EXTRACTION AND CHARACTERIZATION OF OIL FROM

DAUCUS CAROTA (CARROT) SEED

BY

EBEBE ESTHER ADAEZE

HSLT/BC/0369

TO

THE DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY

(SLT), FEDERAL POLYTECHNIC NASARAWA, NASARAWA STATE

APRIL, 2021
 ABSTRACT

The research shows the experimental study and physico-chemical properties of carrot seed oil.
Carrot seed oil is derived from the dried seed of wild carrot plant (Daucus carota) which is a
biannual root plant. Carrot seed contained high carotene a major pigment of carrot. Some
physic-chemical properties of the oil were analyzed and they were found within specification,
except saponification value which was found to be 47.57mg/g and it specific gravity which was
obtained to be 0.000916 which are lower than that of caper seed oil. It can be concluded carrot
seed oil is far removed from the ubiquitous orange vegetable and should not be mistaken for the
cheaper macerated carotene and it can be used in the treatment of dysentery, to expel worms
and for aromatization of food.
Introduction
Carrot (Daucus Carota) is a biannual root plant and one of the most commonly used
vegetables as food for humans. Its taproot which is the consumable part of carrot is
regarded as a very nutritious and healthy food supplement due to its high vitamins and
fiber content (Gonny et al., 2004) and (Öz can and Chalchat, 2007).Carrot is an erect
vegetable plant that flowers every two years. The plant produces the edible root and a
leafy top within the first year. If left in the ground for another year its flower and seeds
are produced (Negi et al, 2000).Oil from carrot seed is an essential oil that is extracted
from the seeds of carrot and should not be confused with the cheaper macerated ―Carrot
oil‖ made when people infuse the carrot material in a base oil (Staniszewska and Kula,
2001).
Scientists Asahina and Tsukamoto in 1925 were the 1st to isolate Carotene pigment. It is
one of the major components found in oil from carrot seed comprising approximately
66% of this essential oil. This sesquiterpene alcohol is thought to be formed in carrot
seeds during the vegetation period. Additional studies have indicated that carotene may
be involved in all elopathic interactions expressing activity as an antifungal, herbicidal
and insecticidal agent (Staniszewka and Kula, 2001).Oliveira et al.(2008)investigated the
antimicrobial activity of Hezelnut oil containing carotene against Gram positive (such as
Staphylococcus aureus), Gram negative (such as Escherichia coli) and fungi (such as
Candida albicans) and the results obtained revealed a high antimicrobial activity against
Gram positive bacteria. Also Hayashi et al. (2013)investigated the effect of carrot oil
extract on the bactericidal activity of a bovine lactoperoxidase system (LPOS) and the
result obtained showed that LPOS antimicrobial activity increased from 1.4 to 3.8 log
units by addition of20folddilutedcarrotextractcontaining carotene This research is focused
on production of local medicated soap from carrot seed oily extract for the treatment of
fungal (Trichophyton rubrum) skin infection.

Carrot Plant
The carrot (Daucus carota subsp. sativus) is a root vegetable, usually orange in colour,
though purple, black, red, white, and yellow cultivars exist. Carrots are a domesticated
form of the wild carrot, Daucus carota, native to Europe and southwestern Asia. The
plant probably originated in Persia and was originally cultivated for its leaves and seeds.
The most commonly eaten part of the plant is the taproot, although the greens are
sometimes eaten as well. The domestic carrot has been selectively bred for its greatly
enlarged, more palatable, less woody-textured taproot.

The carrot is a biennial plant in the umbellifer family Apiaceae. At first, it grows a rosette
of leaves while building up the enlarged taproot. Fast-growing cultivars mature within
three months (90 days) of sowing the seed, while slower-maturing cultivars are harvested
four months later (120 days). The roots contain high quantities of alpha- and beta-
carotene, and are a good source of vitamin K and vitamin B6, but the belief that eating
carrots improves night vision is a myth put forward by the British in World War II to
mislead the enemy about their military capabilities.

The United Nations Food and Agriculture Organization (FAO) reports that world
production of carrots and turnips (these plants are combined by the FAO) for the calendar
year 2013 was 37.2 million tonnes; almost half (~45%) were grown in China. Carrots are
widely used in many cuisines, especially in the preparation of salads, and carrot salads
are a tradition in many regional.

Etymology of Carrot

The word is first recorded in English circa 1530 and was borrowed from Middle French
carotte, itself from Late Latin carōta, from Greek καρωτόνor karōton, originally from the
Indo-European root ker- (horn), due to its horn-like shape. In Old English, carrots
(typically white at the time) were not clearly distinguished from parsnips: the two were
collectively called moru or more (from Proto-Indo-European *mork- "edible root", cf.
German Möhre).

Various languages still use the same word for "carrot" as they do for "root"; e.g. Dutch
wortel.
History of Carrot

Both written history and molecular genetic studies indicate that the domestic carrot has a
single origin in Central Asia. Its wild ancestors probably originated in Persia (regions of
which are now Iran and Afghanistan), which remains the centre of diversity for the wild
carrot Daucus carota. A naturally occurring subspecies of the wild carrot was
presumably bred selectively over the centuries to reduce bitterness, increase sweetness
and minimise the woody core; this process produced the familiar garden vegetable.

When they were first cultivated, carrots were grown for their aromatic leaves and seeds
rather than their roots. Carrot seeds have been found in Switzerland and Southern
Germany dating back to 2000–3000 BC. Some close relatives of the carrot are still grown
for their leaves and seeds, such as parsley, cilantro, coriander, fennel, anise, dill and
cumin. The first mention of the root in classical sources is from the 1st century; the
Romans ate a root vegetable called pastinaca, which may have been either the carrot or
the closely related parsnip.

The plant is depicted and described in the Eastern Roman Juliana Anicia Codex, a 6th-
century AD Constantinopolitan copy of the Greek physician Dioscorides' 1st-century
pharmacopoeia of herbs and medicines, De Materia Medica. Three different types of
carrots are depicted, and the text states that "the root can be cooked and eaten".

The plant was introduced into Spain by the Moors in the 8th century. In the 10th century,
roots from West Asia, India and Europe were purple. The modern carrot originated in
Afghanistan at about this time. The 11th-century Jewish scholar Simeon Seth describes
both red and yellow carrots, as does the 12th-century Arab-Andalusian agriculturist, Ibn
al-'Awwam. Cultivated carrots appeared in China in the 14th century, and in Japan in the
18th century.

Orange-coloured carrots appeared in the Netherlands in the 17th century, which has been
related to the fact that the Dutch flag at the time included orange. These modern carrots
were described by the English antiquary John Aubrey (1626–1697): "Carrots were first
sown at Beckington in Somersetshire. Some very old Man there [in 1668] did remember
their first bringing hither." European settlers introduced the carrot to colonial America in
the 17th century.

Outwardly purple carrots, still orange on the inside, were sold in British stores beginning
in 2002.

Chemistry of Carrot Seed Oil

Polyacetylenes can be found in Apiaceae vegetables like carrots where they show
cytotoxic activities. Falcarinol and falcarindiol (cis-heptadeca-1,9-diene-4,6-diyne-3,8-
diol) are such compounds. This latter compound shows antifungal activity towards
Mycocentrospora acerina and Cladosporium cladosporioides. Falcarindiol is the main
compound responsible for bitterness in carrots. Other compounds such as pyrrolidine
(present in the leaves), 6-hydroxymellein, 6-methoxymellein, eugenin, 2,4,5-
trimethoxybenzaldehyde (gazarin) or (Z)-3-acetoxy-heptadeca-1,9-diene-4,6-diin-8-ol
(falcarindiol 3-acetate) can also be found in carrot.

Cultivation of Carrot

Carrots are grown from seed and can take up to four months (120 days) to mature, but
most cultivars mature within 70 to 80 days under the right conditions. They grow best in
full sun but tolerate some shade. The optimum temperature is 16 to 21 °C (61 to 70 °F).
The ideal soil is deep, loose and well-drained, sandy or loamy, with a pH of 6.3 to 6.8.
Fertilizer should be applied according to soil type because the crop requires low levels of
nitrogen, moderate phosphate and high potash. Rich or rocky soils should be avoided, as
these will cause the roots to become hairy and/or misshapen. Irrigation is applied when
needed to keep the soil moist. After sprouting, the crop is eventually thinned to a spacing
of 8 to 10 cm (3 to 4 in) and weeded to prevent competition beneath the soil.

Extraction of oil
Extraction involves using two immiscible phases to separate a solute from one phase into
the other, therefore extraction is a separation process which involves the removal of a
solute from a mixture by chemical or physical method. Oil extraction is the isolation of
oil from animal by-products, fleshy fruits, olive palm, oil bearing seeds and nuts.
Extraction depends on five basic factors: Preparation, solvent, contact, Time, and
Temperature.
a. Preparation: All kinds of extractors require proper preparation of the oil seed to
achieve good resul begins in preparation” The prepar dehulling, flaking etc.
b. Solvent: Pure, good quality solvent, with enough flow rate for the tonnage
processed, and with a sufficient number of effective counter current stages.
c. Contact: correctly prepared material with high surface area, good porosity, and a
machine providing a good contact for effective washing and drainage.
d. Time: Perhaps 30-50mins for pear depending on bed depth, thickness of the pulp
and other variables to allow the dissolving and flow processes to occur.
e. Temperature: Hot enough to maintain rapid extraction in the time available
(Williams, et al, 1996).
Extraction is of two major types: Mechanical /physical method and Chemical/solvent
method.(Combination of both methods are being practiced in our various industries).

Mechanical/Physical Method of Extraction

This is a physical method that involves a mechanical process. It is an early means of
separation of oil from oil seeds using physical pressure to squeeze the oil out, this process
leaves intact the beneficial nutrient that is naturally contained in the seeds or flesh of the
fruit and eliminate any possibility of solvent residual in the oil. The efficient method is
the modern screw press, more than half of the oil is easily removed but perhaps 7% or 8%
residual oil is left in the solids, the process uses considerable horse power, there is
considerable wear and maintenance (Joe et al 1977).
Basic Principles of Solvent Extraction
In this project work, solvent extraction was the method applied therefore more attention
is needed.
Extraction by solvent involves the dissolution of the soluble constituents from a solid
material by a means of a suitable solvent known as leaching. This type of extraction is a
Liquid-Solid Extraction. It is a unit operation in which components are separated by
 dissolving them in liquids.
The solute may be a solid or a liquid dispersed through the insoluble solids coating their
surfaces dissolved in a liquid adhering (Mc Cade et al, 1985) or entrained in the solids
located within their cellular structure.
In extraction the method used is determined by the:
 Properties of soluble constituent present.
 Its distribution throughout the solid
 The nature of the solid.
 The particle size.
The solvent contacts the solute absorbed in solid and dissolves it and it therefore provides
the product mixture as liquid rich in the solute. Though the process was accomplished in
a variety of ways but its efficiency depends to a very large extent on attaining intimate
contact between the liquid solvent and the solid containing the solute.
Three steps are involved in solvent extraction;
a. Change in the solute by dissolving in the solvent .The solute in the solid changes into
liquid as it dissolves in the solvent.
b. Diffusion through the pores of the solid with the solvent by the solute to the surface
of the solid.
c. The movement from the surface of the solid to the bulk of the solution. This method
is either used to concentrate a solid valuable product or remove impurity from the
solid such as pigments. In designing system for solvent extraction to produce a given
amount of extract from a given quality of solid materials, this is generally
accomplished using a material balance on the various components involved in the
process. Hutchison, and Dalziel, 2015).

Aim of the Study

The aim of this project is extraction and characterization of carrot seed oil This research
is focused on extraction and characterization of carrot seed oil extract for the perfume
industry, aromatization of food and treatment of skin infection.
 Objective of the Study

The objectives of the study are:

i. To extract oil from carrot seed

ii. Characterization of oil

Statement of the Problem

This research work involves the analysis of carrot seed, extraction of the oil.
Furthermore, this project will answer the following questions;
 Solvent extraction by solvent method
 What is the optimum yield of the particle size using n- hexane?
 Chemical properties (Acid value and saponification).

Scope and Limitation of the Study

Many research works has been done in giving a detailed composition found in carrot
seed. But this research account on the optimum route to:
 Extraction of oil from carrot seed.
 Characterization of the carrot seed.
 LITERATURE REVIEW
Carrot Seed oil

Carrot seed oil is the essential oil extract of the seed from the carrot plant Daucus
carota. The oil has a woody, earthy sweet smell and is yellow or amber-coloured to pale
orange-brown in appearance. The pharmocologically active constituents of carrot seed
extract are three flavones: luteolin, luteolin 3'-O-beta-D-glucopyranoside, and luteolin 4'-
O-beta-D-glucopyranoside. Rather than the extract the distilled (ethereal) oil is used in
perfumery and food aromatization. The main constituent of this oil is carotol.

Pressed carrot seed oil is extracted by cold-pressing the seeds of the carrot plant. The
properties of pressed carrot seed oil are quite different from those of the essential oil.

2.2 Carrot

Carrot (D. carota L., Apiaceae) is one of the most commonly used vegetables for human
nutrition. It is a tall robust biennial spiny-fruited herb growing in dried-out fields or
meadows. Carrots are cultivated worldwide. They are characterized by relatively
moderate requirements for climate and soil. Owing to their modest needs for cultivation
and storage, they can be sold fresh throughout the year (Baytop, 1984; Vogel, 1996; Fritz
and Stolz, 1989; Schaller and Schnitzler, 2000; Schieber et al., 2001). Their juices and
blends are among the most popular nonalcoholic beverages. A steady increase in carrot
juice consumption has also been reported from other countries. It is regarded as a healthy
food item because of its high vitamin and fiber content (Nilsson, 1987; Chen and Tang,
1998; Negi and Roy, 2000; Schieber et al., 2001; Gonny et al., 2004).

In the development of new oil seed crops interest has turned to the members of the
Umbelliferae family. The yields per hectare (4-30 dt/ha) and oil content of these oil crops
(8-24%) are not extremely high. But these agricultural crops, which contain spice plants
like caraway, celery, coriander, dill and parsley, are of interest due to their high amounts
of petroselinic acid (Kleiman and Spencer,1982; Rühl, 1993; Hondelman, 1985; Schuster,
1992; Reiter et al., 1998a). Carrot seed essential oil is widely used for its numerous
applications concerning the formulation of certain alcoholic liquors as well as aromatic
and fragrance compositions. It contains about 0.5-1.6% (v/w) essential oil. Its essential
oil is used for medicinal purposes such as diuretic, stomachic (Baytop, 1984; Bauer et al.,
1990; Lawrance 1992-1994; Mazzoni et al., 1999). The high intensity of harsh carrot-like
flavor in fresh plant products is commonly considered to be genetically determined and
has therefore been reduced by breeding in carrots. Nevertheless, unacceptable flavor can
occur at the consumer level and this is connected with suboptimal postharvest storage
conditions and harsh handling during distribution.

In carrots, bitter and harsh as well as flat, insipid flavors have been described in response
to ethylene exposure, mechanical stress or storage in low oxygen atmospheres (Simon,
1985; Lafuente et al., 1989; Seljasen et al., 2001; Seljasen et al., 2004). Various attempts
were made at utilizing carrot pomace in food such as bread (Ohsawa et al., 1994), cake,
dressing and pickles (Ohsawa et al., 1995), and for the production of functional drinks
(Henn and Kunz, 1996; Schieber et al., 2001). Several studies on the chemical
characterization of carrot seed and seed oil have been caried out (Mazzani et al.,1999;
Gonny et al., 2004; Zlatanow, 1994; Seljasen et al., 2004; Lie Ken Jie et al., 1996;
Ochocka and Lamparczyk, 1993; Parker et al., 2003).
Recently, one oil distilled from the blooming umbels of D.corota L. ssp. carota growing
wild in Poland was characterized by a high content of monoterpene hydrocarbons (84%),
and the major components were α-pinene (41%) and sabinene (18%) (Staniszewska and
Kula, 2001). No detailed study on the physical properties, chemical composition,
essential oil composition and mineral contents of the seeds of D. carota has been
performed so far. The aim of this study was to establish physical and chemical properties
of carrot seed and oil and chemical composition of essential and edible oil of the seed oil.
METHODOLOGY
Materials
1. Soxhlet extractor
2. Reflex condenser
3. Thermometer
4. Weighing balance
5. Filter paper
6. Density bottle
7. Burrette
8. Pippet
9. Heating mantle
Reagent
1. N-hexane
2. KOH
3. NaOH
4. Diethyl ether
5. Ethanol
6. Phenolphthalein
Materials Acquisition and Pre-treatment
Carrot seeds used were purchased locally from local farmers in Farin Gada, Jos, Plateau
State. They were cleaned to remove all foreign matter such as dust, dirt, stones, chaff.
Immature and broken seeds were discarded as well. Prior to a chemical analysis, 300 g of
carrot seed were ground to pass a 0.5 mm screen. The analytical grade n-hexane solvent
B. Oil Extraction
150g of crushed carrot seed was placed inside a thimble made from thick filter paper,
which is loaded into the main chamber of the soxhlet extractor.
250ml of n-hexane was poured into a distillation flask and the soxhlet extractor was
placed onto this flask which was equipped with a condenser. The solvent was heated to
reflux at constant temperature of 68oC and the vapour travelled up a distillation arm into
the chamber housing the thimble of solid where the vapour is condensed and the warm
solvent flow under gravity and percolate through the bed of the crushed carrot seed to
extract the oil. When the solvent chamber is almost full, the chamber is automatically
emptied by a siphon side arm, with the solvent running back to the distillation flask. The
cycle was repeated for 8 hours. After the extraction, the extract was separated from the
solvent via a rotary evaporator (Jensen, 2007).
C. Oil Characterization
The following physicochemical properties of the carrot seed oily extract determined
include: colour, density, specific gravity, viscosity, amount of oil yield, boiling point,
refractive index, acid value, iodine value and saponification value.
Boiling Point: The boiling point of the oil was determined by heating the oil in a beaker
on a heating mantle. The oil was observed carefully in the presence of a thermometer,
immediately the oil started circulating and bubbling, the temperature on the thermometer
was read and recorded as the boiling point of the oil.

Density: The density of the carrot seed oil was determined by taking the weight of
sample and divides it by its corresponding volume (TSE, 1971).
Density = Mass
Volume

Specific Gravity: The specific gravity was calculated by dividing the oil density by the
density of water (1000 kg/m3) (TSE, 1971).
Specific Gravity = Density of oil/ Density of water
Determination of Saponification Value: Five (5) g of the oil sample was weighed into a
conical flask. 50 ml of 0.5N KOH was added to the sample, a reflux condenser was
attached to the flask and heated for 30 minutes for perfect dissolution of the sample. It
was allowed to cool then 1 ml of phenolphthalein indicator solution was added and the
content was titrated with 0.5N HCl to an end point. The same process was repeated using
blank sample (Saad et al., 2007).
The saponification value was calculated using the formula;
Saponification value = 56.1 N (V2-V1)
Weight of sample

Where; 56.1 = Molecular weight of KOH N = Normality of the KOH

V1 = Titre value for sample V2 = Titre value for blank

Detrmination of Acid Value: Five (5)g the oil sample was weighed into clean
conical flask and mixture of 25 ml diethyl ether and 25 ml ethanol was added
and used to dissolve the oil in the mixed neutral solvent. 1 ml of phenolphthalein
added and the solution was carefully titrated with 0.1N KOH until a pink colour
which persists for 15 seconds was obtained (Saad et al., 2007)
The acid value is calculated as thus;
Acid Value = 56.1 x N x V
M

Where; 56.1 = molecular weight of KOH

N = normality of KOH
V = volume of KOH used
M = mass of the sample
 RESULT

4.1 Results

Physio-chemical properties Values

Boiling point 1600c
Density 0.916kg/m3
Specific gravity 0.000916
Saponification value 47.57mg/g
Acid value 50.48mg/kOH/g
Colour Amber-coloured.

DISCUSSION, CONCLUSION AND RECOMMENDATION

Discussion
The table above shows the quality standard of a extracted oil produced. The oil was
yellow or amber in colour this may be due to the presence of carotene pigment in carrot
seed oil. Ranken et al (1997) reported that most oil extracted from oil seed are either
chlorophyll or carotene in the seed. The characteristic yellowish-red of natural fat is
caused by the presence of polyene carotenoid, the specific gravity obtained was 0.000916
lower than that of caper seed oil (1.084 – 1.1045) as reported by Akgul and Ozcan
(1999). Ranken et al (1997) reported that the boiling point of most essential oil ranges
from 1500c – 3000c, when they are distilled with water. The boiling point obtained was
1600c, therefore making it suitable for you. The saponification value was 47.57mg/g
which is low therefore making it ability to form soap low. Saponification value are
agreed with pearson’s (1981) ranges from 127 – 196mg/g. acid value was
50.48mg/kOH/g. The result of this work shows that carrot seed oil can be used
industrially for the product of cosmetic and in aromatization of food.

Conclusion
Carrot seed oil is an essential oil extract of the seed from carrot plant Daucus carota. The
oil has a woody earthy sweet smell and yellow or amber in colour. The main constituent
of oil is carotol, it blends well with a wide range of essential oil. The oil of carotene
contain the well known pigment carotene, but does not contain vitamin A or E, or pro-
vitamin A, which are found in the root rather than the extract, the distilled (Ethereal) oil
is used in perfumery and food aromatization. Carrot seed oil perhaps one of the most
underappreciated essential oil, but it is known to have antiseptic, vermifuge
(Antiparasitic) properties. And it can be used in the healing of boils, ringworms and other
skin disorder, it helps to ease ulcers. Carrot seed oil is also widely used in vapotherapy as
a brilliant stress and anxiety buster and an effective enhancer of respiratory and digestive
function.

Recommendation
NAFDAC should create awareness to all companies that manufacture medicated soap to
use carrot seed oil because it can be use for the treatment of fungal infection such as
Trichophyton Rubum and it will go along with in minimizing the cost of additives to
provides antimicrobial property to the common soap. Also manufacturer should be
educated on the use of carrot seed in food aromatization. People should be informed by
the government in the danger of using additives for treatment and should be encourage to
use natural oil without additives like carrot seed oil.
Furthermore the Federal Ministry of Health should recommend carrot seed oil for
treatment of skin disorder. Private sector should also see the need to invest in the farming
and the production of carrot. Also Federal Polytechnic Nasarawa especially the
department of Science Laboratory Technology should find the need to invest more in the
chemical society to create avenue for job creation and reduction of unemployment in the
society. The entrepreneur center should also include these experiment as a means to
generate income and employment.

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APPENDIX
BOILING POINT

Boiling point 1600c

DENSITY

Density = Mass
Volume

Mass of the density bottle = 16.9g (M1)

Mass of the density bottle + the oil = 39.8g (M2)

Mass of the oil = M2 – M1

= 39.8 – 16.9 = 22.9g

Volume of oil = 25ml

Density = mass
Volume

22.5
=0.916 kg /m3
25

SPEFIC GRAVITY
Specific gravity = Density of oil
Density of water
= 0.916
1000
= 0.000916
COLOUR
The oil colour was amber-coloured in appearance
SAPONIFICATION VALUE
Saponification value was calculated using the formula
N
56.1 X (V 2−V 1)
weig h t of t h e sample
Where
56.1 = molecular weight of koH
N = normality of koH
V1 = titre of the sample
V2 = titre value of the blank

Saponification value of oil

Burette reading (cm3) Rough titre value 1st titre value 2nd titre value
First reading (cm3) 2.50 4.80 24.50
Initial reading (cm3) 50.00 47.50 42.70
Volume of acid (cm3) 47.50 42.70 40.20

Average titre volume = 42.70 + 40.20

2
= 82.9
2
V1 = 41. 42cm3

Saponification value for blank

 Burette reading (cm3) Rough titre value 1st titre value 2nd titre value
Final reading (cm3) 49.90 49.90 49.90
Initial reading (cm3) 0.00 0.00 0.00
Volume of acid (cm3) 49.90 49.90 49.90

Average titre value = 49 .90 + 49.90

2
= 49.90cm3
n
Saponification value 56.1 X (V 2−V 1)
weig h of sample
= 56.1 x 0.5 (49.90 – 41.42)
5
= 56.1x 0.5 x 8.48
5
= 237.864
5
= 47.57mg/g

ACID VALUE
The acid value is calculated using a formula
Acid value = 56.1 x N X V
 N
Where
56.1 = molecular weight of KOH
N = normality of KOH
V = volume of KOH
M = mass of sample
N = 0.1
M = 5g
V=?

Burette reading (cm3) Rough titre value 1st titre value 2nd titre value
Final reading (cm3) 2.00 2.00 2.02
Initial reading (cm3) 50.00 48.00 46.00
Volume of acid (cm3) 48.00 46.00 43.98

Average titre value = 46.00 + 43.98

2
= 89.98
2
V = 44.99
Acid value = 56.1 x N x V
M
= 56.1 x o.1 x 44.99
5g
= 252.3939
5
= 50.48mg/koH/g