Phytochemistry & Pharmacognosy

Laboratory Activity No. 7

ISOLATION OF CITRIC ACID

Objectives:
1. Have knowledge about citric acid, its uses, sources, and the
microorganisms employed for its production.
2. Determine the process of isolating and extracting citrci acid from
lemon juice.
3. Identidy the metabolic pathway involving citric acid and the different
methods of its commercial production.

Materials :
Thawed frozen lemon juice Graduated cylinder 10% Sodium hydroxide 10% calcium chloride
250 ml beaker solution Dist. Water
Dropper
Stirring rod Procedure:
Muslin cloth A. Extraction of Citric Acid
Filter paper Buchner funnel Erlenmeyer flask Hot plate
Wire gauze
Reagents: Wash bottle
dessicator

1. Measure 90ml of thawed frozen lemon (Citrus limon, Fam Rutaceae) juice
concentrate into a 250 ml beaker.
2. Carefully add 10% sodium hydroxide solution with stirring, until the mixture is slightly
alkaline. A distinct color change occurs at this point, the solution passing from a clear
yellow to a brownish color.
3. Strain the solution through the fine muslin to remove large particle pulp and then filter
through paper on a buchner funnel. The pores of the filter paper may become
clogged by the extract inspite of the previous straining. Should this occur, change
this paper in the funnel once or twice as required to complete the filtration.
4. Measure the filtrate, place in a beaker and add 5ml strirring constantly, of 10%
calcium chloride solution for each 10ml of the filtrate.
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College of Health Sciences 2 semester Notre Dame of Dadiangas University BS Pharmacy II
Phytochemistry & Pharmacognosy

5. Heat to a boiling and filter off the copious precipitate of calcium citrate from the hot
solution using a buchner funnel.
6. Wash the precipitate thus collected twice with small quantities of boiling water. 7.
Resuspend in a minimum quantity of cold water, heat to boiling at once and collect the
insoluble calcium by filtration.
8. Allow the salt to air dry, weigh, calculate the yield, and turn in the product to the
laboratory instructor.

B. Citric acid may be prepared from the citrate salt as follows:

1. Weigh the air-dried salt, place in a beaker and add 1N sulfuric acid
2. Allow the mixture to stand for a few minutes
3. Filter off the insoluble calcium sulfate, and concentrate the filtrate to small on a
steam bath. Citric acid crystallizes out.
4. Collect the crystals on a buchner funnel, dry, weigh, calculate the yield, and turn in
the product to the instructor.

Results/Observations:

A. Percentage Yield
a. Theoretical yield of citric acid from lemon juice: 4.38 grams
b. Weight of citric acid crystals obtained: 3.62 grams
c. % yield of citric acid from lemon juice: 82.6% or 83%
d. Calculation of % yield
% Yield = ����ℎ� �� ������ ���� x 100
����ℎ� �� ������ ���� ��������

B. Write the balanced chemical reaction

C6H807 (Citric Acid) + 3NaOH (Sodium Hydroxide) 🡪 Na3C6H5O7 (Trisodium
Citrate) + 3H2O (Water)

2Na3C6H5O7 (Trisodium Citrate) + 3CaCl2 (Calcium Chloride) 🡪 Ca3(C6H5O7)2

(Calcium Citrate) + 6NaCl (Sodium Chloride)

Ca3(C6H5O7)2 (Calcium Citrate) + 3H2SO4 (Sulfuric Acid) 🡪 2C6H8O7 (Citric

Acid) + 3CaSO4 (Calcium Sulfate)

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 College of Health Sciences 2nd semester Notre Dame of Dadiangas University BS Pharmacy II
Phytochemistry & Pharmacognosy

Questions:
1. How would you explain the widespread and distribution of citric acid in plants?
Citric acid is widely distributed in nature, found especially in citrus plants such as lemon,
orange, pineapple, currant, raspberry, grape juice fruit, as well as in animal bones,
muscles, and blood. It is is ubiquitous in nature because it is an intermediate in aerobic
metabolism through the TCA cycle whereby carbohydrates are oxidized to carbon
dioxide. Citric acid is synthesized with sugar, starch, molasses, grapes and other sugary
material fermentation, can be divided into anhydrous and two kinds of pure hydrate.
Citric acid is a metabolite in plants and plays an important role in photosynthesis and
cellular respiration. It contains three carboxylic acid (COOH) functional groups (FIG 1)
and can deprotonate to form a negatively charged citrate molecule.

2. In what metabolic pathway is citric acid involved?

Citric acid, 2-hydroxypropane-1,2,3-tricarboxylic acid, is a key metabolic intermediate
and is the starting point of the tricarboxylic acid (TCA) cycle. From the viewpoints of
production volume and utility, citric acid is one of the most important bioproducts.
The citric acid cycle, —also known as the tricarboxylic acid cycle (TCA cycle) or the
Krebs cycle—is a series of chemical reactions used by all aerobic organisms to
generate energy through the oxidation of acetate—derived from carbohydrates, fats,
and proteins—into carbon dioxide. The cycle provides precursors including certain
amino acids as well as the reducing agent NADH that is used in numerous biochemical
reactions. Its central importance to many biochemical pathways suggests that it was
one of the earliest established components of cellular metabolism; it may have
originated abiogenically.
The name of this metabolic pathway is derived from citric acid, a type of tricarboxylic
acid that is first consumed and then regenerated by this sequence of reactions to
complete the cycle. The cycle consumes acetate (in the form of acetyl-CoA) and water,
reduces NAD+ to NADH, and produces carbon dioxide. The NADH generated by the
TCA cycle is fed into the oxidative phosphorylation pathway. The net result of these two
closely linked pathways is the oxidation of nutrients to produce usable energy in the
form of ATP.

3. What Philippine materials could be potential commercial sources of citric acid?

Citric acid is most prevalent in citrus fruits, particularly in lemons and limes, while others
like oranges, grapefruits, berries, tomatoes, broccoli, carrots, and the likes, contain
appreciable amounts. The following are just few of a wide variety of Philippine materials
that could be potential commercial sources of citric acid:
a. Alsem / Wormwood — Artemisia absinthum, family Asteraceae
b. Mandarin Orange / Dalanghita — Citrus reticulata, family Rutaceae
c. Sinturis — Citrus nobilis, family Asteraceae
d. Tangerine Orange / Tangor — Citrus reticulata x Citrus sinesis, family Rutaceae

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 College of Health Sciences 2nd semester Notre Dame of Dadiangas University BS Pharmacy II
Phytochemistry & Pharmacognosy

4. Describe the various commercial processes for citric acid production.

a. Koji Fermentation or Solid State Fermentation Process
Solid-state fermentation (SSF) has been termed as an alternative method to produce
citric acid from agro-industrial residues. Citric acid production by SSF (the Koji
process) was first developed in Japan and is as the simplest method for its
production. SSF can be carried out using several raw materials. Koji fermentation is
conducted using the peels of banana (Musa acuminata) as an inexpensive substrate
for the production of citric acid using Aspergillus niger. Various crucial parameters
that affect citric acid production such as moisture content, temperature, pH,
inoculum level and incubation time were quantified. Moisture (70%), 28 degrees C
temperature, an initial pH 3, 10(8) spores/ml as inoculum and 72h incubation was
found to be suitable for maximum citric acid production by Aspergillus niger using
banana peel as a substrate.
Generally, the substrate is moistened to about 70% moisture depending on the
substrate absorption capacity. The initial pH is normally adjusted to 4.5-6.0 and the
temperature of incubation can vary from 28 to 30°C. The most commonly organism
is Aspergillus niger. However there also have been reports with yeasts. One of the
important advantages of SSF process is that the presence of trace elements may not
affect citric acid production so harmfully as it does in SmF. Consequently, substrate
pre-treatment is not required.

b. Liquid Surface Culture Fermentation Process

The first individual process for citric acid production was the liquid surface culture
(LSC), which was introduced in 1919 by Société des Produits Organiques in Belgium,
and in 1923 by Chas Pfizer & Co. in US. After that, other methods of fermentation,
such as submerged fermentation were developed. Although this technique is more
sophisticated, surface method required less effort in operation and installation and
energy.
In the classical process for citric acid manufacture, the culture solution is held in
shallow trays (capacity of 50-100 L) and the fungus develops as a mycelial mat on
the surface of the medium. The trays are made of high purity aluminum or special
grade steel and are mounted one over another in stable racks. The fermentation
chambers are provided with an effective air circulation in order to control
temperature and humidity. Fermentation chambers are always in aseptic conditions,
which might be conserved principally during the first two days when spores
germinate. Frequent contamination are mainly caused by Penicilia, other Aspergilli,
yeast and lactic bacteria. Refined or crude sucrose, cane syrup or beet molasses
are generally used as sources of carbon. When applied, molasses is diluted to 15-
20% and is treated with hexacyanoferrate (HFC).

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 Phytochemistry & Pharmacognosy

c. Submerged Culture Fermentation Process

The submerged fermentation (SmF) process is the commonly employed technique
for citric acid production. It is estimated that about 80% of world production is
obtained by SmF. Several advantages such as higher yields and productivity and
lower labour costs are the main reasons for this. Two types of fermenters,
conventional stirred fermenters and tower fermenters are employed, although the
latter is preferred due to the advantages it offers on price, size and operation (Rohr
et al., 1983). Preferentially, fermenters are made of high-grade steel and require
provision of aeration system, which can maintain a high dissolved oxygen level.
Fermenters for citric acid production do not have to be built as pressure vessels
since sterilization is performed by simply steaming without applying pressure.
Cooling can be done by an external water film over the entire outside wall of the
fermenter.
In SmF, different kinds of media are employed such as sugar and starch based
media. Molasses and other raw materials demand pre-treatment, addition of
nutrients and sterilization. Inoculation is performed either by adding a suspension of
spores, or of pre-cultivated mycelia. When spores are used, a surfactant is added in
order to disperse them in the medium. For pre-cultivated mycelia, an inoculum size
of 10% of fresh medium is generally required. Normally, submerged fermentation is
concluded in 5 to 10 days depending on the process conditions. It can be carried out
in batch, continuous or fed batch systems, although the batch mode more frequently
used.

5. Conclusion:
In conclusion, citric acid is a tricarboxylic acid naturally concentrated in a variety of fruits
and vegetables, and is particularly concentrated in lemons and limes. Citric acid is vital
in a lot of industries, as it is used in pharmaceutical preparations due to its antioxidant
properties, as a preservative, as an acidulant and anticoagulant, as well as a flavoring
agent in soft drinks and candies. It is also used as a complexing agent and starting
materials for functional compounds in the pharmaceutical and cosmetic industries.
Moreover, commercial methods in the production of citric acid are Koji Fermentation or
Solid State Fermentation Process, Liquid Surface Culture Fermentation Process, and
Submerged Culture Fermentation Process. Citric acid may also be commercially
extracted from a wide variety of microorganisms such as fungi, yeasts, and bacteria.

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College of Health Sciences 2nd semester Notre Dame of Dadiangas University BS Pharmacy II