Table
I.
Concentration, C, moles/liter
Anodic Stripping Analysis of Methylene Blue Solutions
Preelectrolysis time, t, minutes
Average quantity of electricity, Q, pCoulombs"
Q/Ct
384.0 9.60 10-1 1 36.8 9.21 lo-' 1 15.1 9.43 lo-' 4 9.39 9.39 IO-* 25 1.9 9.5 lo-@ 50 Average and average deviation of three replicate determinations. 4.0 X 4.0 X 4.0 X 4.0 X 4.0 X
potential of -0.975 volt us. S.C.E. on the working electrode. At the end of an appropriate electrolysis time the stirring was stopped, and the potential was changed to -0.65 volt us. S.C.E. After 30 seconds, the adsorbed deposit was removed by anodic voltammetry with linearly varying potential. Between replicate run8 the potential of the electrode wm held at +0.28 volt us. S.C.E. while the solution was stirred and deaerated for 1 minute. DISCUSSION
The pre-electrolysis potential of -0.975 volt us. S.C.E. is ostensibly more cathodic than necessary to ensure reproducible mass transfer effects (Figure 1). However, as was observed also by Kemula, Kublik, and Axt (3), such cathodic potentials seem necessary to
Av. dev.,.
%
X 10-6
11.7 11.3 14.3 12.1 13.4
accumulate the adsorbed product a t the electrode. Thus, pending further investigations into the cause of this behavior, the procedure was accepted as a necessary requirement for useful results in this method. The sharp anodic stripping peak obtained a t about +0.24 volt us. S.C.E. (Figure 2) was attributed to the oxidation of the adsorbed product of the methylene blue reduction. The area under this peak was used to determine the stripping coulombs, which in turn could be related to the bulk concentration (Table I). The high residual currents in the stripping curves were attributed to the oxidation of hydrogen, which was simultaneously formed a t the surface during the pre-electrolysis period. The extraneous (first) anodic peak ob-
served at lower concentrations (Figure 2, curve B ) is also present in the blank. More extensive studies of the adsorption and electron transfer processes involved in the methylene blue system are currently underway, utilizing the techniques of cyclic voltammetry and a.c. polarography. Also, the effects of varying certain critical parameterssuch as electrode preparation, surface texture, and surface area-are being investigated. In addition, the general applicability of the analytical method is being tested by the use of other adsorbing systems. LITERATURE CITED
( 1 ) Booman, G. L., ANAL. CHEM.29, 213 (1957). (2) De Ford, D. D., 133rd Meetin$,
A.C.S., San Francisco, Calif., April
1958. (3) Kemula, W., Kublik, Z., Axt, A,, Roczniki Chem. 35, 1009 (1961). (4) Perone, S. P., ANAL. CHEM.35, 2091 (1963). . .
S. P. PERONE T. J. OYSTER
Department of Chemistry Purdue University Lafayette, Ind. RECEIVED for review August 19, 1963. Accepted October 17, 1963. This work supported in part by the American Cancer Society.
A New Spectrophotometric Method for the Determination of (Ethylenedinitri1o)tetraacetic Acid SIR: The following method was developed to meet the need for a rapid spectrophotometric procedure for the determination of (ethy1enedinitrilo)tetraacetic acid (EDTA) in the presence of calcium and magnesium ions. Advantage is taken of the fact that EDTA causes a decrease in the absorbance of Fe(CNS), solutions.
-g
475 mp in a suitable colorimeter or spectrophotometer. The EDTA concentration is estimated b comparison with at least two standardrs.
m
100 r
V
v
9 LL
0
RESULTS AND DISCUSSION
w
0
Z
a
m [r
0
50
v)
m EXPERIMENTAL
a
Ferric thiocyanate (O.lmM) is prepared by mixing 2.0 ml. of 50% NHICKS, 2.0 ml. of 1.0 mM FeCls in 10% acetic acid and diluting to 20 ml. with water. The absorbance of this solution is constant for 40 minutes a t room temperature. Ferric chloride (1.OmM) is prepared fresh each day by diluting 0.1M FeCli in 0.lM HCl with 10% acetic acid. For the determination of EDTA, 1.0 ml. of a solution containing between 0.02 and 0.08 mmoles of EDTA is added to 1.0 ml. of Fe(CNS)s solution. The mixture is diluted to 3.0 ml. with water and absorbance is measured immediately against a water blank a t
w
236
0
ANALYTICAL CHEMISTRY
z 2 w E
0
w n
8
LOG CONCENTRATION
M/L
Log of phosDhale or adenine nucleotide concentration in moles per liter In sample added
Figure 1. Effect of phosphate and phosphate compounds on the absorbance of ferric thiocyanate 0 Phosphate
A Adenylic acid 3 Adenosine diphosphate
0
Adenosine triphosphate
The absorbance of Fe(CNS)a solutions decreases linearly with increasing concentrations of EDTA. The amount of EDTA that can be determined is limited by the amount of Fe(CNS)3(0.1 mmole in the procedure described above). The range can be extended by increasing the amount of FeCla used in preparing the Fe (CKS) solution. The absorbance of Fe(CNS)a is constant between pH 2.5 and 3.5. Above pH 3.5 the absorbance decreases, be coming zero a t pH 5.0. If the sample is a t a pH greater than 3.5, it is necessary to adjust the pH to about 3.0 before analysis. The procedure can be used in the presence of calcium and magnesium. The data presented in Table I show that neither of these ions interfere over the concentration of ranges studied. This
Table 1. Effect of Calcium and Magnesium Ions on Determination of (Ethylenedinitri1o)tetraaceticAcid
(:Ethylenedinitrilo)tetraacetic acid Additions, Added, Found, mmoles mmoles mmoles Ca
+’
hlg+l
1.0 50.0 100.0 200.0
0.040 C.150“
0.042 0.150” 0.041 0.150’
1.0
C.040 0.040
0.042
100.0 a
CI.040 c1.150a
0.042
Ferric thiocyanate concentration used
for analyais, 0.2rnM.
this difficulty. The interference by phosphate and phosphate esters, however, limits the usefulness of the method.
is due to the fact that the stability constant of the iron-EDTA complex is greater than that of the alkali metalEDTA complexes a t pH 3.0. The results presented in Figure 1 show that orthophosphate and adenosine-5’-phosphate interfere with the formation of the red color of Fe(CNS)* a t concentrations in excess of 0.5 mM. Adenosine di- and triphosphate interfere a t concentrations of 0.05 mM. The determination of micro amounts of EDTA by the usual titrametric methods, employing standard solutions of alkali metals (I), is frustrated by the presence of Ca+2 and Mg+2 in the sample. The procedure described herein, aside from being rapid, circumvents
LITERATURE CITED
(1) Martell, A. E., Calvin, M., “Chemistry of the Metal Chelate Compounds,” 488, Prentice-Hall, Englewood Cliffs, . J., 1958. CHARLES J. PARKER, JR. Department of Physiological Chemistry Wayne State University College of
k
Medicine Detroit, Mich. RECEIVED for review September 13, 1963. Accepted October 23, 1963. Research was supported by grants from the Michigan Heart Association and the National Institute of Arthritis and Metabolic Diseases, U.S.P.H.S. (Grant A 5311).
Separaticm of Isomeric Toluidines by Gas Liquid Partition Chromatography Using Ucon Oil as Stationary Phase SIR: A stationary liquid phase that will resolve the individual toluidine isomers (ortho, para, m d meta) suitable for quantitative analpis has not been reported. The separation of the toluidine isomers wa.5 discussed in the recent text of Burchfield and Storrs (1). Separation factors cn several liquid phases that had been reported were too low to resolve all three isomers. Separa-
EXPERIMENTAL
tion was obtained by gas solid chromat,ography using a treated clay but the chromatograms were not suitable for quantitative analysis because of the marked peak asymmetry and tailing. We studied several polyglycol-type stationary liquids and found that a polyalkylene glycol ether gave good resolution of all three toluidine isomers as shown in Figure 1.
Column Preparation. Chromasorb W (trade name for nonacid-washed, flux-calcineddiatomaceous silica)of 60to 80-mesh was treated with alcoholio sodium hydroxide (6% NaOH on dried Chromasorb W) and the alcohol was evaporated (2) The desired weight of Ucon oil (50HB 5100) was dissolved in benzene, slurried with the sodium hydroxide-treated Chromasorb W and I
I
0
I
30
i
0 12
-z
m
0
F 008-
z w
I-
W
w 006
1 U
w
5
004
1 0
0 02
1
40
50
RETENTION T I M E ( M I N I
Figure 1.
o.loI ~
I
Separation of toluidines
I
I
I/Tx3
Figure 2.
o = ortho p = para rn = mefa
Conditions Col., 10 ft., 3/16-in. o.d., 2.5% Ucon (50HB 5100); 100’ C.; flow, 60 ml./min.; sample size, 0.1 MI,
I
2.0
I
I
3.0
Separation vs. temperature
0 porolorfho (20% Ucon col.)
A temp.,
0
X
para/ortho (2.5% Ucon col.) meto/orlho (20% Ucon col.) mefa/ortho (2.5% Ucon col.1
VOL. 36, NO. 1 , JANUARY 1 9 6 4
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