Literature Survey

2.1 About the Compound

Oxalic acid is an organic compound with the formula C2H2O4. It is a white crystalline solid
that forms a colorless solution in water. Its condensed formula is HOOCCOOH, reflecting its
classification as the simplest di-carboxylic acid. Its acid strength is much greater than that
of acetic acid. Oxalic acid is a reducing agent and its conjugate base, known
as oxalate (C2O2−4), is a chelating agent for metal cations. Typically, oxalic acid occurs as
the dihydrate with the formula C2H2O4·2H2O. It occurs naturally in many foods, but
excessive ingestion of oxalic acid or prolonged skin contact can be dangerous. Its name
comes from the fact that early investigators isolated oxalic acid from wood-sorrel
(Oxalis) flowering plants. The chemical formula of oxalic acid is (COOH)2 and its molecular
formula is C2H2O4. Its molar mass is 90.03 g/mol. Its chemical structure is shown below. It is
the simplest dicarboxylic acid, and consists of just two carboxylic acid groups (COOH)
directly attached to each other at the carbon atoms.

2.1.1 Occurrence

(i) Biosynthesis

At least two pathways exist for the enzyme-mediated formation of oxalate. In one
pathway, oxaloacetate, a component of the Krebs citric acid cycle, is hydrolyzed to oxalate
and acetic acid by the enzyme oxaloacetase:

[O2CC(O)CH2CO2]2− + H2O → C2O2−4 + CH3CO−2 + H+

It also arises from the dehydrogenation of glycolic acid, which is produced by the metabolism
of ethylene glycol.

(ii) Occurrence in foods and plant

Calcium oxalate is the most common component of kidney stones. Early investigators
isolated oxalic acid from wood-sorrel (Oxalis). Members of the spinach family and
the brassicas (cabbage, broccoli, brussels sprouts) are high in oxalates, as
are sorrel and umbellifers like parsley. Rhubarb leaves contain about 0.5% oxalic acid, and
jack-in-the-pulpit (Arisaema triphyllum) contains calcium oxalate crystals. Similarly,
the Virginia creeper, a common decorative vine, produces oxalic acid in its berries as well as
oxalate crystals in the sap, in the form of raphides. Bacteria produce oxalates from oxidation
of carbohydrates. Plants of the genus Fenestraria produce optical fibers made from crystalline

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oxalic acid to transmit light to subterranean photosynthetic sites. Carambola, also known as
starfruit, also contains oxalic acid along with caramboxin.The formation of naturally
occurring calcium oxalate patinas on certain limestone and marble statues and monuments
has been proposed to be caused by the chemical reaction of the carbonate stone with oxalic
acid secreted by lichen or other microorganisms.

(iii) Production by fungi

Many soil fungus species secrete oxalic acid, resulting in greater solubility of metal cations,
increased availability of certain soil nutrients, and can lead to the formation of calcium
oxalate crystals.

(iv) Other

Oxidized bitumen or bitumen exposed to gamma rays also contains oxalic acid among its
degradation products. Oxalic acid may increase the leaching of radionuclides conditioned in
bitumen for radioactive waste disposal.

2.1.2 Biochemistry

The conjugate base of oxalic acid is the hydrogenoxalate anion, and its conjugate base
(oxalate) is a competitive inhibitor of the lactate dehydrogenase (LDH) enzyme. LDH
catalyses the conversion of pyruvate to lactic acid (end product of the fermentation
(anaerobic) process) oxidising the coenzyme NADH to NAD+ and H+ concurrently. Restoring
NAD+ levels is essential to the continuation of anaerobic energy metabolism
through glycolysis. As cancer cells preferentially use anaerobic metabolism (see Warburg
effect) inhibition of LDH has been shown to inhibit tumor formation and growth, thus is an
interesting potential course of cancer treatment.

Fig.1 Structure of Oxalic acid

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2.2 Physical properties:

 Colorless, odorless powder or granular solid.

2.3 Chemical properties:

Anhydrous oxalic acid is very hydrophilic and readily absorbs water. It is a strong acid. It is
the simplest dicarboxylic acid, and reacts as a typical carboxylic acid, forming the acid
chloride and ester derivatives. It is a good reducing agent and acts as a good chelating ligand
for various metal cations.
Oxalic acid is widely distributed in the plant in nature, most existing in the form of oxalic
acid salt. C.W. Scheele had for the first time manufactured oxalate in 1776.
Oxalate is the strongest acid among the dicarboxylic acid. Besides having the general
properties of the carboxylic acid, it also has reducing property and can quantitatively reduce
the seven valence manganese to bivalent manganese. This property is often used for
quantitative analysis of potassium permanganate.

C2H2O4 + 2 KMnO4 + 3 H2SO4 →K2SO4 + 2 MNSO4 + 8H2O + 10 CO2;

Oxalic acid can also reduce the trivalent iron into bivalent iron. Because of the high solubility
of the bivalent iron in the water, we can apply this principle to remove rust on the clothes.
Oxalic acid can react with phosphorus pentachloride to generate phosphorus oxychloride.

C2H2O4 + PCl5 → POCl3 + CO + CO2 + 2 HCL.

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Oxalic acid can react with many metals to produce oxalic acid salt. In addition to the alkali
metal salt and bivalent iron salts with the rest of the oxalic acid salt being poorly soluble in
water. Some metal salt, although is poorly soluble in water, can generate complex that is
soluble in water.

Fe2 (C2O4) 3 + 3 K2C2O4 + 6 H2O →2 K3 [Fe (C2O4) 3] • 6 H2O.

Upon heating, alkali metal and alkaline earth metal oxalic acid salt can lose carbon monoxide
and form carbonates with carbonate continuing to be subject to heating to be further
decomposed into oxide and carbon dioxide. The oxalic acid salt of nickel, cobalt and silver
can finally produce metal instead of nonmetal oxide. The decomposing products of the
oxalate are carbon dioxide, carbon monoxide and water.

2.4 Different processes for the manufacturing of the compound

2.4.1 Preparation of oxalic acid by molasses by using nitric acid

1. Laboratary preparation
The sugar beet molasses used in the experiment contained 48.1% sucrose, 3.2% invert sugar
and 19.4% water. The chemicals employed were 65% HNO3 and 93% H2SO4. As catalyst,
7.5% V2O5 on porous silica gel support was used.

2. Process in detail
Three reactor were used in the experiments. The details of the Experimental setup are given
in Fig. 1. While the reactor 1 was used to produce oxalic acid from sugar beet molasses with
mixed nitric acid and Sulphuric acid in the optimum conditions of it was simultaneously
utilized as a source of nitrogen oxides needed as reactant in the second and third reactors. The
reaction gases released from the final reactor were absorbed in the columns at 0°C. All of the
reactants were prepared per 50 g molasses for each reactor. A predetermined amount of
Vanadium Pentoxide catalyst was added to each reactor. The magnetic stirrers, heaters,
cooling water and a cooler were turned on and air started to bubble through. Mixed acid was
added to the reactor at a rate of 6 ml/min through a funnel to the neck of the ask. The
HNO3+H2SO4 mixture was added from the dropping funnel as a thin stream. Intially the

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reaction in the reactor was started. The reaction temperature was controlled by adjusting the
heating or cooling it with water through a water bath. The Oxides of Nitrogen evolving from
the reaction in the reactor were carried, by air to the second and third reactors. These Oxides
were meant to act as oxidizers in the second and third reactors that contained enough H2SO4,
but no HNO3. In order to see how the conditions in the second reactor ejected the Oxalic acid
yield, temperature, quantity of sulphuric acid, catalyst, additional water and total air flow rate
for the two reactors were chosen as parameters. A similar study was carried out for the third
reactor as well. After the addition of acid-mixture to the reactor, evolution of brown-coloured
Nitrogen Oxides was visible immediately that indicates a rapid reaction. However, it took the
reaction in the second reactor 16 min to give visible gaseous products, and another 45 min
period elapsed before any coloured gas evolution started in the third
reactor.
Fig. 2. Flow diagram of experimental setup; a1; a2: absorption column; C1;C2;C3:
condenser; m: manometer; R1;R2;R3: reactor; r: rot meter; S1; S2; S3: sampling valve; t1; t2;
t3: thermometer.
Based on these observations the delay for the start of the reaction in the third reactor was
assumed to be 61 min and this was used in the kinetic studies. The reaction was assumed to
be completed when the colour of the exhaust gases turned pale yellow. The reaction mixture
was cooled to about 0°C and Oxalic acid crystals were separated by vacuum . Approximately
eighty percent of the filtrate was evaporated gradually in a dish at about 45°C and then it was
cooled to 5°C. The needle-shaped crystals of oxalic acid were separated again by vacuum
filtration, and crystals were combined with the previous lot. For recrystallization, the crude
product was dissolved in hot water, filtered and cooled to 0°C. Then, pure crystals were
separated, and the residual filtrate was again evaporated in a dish at about 45°C by the same
manner. The saturated solution was cooled to 0°C and filtered. The crystals were dried at
about 60°C and weighed. The product was checked by determining the melting point,
elemental composition and percent purity.

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Fig.2. Flow diagram of experimental setup

2.4.2 FERMENTATION BY USING POTTASIUM FERROCYANIDE

Optimum conditions for treatment of this molasses sample, which in many respects is similar
to the procedure of Eisenman, were found to be as follows: To 340 g of beet molasses
partially diluted were added 0.60 gm of Potassium Ferrocyanide (in solution), and the whole
was made to 1 liter with distilled water. Ten g of diatomaceous earth were added and mixed
thoroughly. The medium thus prepared was allowed to stand overnight in a graduated
cylinder or similar container at approximately 6O C. The medium was placed in the
fermentation containers and autoclaved at 120O C for 15 minutes. Then it was kept still for 4
days. The final medium thus prepared contained approximately 15 per cent sugar. A total of
four samples of straight house beet molasses from different localities were treated with
Ferrocyanide. All of the samples gave satisfactory Oxalic acid& citric acid yields, although
optimum conditions were established for only one sample, no. 1. The Ferrocyanide level, pH,
and sterilization conditions required for maximum yields with the different samples was
varied considerably. Thus yields obtained with the four samples of molasses were sterilized

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with the pH unadjusted . Under these conditions, molasses no. 1 gave better results when
sterilized, the other three apparently giving better results in an unsterile condition. No trouble
was encountered from contamination of the unsterile media in the course of the fermentation,
presumably because of the high concentration of acid formed. Further evidence of the
necessity for determining the optimum treatment for a given lot of molasses in large-scale
production became apparent in the case of molasses no. 2. The pH of this sample was 8.4, as
compared to the nearly neutral reactions of the other samples. By adjusting the pH of this
sample before Ferrocyanide treatment to an optimum, pH 6.0, and sterilizing the medium,
total acidity yields as citric were increased from 90.5 to 91.5 per cent, and Oxalic acid yields,
from 44.8 to 54.0 per cent.

2.4.3 BY USING ASPERGILLUS NIGER

Materials – Microorganism Aspergilus Niger NCIM 548 strain used in this study . Medium
composition- The medium contained the following components in (g/l): glucose (105.5) or
sucrose (100) or lactose (102.6) plus glucose (2.51)—all with the same carbon content;
NaNO3 (1.5); KH2PO4 (0.5); MgSO4*7H2O (0.025); KCl (0.025); and yeast extract (1.6)
Medium pH was adjusted to 6 with 4 M NaOH before sterilization. 20 ml of universal pH
indicator solution was added per litre medium for observing its pH which was maintained
within 6 to 7 by adding alkali during fermentation. Spore suspensions -Spores from 7-day
stationary culture at 30 0C in potatodextrose broth were suspended in 0.001% (v/v) Triton X-
100 solution, and were counted under a light microscope in a Neubauer chamber.

METHOD-:
Fifty milliliter culture medium taken in a 250-ml Erlenmeyer flask was inoculated with
spores of Aspergillus Niger (range: 5 to 6 per ml medium), and was incubated at 30 0C on an
orbital shaker at 215 rpm , & for 7 days it was kept in a dry condition . Culture filtrate from
each triplicate set was analyzed for oxalic acid content. During fermentation, the pH of the
medium was maintained at 6.5 with 4M NaOH. Growth and oxalic acid yield were monitored
during the course of fermentation. Broths after fermentation were filtered through a 0.2-mm
membrane. Oxalate in the filtrate was estimated by titration with KMnO4 after precipitating
with CaCl2. Sugars were estimated calorimetrically using Orcinol . Briefly, 1.5 ml of cold
orcinol reagent (200 mg orcinol dissolved in 100 ml of 70% (v/v) H2SO4 in water) was

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added to 0.5 ml of diluted sample in a test tube, heated in a boiling water bath for 20 min, and
then cooled. Biomass of Aspergillus Niger grown under different conditions was measured
gravimetrically after separating the mycelia from fermented broth by filtration,
washing with wa-ter, and drying at 80 0C to constant weight.

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