantitation

While instrumentational advancements were important, the historical

backdrop of HPLC is principally about the history and development of molecule

technology. After the presentation of permeable layer particles, there has been a

steady pattern to reduced molecule size to enhance efficiency. However, by

decreasing molecule size new issues arrived. The disadvantage from the

unnecessary pressure drop is expected to drive versatile liquid through the segment

and the trouble of setting up a uniform pressing of to a great degree fine materials.

Every time molecule size is diminished altogether, another round of instrument

advancement normally should occur to handle the pressure (Thammana, 2016).

2.2.2 Operation

The sample blend to be isolated and dissected is presented, in a discrete little

volume (commonly microliters), into the stream of mobile phase permeating

through the column. The segments of the sample travel through the segment at

various speeds, which are a component of particular physical connections with the

adsorbent (likewise called stationary stage). The velocity of every component relies

on upon its compound nature, composition of mobile phase. The time at which a

particular analyte elutes (rises up out of the column) is called its retention time. The

retention time measured under specific conditions is a distinguishing normal for a

given analyte.

Various sorts of columns are available, loaded with adsorbents varying in

molecule size, and in the nature of their surface (surface science). The utilization of

small molecule size packing materials requires the utilization of higher operational

pressure (back pressure) and regularly enhances chromatographic resolution (i.e.

the degree of division between sequential analytes rising up out of the column).

Sorbent particles might be hydrophobic or polar in nature. Basic mobile phases

utilized incorporate any miscible mixture of water with different natural solvents

(the most widely recognized are acetonitrile and methanol). Some HPLC systems

use without water mobile phases. The aqueous segment of the mobile phase may

contain acids, (for example, formic, phosphoric or trifluoroacetic corrosive) or salts

to help with the seperation of the sample components. The composition of the

mobile phase might be kept constant (isocratic elution mode) or changed

(inclination elution mode) during the chromatographic examination. Isocratic

elution is normally successful in the partition of sample components that are not

altogether different in their proclivity for the stationary stage. In gradient elution

the organization of the mobile phase is fluctuated ordinarily from low to high
eluting quality. The eluting quality of the mobile phase is reflected by analyte

maintenance times with high eluting quality delivering quick elution.

The selected structure of the mobile phase (additionally called eluent) relies

on upon the force of connections between different example parts ("analytes") and

stationary stage (e.g. hydrophobic connections in turned around stage HPLC).

Dependent upon their partiality for the stationary and mobile stages analytes

partition between the two. During the detachment procedure occurring in the

sample. This procedure is like what happens amid a liquid–liquid extraction

however is continuous, not step-wise. In this case, utilizing a water/acetonitrile

angle, more hydrophobic parts will elute (fall off the column) late, once the mobile

stage gets more packed in acetonitrile (i.e. in a versatile period of higher eluting

quality).

2.2.3 Principle of HPLC

With the help of a mobile phase pump water is flowed through the detectors

column. The snippet is inserted into the mobile phase flow by injection. In the

column separation of mixed components occurs. Because of the difference in the

strength of the interaction between the solutions to the stationary phase.

Solutions that have less interaction with the stationary phase come out of the

column first. Conversely, strong solutes interact with the stationary phase so the

solutes will come out of the column for longer. Each component of the mixture that

exits the column is detected by the detector and then recorded in the form of a

chromatogram.
2.2.4 Instrumentation

The HPLC instrumentation involves pump, injector, column, detector,

integrator and display system. In the column the separation occurs. The parts

include:

1. solvent reservoir: The contents of mobile phase are present in glass container. In

HPLC the mobile phase or solvent is a mixture of polar and non-polar liquid

components. Depending on the composition of sample, the polar and non-polar

solvents will be varied.

2. pump: The pump suctions the mobile phase from solvent reservoir and forces it

to column and then passes to detector. 42000 KPa is the operating pressure of

the pump. This operating pressure depends on column dimensions, particle size,

flow rate and composition of mobile phase.

3. sample injector: The injector can be a solitary infusion or a computerized

infusion framework. An injector for a HPLC framework should give 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).

4. columns: Columns are typically made of cleaned stainless steel, are somewhere

around 50 mm and 300 mm long and have an inward distance across of

somewhere around 2 and 5 mm. They are generally loaded with a stationary

phase with a molecule size of 3 μm to 10 μm. Columns with inner diameters of

< 2 mm are regularly alluded to as microbore segments. Preferably the

temperature of the mobile phase and the column should be kept consistent during

investigation.
5. 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,

massspectrometric and electrochemical identifiers.

6. data collection devices or integrator: 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 PC coordinates the reaction of the indicator to every part and

places it into a chromatograph that is anything but difficult to interpret.

The schematic representation of a HPLC instrument ordinarily incorporates

a sampler, pumps, and a locator. The sampler brings the sample into the mobile

phase stream which conveys it into the column. The pumps convey the mobile phase

through the column. The detector generates a sign relative to the measure of sample

component rising up out of the segment, consequently taking into consideration

quantitative investigation of the example parts. A computerized microchip and

software control the HPLC instrument and give information data. A few models of

mechanical pumps in a HPLC instrument can combine numerous solvents in

proportions changing in time, producing a sythesis slope in the portable stage. Most

HPLC instruments likewise have a column broiler that considers altering the

temperature at which the partition is performed.

 Image 1. Principle Scheme of HPLC

2.2.4 Types Of HPLC

Depending on the substrate used i.e. stationary phase used, the HPLC is

divided into following types:

1. Normal Phase HPLC: In this method the separation is based on polarity. The

stationary phase is polar, mostly silica is used and the non-polar phase used is

hexane, chloroform and diethyl ether. The polar samples are retained on column.

2. Reverse Phase HPLC: It is reverse to normal phase HPLC. The mobile phase is

polar and the stationary phase is non polar or hydrophobic. The more is the non-

polar nature the more it will be retained.

3. Size-exclusion HPLC: The column will be incorporating with precisely

controlled substrate molecules. Based on the difference in molecular sizes the

separation of constituents will occur.

4. Ion-exchange HPLC: The stationary phase is having ionically charged surface

opposite to the sample charge. The mobile phase used is aqueous buffer which

will control pH and ionic strength

2.2.5 APPLICATIONS OF HPLC

The HPLC has several applications in the fields of pharmacy, forensic,

environment and clinical. It also helps in the separation and purification of

compound.

1. Pharmaceutical Applications: The pharmaceutical applications include

controlling of drug stability, dissolution studies and quality control.

2. Environmental Applications: Monitoring of pollutants and detecting

components of drinking water.

3. Forensic Applications: Analysis of textile dyes, quantification of drugs and

steroids in biological samples.

4. Food and Flavour Applications: Sugar analysis in fruit juices, detecting

polycyclic compounds in vegetables, analysis of preservatives.

5. Clinical Applications: Detecting endogeneous neuropeptides, analysis of

biological samples like blood and urine

2.3 Analysis Polycyclic aromatic hydrocarbon (PAH) using HPLC

2.3.1 Comparison Of Fve Diferent HPLC Columns With Diferent Particle

Sizes, Lengths And Make For The Optimization Of Seven Polycyclic

Aromatic Hydrocarbons (PAH) Analysis

The aim of this study is to evaluate the performance of fve diferent

analytical columns for the analysis of seven carcinogenic polycyclic aromatic

hydrocarbons namely Benz[a]anthracene, Chrysene, Benzo[j]fuoranthene,

Benzo[e]Pyrene, Benzo[k]fuoranthene, Benzo[a]Pyrene, Dibenzo[a,h]anthracene

and select the best column in terms of separation, peak shape and analysis time and

to use the selected column under optimized analytical condition for the detection

and estimation of polycyclic aromatic hydrocarbons (PAHs) in light cycle oil; one

of aromatic rich petroleum stream. The performance of fve diferent analytical

columns with diferent lengths, particle sizes and make were compared for analysis

of above PAHs using HPLC with PDA detector and monitoring the HPLC–UV

signals at 254 nm wavelength. Chromatographic parameters including retention

time (tR), resolution (R), limit of detection, limit of quantifcation, number of

theoretical plates (N), height equivalent to theoretical plate (HETP) and reduced

plate height (h) were evaluated and compared on mentioned columns for analysis

of PAHs under study. The mobile phase acetonitrile (95%):water (5%) was used at

fow rate of 1.5 mL/min and the injection volume in all the case was 20.0 µL. The

best results w.r.t time and acceptable resolution were obtained when these PAHs

were analyzed using the shorter Agilent Eclipse PAH column having 1.8 µm,

100 mm×4.6 mm id.

A. Materials and methods

1. Reagents and standards

Acetonitrile, dicholromethane and n-Hexane (free from PAHs) were

obtained from M/s Rankem, India. Highly pure (> 99%) Benz[a]anthracene,

Chrysene, Benzo[j]fuoranthene, Benzo[e]Pyrene, Benzo[k]fuoranthene, Benzo[a]

Pyrene, Dibenzo[a,h]anthracene were procured from M/s Aldrich Chemical

Company, Inc., Milwaukee, WI, USA and used directly without any purifcation.
All solvents were of HPLC grade. Pure water was obtained from a Milli-R/Q water

system (Millipore, Bedford, MA, USA).

2. Standard preparation

Stock solution of above PAH standards were prepared in the range of

90–110 ppm in hexane at 30 °C by weighing appropriate weight. Appropriate

volume of stock solutions of these polycyclic aromatic hydrocarbons were placed

in 10 mL volumetric fask and were let to dry under gentle stream of N2 at 35 °C.

After drying these STD were diluted (1.0–0.0625 ppm) using Acetonitrile:water

mixture in the ratio of 80:20.

2.3.2 Analytical Method Validation and Rapid Determination of Polycyclic

Aromatic Hydrocarbons (PAHs) in Cocoa Butter Using HPLC-FLD

A simple, fast and ecological analytical method using a semi-automatic fat

extractor and HPLC-FLD (fluorescence detection) for determination of polycyclic

aromatic hydrocarbon markers i.e. benzo(a)anthracene (BaA), chrysene (Chr),

benzo(a)pyrene (BaP) and benzo(b)fluoranthene (BbF) in cocoa butter has been

validated. Validation’s procedure performed out in concordance with French

standard NF V03-110 (2010) was based on existing polycyclic aromatic

hydrocarbon (PAH) determination methods in various smoked foodstuffs and

edible vegetable oils. Determination of correlation coefficients for specific PAHs

ranged from 0.9992 to 0.9998. Respective values of limits of detection were 0.010,

0.011, 0.033 and 0.029 μg kg−1 and those of quantification were 0.035, 0.038,

0.111 and 0.098 μg kg−1 for BaA, Chr, BbF and BaP. Both values of repeatability

and intermediary precision tests coefficients of variation were less than 5%.

Recovery scores of four PAH markers matched EU standard 836/2011

recommendations. Sum of four PAH markers (BaA, Chr, BbF, BaP) contents varied

from 5.42 ± 0.58 to 11.37 ± 0.01 μg kg−1 whereas those of BaP was comprised

between 0.26 ± 0.00 and 1.75 ± 0.13 μg kg−1 in 20 cocoa butter samples extracted

from raw cocoa bean stored at Ivorian cocoa farmer levels.

A. Material and Methods

1. Sample Collection

A total of 20 raw cocoa beans samples (250 g each) were collected at farmer’s

level. Cocoa beans were fermented in banana leaves for 6 days and then sun-dried

on plastics tarpaulins before storing at a traditional smokeless warehouse. Cocoa

beans samples were packed in aluminium foil and then transported for analysis to

Montpellier (France).

2. Reagents and Standards

Solvents such as acetonitrile, cyclohexane, and ethanol used for analysis were

of HPLC grade. They were purchased from Sigma (St. Louis, MO, USA).

Petroleum ether and potassium hydroxide were purchased, respectively, from Ca rl

o E r ba, (Val de Re uil, F ra nce ) a n d Pa n reac (Barcelona, Spain). Ultra-pure

deionised water was purified by Millipore ultra-pure system (#1694 Millipore

Milli-Q Plus Water Purification System). A controlled quality cocoa butter

containing standards of benzo(a)anthracene (BaA) (1.00 μg kg−1), chrysene (Chr)

(1.34 μg kg−1), benzo (b) fluoranthene (BbF) (0.74 μg kg−1), benzo(a)pyrene

(BaP) (5.44 μg kg−1), indeno(1,2,3-cd)pyrene (Id) (0.39 μg kg−1) and

benzo(g,h,i)perylene (BghiP) (0.45 μg kg−1 ) were purchased from FAPAS. The

mixture of 16 PAHs standard such as BaA (10.720 μg ml−1), Chr (10.493 μg ml−1),

BbF (10.683 μg ml−1) and BaP (10.725 μg ml−1) in 1 ml of acetonitrile (Supelco,

Bellefonte, PA, USA) was used for the calibration of HPLC analytical conditions.

Glasswares were carefully washed and rinsed with distilled solvents (hexane)

before using.

3. Apparatus

An automatic extraction system equipment (Soxtec™ AVANTI 2050) was

used for fat extraction from cocoa beans. An HPLC chains DIONEX of

ULTIMATE 3000 type coupled to a fluorescence detector (RF 2000) was used for

PAHs determination. The column thermostat was at 30 °C and was C18 reverse

phase (Uptisphere 300 Å WTF), 250 × 4.6 mm ID, 5-μm particle size (Interchim,

Montluçon, France).

4. Determination of Moisture Content

Moisture content was determined as per ISO (1980-12-01). Cocoa powder

sieved at 0.5 mm was dried in an oven at 103 °C for 16 h. All moisture contents

determinations were performed in triplicate Servent et al. (2017)

5. Soxtec Fat Extraction

The fat was extracted from cocoa nibs using a previously described method

by Rosalía et al. (2014) and Servent et al. (2017). The Soxtec-Avanti 2055 semi-

automatic device (Foss, Hillerod, Denmark), with petroleum ether as solvent, was

used for fat extraction. After optimization, the extraction time was set at 1 h at

110 °C. In order to reach this purpose, about 3 g of cocoa powder sourced from

crushing (25 μm) of previously liquid nitrogen frozen shelled cocoa beans were put

in extraction cartridges containing 65 ml of extraction solvent. The defatted matter

was discarded, and the butter without solvent was stored at − 20 °C until analysis.

The butter must be heat at 60 °C for 20 min to be fully liquid and homogeneous.
6. Extraction of PAHs from Cocoa Butter

Extraction and analysis of PAHs were according to Cirad method which

aimed at the determination of four PAHs in smoked meat products. This method

was optimized and validated in-house for the determination of four PAH markers

named benzo(a)anthracene (BaA), chrysene (Chr), benzo(a)pyrene (BaP) and

benzo(b)fluoranthene (BbF) in the cocoa butter. (1) A test specimen of 1 g of cocoa

butter resulting from a sample or a reference material is weighed in a tube to

centrifuge out of glass, to which 6-ml solution of potassium hydroxide in ethanol

(KOH, 1 N) and a bar magnet there were added. (2) The unit is put to saponify in a

hoting bath with 80 °C during 1 h under agitation with 450 rotaries per minute

(rpm). (3) The tube is then withdrawn from the hoting bath and 6 ml of cyclohexane

are added there. (4) The unit is given to saponify with the hoting bath with 80 °C

during 5 min under agitation with 450 trs min−1 . (5) At the end of saponification,

the tube is withdrawn from the hoting bath and 4 ml of ultra-pure water are added

there. (6) The whole is vortexed during 1 min to 1500 rpm then centrifuged during

5 min with 3000 trs min−1 . (7) The higher phase is taken using a Pasteur pipette

and transferred in another tube out of glass. (8) Three millilitres of cyclohexane are

added to the remaining phase to take again processes (6) and (7). (9) This operation

was repeated twice and the supernatants were eliminated by drying under nitrogen

flow in a bath at 40 °C. (10) The dried extract was taken again in 1 ml of acetonitrile

(ACN), vortexed at the same centrifugal force and then placed in a syringe equipped

with a PTFE filter to threshold of 0.22 μm. Then, it was filtered in 2-ml HPLC vials.
7. Method of PAHs Detection and Quantification

PAHs are proportioned by the use of chains HPLC DIONEX of type

ULTIMATE 3000 coupled to a fluorescence detector RF 2000. Chromatographic

determination of PAHs was performed using the following gradient: ACN/water

(60:40, v/v) at 100% ACN during 5 min, followed by a 40 min ramp to 100% water,

and finally, 100% ACN during 15 min. The flow rate was 1.0 ml min−1 , and the

detection was performed at previously selected excitation and emission

wavelengths as follows: 0–29 min, 270/385 nm; 29–34 min, 270/ 420 nm and 34–

60 min, 381/405 nm. An aliquot of 10 μl was injected into HPLC using an auto-

sampler. A curve of calibration at five points of concentration (2, 4, 8, 16, 32 ng

ml−1 ) carried out starting from a PAH mix standards solution. This curve permitted

to establish an adequate correlation between surfaces of the peaks and PAH

concentrations found in tested samples. The determination of PAH content of each

sample was carried out in triplicate and the average was calculated. Validation of

the Method for PAHs Content Determination The validation of the method for

PAHs content determination was performed according to the EU indications

(European Commission 2011) and to the specific recommendations of the French

standard (NF V03-110 1998). This procedure included successive steps and the

determination of various parameters: linearity of the calibration interval, limits of

detection and quantification, coefficients of variation for repeatability and

intermediary precision tests and accuracy tests for recovery rate.

8. Validation of the PAH Determination Method in Butter Extracted from Raw

Cocoa
 Figure 1 shows the chromatogram of four PAHs markers analysed at a

concentration of 8 μg l−1 with different times of retention for each marker. The pics

of benzo(a)anthracene (BaA), chrysene (Chr), benzo(b)fluoranthene (BbF) and

benzo(a)pyrene (BaP) appeared, respectively, at 22.3, 24.39, 28.8 and 34.23 min.

Values of the coefficient of linearity of all PAH markers are comprised between

0.9992 for BaP and 0.9998 for BaA (Table 1). The linearity of the calibration range

was validated by Fisher-Snedecor test at 1% risk (Table 2). The limits of detection

were 0.010, 0.011, 0.033 and 0.029 μg kg−1 and the limits of quantification were

0.035, 0.038, 0.111 and 0.098 μg kg−1 , respectively for BaA, Chr, BbF and BaP

(Table 3). Calculated variation coefficients of the repeatability tests were ranged

from 1.31 to 2.29% for a reference cocoa butter (Table 4). Variation coefficients

for intermediary precision tests were comprised between 1.54 and 4.12% for three

cocoa butter samples A–C extracted from different cocoa beans samples (Table 5).

The results of these tests clearly reflect accuracy of the PAH determination method

and the precision of HPLC-FLD technique. The recovery rates obtained were

between 70.89% (Chr) and 98.00% (BaA). The measured values of PAH marker

contents are significantly similar to those present in reference cocoa butter through

compliance tests of the accuracy at risk of 1% (Table 6).

9. Fat and Moisture Contents of Tested Raw Cocoa Samples

Fat and moisture contents of tested cocoa samples collected from cocoa

farmers level in Côte d’Ivoire varied, respectively, from 38.10 ± 0.42 to 53.88 ±

0.14% and 6.89 ± 0.01 to 7.92 ± 0.05%. The average values of fat and moisture

contents were about, respectively, 48.83 and 7.40% (Fig. 2).

10. PAH Contents of Butter Samples Extracted from some Ivorian Raw Cocoa

Collected

Average values of PAHs contents in butter extracted from cocoa beans

samples collected from cocoa farmers level in Côte d’Ivoire were comprised

between 5.42 ± 0.58 and 11.37 ± 0.01 μg g−1 and ranged from 0.26 ± 0.00 to 1.75

± 0.13 μg kg−1 , respectively, for Σ4PAHs (Σ4PAHs = sum of benzo(a)pyrene,

benzo(a)anthracene, benzo(b)fluoranthene and chrysene) and BaP (Fig. 3). All

analysed butter samples were characterized by PAH contents below European

specifications and standards.

B. Conclusion

The results of our study showed acceptability and reliability of an analytical

method for the detection and quantification of four major markers of PAHs named

benzo(a)anthracene (BaA), chrysene (Chr), benzo(b)fluoranthene (BbF) and

benzo(a)pyrene (BaP) in cocoa butter extracted from raw cocoa. A simple, reliable,

fast, safe and ecological analytical method for analysis of PAHs in cocoa butter is

validated. This method in accordance with the standards of Commission Regulation

(EU) No. 836/2011 could be used for efficient and reliable measurement of PAH

marker contents in cocoa butter.

2.3.3 Assessment of Polycyclic Aromatic Hydrocarbons (PAH) Concentration

in Sediments and Soils around Ibadan, Southwestern Nigeria

The concentrations of polycyclic aromatic hydrocarbons (PAHs) in

sediments and surface soils under different land use patterns were studied using a

High Performance Liquid Column-Fluorescence Detector (HPLC-FD) and Gas

Chromatography-Mass Spectrometry (GC-MS) systems. Two sets of separate

samples were collected. Set A involved ten surface soil samples and set B contained

five top soil and two sediment samples. The results show that the sum of 15US

Environmental Protection Agency (EPA) priority pollutant PAHs are present. The

concentration of PAHs in the sediment and surface soils range from 1.68 to 919 μg

kg -1 and 1.76 to 2926.68 μg kg -1 respectively. The results show also that the low

molecular weights are more than the high molecular weight, and the calculated

ratios of PHen/ANth , FllTH/PYR, and FLth/ (FLth+PYr) exceed the background

values, all these suggest that the PAHs are more of petrogenic sources. These results

show more of petrogenic origin than pyrogenic and others of mixed nature. The

average PAH-homologue concentrations are 3 rings >4 rings>5 rings 2 rings. PAHs

concentrations are higher in engine oil impregnated soil, incinerated soils and

stream sediments as against a non - oil industrialized areas. Most of the PAHs like

benzo[a] anthracene, chrycene, benzo [b] fluoranthene, benzo[ ghi] perylene have

properties that are carcinogenic, toxic, mutagenic and teratogic and are therefore

dangerous to health. The public is advised not to take in food that are derived from

such contaminated surface soils.

 1. Analytical Procedures

Analytical procedures involved extraction, separation and analyses of the

samples using High Performance Liquid Chromatography (HPLC) –Fluorescence

Detector (FD) in the laboratory. PAHs in the soils are extracted using Soxhlet

extraction. 5g of freeze-dried sample are filtered in a porous cellulose thimble

(25×70 mm) and placed in a Soxhlet extractor. The extractor is then fitted to a 100

mL flask containing 60 mL dichloromethane. The extraction is performed for 24

hrs. The PAHs are mixed with 50 mL dichloromethane in a 500 mL brown glass

bottle with Teflon lined caps and the mixture was shaken on a rotary shaker (200

rpm) at 25 °C. Thereafter, 20 mL of the dichloromethane layer is transferred to a

100 mL in the flask. All the extracts in the round bottom flask are dried by rotary

evaporation. The residues are then dissolved in 2 mL of cyclohexane while 0.5 mL

of the solute is transferred and purified with a silica gel column (8×220 mm) and

washed with a mixture of 1:1 of hexane and dichloromethane. The first 1 mL of

eluate is discarded because it contained non-polar saturated hydrocarbons and is

less retained than PAHs by silica gel. The second 2-mL aliquot of eluate is

collected, dried by sparging with N2 and then redissolved in 1 mL acetonitrile for

HPLC determination. The first set of samples collected is analysed using Gas

Chromatography-Mass Spectrometry.

2. Quantification of PAHs

Quantitative analysis of the soil and sediments extracts is performed by

using the high performance liquid chromatography (HPLC). 15 US Environmental

Protection Agency (USEPA) priority PAHs analyzed are Naphthalene (Na),

Acenaphthene (Ace), Fluorene (Flu), Phenanthrene (Phe), Anthracene (An),

Fluoranthene (FluA), Pyrene (Pyr), Benzo[a]anthracene(BaA), Chrysene(Chry),

Benzo[b]fluoranthene(BbF), Benzo[k]fluoranthene -(BkF), Benzo[a]pyrene (Bap),

Dibenz[a,h]anthracene (DBA), Benzo[g,h,i]perylene (BghiP), and Indenol[1,2,3-

cd]pyrene (IP) (EPA1984). Acenaphthylene was excluded from this study due to its

low fluorescence properties.The total PAHs concentration is regarded as the sum of

the concentration of 15 PAHs for each collected sample. In order to understand

PAH species the Low Molecular Weight (LMW) containing 2-4 ring and High

Molecular Weight (HMW) containing 5-6 ring are also determined The Shimadzu

Class-VP HPLC system equipped with a fluorescence detector (RF10AXL), a

gradient pump (LC-10AT) and a reversed phase column C18 (Varian ChromSpher

5 PAH, 250×4.6 mm) was used for the determination of PAHs in the soils and

sediments. An external standard mixture was used for quantification of the 15

PAHs.

3. Quality Control

The detection limit of the HPLC method for the 15 PAHs was in the range

of 0.12 μg g−1 to 1.57 μg kg−1. Method blanks (solvent) and spiked blanks

(standards of EPA610 PAH mixture, LA 96245, Supelco, USA spiked into soil)

were extracted and analyzed as described above. The recoveries and the relative

standard deviations of this method for 15 PAHs were in the range of 74 to 110%

and 0.53 to 0.57%, respectively. Results of blanks extracted under the same

conditions were below detection limits and analytical results were without recovery

ratio correction. Data were analyzed using known standard and current used

geochemical software package and statistical SPSS software.

4. Conclusions

Accumulation of PAH in topsoils and sediments is investigated in some

areas of Ibadan Southwestern Nigeria. The sum of 15 PAHs concentration range

from 1.68 to 919 μg kg for topsoil and 1.76 to 2926.68 μg kg for sediment. The

lower molecular weight (LMW) makes up the largest proportion of all the PAHs

while the high molecular weight (HMW) shows ing the lowest concentration. The

higher concentration of PAHs is found in sediment and soils collected from dump

site and mechanic village. The sources of the PAH into the environment are mainly

petrogenic.
 BAB III

CONCLUSION

Polycyclic aromatic hydrocarbons (PAH) or also known as polycyclic

organic matter (POM) are aromatic molecules consisting of two or more aromatic

ring molecules arranged by carbon and hydrogen atoms. HPLC is a technique in

analytical chemistry used to separate, identify, and measure each component in a

mixture. This method is suitable for compounds that are not volatile, unstable, and

have a large molecular mass. HPLC is most often used to: regulate levels of certain

substances such as amino acids, nucleic acids, and proteins in physiological fluids

in pharmaceutical preparations. For example of PAH analysis using HPLC is

“Comparison Of Fve Diferent HPLC Columns With Diferent Particle Sizes,

Lengths And Make For The Optimization Of Seven Polycyclic Aromatic For

example of PAH analysis using HPLC is a “Hydrocarbons (PAH) Analysis”

and “Analytical Method Validation and Rapid Determination of Polycyclic

Aromatic Hydrocarbons (PAHs) in Cocoa Butter Using HPLC-FLD” nznzx

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