Iodometric Estimation of Copper in the Presence of Citrate J. F. SCAIFE' l o w Temperafure Station for Research in Biochemistry and Biophysics, University of Cambridge, England
b The method presented permits copper to be estimated iodometrically in the presence of citrate without recourse to titrating in solutions of very low pH values where inaccuracies due to the nature of other materials present, precipitated cuprous iodide, or difficult starch end point can occur. Tedious wet digestion of the organic material present i s also avoided.
T
HE volumetric method for the iodometric estimation of copper has been investigated ( I - 4 ) , and the conditions have been defined under which it gives highly accurate results. However, under certain conditions when copper complexing compounds are present the reaction does not go to completion. A limited reaction takes place with some of the copper, without the usual precipitation of cuprous iodide. The extent to which this reaction proceeds is governed b y the concentration of copper, ligand, and potassium iodide, and by PH. Investigations have shown that the addition of dilute acetic acid to lower the p H favors completion of the reaction in the presence of a copper ligand b y causing a n increased dissociation of the complex. Mineral acids have been used t o achieve this effect in special cases (3). On the other hand an increase in the concentration of the complexing agent by reducing the amount of free copper in solution reduces the extent of the reaction. Higher concentrations of potassium iodide also favor a more complete reaction of the copper. If an attempt is made to titrate the liberated iodine in a solution from which no cuprous iodide has been precipitated, iodine color is rapidly discharged and only s l o ~ l yreappears as more copper dissociates from the complex and reacts with potassium iodide. It is thus impossible to titrate the solution in the normal manner. If the concentration of ligand is not too great, however, it may be possible to complete the titration b y adding a large excess of potassium iodide and increasing the 1 Present address, Chemistry Division, Science Service, Department of Agriculture, Ottawa, Canada.
1224
ANALYTICAL CHEMISTRY
acidity of the solution by thc addition of acid, according to the principles outlined abovc. Thc effect of pFI on the course of such :Ltitration in the presence of citrate is slion n in Tablc I. Appreciable disiociation of the complexed copper dors not begin until p H values below approximately 3.8. Even so, it is not possible to titrate to a satisfactory end point under these conditions, in :I reasonable amount of time. When the end point p H is 2.0 or lower, titration can be made in the normal manner but the undesirable precipitation of cuprous iodide also begins to occur under thcse conditions.
Table I. Effect of End Point pH on Titration of Complexed Copper
(8mmonium citrate 2m.M. Addcd copp(~i' 0.lm.lf.)
No Pot:miuni
Cyanide Copper recovered. PH mM 5.95 0.008 0.030 5.20 0.061 4.40 0.067 3.90 0.097 3.50 0.099 3.00 1.80 _
_
0.100 _
~~
~
Potassium Cyanide, 20 M g . Copper reco\,ered, PH mX 6 25 0.091 0.095 5.75 0.096 5.40 0,099 4.90 0.100 4.20 0,100 4.10 2.00 0.100
iodiw color even in the presence of high concentrations of complexing agent. The solution is now titrated in the normal way with thiosulfate using starch a t the end point. The effect of cyanide, as seen from Table I, is to permit t h e titration to be made accurately at a much higher pH than is possible it,, absence and, as no cuprous iodidc mecipitates under these conditions, adsorption errors are eliminated. I n the absence of any complexing agent, titration of copper must be made a t p H values less than 6.3 (4). If the amount of ligand present is high, the reaction of the copper with potassium iodide as the end point is approached may still be rather slow, but careful titration gives complete and accurate results. The amount of potassium cyaiiide added to the solution, however, must not be in too great an excess Over the amount of copper present, as potassium cyanide is not wholly without effect upon the titration values and in large amounts gives rise to a low value (Table 11).
Table 11. Titration of Complexed Copper in Presence of Potassium Cyanide
(End point pH 4.2)
~
Such a procedure is not always convenient or economical, since the copper complexing agent may be particularly powerful or present in high concentration. The separation of cuprous iodide during the titration, with the consequent necessity for thiocyanate addition to liberate adsorbed iodine, is a major factor leading to inaccuracies (4). Moreover, a high acidity is undesirable, as it may lead to inaccuracies due to oxidation, and the starch end point does not work satisfactorily in strongly acid solutions. A simple and effective solution to the problem has been found, which allows fairly rapid titration even in the presence of such a powerful copper complexing agent as ammonium citrate. The method consists of the addition of a small amount of potassium cyanide to the solution after the addition of potassium iodide, so that the immediate visible result is an intensification of the
0 0 0 0 0.5 0.5 1 0 1 0 2.0 2.0
4.0 40 4.0
10-40 Excess 5-20 20-60 5-20 20-60 20-60 20-60 20-60 Excess Sone
0 0 0 0 0 0 0 0 0 0
100 100 100 100 100
100 100 100
100 100
0 100
0 0 0 0
0 0 0 0 0 0 0
100 098 100 099 100 099 100 098 098 097 010
PROCEDURE
The method of estimation outlined is illustrated in the following specific example for a solution containing copper sulfate and ammonium citrate. I n a 100-ml. conical flask containing 10 ml. of 0.01M copper sulfate and 1 ml. of lill ammonium citrate are placed 2 ml. of dilute acetic acid (1 to 4), followed by 1 gram of solid potassium iodide. After 30 seconds, 2 nil. of 1%
pota.siuni cyanide solution are added and the liberated iodine is titrated n-ith standard thiosulfate, proceeding slowly toward the end point of the titration, where if the find stages of the reaction are too slow the addition of a further quantitj- of potassium iodide is advisable. Starch is used to determine the end point.
notably the amino acids. The method is believed to be npplicable in principle to any solution containing complexed copper. It fails only when the copper complex is insoluble-for example, methionine. ACKNOWLEDGMENT
Tlicx r c w l t i tabled in thi- nork were obtaincd \I ith the powerful copper compleving agent citrate. Using the same techniyucJs excellent results have been achierctl with other compleving agentq,
This work was undertaken as part of the program of food iiivestigation of the Department of Scientific and Industrial Resenrch, Food Investigation Board, L o r Trmperature Station for Research
in Biochemistry and Biophysics. University of Cambridge, England. LITERATURE CITED
(1) Biuhns, G., J . Soc. Chem. Znd. (Lond o n ) 33, 445.4 (1918). (2) Foote, H. IT.,J Am. Chem. Sac. 60, 1349 (1938). (3) Kolthoff, I. AI., Sandcll, E. B., "Textbook of,. Quantitative Inorganic hnslysis, p. 630, LIacmillan, Sew Tork, 1946. (4) LlC'iteS, L., - 1 N A L . CHEM. 24, 1618
(1952).
RECEIVEDfor review October 8, 1936. 12cceptc.d February 9,1957.
Carbon Determination in Biological Material with a Persulfate Oxidation Method S. L. CHEN and K. J. H. LAUER Biochemistry laboratory, Research Department, Red Star Yeast and Products Co., Milwaukee, Wis.
b l n a study of the assimilation of radioactive carbon in biological material, primarily microorganisms, a method was developed which simplifies routine carbon determination and extends the application of the persulfate combustion method.
A
convenient method for the determination of carbon content in organic compounds by persulfate oxidation has recently been published by Katz, Abraham, and Baker ( 1 ) . T n fortunately, this method can be used only for water-soluble compounds. LOF recoveries were obtained with biological materials which are insoluble in water. With a slight modification in the preparation of the samples, satisfactory results may be obtained with biological materials as well as with some waterinsoluble organic compounds of biological interest. YCRI
PROCEDURE
T l i ~saniple to be analyzed was first ground to pass SO-mesh, if necessary. and suspended in 2.5 ml. of distilled water, to which concentrated sulfuric acid \vas added to make up to 25 ml. in volume. Care was taken to avoid charring of the biological material by cooling in a n ice bath during the preparation of the sample solution, which contained a!iout 1 to 5 mg. of carbon per ml. Into each combustion-diffusion vessel ( I ) , 500 nig. of potassium persulfate. 4 to 5 ml. of distilled water, and 0.1 to 0.6 ml. of acid sample solution were introduced. A micropipet was used for
measuring sample solutions. One milliliter of 5% silver nitrate solution was added after the contents were mixed. The vessel was then capped with a serum bottle stopper and evacuated immediately for 30 seconds through a n inserted hypodermic needle. The evacuated vessels were incubated at 80" C. for 1.0 t o 1.5 hours. After complete combustion. the vessels were cooled to room temperature. Two milliliters of 5iV carbonate-free sodium hydroxide solution were introduced to the center well of the vessel for the absorption of the liberated carbon dioxide, which was determined gravimetrically in the form of barium carbonate following ammonium chloride-barium chloride precipitation. EXPERIMENTAL
Owing to the rapid decomposition of potassium persulfate in concentrated
Table 1. Effect of Sulfuric Acid Concentration on Carbon Determination in Yeast, Saccharomyces cerevisiae, by Persulfate Oxidation Method
Coned. H2S04 Added, Vessela 111. 1
2 3 4 5 6
0.0
0.1 0.3
0.5 1.0
1.5 7 2.0 Each vessel sample solution.
Concn. of HzSO, in Digestion BaCOl Mixture, Recovered, s Mg. 0.55 9.7 1.15 9.9 2.35 9.8 3.55 10.0 6.55 5.8 9.55 3.4 12,50 0.9 contained 0.1 ml. of
sulfuric acid, the final acid concentration in the digestion mixture is important. The results in Table I showed that the recovery of the total carbon in a sample of yeast, Saccharomyces cereiiisiae, declined rapidly as the sulfuric acid con-
Table II. Determination of Carbon Content in Biological Material and Certain Organic Compounds of Biological Interest with a Modified Procedure of the Persulfate Oxidation Method
Material
Size of Sample, 75 of N g . of Carbon Carbon Recoverya
Saccharomyces cerevisiae 1.0-2.4 Candida utzlis 1 0-2 0 Horse liver, acetone uowder 1 5-2 0 Ja'ck bean meal 1.3-2 0 Oleic acid 1.0-2 0 Adenine 1.0-2.0 Guanine 1 0-2 0 Xanthine 1 0-2 0 Tyrosine 1 0-2 0 Tryptophan 1 0-2 0 Cysteine HC1 1 0-2 0 Cystine 1 0-2 0
99 97
101 98
96 97
101
96 96 96
99 96
a For biological material and oleic acid, per cent of carbon recovery was calculated against results obtained with a wet combustion method by Van Slyke-Folch combustion mixture ( 2 ) . For other organic compounds, calculations were based upon nitrogen analysis of these compounds xith a micro-Kjeldahl method. For cysteine and cystine, the COz absorbed in SaOH was regenerated once with 5N H&04 containing 1% HzOz as a precaution.
VOL. 29, NO. 8, AUGUST 1957
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