FIELD OF THE INVENTION

The present invention relates to an advanced process for preparing pharmaceutical grade ferric citrate.

BACKGROUND

Ferric citrate was approved by US FDA in September 2014 with the trade name Auryxia. The compound is a phosphate binder, indicated for the control of serum phosphorus levels in patients with chronic kidney disease on dialysis.

Method for preparing ferric citrate are known in the art. For example, US Patent No.

7,767,851 discloses a process for preparing ferric citrate having an intrinsic dissolution rate between 1.9 and 4.0 mg/cm ² /min. it is also disclosed that product prepared by said process has a BET active surface area exceeding 16 m ² /g. US Patent No. 8,338,642 discloses in particular Ferric citrate having a BET active surface area greater than 16 m ² /g, and a pharmaceutical composition and a method of treating hyperphosphatemia comprising administering ferric citrate having a BET active surface area greater than 16 rn ² /g to a patient in need of such treatment.

US Patent No. 8,609,896 discloses an orally administrabie form of ferric citrate, prepared from ferric citrate having a BET active surface area greater than about 16 m ² /g.

US Patent No. 8,754,258 discloses an orally administrabie form of ferric citrate, prepared from a form of ferric citrate having an intrinsic dissolution rate of at least 1.88 mg/cm ² /rniri.

In these prior art documents, a BET active surface area of ferric citrate synthesized by the disclosed process is at least 26 times greater than commercially available ferric citrate (> 16 m ² /g) , as compared with >0.61 m ² /g of commercial ferric citrate). Further, the mean dissolution rate is lower i.e. 0.83 mg/cm ² /rnin when commercially available ferric citrate with less BET active surface area is used, compared with ferric citrate obtained by the prior art methods, which result in ferric citrate with a mean dissolution rate between 1.9 to 4.0 mg/cm /rnin with a BET active surface area greater than 16 m /g. The inventors of present invention have surprisingly found a process for preparing ferric citrate whereby an increased dissolution rate can be achieved for a pharmaceutical grade ferric citrate having BET active surface area less than 16 m ² /g. SUMMARY

The present invention in a first aspect provides a method for preparing ferric citrate. The method comprises steps of

(a) treating a solution of a ferric salt with an alkali metal carbonate;

(b) treating the resulting reaction mass with a coagulating agent to obtain a ferric oxohydride mass;

(c) treating the resulting ferric oxohydride mass with citric acid;

(d) heating the resulting mixture to obtain a solution; and

(e) add an organic solvent to the solution to obtain ferric citrate.

The ferric citrate obtained by the process is a pharmaceutical grade ferric citrate, that is preferably characterized by a BET active surface area less than 16m ² /g. The ferric citrate is also preferably characterized by a mean dissolution rate in the range of 4 to 9 mg/cm ² /min.

BET active surface area, in the present context, represents surface area that is based on the Brunauer-Emmett-Teller (BET) theory of physical adsorption of gas molecules on solid surfaces.

The invention therefore also provides pharmaceutical grade ferric citrate having a BET active surface are of less than 16m ² /g. Further provided is pharmaceutical grade ferric citrate having a mean dissolution rate of 4 to 9 mg/cm ² /min. The invention also provides pharmaceutical grade ferric citrate having a BET active surface are of less than 16m ² /g and a mean dissolution rate of 4 to 9 mg/cm ² /min.

A further advantage of the invention is that the invention provides a process for preparing pharmaceutical grade ferric citrate that is feasible on a laboratory small scale as well as at industrial large scale.

DETAILED DESCRIPTION

Before describing particular embodiments of the present invention, it is to be understood that the invention is not intended to be limited to the particular embodiments that are described in the following. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting in any way. It should be noted that as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also contemplated, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also contemplated.

It should be understood that the particular methodology, protocols, material, reagents, and substances, etc., described herein can vary. Thus, variations that are within the skills of the ordinary practitioner are also contemplated. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined by the claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The present invention provides a process for preparing pharmaceutical grade ferric citrate that has distinct advantages in that the ferric citrate obtained by the process has a BET surface area of less than 16 m ² /g. Further, the ferric citrate has the pharmaceutically desirable property of a high dissolution rate that is preferably in the range of 4 to 9 mg/cm ² /min. Step a) In a first step of the process, a solution of a ferric salt is reacted with an alkali metal carbonate. The ferric salt can for example be a halogenic ferric salt, such as ferric chloride or ferric iodide. The ferric salt can also be ferric nitrate or ferric sulfate. In some embodiments, the ferric chloride solution can be a solution of ferric chloride or ferric chloride hexahydrate (FeCI ₃ -6H ₂ 0) .

The reaction temperature can be in the range of about 15°C to 4Q°C In some embodiments, the reaction temperature is 20°C to 30°C. The pH of the reaction mixture can be in the range from 7,5 to 9, such as in the range from 7.5 to 8.5. The reaction mixture can be stirred at the set temperature for 30 minutes to 120 minutes. In some embodiments, the reaction can be stirred for 30 minutes to 90 minutes, or from 30 minutes to 60 minutes. In certain embodiments, the reaction can be stirred for about one hour. In some embodiments, water is added to the reaction following the initial reaction step, and the reaction mixture heated to a temperature of about 40°C to 60°C, and preferably to a temperature of about 45°C to 55°C. The reaction mixture can be stirred at this temperature for 10 minutes to 90 minutes., and preferably for 20 minutes to 40 minutes.

Step b) In a second step of the process, an aqueous solution of a coagulating agent is added to the reaction mixture from the first step. The addition of the coagulating agent can suitably be performed at 40°C to 60°C, and preferably at 45°C to 55°C It can be preferable to add the coagulating agent slowly to the solution, for example over a time period of 5 minutes to 60 minutes, or over a time period of 10 to 40 minutes. The subsequent reaction mixture can be stirred for about 5 minutes to 60 minutes, for 5 minutes to 40 minutes, for 5 minutes to 20 minutes or for 5 minutes to 15 minutes. In some embodiments, the reaction mixture can be stirred for about 20 minutes, for about 30 minutes for about 40 minutes, for about 50 minutes, or for about 60 minutes.

Coagulation, sometimes also referred to as flocculation, is the process whereby colloid compounds come out of a suspension in the form of a flake or a floe. This can occur spontaneously or can be the result of, or be aided by, the addition of an agent. The coagulants used in present invention can be suitably selected polymeric coagulants, such as hydrophobically modified polymer coagulants. For example, the coagulants can be hydrophobic coagulants selected from the group consisting of hydrophobically modified copolymers of diallyldimethyl ammonium halide and hydrophobically modified copolymers of acrylamide. The diallyldimethyl ammonium halide can be diallyl dimethyl ammonium chloride (DADMAC), or it can be the corresponding bromide or iodide compounds.

The coagulant can preferably be a homopolymer of diallyl dimethyl ammonium halide. For example, the coagulant can be a homopolymer of diallyl dimethyl ammonium chloride

(DADMAC), a homopolymer of diallyl dimethyl ammonium bromide, or a homopolymer of diallyl dimethyl ammonium fluoride. In some preferred embodiments, the hydrophobically modified diallyldimethyl ammonium chloride polymer is a copolymer selected from the group consisting of diallyldimethyl ammonium chloride-co-dimethylaminoethylacrylate benzyl chloride quaternary, diallyldimethyl ammonium chloride-co-dimethylaminoethylacrylate cetyl chloride quaternary, diallyldimethyl ammonium chloride-co-dimethylaminoethylmethacrylate benzyl chloride quaternary, and diallyldimethyl ammonium chloride-co-dimethylaminoethylmethacrylate cetyl chloride quaternary.

In some embodiments, the coagulant can be a quaternized dimethylaminoethylacrylate (DMAEA) or a quaternized dimethylaminoethylmethacrylate (DMAEM). The coagulant can also comprise quaternized DMAEM salts of other mineral acids such as DMAEM»hydrochloride. The quaternized DMAEA and DMAEM monomers can include C ₄ to C20 chloride quaternaries. The C ₄ to C20 chloride quaternaries can be either aliphatic, for example cetyl chloride quaternary (CCQ). The C to C20 chloride quaternaries can also be aromatic, for example benzyl chloride quaternary (BCQ) . In general, the quaternaries can be halide quaternaries. Accordingly, the quaternaries can be sulfate, bromide or other similar quaternaries.

In some preferred embodiments, the preferred ester of acrylic acid or methacrylic acid is ethylhexyl acrylate. The hydrophobically associating monomers can in some embodiments be selected from vinylpyrolidone, styrene, vinylformamide, vinylacetamide, vinylpyridine, and vinylmaleimide monomers.

The skilled person will appreciate that coagulants with alternative chemical formulas can be used to achieve the advantageous effect of facilitating the preparation of ferric oxohydride according to the disclosed process, and such alternative coagulants are therefore also within scope of the present invention.

Following stirring of the reaction mixture containing coagulating agent, the stirring is stopped and the reaction allowed to rest for a period of about two to ten hours, preferably four to ten hours and more preferably six to nine hours. During this step, the solid ferric oxohydroxide material settles as a solid mass. The upper aqueous layer of the resulting mixture can then be decanted and the resulting solid product filtered. Filtration can be done by conventional means, for example by use of Agitated Nutsche Filter Dryer (ANFD) . Alternatively, the solid product can be obtained without filtration, i.e. by decantation.

It is also possible that the ferric oxohydride be obtained as a solid mass, either by decantation or by decantation and/or filtering, and water added to the wet mass for a second stirring step, which is then foliowed by steps of allowing the mass to settle, and decantation and/or filtering of the reaction to obtain wet ferric oxohydride.

Step c) Citric acid is then combined with the wet ferric oxohydride material. In some embodiments, the citric acid can be added as a solid . It is also possible to add the citric acid as an aqueous solution of citric acid. Step d) The subsequent reaction mixture of ferric oxohydride and citric acid is heated, preferably to about 50°C to 100°C, about 60°C to 100°C, or about 75°C to 95°C for about 30 minutes to 6 hours, for about 1 hour to 5 hours, for about 2 hours to 5 hours, or for 3 to 4 hours. The reaction can then be cooled to a suitable temperature, and filtered at that temperature. In some embodiments, the reaction is cooled to about 30°C to 60°C, to about 40° to 60°C, or to about 40°C to 50°C, and filtered at that temperature.

Step e) Ferric citrate is subsequently obtained by addition of an organic solvent to the filtrate. The solvent is preferably a polar, water soluble solvent. For example, the solvent can be a protic solvent, for example an alcoholic solvent. In some embodiments, the solvent is selected from the group consisting of ethanol, methanol, isopropyl alcohol, butanol, acetone and tetrahydrofuran. In some embodimens, the solvent is isopropyl alcohol. In some embodiments, it is preferable to add a large volume of the solvent to the filtrate to obtain a solid ferric citrate with the desirable properties described herein. In some embodiments, from 5 to 20 volumes of the solvent are added to the filtrate, thus resulting in a ratio of solvent to filtrate from 5: 1 to 20: 1. It can be preferable to add 6 to 15 volumes of solvent, 8 to 14 volumes of solvent, or 10 to 12 volumes of solvent to the filtrate. In some embodiments, about 12 volumes of solvent are added to the filtrate.

It can be preferable to add the solvent slowly to the filtrate. The solvent can for example be added dropwise, or it can be added in a slow stream. It can also be preferable to add the solvent at a constant rate to the filtrate. This means that a constant volume of solvent is added to the reaction with time. Thus, in some embodiments, the solvent is added over a time period of 1 to 10 hours, 2 to 9 hours, 3 to 8 hours, 4 to 7 hours, or 5 to 6 hours. The solvent can be added at a suitable temperature that can in certain embodiments range from 20°C to 60°C, 30°C to 50°C, 30°C to 40°C or 40°C to 50°C. In some embodiments, the solvent is added at room temperature. Without being bound by theory, it is believed that the surface area and solubility

characteristics of the ferric citrate that is obtained by the process are influenced by the nature of the solvent used in the last step of the process, the ratio of solvent to filtrate, the speed at which the solvent is added and the temperature at which the addition occurs. Thus, the amount of solvent, the rate of addition of the solvent and the temperature at which the solvent is added are believed to represent critical parameters for the properties of the final product, which preferably has a low BET (< 16 m ² /g) and a high dissolution rate (4 - 9 mg/cm ² /min).

In certain embodiments the final ferric citrate product has a BET of less than 16 m ² /g, such as less than 15 m ² /g, less than 14 m ² /g, less than 13 m ² /g, less than 12 m ² /g, less than 11 m ² /g, less than 10 m ² /g, less than 9 m ² /g, less than m ² /g, less than 8 m ² /g, less than 7 m ² /g or less than 6 m ² /g. The ferric citrate product can also have a BET in the range of about 2 to 15 m ² /g, in the range of about 3 to 14 m ² /g, in the range of about 4 to 14 m ² /g, in the range of about 4 to 12 m ² /g, in the range of about 4 to 10 m ² /g, in the range of about 4 to 8 m ² /g, or in the range of about 4 to 6 m ² /g. The ferric citrate can also have a BET in the range of about 2 to 10 m ² /g, in the range of about 2 to 8 m ² /g, or about 2 to 6 m ² /g. Alternatively, the ferric citrate can have a BET in the range of about 6 to 15 m ² /g, about 6 to 14 m ² /g or about 6 to 12 m ² /g. In some embodiments, the ferric citrate product has a BET of about 5 m ² /g.

The ferric citrate can in some embodiments have a mean dissolution rate of about 2 to 10 mg/cm ² /min, about 3 to 10 mg/cm ² /min, about 4 to 9 mg/cm ² /min, about 5 to 8

mg/cm ² /min, or about 6 to 8 mg/cm ² /min. The final ferric citrate product can be collected by filtration and dried by conventional means to obtain pharmaceutical grade ferric citrate. In some embodiments, the solid ferric citrate is collected by filtration, washed with a suitable solvent such as acetone, and dried under vacuum.

A distinct advantage of the invention is that the BET surface area of the ferric citrate can in part be regulated by the temperature at which the organic solvent is added. Thus, to provide a representative example, a pharmaceutical grade ferric citrate having BET active surface area from 6 m ² /g to 16 m ² /g can be obtained when the organic solvent in step e) is added at room temperature. Accordingly, in some embodiments, the invention provides a

pharmaceutical grade ferric citrate having a BET active surface are of 6 to 15 m ² /g, 6 to 14 rn ² /g, 6 to 12 m ² /g or 6 to 10 m ² /g when the organic solvent in step e) is added at room temperature. In another example, a pharmaceutical grade ferric citrate having BET active surface area from 2 rn ² /g to 6 m ² /g can be obtained when the organic solvent is added at a temperature of 40 to 50°C. Accordingly, in some embodiments, the invention provides a pharmaceutical grade ferric citrate having a BET active surface are of 2 to 6 m ² /g, 3 to 6 rn ² /g, 4 to 6 m ² /g or about 5 m ² /g when the organic solvent in step e) is added at a temperature of 40 to 50°C.

These and other advantages of the present invention, as described in the foregoing and illustrated by the Examples provided herein, should be apparent to the skilled person.

Exemplary embodiments of the invention are described in the following clauses:

1. A process for preparing ferric citrate, comprising steps of

a) Treating a solution of a ferric salt with an alkali metal carbonate; b) Treating the resulting reaction mixture with a coagulating agent to obtain a ferric oxohydride mass;

c) Treating the ferric oxohydride mass obtained in step b) with citric acid;

d) Heating the reaction mixture to obtain a solution;

e) Add an organic solvent to the solution to obtain ferric citrate.

2. The process of clause 1, wherein the ferric salt is ferric chloride.

3. The process of clause 2, wherein the ferric chloride is an aqueous solution prepared from ferric chloride or ferric chloride hexahydrate.

4. The process of any one of clauses 1 to 3, wherein the reaction of ferric chloride with the alkali metal carbonate in step a) is performed in an aqueous solution at a pH between 7.5 and 9.0 and a temperature between 15 and 40°C for 30 to 120 minutes.

5. The process of clause 4, wherein step a) further includes a subsequent step of adding water and heating the solution to a temperature of 40 to 60°C for 10 to 90 minutes. The process of any one of the previous clauses, wherein the coagulating agent is a hydrophobically modified copolymer of a polydiallyldimethylammonium halide or a hydrophobically modified copolymer of acrylamide.

The process of clause 6, wherein the coagulating agent is a homopolymer of a diallyl dimethyl ammonium halide.

The process for of clause 7, wherein the coagulating agent is a homopolymer of diallyl dimethyl ammonium chloride.

The process of any one of the clauses 6 to 8, wherein the coagulating agent is selected from the group consisting of diallyldimethyl ammonium chloride-co- dimethylaminoethylacrylate benzyl chloride quaternary, diallyldimethyl ammonium chloride-co-dimethylaminoethylacrylate cetyl chloride quaternary, diallyldimethyl ammonium chloride-co-dimethylaminoethylmethacrylate benzyl chloride quaternary, and diallyldimethyl ammonium chloride-co-dimethylaminoethylmethacrylate cetyl chloride quaternary.

The process of any one of the previous clauses, wherein after addition of coagulant agent in step b), the reaction is stirred at a temperature of 40 to 60°C for 5 to 30 minutes.

The process of clause 10, wherein the ferric oxohydride is collected by allowing the reaction to settle for 2 to 10 hours after the stirring, followed by filtration.

The process of any one of the preceding clauses, wherein step d) is performed at a temperature of 50 to 100°C for 1 to 6 hours.

The process of clause 12, wherein the reaction is subsequently cooled to a

temperature of 30 to 60° and filtered at that temperature.

The process of any one of the previous clauses, wherein the organic solvent in step e) is selected from ethanol, methanol, isopropyl alcohol, butanol, acetone and tetrahydrofuran.

The process of clause 14, wherein the ratio of organic solvent to reaction mixture from step c) is in the range of 6: 1 to 15: 1 (v/v).

The process of clause 14 or clause 15, wherein the organic solvent is added over a time period of 3 to 8 hours.

The process of any one of the clauses 14 to 16, wherein the organic solvent is added at room temperature.

The process of any one of the clauses 14 to 16, wherein the organic solvent is added at a temperature of 40 to 50°C. 19. The process of any one of the previous clauses, further comprising collecting the ferric citrate from step e) by filtration.

20. Ferric citrate obtained by the process of any of the clauses 1 to 19.

21. Ferric citrate according to clause 20 that as a BET active surface are of less than 16 m ² /g.

22. The ferric citrate of clause 20 or clause 21 that has a BET active surface area in the range of 2 to 15 m ² /g.

23. The ferric citrate of any one of clauses 20 to 22 that has a BET active surface area in the range of 2 to 6 m ² /g. 24. The ferric citrate of any one of clauses 20 to 22 that has a BET active surface area in the range of 6 to 15 m ² /g.

25. Ferric citrate according to any one of clauses 20 to 24 that has a mean dissolution rate of 4 to 9 mg/cm ² /min.

26. Pharmaceutical grade ferric citrate having a BET active surface area of less than 16 m ² /'g.

27. The pharmaceutical grade ferric citrate of clause 26 _; having a BET active surface area in the range of 2 to 15 m ² /g.

28. The pharmaceutical grade ferric citrate of clause 26, having a BET active surface area in the range of 2 to 6 m ² /g.

29. The pharmaceutical grade ferric citrate of clause 26, having a BET active surface area in the range of 6 to 15 m ² /g.

30. The pharmaceutical grade ferric citrate of any one of the clauses 26 to 29, further characterized by having a mean dissolution rate from 4 to 9 mg/cm ² /min.

31. Pharmaceutical grade ferric citrate having a BET active surface area of less than 16 m ² /g and a mean dissolution rate of 4 to 9 mg/cm ² /min.

The invention is exemplified by the following non-limiting examples.

Example 1: Synthetic process for preparation of pharmaceutical grade ferric citrate

A solution of sodium carbonate ( 19.5kg in 45L water) was added to a Ferric Chloride solution ( 15kg in 75L water) and reaction mass was stirred for one hour at room temperature. Water ( 150L) was added and the reaction mass was heated to about 40- 60°C followed by addition of Polydiallyldimethylammonium chloride solution ( 15gm in 15L water) over a period of 10-40 minutes. Reaction mixture was further stirred for 20-60 minutes at about 40- 60°C. Stirring was stopped and the reaction mass allowed to settle down. Aqueous layer was decanted and the solid material was filtered at 40- 60°C. To the resulting wet cake water (200L) was added and followed by stirring for 12-60 minutes at 40- 60°C. Stirring was stopped and the reaction mass allowed to settle down. Aqueous layer was decanted and solid material was filtered at 40- 60°C. To the wet solid material citric acid (22.20kg) was added and the reaction mixture heated to 60-70°C and stirred for one hour at about 85- 100°C. The reaction mixture was cooled to about 40- 60°C, and filtered at this temperature. To the obtained filtrate, isopropyl alcohol ( 180L) was added over a period of three to six hours at room temperature. Reaction mass was stirred for 30 minutes at room temperature and filtered to get wet cake. Acetone ( 180L) was added to the wet cake and slurry was stirred for 15 to 30 minutes. Reaction mass was filtered and dried under vacuum to get 15.88kg dry product.

BET active surface area of product was determined as 5.1 rn ² /g and dissolution rate (IDR) is 7.7 (at pH 8) .

Example 2: Synthetic process for preparation of pharmaceutical grade ferric citrate A solution of sodium carbonate ( 19.5kg in 45L water) was added to a Ferric Chloride solution ( 15kg in 75L water) and the reaction mass stirred for one hour at room temperature. Water ( 150L) was added and the reaction mass was heated to about 40- 60°C followed by addition of Polydiallyldimethylammonium chloride solution ( 15gm in 15L water) over a period of 10-40 minutes. Reaction mixture was further stirred for 20-60 minutes at about 40- 60°C. Stirring was stopped and the reaction mass allowed to settle. Aqueous layer was decanted and solid material was filtered at 40- 60°C. To this wet cake water (200L) was added and followed by stirring for 12-60 minutes at 40- 60°C. Stirring was stopped and the reaction mass allowed to settle. Aqueous layer was decanted and solid material was filtered at 40- 60°C. To the wet solid material citric acid (22.20kg) was added and the reaction mixture was heated to 60- 70°C and stirred for one hour at about 85- 100°C. Reaction mixture was cooled to about 40- 60°C, and filtered at 40- 60°C. To the obtained filtrate, isopropyl alcohol ( 180L) was added over a period of three to six hours at 40- 50°C. Reaction mass was further stirred for 30 minutes at 40- 50°C and filtered to get wet cake. Acetone ( 180L) was added to the wet cake and the slurry was stirred for 15 to 30 minutes. Reaction mass was filtered and dried under vacuum to get 15kg dry product.

BET active surface area of product was determined as 5.1 m ² /g.

Example 3. Determination of dissolution rates

1.1 Dissolution Media

· pH 1.0 : Take 5000 mL of purified water & pH adjusted to 1.0 with concentred HCI solution & mixed media

• Purified water:

• pH 8.0:

Weighed 34. Og of KH ₂ P0 ₄ and 5000 mL water and mixed well. pH adjusted to 8.0 with NaOH Solution

2 Dissolution Parameters:

Medium : Water

pH 1.0 : Hydrochloride Acid

pH 8.0 : Phosphate buffer pH 8.0

Volume : 1000 mL

RPM : 100 rpm

Volume sampled : 10 mL

Volume replaced : 10 mL

Temperature : 37.0°C ± 0.5°C.

Time points : 5, 10,15,20,30,45,60,90,120,150,180,240 minutes

3 Sample preparation :

Weighed API into die cavity and remove the air compressed up to 3 ton pressure to make pellet.

4 Standard solution : For Respective Media

Weigh API accurately 100 mL of ferric citrate standard into 50 mL volumetric flask; add about 30 ml of Mcllvaine buffer pH 4.0. Heat on a heating mantle at about 80°C, with intermittent swirling, until the standard dissolves. Cool to room temperature and dilute up to mark with Mcllvaine buffer (pH=4.0).Mix well. (Prepare Standard in duplicate and label as standard preparation 1 and standard preparation 2).

5 Color development :

Pipet 1.0 mL of medium, standard, sample solution in to 50 mL flask, added 4 mL of 10% Hydroxylamine HCI 5 mL & 10 mL 0.3% 1-10 Phenanthroline and, mixed & kept for 45 min. Then volume make up to the mark with corresponding dissolution medium & mix well.

6 Procedure Absorbance of blank, standard & sample were taken at 510 nm using 1cm cuvette.

Example 4. Determination of surface area

Method uses Thermo E lectro n So rpto matic 1990 ve rsio n 1 .03.

Sample density: lg/cm ³

Gas: Nitrogen

Acquisition parameters:

Adsorption:

Maximum Ads pressure: 250

Minimum Sat pressure : 240

Initial load pressure: 300

Pre-run time (min) : 5

Analysis type: Ads

Sat pressure: 760

Analytical conditions:

Gas molecular weight g/mol : 28.01

Gas molecular area (A ² ) : 16.2

Liquid gas density (g/cm ³ ) : 0.8086

Liquid gas surf. Tens (Dyne/cm ³ ) : 8.85

Monolayer thick (A) : 3.54

2 ^nd virial burette temperature: -262

2 ^nd virial piston temperature: -2.5

Burette temperature (°C) : -196 Instrument hysteresis: 1

Correct slope/intercept: Yes

Instrument parameters:

Total stroke volume (cm ³ ) : 17.081

1/2 stroke volume (cm ³ ) : 8.601

1/2 stroke volume (cm ³ ) : 4.38

Piston dead volume (cm ³ ) : 0.21

Piston temperature (°C) : 37

Calculation parameters:

Surface area calc method : B.E.T.

Pore: Radius

Pore size calculation method : DolliumJHeal

Smoothing pores distribution convolution points (0-20) : 3

Initial P/P0 for B.E.T: 0.07

Pore specific volume at P/P0: 0.9997

Final P/P0 for B.E.T: 0.18

Sample preparation : Weigh 2g of sample and transfer into 5 mL burette. Degas the sample at 40°C for 2 hrs in furnace slot.

Blank: Perform blank analysis omitting the sample.