FORM - 2 THE PATENTS ACT, 1970
(39 of 1970) (
& THE PATENTS RULES, 2003
COMPLETE

(See section 10 and rule 13)
PROCESS FOR EXTRACTION AND ISOLATION OF 4-HYDROXYISOLEUCINE FROM FENUGREEK SEEDS
INNOVASSYNTH TECHNOLOGIES (INDIA) LTD.,
an Indan Company of Paragon Condominium, 3rd floor, Pandurang Budhkar Marg, Mumbai 400 013,
Maharashtra, India,
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.


Title
Process for extraction and isolation of 4-Hydroxyisoleucine (HIL) from fenugreek seeds.
Field of the Invention
This invention relates to the process for the extraction of biologically useful molecule from plant material.
More specifically, this invention relates to the extraction and isolation of 4-Hydroxyisoleucine from fenugreek seeds (Trigonella foenum graecum L).
Definitions
As used in the present specification, the following words and phrases are
generally intended to have the meanings as set forth below, except to the
extent that the context in which they are used indicates otherwise.
"C-1" means first Column.
"C-II" means second Column.
"Defatting" means a process of removing lipophilic substances from the plant
material using a suitable solvent.
"DM" means demineralized water.
"Elution" means a solventsystem that elutes a substance bound on a stationary
phase with respect to the flow rate of the respective solvent.
"Extraction" means a process of treating a natural raw material with an
organic solvent in which the desired compounds are solubilized at a particular

temperature and volume. Thus obtained solution containing the solutes is filtered free of insolubles.
"Fenugreek seeds" means seeds of Trigonella Foenum Graecum L. "4-ML" means "4-Hydroxyisoleucine". "KL" means Kilolitre or one thousand liter "MDC" means "Methylene di chloride" "MeOH" means Methyl alcohol. "TDS" means "Total dissolved solids".
Background of the Invention
Introduction
Fenugreek
Botanical Name: Trigonella Foenum graecum L.
Habitat: Fenugreek is herbaceous plant of the leguminous family and is
native to Western Asia, from where it has spread widely over Europe, the
Mediterranean and rest of Asia.
It is one of the oldest cultivated plants and through the ages has found wide application as a food, a food additive and in traditional medicines of every region in which it has been cultivated.
Fenugreek seeds are a rich source of protein, the overall protein content being about 26% in raw seeds, which gets concentrated to about 43% in the residual dietary fiber obtained upon extraction of the seeds.

All the amino acids are represented in fenugreek seeds and the distribution and content thereof is almost equal to that of the other proteinaceous leguminous seed, soybean.
Fenugreek also contains an important amino acid called 4-hydroxyisoleucine (HIL).
Chemical Structure: OK MHj+
a ■
4-Hydroxyisoleucine
4-HIL is a very useful constituent of fenugreek seeds. For medical as well as nutritional purposes, fenugreek seeds should be consumed in sufficient quantities regularly to produce the desired effects. However, raw, soaked or cooked fenugreek seeds cannot be consumed in sufficient quantities regularly due to their bitter taste.
Therefore, there is a need for a process for the preparation of an acceptable form of de-bitterized extract, with high concentration of 4-HIL, from fenugreek seeds.
Prior Art
WO03094948A1 describes a method, involving the following steps: (1) Providing a plurality of fenugreek seeds;

(2) Preparing the fenugreek seeds &
(3) extracting a composition of bio-active compounds from the fenugreek seeds, wherein the bio-active compounds comprise 4-hydroxyisoleucine and other amino acids like arginine, aspartate, threonine, serine, glutamate, praline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tryptophan, phenylalanine, ornithine, lysine, histidine, gamma-amino butyrate, and trimethylhistidine.
The composition obtained from this method contains about 10% to 70% of 4-hydroxyisoleucine and other amino acids.
In accordance with another known method for isolation of 4-HIL, Fenugreek seed powder is defatted with hexane and extracted with 70% ethanol at room temperature. The extract is concentrated and passed through a cation exchange resin and eluted with N or 2N ammonia solution. The eluted fraction is concentrated and subjected to further elution using a silica gel column with 70% ethanol. The eluted fraction is concentrated and crystallized using diethyl ether.
However, these above mentioned methods involve usage of column chromatography, in which silica and alumina are used, which is not desirable and furthermore these processes are expensive.
Summary of the Invention
This invention provides a process for preparation of an acceptable form of de-bitterized 4-HIL from fenugreek seeds.

Further object of this invention is to isolate and extract 4-HIL from fenugreek seeds, with greater than 80% concentration by subjecting the eluted solution, obtained from a cation exchange resin, to reverse osmosis at least two times.
A still further object of this invention is to provide an economical process for extraction and isolation of 4-HIL from fenugreek seeds.
Another object of this invention is to provide a process, which does not use column chromatography using silica and alumina for extraction and isolation of 4-HIL from fenugreek seeds.
According to this invention there is provided a process for extraction of 4-HEL fenugreek seeds, said process comprising the following steps:
(a) De-stoning and pulverizing the fenugreek seeds and sieving the resulting powder,
(b) Subjecting the sieved powder, to de-fatting, using a first solvent, at least twice; to obtain a defatted powder,
(c) Subjecting the defatted powder, to extraction with a second solvent to obtain a viscous extract,
(d) Concentrating and spray-drying the viscous extract to obtain a free flowing dry powder containing 5% of 4-HDL,
(e) Dissolving the free flowing dry powder in demineralized water to give a first solution,
(f) Subjecting the first solution, to a cation exchange resin column for adsorption of 4-HIL to obtain a cation exchange resin column with 4-HIL adsorbed thereon,

(g) Washing the cation exchange resin column with 4-HIL adsorbed thereon,
with demineralized water and eluting 4-HIL there from, using the Ammonia
Solution resulting in a second solution,
(h) Concentrating and drying the second solution to obtain a concentrate,
(i) Treating the concentrate, with Methyl alcohol and MDC, followed by
filtration to give a filtrate; and
(j) Concentrating and drying the filtrate, to yield the de-bitterized 4-HIL.
Typically, a sieve is selected from a group of sieves consisting of sieve no 20, sieve no 40, sieve no50, and sieve no 60.
Typically, the first solvent is selected from a group of solvents consisting of hexane, methanol, aqueous methanol, ethanol and aqueous ethanol.
Typically, the second solvent is 50% aqueous ethyl alcohol.
Typically, ammonia solution is 1M Ammonia solution.
Typically, ammonia is stripped off after elution and the resulting eluted solution is passed through RO PES Spiral 2.5 m2 to give the second solution in the from of a permeate.
Typically, the second solution in the form of permeate is recirculated during the RO process.
Typically, the elution is carried out two times with ammonia solution till the HIL spot is absent in TLC.

Typically, methyl alcohol and MDC are used in the ratio of 4:6.
Typically, a strong acid cation exchange resin based on cross linked polystyrene matrix, containing the sulfonic acid as a functional group is used. Alternatively, a weak acid [poly (methacrylic acid)] cation exchanger containing the "-COOH" group as a functional group is used.
Detailed description of the invention
According to this invention there is provided a process for extraction of 4-HIL fenugreek seeds, said process comprising the following steps:
(a) Pulversing fenugreek seeds and sieving the resulting powder and
demagnetizing the resultant powder.
Typically, fenugreek seeds are pulverized using disc attrition mill to obtain a powder, alternatively a hammer mill or a micro pulveriser can be used. This powder is then further sieved, typically using sieve no.40. Alternatively, sieve no50, and sieve no 60 can also be used.
(b) Subjecting the sieved powder obtained from step (a), to defatting, using a
first solvent, at least two times.
The sieved powder is then treated with a solvent selected from a group of solvents consisting of hexane, methanol, and ethanol or isopropyl alcohol, preferably with methanol.
This step is performed at least twice to remove any remaining residues of the fatty constituents.
(c) Subjecting the defatted powder, to extraction with a second solvent.

The defatted powder obtained from step (b) is treated with 50% aqueous ethanol for extraction purpose and the resulting slurry is then filtered to obtain a viscous extract containing 4-HIL.
(d) concentrating and spray-drying the viscous extract obtained from step
(c), to obtain a free flowing dry powder containing at least 5% of 4-HIL.
The viscous extract is further concentrated and subjected to spray drying to
obtain a free flowing fenugreek powder extract.
Fenugreek seed extract is a free flowing yellowish to yellowish brown
powder. It is bitter in taste and has a characteristic odor. Moisture content of
this powder is less than 5%. This powder contains a range of 4 to 5% 4-HIL
byHPLC.
This powder is soluble in water and a 1% solution has a pH of 5 to7. This dry
form increases the shelf life of the extract.
(e) Dissolving the free flowing powder obtained in step (d), in demineralized water to give a first.
(f) Subjecting the first solution obtained from step (e), to a cation exchange resin column for adsorption of 4-HIL.
Typically, a strong acid cation exchange resin based on cross linked polystyrene matrix, containing the sulfonic acid as a functional group is used. Alternatively, a weak acid [poly (methacrylic acid)] cation exchanger containing the "-COOH" group as a functional group can also be used. This 4-HIL solution is allowed to adsorb on the resin for 30 minutes.

(g) Washing the cation exchange resin column, with 4-HIL adsorbed thereon with demineralized water and eluting 4-HIL there from, using Ammonia Solution resulting in a second solution.
The column is washed with DM water to remove the saponins whereas 4-HIL remains adsorbed on to the resin.
(h) Concentrating and drying the second solution, obtained from step (g) to obtain a concentrate.
(i) Treating the concentrate, with Methyl alcohol and MDC, followed by filtration to give a filtrate.
The concentrate obtained from step (h) is then treated with Methyl alcohol and MDC in 4:6 ratios.
(j) Concentrating and drying the filtrate, to yield the De-bitterized 4-HIL. The non-odorous and de-bitterized extract of fenugreek seeds containing more than 80% of 4-HIL is obtained in this step.
Examples
Example 1
1100 kg of Fenugreek seeds were pulverized. The powder so obtained was
then sieved using sieve no.40.
Sieved Powder was defatted by treating it with 2200 liters of methanol to
remove fatty material from the powder and this step was repeated again
to remove any traces of fatty substances.

The defatted powder so obtained from the above step was subjected to
extraction with 2000 liters of aqueous ethanol twice, and was filtred to
obtain the viscous extract.
The viscous extract obtained from the above step was concentrated to
30TDS and was subjected to spray drying to obtain free flowing
fenugreek dry powder extract of lOOKg containing 5.5% HIL.
lOOKg spray dried fenugreek extract containing 5.5% HIL was dissolved in
lOOLitres of water and was passed through 200Kg of cation exchange resin
packed in a column. The material was allowed to adsorb on the Resin-column
for 30 minutes and the resin was washed with 1.6KL of DM water till a clear
elutant was obtained.
Then the adsorbed 4-HIL was eluted with 2.4KL of 1M ammonia solution.
The ammonia was stripped off at 70-80°C and the resulting ammonia free
aqueous elutant was concentrated under vacuum at 50mm of Hg to obtain a
thick mass of 17 Kg of 29.4% of 4-HIL.
The 17 Kg of the above mass was treated with 45Litres of MeOH : MDC (4:6
volume) and filtered in a Nutsch filter under nitrogen in a dry room to get 8Kg
of 4-HIL, of 60% assay. (Recovery is 87%)
Example 2
1100 kg of Fenugreek seeds were pulverized. The powder so obtained was
then sieved using sieveno.40.
Sieved Powder was defatted by treating it with 2200 liters of methanol to
remove fatty material from the powder and this step was repeated again
to remove any traces of fatty substances.

The defatted powder so obtained from the above step was subjected to extraction with 2000 liters of aqueous ethanol twice, and was filtered to obtain the viscous extract.
The viscous extract obtained from the above step was concentrated to 30TDS and was subjected to spray drying to obtain free flowing fenugreek dry powder extract of lOOKg containing 6% HIL. lOOKg spray dried fenugreek extract containing 6% HIL was dissolved in lOOLitres of water and passed through 200Kg of cation exchange resin. The material was then allowed to adsorb on the column for 30 minutes and the resin was washed with 1.6KL of DM water till clear elutant was obtained. Then the adsorbed 4-HIL was eluted with 2.4KL of 1M ammonia solution. The ammonia was stripped off at 70-80°C and the resulting ammonia free aqueous elutant was passed through polyether sulphone (PES) membrane spiral 2.5m2 in a Reverse Osmosis system. Permeate was re-subjected to one more cycle of Reverse Osmosis to get a retentive. The retentive was concentrated to 12.5Kg powder of assay 45% 4-HIL.
This 40-45% HIL was treated with MeOH: MDC (4:6 volume) and filtered in a Nutsch filter under nitrogen in dry room to get 5.8Kg of 90% 4-HIL. 4-HIL thus obtained was non-odorous and sweetish. There was no significant loss of 4-HIL during this process and was totally recovered. (Recovery 87%)
Example 3
1100 kg of Fenugreek seeds were pulverized. The powder so obtained was
then sieved using sieveno.40.
Sieved Powder was defatted by treating it with 2200 liters of methanol to
remove fatty material from the powder and this step was repeated again
to remove any traces of fatty substances.

The defatted powder so obtained from the above step was subjected to
extraction with 2000 liters of aqueous ethanol twice, and was filtered to
obtain the viscous extract.
The viscous extract obtained from the above step was concentrated to
30TDS and was subjected to spray drying to obtain free flowing
fenugreek dry powder extract of lOOKg containing 5% HDL.
lOOKg spray dried fenugreek extract containing 5% HIL was dissolved in
lOOLitres of water and passed through 200Kg of cation exchange resin.
The material was allowed to adsorb on the column for 30 minutes and the
resin was washed with 1.6KL of DM water till clear elutant was obtained.
Then the adsorbed 4-HIL was eluted with 2.4KL of 1M ammonia solution.
The elutant was passed through polyether sulphone (PES) membrane spiral
2.5m in a Reverse Osmosis system.
Permeate was re-subjected to one more cycle of Reverse Osmosis to get a
retentive. The retentive was concentrated to ll.SKg powder of assay 43%
HIL.
This 43% HIL was treated with MeOH: MDC (4:6 volume) and filtered in a
Nutsch filter under nitrogen in dry room to get 6.1 Kg of 80 %4- HDL4-HIL
thus obtained was non-odorous and sweetish. There was minimal Loss of 4-
HIL during this process.
Example 4
110 kg of Fenugreek seeds were pulverized. The powder so obtained was
then sieved using sieveno.40.
Sieved Powder was defatted by treating it with 220 liters of methanol to
remove fatty material from the powder and this step was repeated again
to remove any traces of fatty substances.

The defatted powder so obtained from the above step was subjected to
extraction with 200 liters of aqueous ethanol twice, and was filtered to
obtain the viscous extract.
The viscous extract obtained from the above step was concentrated to
30TDS and was subjected to spray drying to obtain free flowing
fenugreek dry powder extract of 10Kg containing 5% HIL.
lKg spray dried fenugreek extract containing 5% HIL was dissolved in
300mL of water and was passed through 2Kg of cation exchange resin.
The material was allowed to adsorb on the column for 30minutes and the resin
was washed with lOLitres of DM water till clear and saponin free elutant was
obtained.
Four Liters of 1M ammonia solution was passed through the column till the
elutant was alkaline. (pH8)
Alkaline fraction al (containing 4-HIL) was collected and then
al was re-circulated through the col-I (collected as a2)
a2 was passed through 2nd column(collected as a3).
a3 was re-circulated through C-II (collect as a4);
a4 was re-circulated through C-II (collect as a5)
4-Litres of Fresh 1M ammonia solution was passed through C-I (b) and
collected as bl.
The bl is re-circulated through C-I Elutant is b2. This was passed through C-
II and collected as b3.
Then 2Litres of fresh 1M ammonia solution (c) was passed through C-I and eluted as cl which was passed through C-II and the elutant collected as c2. 2Litre water wash was done to C-I and passed the same elutant through C-II (containing 4-HIL).

Thus a total volume of 9.5 L (9-10 volumes) of 1M solution was used to get
134g of 36%4-HIL.
The resultant amnionic solution was taken into a 20L Rotavac and the
ammonia was stripped off at 50°C and the ammonia free aqueous elutant was
passed through polyether sulphone (PES) membrane spiral 2.5m in a Reverse
Osmosis system.
Permeate obtained from the Reverse Osmosis system was re-subjected to one
more cycle of Reverse Osmosis to get a retentive. The retentive was
concentrated to 1 OOg powder of Assay 46% 4-HIL.
This 46% 4-HIL was treated with MeOH: MDC (4:6 volume) and filtered in a Nutsch filter under nitrogen in a dry room to get 50gms of 91%4-HIL. 4-HIL thus obtained was non-odorous and sweetish. There was minimal Loss of 4-HIL during this process.
Example 5
110 kg of Fenugreek seeds were pulverized. The powder so obtained was
then sieved using sieveno.40.
Sieved Powder was defatted by treating it with 220 liters of methanol to
remove fatty material from the powder and this step was repeated again
to remove any traces of fatty substances.
The defatted powder so obtained from the above step was subjected to
extraction with 200 liters of aqueous ethanol twice, and was filtered to
obtain the viscous extract.
The viscous extract obtained from the above step was concentrated to
30TDS and was subjected to spray drying to obtain free flowing
fenugreek dry powder extract of lOKg containing 5% HIL.

lOKg spray dried fenugreek extract containing 5% 4-HIL was dissolved in
3Litres of water and passed through 20Kg of cation exchange resin.
The material was allowed to adsorb on the column for 30 minutes and the
resin was washed with lOOLitres of demineralized water till the elutant was
clear and free of saponins.
Forty Liters of IM ammonia solution was passed through the column till the
elutant was alkaline. (PH range needs to be mentioned)
Alkaline fraction al containing4- HIL was collected, then
al was re-circulated through the col-I (collected as a2)
a2 was passed through 2nd column(collected as a3).
a3 was re-circulated through C-II (collected as a4);
a4 was re-circulated through C-II (collected as a5)
40Litres of Fresh IM ammonia solution was passed through C-I (b) and
collected as bl.
The bl was re-circulated through C-I (Elutant b2).
This was passed through C-II and collected as b3.
Then 20Litres fresh IM ammonia solution (c) passed through C-I and eluted
as cl which was passed through C-II and the elutant collected as c2.
20Litre water wash was done to C-I and passed the same elutant through C-II
(contain 4- HIL).
Thus a total volume of 103 Litres (9-10 volumes) of IM solution was used
(instead of 24 volumes as per the previous process) to get 1.3Kg of
35% 4-HIL.
The resultant amnionic solution was passed through polyether sulphone (PES)
membrane spiral 2.5m in a Reverse Osmosis system.
Permeate obtained from the Reverse Osmosis system was re-subjected to one
more cycle of Reverse Osmosis to get a retentive.

The retentive was concentrated to 1.12Kg powder of Assay 40% 4-HEL. This 40% 4-HIL was treated with MeOH: MDC (4:6 volume) and filtered in a Nutsch filter under Nitrogen in dry room to get 505g of 88% non-odorous and sweetish 4-HIL There was minimal Loss of 4-HIL during this process.
Example 6
110 kg of Fenugreek seeds were pulverized. The powder so obtained was
then sieved using sieveno.40.
Sieved Powder was defatted by treating it with 220 liters of methanol to
remove fatty material from the powder and this step was repeated again
to remove any traces of fatty substances.
The defatted powder so obtained from the above step was subjected to
extraction with 200 liters of aqueous ethanol twice, and was filtered to
obtain the viscous extract.
The viscous extract obtained from the above step was concentrated to
30TDS and passed through 20Kg of cation exchange resin.
The material was allowed to adsorb on the column for 30 minutes and the
resin was washed with lOOLitres of demineralized water till the elutant was
clear and free of saponins.
Forty Liters of 1M ammonia solution was passed through the column till the
elutant was alkaline. (PH range needs to be mentioned)
Alkaline fraction al containing4- HIL was collected, then
al was re-circulated through the col-I (collected as a2)
a2 was passed through 2nd column(collected as a3).
a3 was re-circulated through C-II (collected as a4);
a4 was re-circulated through C-II (collected as a5)

40Litres of Fresh IM ammonia solution was passed through C-I (b) and
collected as bl.
The bl was re-circulated through C-I (Elutant b2).
This was passed through C-II and collected as b3.
Then 20Litres fresh IM ammonia solution (c) passed through C-I and eluted
as cl which was passed through C-II and the elutant collected as c2.
20Litre water wash was done to C-I and passed the same elutant through C-II
(contain 4- HIL).
Thus a total volume of 103 Litres (9-10 volumes) of IM solution was used
(instead of 24 volumes as per the previous process) to get 1.38Kg of
36% 4-HIL.
The resultant amnionic solution was passed through polyether sulphone (PES)
membrane spiral 2.5m2 in a Reverse Osmosis system.
Permeate obtained from the Reverse Osmosis system was re-subjected to one
more cycle of Reverse Osmosis to get a retentive.
The retentive was concentrated to 1.16Kg powder of Assay 42% 4-HIL.
This 40% 4-HIL was treated with MeOH: MDC (4:6 volume) and filtered in
a Nutsch filter under Nitrogen in dry room to get 530g of 91% non-odorous
and sweetish 4-HIL There was minimal Loss of 4-HIL during this process.
HEL appears to increase the body's production of insulin when blood sugar
levels are high. Fenugreek lowers cholesterol levels as well as blood sugar
levels.
In doses of 200mg/day, it is a supplement of choice to lower glucose levels in
diabetic patients due to its safety profile, documented potency & inexpensive
ability.
4-Hydroxyisoleucine (HIL) is safe & an effective nutraceutical that enhances
the performance in sports persons without affecting the glucose homeostasis

HIL is also found to be useful in weight reduction.
HIL can reduce formation of kidney stones by reducing the amount of calcium
oxalate in the kidneys.
4-Hydroxyisoleucine is also used in the process of making a flavorant with the
help of microorganisms which have L-amino acid oxidase activity or with an
L-amino acid oxidase.
4-HIL is also very effective in the treatment of chronic diseases such as
coronary artery diseases, diverticultis, and cancer of the colon, piles, fissures,
chronic constipation & the like.
While considerable emphasis has been placed herein on the specific steps of the preferred process, it will be appreciated that many steps can be made and that many changes can be made in the preferred steps without departing from the principles of the invention. These and other changes in the preferred steps of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.
The isolation and purification procedures described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or preparative chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples herein below. However, other equivalent separation or isolation procedures can, of course, also be used.

We Claim:
1. A process for isolation of 4-Hydroxyisoleucine from trigonelles
(Trigonella SP) comprising the following steps:
(a) Pulversing fenugreek seeds and sieving the resulting powder;
(b) Subjecting sieved powder, to defatting, using a first solvent, at least twice; to obtain a defatted powder;
(c) Subjecting the defatted powder, to extraction with a second solvent to obtain a viscous extract;
(d) Concentrating and spray-drying the viscous extract to obtain a free flowing dry powder containing at least 5% of 4-HIL;
(e) Dissolving the free flowing dry powder in demineralized water to give a first solution;

(f) Subjecting the first solution, to a cation exchange resin column for adsorption of 4-HIL to obtain a cation exchange resin column with 4-HIL adsorbed thereon;
(g) Washing the cation exchange resin column with 4-HIL adsorbed thereon with demineralized water and eluting 4-HIL there from, using Ammonia Solution resulting in a second solution;
(h) Concentrating and drying the second solution to obtain a concentrate; (i) Treating the concentrate, with Methyl alcohol and MDC, followed by filtration to give a filtrate; and (j) Concentrating and drying the filtrate, to yield De-bitterized 4-HIL.
2. A process as claimed in claim 1,wherein the sieve is selected from a group
of sieves consisting of sieve #20 , sieve # 40, and sieve# 60.

3. A process as claimed in claim 1, wherein the first solvent is selected from a
group of solvents consisting of hexane, methanol, aqueous methanol, ethanol
and aqueous ethanol.
4. A process as claimed in claim 1, wherein the second solvent is 50%
aqueous ethyl alcohol.
5. A process as claimed in claiml, wherein ammonia solution is 1M Ammonia
solution.
6.A process as claimed in claim 5, wherein ammonia is stripped off after elution and the resulting eluted solution is passed through RO PES Spiral 2.5 m2 to give the second solution in the from of a permeate.
7. A process as claimed in claim 5, wherein the second solution in the form of permeate is recirculated during the elution process.
8. A process as claimed in claim 6, wherein the elution is carried out at least twice with ammonia solution.
9. A process as claimed in claim 1, wherein Methyl alcohol and MDC are used in the ratio of 4:6.
10. A process as claimed in claim 1, wherein the cation exchange resin is a
strong acid cation exchange resin based on cross linked polystyrene matrix,
containing the sulfonic acid as a functional group.

11. A process as claimed in claim 1, wherein the cation exchange resin is a weak acid[poly(methacrylic acid)] cation exchanger containing the "-COOH" group as a functional group.
Dated this 27th day of March, 2006.
MOHAN DEWAN
OF R.K.DEWAN & COMPANY
APPLICANTS' PATENT ATTORNEYS