Does calcium oxalate react with acetic acid?

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the equation is as follows: CaC2O4 + CH3COOH ----> ??? Some say no because acetic acid is a weak acid and calcium oxalate is not soluble in acetic acid or something. I need ...show more
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I think it does
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  • cartier95 answered 8 years ago
    Notes for the Practical Problems:
    In the practical preparatory problems we have not included specific details of handling or disposal of labaratory materials and wast as regulations vary greatly from country to country.
    We take it for granted that the students are able to perform basic experimental techniques such as titration, filtration, recrystallisation, distillation.
    As mentioned in the preface we attach great importance to safety.
    The rules below have to be followed during laboratory work at the 36th IChO in Kiel.
    - The students have to bring their own labaratory coats.
    - When the students enter the labs they must familiarise themselves with the locations of
    emergency exits
    safety shower, fire blanket and eye wash.
    - Laboratory coats, eye protections and closed shoes must be worn at all times in the laboratories. Long hair has to be tied.
    - Coats and bags are forbidden in the laboratory. They have to be deposited in the cloakroom.
    - Eating, drinking or smoking in the laboratory or tasting any chemicals are strictly forbidden.
    - Pipetting by mouth is strictly forbidden.
    - All potentially dangerous materials will be labelled by international symbols. Each student is responsible for recognizing these symbols and knowing their meaning.
    - Do not dispose of chemicals down the sink. Follow all disposal rules provided by the organizer.
    - Do not hesitate to ask an instructor if you have any questions concerning safety issues.
    A brief instruction will be given on the day preceding the examination.
    Apologies for all the do’s and don’t’s - we guarantee that the students will still be allowed to perform the experiments and we hope they will enjoy it!
    45
    Problem 35: Preparation and volumetric determination of strontium peroxide octahydrate
    Introduction
    Peroxo compounds play an important role in many areas including e. g. perborates or percarbonates in the detergent industry or peroxo compounds for the whitening of a variety of products.
    Barium peroxide is one of the best-known peroxides. It can be prepared by the oxidation of barium oxide with oxygen in a reversible reaction. However, the peroxide content of BaO2 is always lower than that calculated.
    2 BaO + O22 BaO2500 °C700 °C
    Because of the reversibility of this reaction, barium peroxide provides a means of storage of elemental oxygen and several years ago, it was the only source of oxygen gas.
    The peroxide content of such compounds can be determined by reaction with an excess of acid to give dihydrogen peroxide followed by a titration with a standard solution of potassium permanganate. This quantitative method is widely used in all areas where peroxides are of importance.
    This practical exercise involves the preparation of strontium peroxide, determinimation of the strontium content by a complexometric titration and determination of the peroxide content by manganometric analysis.
    List of chemicals
    reagent
    concentration
    R phrases
    S phrases
    ammonia
    w(NH3) = 25 %
    34-50
    26-36/37/39-45-61
    EDTA disodium salt
    c(Na2EDTA) = 0.1 mol L-1
    ethanol
    w(C2H5OH) = 96 %
    11
    7-16
    hydrogen peroxide
    w(H2O2) = 3%
    34
    3-26-36/37/39-45
    methyl red
    solid
    naphthol green B
    solid
    perchloric acid
    w(HClO4) = 10%
    10-35
    23.2-26-36/37/39-45
    phthalein purple
    solid
    potassium perman-
    ganate
    c(KMnO4) = 0.1 mol L-1
    strontium chloride
    hexahydrate
    solid
    Procedure 1: Preparation of strontium peroxide
    5.0 g of strontium chloride hexahydrate are dissolved in about 2.5 mL of distilled water and 25 mL of dihydrogen peroxide (w(H2O2) = 3%) are added. A solution of 3.5 mL of ammonia (w(NH3) = 25%) in 50 mL of distilled water is added to the mixture to give strontium peroxide octahydrate on standing. The precipitate is filtered off, and dried at about 150°C. In this procedure, the octahydrate transforms nearly completely into the anhydrous compound. An extremely small amount of water remains in the product and the peroxide content is slightly lower than calculated for SrO2. At higher temperatures, strontium peroxide decomposes rapidly. Note: calcium peroxide can be prepared similarly.
    Record the yield of the product in g. 46
    Procedure 2: Manganometric determination of the peroxide content
    About 100 mg of the product prepared in procedure 1 (record the exact weight) are transferred into a 300 mL Erlenmeyer flask and the contents dissolved in 5 mL of perchloric acid. The volume of the solution is increased to about 100 mL by addition of water. The determination of the peroxide content is performed by titration with potassium permanganate solution (c(KMnO4) = 0.02 mol·L-1), until the solution is slightly pink in colour. At the beginning, the solution has to be titrated slowly because of the slow rate of reaction. The latter can be accelerated by the addition of a small amount of a manganese(II) compound.
    Record the volume of the potassium permanganate solution used in the titration in mL.
    Procedure 3: Complexometric determination of the strontium content
    About 100 mg of the product prepared in procedure 1 (record the exact weight) are transformed into a 300 mL Erlenmeyer flask and the contents dissolved in 5 mL of perchloric acid. The solution is made up to a volume of 50 mL and 15 mL of ammonia solution, 60 mL of ethanol and 2 mL of phthalein purple indicator are added. The resulting deep purple solution is titrated with disodium EDTA (c(Na2EDTA) = 0.1 mol L-1) until the solution is intense light-green in colour.
    Record the volume of the Na2EDTA solution in mL
    Preparation of the phthalein purple indicator
    100 mg of phthalein purple, 5 mg of methyl red and 50 mg of naphthol green B are dissolved in 2 mL of ammonia solution. The solution is filled up to a volume of 100 mL. The indicator is stable for up to a period of one week.
    35.1 Calculate the yield (%) of the product based on the theoretical yield of strontium chloride hexahydrate.
    35.2 Calculate the content of the liberated dihydrogen peroxide in percent for the manganometric analysis and compare this value with the theoretical value of SrO2.
    35.3 Calculate the strontium peroxide content in percent determined by the manganometric analysis.
    35.4 Calculate the strontium peroxide content in percent determined by the complexometric determination
    35.5 Write down the equation of the formation of SrO2 from SrCl2, H2O2
    and NH3.
    35.6 Write down the equation for the reaction of permanganate anions with dihydrogen peroxide in an acidic solution
    35.7 Why will the reaction in the manganometric analysis proceed faster if a manganese(II) salt is added to the mixture?
    Problem 36: Preparation and iodometric determination of potassium iodate
    Introduction
    Iodometric analysis is one of the most important volumetric procedures, because concentrations of both oxidizing and reducing agents, can be accurately determined using
    47
    this approach. The reaction between thiosulfate dianions and elemental iodine in a neutral or acidic solution is the basis of this method.
    2 S2O32– + I2S4O62– + 2 I–colourlessblue
    For the determination of oxidizing agents an excess of potassium iodide and a small amount of an acid are added to the sample solution. The iodine formed in this reaction is titrated with sodium thiosulfate solution.
    In contrast a back titration is typically performed for the determination of reducing agents in which a well defined excess of an iodine solution is added to the sample solution and the unreacted iodine is titrated with thiosulfate solution. Potassium iodate is used as a titrimetric standard for the standardization of the thiosulfate solution, because of its high stability and the fact that it can be produced in a very pure state. If an excess of potassium iodide is added to a well defined amount of potassium iodate in an acidic solution, an equivalent amount of iodine will be generated which can be titrated with sodium thiosulfate solution.
    The practical exercise involves the preparation of potassium iodate and the determination of its purity by iodometric titration.
    List of chemicals
    reagent
    concentration
    R phrases
    S phrases
    acetic acid
    w(H3CCOOH) = 5%
    ethanol
    w(C2H5OH) = 96 %
    11
    7-16
    hydrochloric acid
    c(HCl) = 2 mol L-1
    34
    26-36/37/39-45
    potassium iodide
    solid
    63-36/38-42/43
    26-36/37/39-45
    potassium permanganate
    solid
    8-22
    2
    sodium thiosulfate
    c(Na2S2O3) = 0.1 mol L-1
    Procedure 1: Preparation of potassium iodate
    6 g of potassium permanganate are dissolved in 150 mL of hot distilled water. 3 g of potassium iodide dissolved in a small amount of distilled water are added to the solution.
    The reaction mixture is heated on a boiling water bath for 30 min. The unreacted potassium permanganate is removed by the addition of ethanol. During this procedure, the supernatant liquid becomes colourless.
    The resulting precipitate of manganese(IV) oxide is filtered off and the filtrate is acidified by the addition of acetic acid. The solution is concentrated by heating on a water bath until the product begins to crystallize. The solution is allowed to cool to room temperature. The crystalline product is filtered off and washed with a small amount of ethanol. More product can be isoIated by further concentration of the mother liquor. The product can be recrystallized from water and dried at 110°C.
    Record the yield of the product in g
    Procedure 2: Iodometric determination of the purity of the isolated potassium iodate.
    If a 25 mL burette is to be used in the determination take about 60 mg of the product prepared in procedure 1 (record the exact weight) and dissolve it in about 100 ml of distilled water. Add 1 g of potassium iodide to the solution and slightly acidify with dilute hydrochloric acid. The solution is titrated with sodium thiosulfate solution (c(Na2S2O3) = 0.1 mol L-1) until it
    48
    becomes colourless. Just before the end point 2 - 3 mL of starch solution are added as an indicator.
    Record the volume of the sodium thiosulfate solution used in mL
    Preparation of the starch solution:
    About 2 g of starch are suspended in 3 mL of distilled water and the suspension vigorously stirred. The mixture is added to 300 mL of boiling water and heated for about two min. Any undissolve starch should be removed by decanting.
    The starch solution should be prepared as required, however, it can be kept for a longer period by the addition of a small amount of a mercury(II) iodide solution.
    36.1 Calculate the yield (%) of the product.
    36.2 Calculate the purity of your product in a percentage.
    36.3 Give the equation for the reaction between iodate and iodide anions in an acidic solution.
    36.4 What name is given to the redox reaction in 36.3?
    36.5 Why should an iodometric determination not be performed in an alkaline solution ?
    36.6 What is the expected trend in oxidising ability on going from fluorine to iodine? Givew the explanation for this trend.
    36.7 How can the following ions be determined iodometrically? In each case give the appropriate equation:
    a) iron(III) cations
    b) copper(II) cations
    c) sulfide anions
    Problem 37: Qualitative analysis of anions in an unknown mixture
    Introduction
    Besides the quantitative analysis of chemical compounds, the qualitative analysis of unknown substances or mixtures of substances in order to identify the cations and/or anions is also an important procedure in analytical chemistry.
    Cations have to be seperated prior to identification, however, this is not the case for anions.
    In this exercise, the anions in an analytical sample are to be identified. Some of these anions can be identified by direct analysis of the solid sample, however, for other it is necessary to identify them in the filtrate of a soda extract. Several reagents are provided that can either be used in the initial identification of the anions present, or to perform the necessary confirmation tests for a particular anion.
    The reactions of the anions with the reagents that are available, as far as is necessary for your analysis, are described below.
    List of potential anions:
    acetate H3CCOO-
    nitrate NO3-
    carbonate CO32-
    oxalate C2O42-
    chloride Cl-
    perchlorate ClO4-
    chromate CrO42-
    sulfate SO42- 49
    Preparation of the soda extract
    One spatulaful of the sample (about 1 g) is mixed with 2 – 3 times the amount of sodium carbonate. The mixture is suspended in water and heated for 10 minutes. After cooling, the residue is filtered off and washed with water. The filtrate is used in the anion identification. It is always a good idea to use blind samples for comparison and to check the purity of soda.
    Selected reactions of the anions that may be present:
    Acetate
    Theory: Acetate anions react with potassium hydrogensulfate to form acetic acid:
    H3CCOO– + HSO4–H3CCOOH + SO42–
    Dilute sulfuric acid also forms acetic acid upon reaction with acetate anions.
    Procedure: The solid sample is grinded with four times the amount of potassium hydrogensulfate in a mortar. In the presence of acetate anions, there is the characteristic smell of acetic acid.
    Carbonate
    Theory: Carbonate anions react with dilute hydrochloric acid to form unstable carbonic acid that decomposes into water and carbon dioxide:
    CO32– + 2 H+{H2CO3}CO2 ↑ + H2O
    Carbon dioxide reacts with barium hydroxide to form barium carbonate:
    CO2 + Ba(OH)2BaCO3 + H2O
    Procedure: In a test tube, dilute hydrochloric acid is added to a small amount of the sample. The test tube is closed immediately connected to a fermentation tube filled with freshly prepared barium hydroxide solution. The test tube is gently heated. In the presence of carbonate anions, white flakes of barium carbonate are observed in the solution in the fermentation tube within 3 - 5 minutes.
    Ba(OH)-solution2
    schematic representation of a fermentation tube
    Chloride
    Theory: Chloride anions in a nitric acid solution react with silver nitrate to form silver chloride:
    Ag+ + Cl–AgCl
    Silver chloride is soluble in concentrated ammonia solution. It is insoluble in concentrated nitric acid.
    Procedure: An aqueous solution of silver nitrate is added to 5 mL of the soda extract acidified with dilute nitric acid. In the presence of chloride anions, white silver chloride precipitates from solution. The latter decomposes into elementary silver within a few hours if it is exposed to sunlight. 50
    Chromate
    Theory: Chromate anions react with silver nitrate in a neutral or dilute nitric acid solution to form silver chromate:
    2 Ag+ + CrO42–Ag2CrO4
    Silver chromate is soluble in acids and ammonia solution.
    Procedure: An aqueous solution of silver nitrate is added to 5 mL of the soda extract that is acidified with dilute nitric acid. In the presence of chromate anions, reddish brown silver chromate precipitates from the solution.
    Theory: Chromate anions react with barium chloride in an acetic acid solution buffered by ammonium acetate to form barium chromate:
    Ba2+ + CrO42–BaCrO4
    Barium chromate is soluble in strong mineral acids.
    Procedure: A spatulaful of ammonium acetate is added to 5 mL of the soda extract that has been acidified with acetic acid. An aqueous solution of barium chloride is added and the mixture boiled for 2 minutes. In the presence of chromate anions, yellow barium chromate precipitates from the solution.
    Concentrated, yellow coloured, chromate containing solutions form orange coloured dichromates upon acidification with dilute sulfuric acid. The addition of more highly concentrated sulfuric acid leads to the formation of dark coloured oligo- and polychromates.
    Nitrate
    Theory: Nitrate anions are reduced to nitrogen monoxide (NO) by iron(II) sulfate in solutions acidified with sulfuric acid. Nitrogen monoxide reacts with iron(II) cations to form the brownish nitrosyl complex [Fe(NO)(H2O)5]2+.
    Procedure: 2.5 mL of an iron(II) sulfate solution acidified with sulfuric acid is added to 2.5 mL of the soda extract. After mixing, the test tube is brought into a skew position and concentrated sulfuric acid is poured carefully along the inner surface. In the presence of nitrate anions, a brownish ring forms at the phase boundary between the soluton and the sulfuric acid.
    Oxalate
    Theory: In a neutral solution, oxalate anions react with silver nitrate solution to form silver oxalate:
    2 Ag+ + C2O42–Ag2C2O4
    Silver oxalate is sparingly soluble in acetic acid. It is soluble in nitric acid and ammonia solution.
    Procedure: An aqueous solution of silver nitrate is added to 5 mL of the soda extract neutralized with acetic acid. In the presence of oxalate anions, a white precipitate of silver oxalate is formed.
    Theory: Oxalate anions react in an ammoniacal or acetic acid solution that is buffered by sodium acetate, with calcium chloride to form calcium oxalate:
    Ca2+ + C2O42–CaC2O4 51
    Calcium oxalate is insoluble in dilute acetic acid. It is soluble in strong mineral acids. Calcium oxalate is oxidized to carbon dioxide by potassium permanganate in an acidic solution. In this reaction, the manganese(VII) cations are reduced to manganese(II) cations.
    Oxalates and oxalic acid decompose by reaction with concentrated sulfuric acid into carbon monoxide and carbon dioxide:
    H2C2O4H2SO4H2O + CO ↑ + CO2 ↑
    Procedure: 5 mL of the soda extract are acidified with acetic acid. Ammonia solution is added until the mixture is slightly ammoniacal followed by the addition of an aqueous solution of calcium chloride. In the presence of oxalate anions, white calcium oxalate precipitates from solution. The precipitate is filtered off and dissolved in sulfuric acid. A solution of potassium permanganate is added dropwise to the solution. The potassium permanganate solution rapidly decolourizes and a gas is formed.
    Theory: In a neutral solution, oxalate anions react with barium chloride to form barium oxalate:
    Ba2+ + C2O42–BaC2O4
    Barium oxalate dissolves in dilute acetic acid.
    Procedure: An aqueous solution of barium chloride is added to 5 mL of the soda extract neutralized with dilute hydrochloric acid. In the presence of oxalate anions, white barium oxalate precipitates from the solution.
    Perchlorate
    Theory: In a solution slightly acidified with nitric acid, perchlorate anions react with potassium nitrate to form potassium perchlorate:
    ClO4– + K+KClO4
    Potassium perchlorate is insoluble in cold water and cold dilute acid.
    Procedure: An aqueous solution of potassium nitrate is added to 5 mL of the soda extract slightly acidified with nitric acid. In the presence of perchlorate anions, a white precipitate of potassium perchlorate forms.
    Theory: In a neutral and slightly alkaline solution perchlorate anions are reduced by iron(II) hydroxide (formed by the reaction of iron(II) sulfate with sodium hydroxide) to chloride anions.
    Procedure: 4 mL of an aqueous iron(II) sulfate solution are added to 5 mL of the soda extract acidified with dilute nitric acid. Dilute sodium hydroxide solution is added until some iron(II) hydroxide begins to precipitate from solution or the solution is slightly alkaline. The mixture is boiled for a few minutes and the resulting precipitate is filtered off. In the presence of perchlorate anions, the filtrate of the reaction contains chloride anions, which can be confirmed by reaction with silver nitrate in a solution acidified with nitric acid .
    Sulfate
    Theory: In an acidic solution acidified with hydrochloric acid sulfate anions react with barium chloride to form barium sulfate:
    Ba2+ + SO42–BaSO4 52
    Barium sulfate is insoluble in concentrated hydrochloric acid and in concentrated nitric acid. It is sparingly soluble in hot concentrated sulfuric acid, 12 percent of barium sulfate dissolves.
    Procedure: An aqueous solution of barium chloride is added to 5 mL of the soda extract acidified with dilute hydrochloric acid. In the presence of sulfate anions white barium sulfate precipitates from the solution.
    Theory: In an acidic solution acidified with hydrochloric acid, sulfate anions react with calcium chloride to form calcium sulfate:
    Ca2+ + SO42–CaSO4
    Calcium sulfate dissolves in concentrated sulfuric acid and concentrated hydrochloric acid.
    Procedure: An aqueous solution of calcium chloride is added to 5 mL of the soda extract acidified with dilute hydrochloric acid. In the presence of sulfate anions, white calcium sulfate precipitates from the solution. The precipitation is not quantitative!
    37.1 Which anions are present in your sample?
    37.2 Give the equations of the reaction of nitrate anions with iron(II) cations and of the subsequent formation of the nitrosyl complex.
    37.3 Why does the brownish coloured complex form directly at the phase boundary between the solution and concentrated sulfuric acid?
    37.4 Write the equation of the reaction of permanganate anions with oxalate anions in an acidic solution.
    37.5 Write the equation of the reaction of perchlorate anions with iron(II) hydroxide in a neutral solution.
    List of chemicals
    reagent
    concentration
    R phrases
    S phrases
    acetic acid
    w(H3CCOOH) = 99%
    10-35
    23-26-45
    acetic acid
    w(H3CCOOH) = 5%
    ammonia
    w(NH3) = 25%
    34-50
    26-36/37/39-45-61
    ammonium acetate
    solid
    barium chloride
    c(BaCl2 · 2 H2O) ~ 1.5 mol L-1
    20/22
    28
    barium hydroxide
    w(Ba(OH)2 · 8 H2O) ~ 2 %
    20/22-34
    26-36/37/39-45
    calcium chloride
    c(CaCl2 · 2 H2O ) = 1 mol L-1
    36
    22-24
    hydrochloric acid
    w(HCl) = 36%
    34-37
    26-45
    hydrochloric acid
    c(HCl) = 2 mol L-1
    34
    26-36/37/39-45
    iron(II) sulfate
    c(FeSO4 · 7 H2O) = 1 mol L-1
    22
    24/25
    nitric acid
    w (HNO3) = 65%
    8-35
    23-26-36-45
    nitric acid
    c (HNO3) = 2 mol L-1
    8-35
    23-26-36-45
    potassium hydrogen-sulfate
    solid
    34-37
    26-36/37/39-45
    potassium nitrate
    saturated
    36/38
    26
    potassium perman-ganate
    c(KMnO4) = 0.02 mol L-1
    52/53
    61
    silver nitrate
    c(AgNO3) = 0.2 mol L-1
    34-51/53
    26-36/37/39-45-61
    sodium acetate
    solid
    53
    sodium carbonate
    solid
    36
    22-26
    sodium hydroxide
    w(NaOH) ~ 5 %
    35
    26-37/39-45
    sulfuric acid
    w(H2SO4) = 95-97 %
    35
    26-30-45
    sulfuric acid
    c(H2SO4) = 2 mol L-1
    35
    26-30-45
    Preparation of the sample:
    To avoid interferences in the qualitative determinations only certain selected counter ions should be present in the analytical sample. The following salts guarantee the determination of anions without any interference: LiCl, LiClO4, Na(OOCCH3), Na2CO3, NaCl, NaNO3, Na2C2O4, NaClO4, Na2SO4, K2CO3, K2Cr2O7, KNO3, K2SO4, AlCl3, Al2(SO4)3, FeCl2, FeSO4, CoCl2, Co(NO3)2, CoSO4, NiCl2, Ni(NO3)2, NiSO4. Certain other salts can be used. The salts must not form sparingly soluble residues. If salts are to be used that are not mentioned in the following table, then the hazard and safety data sheets for the compounds must first be consulted.
    salt
    formula
    R phrases
    S phrases
    aluminium chloride
    AlCl3 · 6 H2O
    36/38
    aluminium sulfate
    Al2(SO4)3 · x H2O
    24/25
    cobalt(II) chloride
    CoCl2 · 6 H2O
    49-22-42/43-50/53
    53-22-45-60-61
    cobalt(II) nitrate
    Co(NO3)2 · 6 H2O
    22-20-43
    36/37
    cobalt(II) sulfate
    CoSO4 · 7 H2O
    49-22-42/43-50/53
    53-22-45-60-61
    iron(II) chloride
    FeCl2 · 4 H2O
    22-38-41
    26-39
    iron(II) sulfate
    FeSO4 · 7 H2O
    22
    24/25
    lithium chloride
    LiCl
    22-36/38
    lithium perchlorate
    LiClO4
    8-22-36/37/38
    17-26-36
    nickel(II) chloride
    NiCl2 · 6 H2O
    25-43-50/53
    24-37-45-61
    nickel(II) nitrate
    Ni(NO3)2 · 6 H2O
    8-22-43
    24-37
    nickel(II) sulfate
    NiSO4 · 6 H2O
    22-40-42/43-50/53
    22-36/37-60-61
    potassium carbonate
    K2CO3
    36/37/38
    22-26
    potassium dichromate
    K2Cr2O7
    49-46-21-25-26-37/38-41-43-50/53
    53-45-60-61
    potassium nitrate
    KNO3
    36/38
    26
    potassium sulfate
    K2SO4
    sodium acetate
    NaH3CCOO
    sodium carbonate
    Na2CO3
    36
    22-26
    sodium chloride
    NaCl
    sodium nitrate
    NaNO3
    8-22-36
    22-24-41
    sodium oxalate
    Na2C2O4
    21/22
    24/25
    sodium perchlorate
    NaClO4 · H2O
    9-22
    13-22-27
    sodium sulfate
    Na2SO4
    54



    IMPROVEMENTS IN THE METHODS FOR CALCIUM
    DETERMINATION IN BIOLOGICAL MATERIAL
    BY CHI CHE WANG
    (Prom the Children’s Hospital Research Foundation and the Department of
    Pediatrics, University of Cincinnati, Cincinnati)
    (Received for publication, June 10, 1935)
    It is well known that the principal disadvantage of any method
    for the determination of calcium involving precipitation as calcium
    oxalate lies in washing the precipitate. The solubility of
    the precipitate in water or in dilute ammonium hydroxide is appreciable
    and, in centrifuging, a small amount of calcium oxalate
    tends to float on the surface of the liquid and is lost in decanting.
    The satisfactory results obtained by the oxalate method are due
    largely to the compensation of errors, the loss of the precipitate
    of calcium oxalate being balanced by incomplete removal of ammonium
    oxalate in the washings. It is apparent that a washing
    fluid which would remove the excess of oxalate solution with no
    appreciable solution of the precipitate and which prevents any of
    the precipitate from floating on the surface of the liquid would
    increase the accuracy of the method. It has been found that a
    mixture of 2 per cent ammonia in equal parts of alcohol, ether,
    and distilled water meets these requirements, provided that the
    ammonium oxalate used for precipitation is not too concentrated.
    0.1 M ammonium oxalate is substitutued for the usual saturated
    ammonium oxalate (4 per cent). This is about one-third the
    usual concentration but insures an adequate oxalate concentration
    for complete precipitation of calcium oxalate. Two washings
    with the ammoniacal alcohol-ether-water mixture are needed but
    there is no appreciable loss of calcium with three washings. Three
    washings are not sufficient when more concentrated ammonium
    oxalate is used.
    In applying the new washing solution to calcium determinations
    we have used Kramer and Tisdall’s (1) permanganate titration,
    443
    Downloaded from www.jbc.org by on September 19, 2006
    TABLE I
    Calcium Determinations with New Washing Technique
    All precipitations stood overnight.
    On calcium chloride solutions (Iceland *pm in HCI)
    --
    c?. ,mg. pm cent mg.
    0.224 0.228 +1.8 0.124
    0.224 0.227 +1.3 0.124
    0.224 0.224 0.0 0.124
    0.224 0.227 f1.3 0.124
    0.224 0.228 +1.8 0.124
    0.224 0.225 +0.4 0.124
    __-__
    Average.0.224 0.226 +l.l 0.124
    Ca
    determined
    ms. per cm1
    0.123 -0.8
    0.123 -0.8
    0.122 -1.6
    0.123 -0.8
    0.124 0.0
    0.124 0.0
    0.123 -0.7
    ---
    %?. mg. per cent
    0.062 0.061 -1.6
    0.062 0.060 -3.2
    0.062 0.062 0.0
    0.062 0.061 -1.6
    0.062 0.062 0.0
    0.062 0.063 -1.6
    --~
    0.062 0.062 f1.3
    Recovery of Ca from artificial tissue extract containing per 100 cc. 8 mg. Ca, 20 mg. Mg,
    152 mg. P, 150 mg. Na. and 190 mg. K
    Diluted 0.08800 5 mcgc.. sCaam ple = Diluted 80 5 cc. sample = 0.200 mg. Ca
    Pptd. directly
    mg.
    0.079
    0.080
    0.078
    0.078
    0.082
    0.078
    0.081
    0.082
    Pptd. directly
    w7.
    0.200
    0.202
    0.201
    0.198
    0.197
    0.197
    Average.0.080 0.199
    Ashed with 5 dro s HzSOa.
    taken up mt. % HCl
    “8.
    0.201
    0.201
    0.197
    0.198
    0.200
    0.201
    0.200
    Dog 8erum (1 cc;~$valent of serum Recovery of Ca added to dog mm (trichloroacetic
    acid filtrate)
    Ashed with
    Trichloroacetic acid Cs in serum
    filtrate Ell~g%& detgp- Ca *dckJd
    w7. mg. m?. “9.
    0.099 0.097 0.021 0.062
    0.098 0.098 0.069 0.062
    0.100 0.100 0.080 0.062
    0.101 0.101 0.059 0.124
    0.100 0.102 0.060 0.124
    0.103 0.099 0.042 0.186
    0.097 0.097 0.128 0.124
    0.098 0.097
    Avcrage.0.100 0.099
    444
    Added Ca
    recovered
    m7. per cent
    0.063 $1.6
    0.062 0.0
    0.063 f1.6
    0.126 f1.6
    0.127 +2.4
    0.187 $0.5
    0.127 +2.4
    +1.5
    Downloaded from www.jbc.org by on September 19, 2006
    C. C. Wang 445
    Clark and Collip’s (2) method of removing the washing fluid, and
    Van Slyke and Sendroy’s (3) principle of removing proteins with
    trichloroacetic acid.
    The method, as applied to samples containing between 0.06
    and 0.22 mg. of calcium, gave an average deviation of ~1.1 per
    cent of theoretical values with standard CaC& solution or with
    artificial tissue extract solution, and an average deviation of
    $1.5 per cent when calcium was added to the filtrate from blood
    serum (Table I). After a little experience, one person can easily
    run twenty-four calcium determinations during a working day.
    The author has run as many as forty-eight in a day. In connection
    with our metabolic work, this method has been used by the
    writer in approximately 2500 determinations of calcium, in blood,
    urine, food, and feces, with an error of approximately 2 per cent.
    The procedure also has been successfully used by associates in
    other departments of this institution. The exact techniques for
    serum, urine, food, and feces are.given below.
    Reagents
    Trichloroacetic acid, 20 per cent; 20 gm. dissolved and diluted
    to 100 cc.
    Sodium acetate, 20 per cent; 20 gm. dissolved and diluted to
    100 cc.
    Brom-cresol green, 0.016 per cent; prepared from stock solution
    as described by Clark (4).
    Ammonium oxalate, 0.1 M; 1.42 gm. of ammonium oxalate dissolved
    and diluted to 100 cc. Add a crystal of thymol and keep
    the bottle in the refrigerator.
    Acetic acid, approximately 1.5 M; 42 cc. of 99.5 per cent acetic
    acid, diluted to 500 cc.
    Ammonium hydroxide, 1: 1; 1 volume of ammonium hydroxide
    to 1 volume of distilled water.
    Ammonium hydroxide, 1: 3; 1 volume of ammonium hydroxide
    to 3 volumes of distilled water.
    Washing solution; 20 cc. of concentrated ammonium hydroxide
    in 980 cc. of a mixture of 1 volume of redistilled alcohol, 1
    volume of redistilled ether, and 1 volume of distilled water.
    Sulfuric acid, approximately 1 N; 27 cc. of concentrated sulfuric
    acid diluted to 1000 cc.
    Downloaded from www.jbc.org by on September 19, 2006
    446 Cn in Biological Material
    Sulfuric acid, 1: 1; 1 volume of concentrated sulfuric acid to
    1 volume of distilled water.
    Nitric acid, concentrated.
    Nitric-perchloric acid mixture; 100 cc. of fuming nitric acid
    (sp. gr. 1.49), 50 cc. of perchloric acid (sp. gr. 1.615), 100 cc. of
    distilled water.
    Hydrochloric acid, approximately 20 per cent; 1 volume of
    concentrated hydrochloric acid to 1 volume of distilled water.
    Hydrochloric acid, approximately 1 N; 100 cc. of concentrated
    hydrochloric acid diluted to 1000 cc. with distilled water.
    Acid-washed charcoal.’
    Standard potassium permanganate, 0.01 N (5).
    Standard sodium oxalate, 0.02 N; 0.134 gm. of sodium oxalate
    dissolved and diluted to 100 cc.; Merck’s reagent, dried to constant
    weight and kept in a vacuum dessicator. It keeps about
    1 month in the refrigerator.
    Blank determinations must be made including all reagents in
    the quantities used in the actual determination. In the results
    reported here there was no detectable calcium in the reagents used.
    All glassware, including reagent bottles, should be of Pyrex, except
    the burettes and pipettes. In this study Thomas pipettes
    were used.
    Procedures
    Blood Serum-To 1 volume of serum and 3 volumes of distilled
    water in an Erlenmeyer flask, is added, drop by drop with constant
    shaking, 1 volume of 20 per cent trichloroacetic acid. The mixture
    is allowed to stand for about + hour, transferred t.o a centrifuge
    tube, and centrifuged for 5 minutes. The supernatant
    liquid is poured off through a small ashless filter paper.
    Into a scrupulously clean2 15 cc. conical centrifuge tube, with
    1 We used Darco decolorizing carbon from the Darco Sales Corporation,
    Sew York, but have not tested other charcoals. This carbon contained
    considerable calcium which was removed as follows: 10 gm. of the carbon
    were boiled with about 150 cc. of 10 per cent HCl for about 10 minutes,
    poured on an ashless filter paper in a Buchner funnel, and washed with distilled
    water until free of chlorides. It was then dried in an oven at about
    100”.
    2 The centrifuge tubes must be thoroughly clean, so that no liquid will
    pling to the wall of the tube after decanting. It is advisable t.o boil the
    Downloaded from www.jbc.org by on September 19, 2006
    C. C. Wang
    a proper point,3 5 cc. of the filtrate, 1 cc. of 20 per cent sodium
    acetate, 6 to 8 drops of 0.016 per cent brom-cresol green indicator,
    and 1 cc. of an approximately 0.1 M ammonium oxalate” are measured.
    In adding the oxalate, care must bc taken that it drops
    directly into the solution and does not touch the wall of the tube,
    from which it will be difficult to remove by subsequent washings.
    The mixture is stirred with a fine glass rod and the pH is adjusted
    to match a phosphate buffer solution of pH 5 containing the same
    number of drops of the indicator in a similar volume, with 1: 1
    ammonia at first and 1: 3 ammonia for the end-point.5 If the
    end-point is passed, the reaction is brought back to acid by means
    of dilute acetic acid, approximately 1.5 M. The rod is rinsed
    with water and the tube covered with a nursing bottle rubber cap.
    The mixture is permitted to stand for at least 1 hour, preferably
    overnight, and then centrifuged for 8 minutes at 2000 R.P.M. The
    supernatant solution is carefully decanted and while the tube is
    still inverted, it is placed in a rack on a few sheets of filter paper
    and allowed to drain for 5 minutes. (The supernatant liquid may
    be removed by suction with an immersion filter of a mesh that
    will hold back the precipitate and the immersion filter can be
    used afterwards as a stirring rod during titration.) The mouth
    of the tube is wiped with a strip of ashless filter paper and any
    drops that cling to the wall of the tube are also removed in this
    way. The precipitate is washed with approximately 3 cc. of
    tubes first in a soap solution for a few minutes (Dreft or Orous are much
    better than ordinary soap), after which they are rinsed and heated at approximately
    100” for a few minutes in a mixture of chromate and sulfuric
    acid.
    3 The shape of the tube is important, because if the point is too narrow, a
    part of the fluid will cling to the bottom of the tube when being decanted.
    If it is too wide, the precipitate will break. Since it is difficult to get the
    ideal shape and size of point, the simplest way is to test each tube with
    distilled water and see that all of the liquid can be removed by inverting the
    tube slowly.
    * 0.1 M (NH&C201 deteriorates easily on standing at room temperature.
    It should be kept in a refrigerator and preserved with a crystal of thymol.
    5 In case the mixture is strongly acid, as in oxidizing food and feces, concentrated
    NH,OH should be used first, and if the reaction of the mixture is
    alkaline, it should be made acid with concentrated acetic acid before the
    addition of (SH4)nCr04.
    Downloaded from www.jbc.org by on September 19, 2006
    Ca in Eiological Material
    2 per cent ammonium hydroxide in a mixture of equal parts of
    alcohol, ether, and distilled water from a wash bottle with a very
    fine jet, the stream being directed slightly above the point on the
    wall reached by the supernatant solution, while the tube is held at
    an angle of about 60” and is kept constantly rotating. The precipitate
    is thoroughly broken with a fine Pyrex glass rod and mixed
    with the washing mixture. The glass rod is rinsed, removed,
    and the tube centrifuged for 5 minutes. The supernatant solution
    is again decanted and drained as above. This washing is repeated
    once and the tube is placed in an oven for about 1 hour at a temperature
    between 85-100’ in order to remove the last trace of the
    organic solvents. At the end of an hour, 2 cc. of approximately
    N sulfuric acid are delivered from a pipette into the tube, with care
    to wash the sides of the tube with the acid. The mixture is stirred
    with the fine glass rod and the tube is placed in a boiling water
    bath for 1 minute. The oxalic acid is immediately titrated with
    0.01 N potassium permanganate,6 from a microburette graduated
    in 0.01 cc. and with a tip delivering not more than 0.015 cc. at a
    drop. If the oxalic acid requires more than 0.5 cc. of potassium
    permanganate, it is advisable to carry out the titration in a water
    bath at 70-75”. With the aid of a stirring rod it is possible to
    detect the end-point with not more than 0.008 cc. of the potassium
    permanganate. Thus the method is accurate from approximately
    0.0016 to 0.002 mg.
    Urine-Fiske and Logan (6) have pointed out that calcium
    may be determined directly on urine only when it is quite free
    from material (other than calcium oxalate) which precipitates
    under the conditions used, and, that whenever such material is
    present ashing is either more convenient or necessary. Our experience
    has been that, although in some cases it may be possible
    to determine calcium directly on fresh urine, it is not satisfactory,
    as high values frequently result. However, if the urine is treated
    with trichloroacetic acid, calcium values agreeing with those on
    urine ash are obtained. Even in apparently normal urine, without
    detectable traces of albumin, this treatment may remove
    interfering substances. One objection to using trichloroacetic
    6 The author prefers using 0.01 N KMn04 made according to Halverson
    and Bergeim (5). When this solution is kept in a dark bottle, the concentration
    is not appreciably changed for at least a month and there is no
    necessity to restandardize in less than that time.
    Downloaded from www.jbc.org by on September 19, 2006
    C. C. Wang 449
    acid filtrate for the permanganate titration method is the frequent
    presence of color in the filtrate which interferes with the adjustment
    of pH by brom-cresol green indicator. This difficulty is
    overcome by adding a small amount of acid-washed charcoal’
    to the mixture of urine and trichloroacetic acid before it is filtered
    but after the volume has been made up. The filtrate from this
    mixture is usually clear and almost colorless.
    Tests were made which proved that charcoal did not remove
    calcium from pure CaCL solution. In Table II, the results of
    parallel determinations on urine ash, trichloroacetic acid filtrate,
    and filtrate from trichloroacetic acid plus carbon are given. Practically
    colorless urines were selected for this series. In the latter
    part of Table II are given a few representative parallel determinations
    on ashed urine and on trichloroacetic acid-carbon filtrate
    of urine so highly colored that determinations on the filtrate without
    carbon were impossible. It is evident that this treatment
    with trichloroacetic acid and carbon allows determinations of
    calcium in urine to within the accuracy of the titration technique.
    Incidentally, the use of carbon removes the objectionable urine
    odors.
    Urine is treated with 20 per cent trichloroacetic acid in the same
    manner as is blood, except that the dilution should be varied according
    to the calcium content expected and carbon is added
    before filtering. For instance, in the urine of infants and that of
    patients suffering from certain diseases such as nephrosis, the
    total per 24 hour urinary calcium may be as low as 10 mg. On
    the other hand, the urinary calcium of adults and that of children
    suffering from progressive pseudohypertrophic muscular dystrophy
    or in conditions of highly acid urine may be as high as 300
    mg. per 24 hours. In the former case, 1 volume of 20 per cent
    trichloroacetic acid is added slowly to 4 volumes of urine in an
    Erlenmeyer flask. In the latter case, 1 volume of the acid is
    added to a mixture of 1 volume of urine and 3 volumes of water.
    For every 25 cc. of this trichloroacetic acid-urine mixture, about
    0.4 gm. of acid-washed, dry charcoal is added. The mixture is
    shaken and allowed to stand for about 15 to 20 minutes with occasional
    shaking and then filtered through an ashless filter. The
    filtrate should be clear and almost colorless. Calcium is determined
    on an aliquot 5 to 10 cc. portion of the filtrate as in the
    Downloaded from www.jbc.org by on September 19, 2006
    450 Ca in Biological Material
    case of blood serum. Thus in the lowest output found, 10 mg. of
    Ca in 1000 cc. of urine per day, 10 cc. of the filtrate gave 0.08 mg.
    TABLE II
    Comparison oj Calcium Determinations on Ashed Urine with Those on
    Trichloroacetic Acid Filtrale with and without Charcoal
    All precipitations stood overnight. The values are given in mg. per
    100 cc
    Faintly colored
    urines, 6 determinations
    on each
    Highly colored
    urines, 2 determinations
    on each
    LJrine
    NO.
    1
    2
    3
    4
    5
    6
    7
    8
    9
    LO
    11
    12-a*
    12-b
    13-a
    13-b
    14-a
    14-b
    15
    16
    17
    18
    19-ai
    19-b
    T i Med with 5 drops l?iltrate from
    concentrated Trichloroacetic trichloroacetic
    HzS$h;a$ze;ec; UP acid filtrate acid filtrate +
    charcoal
    Range
    11.4-11.1
    Ll.6-ll.!
    Ll.l-Il.‘
    LO.9-11.:
    8.7- 9.t
    8.6- 9.1
    7.8- 8.:
    7.8- 8.
    _____
    4verwe
    RrtIl,BC?
    11.6 11.5-ll.!
    11.711.3-ll.!
    11.3 10.5-11.
    11.2 10.7-11.t
    8.9 8.5- 8.1
    8.8 8.5- 8.1
    8.1 7.7- 8.
    8.1 7.8- 8.
    30.0
    24.3
    22.1
    11.4
    11.3
    11.3
    11.2
    8.2
    8.1
    4.8
    4.1
    3.2
    2.1
    1.6
    1.5
    9
    9
    1
    3
    3
    3
    1
    1 7
    _____
    4verwe
    Range
    __-___
    11 .8 11.6-12.f
    11.7 11.7-12.t
    10.9 11 .o-11.:
    10.9 11.2-11.:
    8.7 8.5- 8.!
    8.6 8.6- 9.f
    7.9 7.7- 8.
    7.8- 8.:
    -
    _
    4verage
    11.8
    11.8
    11.2
    11.2
    8.7
    8.8
    8.0
    7.9
    30.4
    24.4
    22.1
    11.4
    11.2
    11.3
    11.2
    8.2
    7.9
    4.9
    4.0
    3.1
    2.0
    1.6
    1.6
    * The a and b indicate separate days for the same subject.
    t Urine samples, from 2 successive days, from a child with nephrosis, on
    an ordinary hospital diet. There were about 2 gm. of albumin per liter
    of urine.
    of Ca, and in the adult urine of 300 mg. of Ca in 1500 cc. of urine
    per day, 5 cc. of filtrate gave 0.2 mg. of Ca. As shown in Table
    I, these amounts of calcium are accurately determinable.
    Downloaded from www.jbc.org by on September 19, 2006
    C. C. Wang 451
    Food and Feces-About 2 gm. of dry, finely ground food or
    0.5 gm. of feces is placed in a 250 cc. Kjeldahl flask (or 800 cc.
    flask if perchloric acid is employed). It is oxidized with either
    sulfuric and nitric acids or by a nitric-perchloric acid mixture according
    to the method of Lematte, Boinot, and Kahane (7).
    Sulfuric Acid Method-10 cc. of 1: 1 sulfuric acid are placed in
    a flask containing the dried material and the flask is heated over
    a water bath until the mixture is thoroughly charred. Nitric
    acid, 8 to 10 drops at a time, is added from time to time and the
    flask shaken and heated over a microburner until the mixture is
    thoroughly oxidized and becomes colorless on further heating.
    The solution is cooled and rinsed into a 500 cc. volumetric flask
    and made up to the mark with distilled water. In order to remove
    the excess of sulfate, an aliquot part, usually 5 cc., of the
    solution is pipetted into a 30 cc. Pyrex beaker, evaporated down
    to dryness on a water bath, and placed in a muffle furnace at
    600' for 15 to 20 minutes. Onto the residue 1 cc. of 20 per cent
    hydrochloric acid is measured and evaporated just to dryness on
    a sand bath on a electric plate, care being taken to avoid spattering.
    About 10 minutes are usually required to bring the solution
    just to dryness. This procedure is repeated four times, the
    beaker being cooled each time before the addition of hydrochloric
    acid. The final residue is dissolved in 2 cc. of N hydrochloric acid
    and is quantitatively transferred into a 15 cc. conical centrifuge
    tube. The procedure for the precipitation of calcium is then carried
    out as described above for serum.
    Nitric-Perchloric Acid Method-To the flask containing the dry
    material are added about 40 cc., or 20 cc. in the case of feces, of
    the nitric-perchloric acid mixture and the flask is heated over a
    microburner with constant shaking in order to avoid explosion.
    When white fumes begin to appear and the liquid starts to
    darken, more acid mixture, 10 to 20 cc., is added. The digestion
    is continued until the mixture becomes colorless or only slightly
    colored. The flask is cooled and the mixture is quantitatively
    transferred into a 500 cc. volumetric flask. Calcium is determined
    on 5 cc. portions of the solution. Although it requires
    considerable care to prevent explosion, this method saves the step
    of removing the excess of sulfate ion.
    Interference of Sulfate-One of the difficulties encountered in
    Downloaded from www.jbc.org by on September 19, 2006
    452 Ca in Biological Material
    determining calcium in urine or in the ash of the digestion by
    means of sulfuric and nitric acids is the interference of the sulfate
    ion in the precipitation of calcium oxalate noted by Fiske. In
    order to find the proportion of sulfate to calcium at which the
    former would not interfere appreciably with the precipitation
    under the conditions of oxalate concentration suitable for this
    new washing technique, experiments were made in which various
    amounts of sulfuric acid were added to serum filtrates of known
    calcium value. As shown in Table III, the interference decreases
    TABLE III
    Effect of Sulfate Ion on Calcium Oxalate Precipitation
    Calcium and HzSOa were added to tubes containing 5 cc. of trichloroacetic
    acid filtrate of dog serum equivalent to 1 cc. of serum, containing
    0.098 mg. of calcium oxalate added; pH adjusted to 5, and allowed to stand
    overnight.
    Ca added
    Total Ca present
    w. mg.
    0.062 0.160
    0.124 0.222
    0.186 0.284
    0.062 0.160
    0.124 0.222
    0.186 0.284
    0.062 0.160
    0.124 0.222
    0.186 0.284
    0.124 0.222
    0.186 0.284
    mg.
    432.5 2703
    432.5 1948
    432.5 1523
    288.3 1802
    288.3 1299
    288.3 1015
    216.3 1352
    216.3 974
    216.3 761
    144.2 649
    144.2 506
    -
    w?.
    0.080
    0.148
    0.230
    0.131
    0.201
    0.272
    0.147
    0.214
    0.275
    0.218
    0.281
    per cent
    50.0
    33.3
    19.0
    18.1
    9.5
    4.2
    8.1
    3.6
    3.2
    1.8
    1.1
    3ulfate added
    -7 - --
    Deviation
    from total 104 : Cs ratio
    with the ratio of sulfate to calcium, and when the ratio is approximately
    500: 1, the interference becomes negligible. When interference
    occurs, multiple determinations disagree among themselves
    and failure to get good checks should raise suspicions as
    to completion of precipitation. In our experience, whenever
    sulfuric acid is used in oxidation, unless the ratio can be dennitely
    established by calculation, it is wise to remove the excess of sulfate
    by means of heat and to convert the insoluble metaphosphates
    to soluble orthophosphates first, otherwise the results may be
    considerably lower than the actual values. Although a ratio of
    Downloaded from www.jbc.org by on September 19, 2006
    C. C. Wang
    50O:l seems rather large, it does not take much sulfuric acid to
    reach this point. For instance, in Fiske’s alkalimetric calcium
    method (6), when he oxidizes a urine specimen containing from
    0.25 to 0.75 mg. of calcium with 1 cc. of 10 N sulfuric acid, not
    including the sulfate already present in the specimen, the ratio
    lies between 1920 and 640 to 1.
    This high sulfate-calcium ratio does not interfere with accurate
    results in Fiske’s method, but it is apparent that it does under
    the conditions of lower oxalate concentration used with the
    present method. Since the sensitivity to sulfate interference is
    increased under these conditions, it is possible that the interfering
    action of other substances, as P and Mg discussed by Fiske,
    might likewise be intensified. The result on artificial tissue salt
    mixture, given in Table I, shows that salt relations in tissue extract
    or ash do not interfere and the results with urines given in
    Table II indicate that there is no interference in ordinary human
    urine. We have not yet tested it against Fiske’s double precipitation
    method for urines containing unusually high phosphorus
    and magnesium.
    SUMMARY
    1. A new washing solution for calcium oxalate precipitation
    of 2 per cent ammonia in equal parts of alcohol, ether, and water
    prevents flotation and permits washing of the precipitate wit.hout
    appreciable loss of calcium.
    2. The treatment of urine with trichloroacetic acid and carbon
    allows direct calcium determination on urine.
    BIBLIOGRAPHY
    1. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 47, 475 (1921); 48, 223
    (1921).
    2. Clark, E. P., and Collip, J. B., J. Biol. Chem., 63,461 (1925).
    3. Van Slyke, D. D., and Sendroy, J., Jr., J. Biol. Chem., 84,217 (1929).
    4. Clark, W. M., The determination of hydrogen ions, Baltimore, 3rd edition,
    61, 94, 126 (1928).
    5. Halverson, J. O., and Bergeim, O., J. Ind. and Eng. Chem., 10,119 (1918).
    6. Fiske, C. H., and Logan, M. A., J. Biol. Chem., 93,211 (1931).
    7. Lematte, L., Boinot, G., and Kahane, E., J. pharm. et chim., 6, 325, 361
    (1927).
    Downloaded from www.jbc.org by on September 19, 2006
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