The kinetics of the reaction between -4g+ and the -SH groups of hemoglobin can easily be followed as expressed in the curve relating current to time after the intrcduction of hemoglobin into the vessel. Figure 1 shows that there is a linear relationship between the current and the amount of thiol added down to about one third of the maximal current. The deviation from linearity indicates dissociation of the silver mercaptides. In Table I are s h o w the results of titrations of about 0.1 pmole of the 3 substituted thiols-glutathione, cyskine, and cysteamine. The relative standard deviations Lire about 1% of the mean. This reprolucibility appears to be better than the one obtained by Burton (2) in his ccnventional titrations, in spite of tfle fact t h a t the amounts of thiols di?termined in his titrations were about 10 times greater than the quantities measured by us. The average time spent on the titrations reported in Table I was 2.7 minutes. This figure includes the preparation of the apparatus for each titration. The stoichi1,metric relationships are similar t o those reported for conventional amperometric titrations (3). The last row of Table I shows t h a t more than one P.g+ is apparently consumed per -SH group, presumably because the silver mercaptides (RShg)
In1 O
1
L
CYSTEAMINE CYSTEINE GLUTATHIONE
z -
40
0-0
-* A-A
120
80
160
pl.OF THIOL Figure 1. Demonstration of linear dependence of current on amount of thiol added Initial concn. of AgNOa was 0.9.10-jM. in 30 ml. of Tris, pH 7.4. Below about one third of initial current reading, linear relationship is no longer obeyed, indicating dissociation of silver mercaptides
bind some of the excess silver (3). I n this respect our method consequently does not represent any advantage. I n our modified titration procedure the concentration of .4g+ exceeds t h a t of the thiol during the entire titration. Consequently, errors due to spontaneous oxidation of the thiol in the titration vessel are ruled out. Thus it was unnecessary to remove oxygen from the solution by bubbling nitrogen through it. Furthermore, one i. free
to choose a pH higher than 7.4 in t,he buffer, since a low p H is no longer needed to counteract the spontaneous oxidation. Sluggishness and instability of the silver-coated platinum electrode due to “intoxicating effects” of the thiols are reduced or eliminated in our system. The excess of silver counteracts the dissociation of the silver mercaptide~. -kt the end point, however, this excess is abolished and the dissociation ia considerable (see Figure 1). I n contrast to the conventional method, the sharpness of the calculated end point of our modified method is not affected by such a dissociation, since the calculation is not based upon the actual attainment of the end point during the tibration. LITERATURE CITED
(1) Benesch, R. E., Lardy, H. A., Benesch, R.. J . Biol. Chem. 216. 663 (1955).
HANSCHR.BORREGEN’ Institute of Clinical Biochemistry Rikshospitalet, University of Oslo Oslo, Korway
1 Fellow of The Torwegian Cancer Society.
The Quantitative Recovery of Germanium by Distillation from Hydrochloric Acid in the Presence of Sodium Chloride SIR: In Cluley’s n ell-known analytical procedure, germanium is brought into solution by fusion with sodium carbonate, and separated as the tetrachloride by distillation from hydrochloric acid of approx mately constantboiling composition. The constantboiling acid is produced by adding to the neutralized germanium solution an equal volume of concentrated acid. The specific gravity of standard reagent grades of concentrated hydrochloric acid varies be tween 1.18 m d 1.19; Cluley stipulates the use of 1.18 sp. gr. acid, whereas Sandell states only that the mixture should be 6il.r in hydrochloric acid ( 2 , 3). The mixture will be approximately 5.8M in HCl when concentrated acid of sp. gr. 1.18is used, and approiimatelp 6.2.11 in HCI in the case of acid of 1.19 sp. gr. It is the purpose of this note to show that in the presence of sodium chloride, these slight variations in the initial concentration of the acid in the mixture undergoing distillation may exert a profound effect on the recovery of the germanium.
RESULTS
I n the presence of 2.2 grams of sodium chloride-which is necessarily produced as a result of the sodium carbonate (2.0 grams) fusion and subsequent neutralization procedure-distillation of a germanium solution 6.2M in HCl resulted in a slight escape of fumes for a few moments immediately prior to the appearance of the first drop of distillate, and in a very severe loss of germanium. No significant loss occurred if the solution n-as only 5 . W in HCI, or in either case in the a b e n c e of sodium
Table I.
Sp. gr., concd. HCI 1.18 1.19 1.18 1.19 1.19
DISCUSSION
AlniAsy and Rarchfalvi found that the vapor-liquid equilibrium of the GeCl4/HC1/H$O system is shifted in
Effect of HCI/NaCI Concentration on G e Recovery
Molarity, diluted SaCl HC1 !grams) 5.8 6.2 5.8 6.2 6.2
chloride. This loss was obviated bv employing a long delivery tube, which initially dipped into a few milliliters of water in the receiver, and subsequently was maintained just below the surface of the distillate. Typical experimental results are illustrated in Table I.
0 0 2.2 2.2 2.2
Delivery tube Short
Short Short Short
Long
Fumes apparent before distillate YO
10 T O
Slight
...
Ge recovery ( p g ) from: 50 pg. 500pg. Ge Ge 50.5 509 49.1 494 48.3 489 14.0 153 49.9 495
VOL. 35, NO. 8, JULY 1963
1097
favor of an increase of GeC14 (and presumably HCl also) in the vapor phase by the presence of calcium chloride in the liquid phase (1). Sodium chloride evidently produces a similar effect, and germanium may therefore be lost easily in the vapor initially evolved from boiling solutions containing sodium chloride, which escapes condensation when the HCl concentration in the liquid phase is as high as 6.1M (the constant-boiling
concentration for the HCl/H20 system in the absence of sodium chloride). Loss probably also occurs at lower HCl concentrations, depending upon the concentration of the sodium chloride present. LITERATURE CITED
(1) AlmBsy, A,, Barchnfalvi, F., NehBzvegyip. Kut. Int. KozlemBn. 1, 293-301 (1959); C . A . 54, 6236.f (1960).
( 2 ) Cluley, H. J., Analyst 76, 523-35
(1951). (3) Sandell, E. B., "Colorimetric Determination of Traces of Metals," 3rd ed., pp. 482-93, Interscience, New York, 1959. HUGHB. RAYNER
British Columbia Research Council, at the University of British Columbia, Vancouver 8, British Columbia, Canada.
Determination of Aluminum in Organo-Aluminum Compounds by X-Ray Fluorescence SIR: We have developed an x-ray fluorescence method for the determination of aluminum in organo-aluminum compounds. The method is applicable over the range from 0.05 to 10% and possibly even higher. The method has been developed especially for aluminum analysis of highly reactive pyrophoric compounds. Recent publications (1, 3) point u p the application of x-ray fluorescence to aluminum concentration determination. However, our method differs significantly from any previously reported, since i t applies to liquid systems which are highly reactive and require special handling techniques and equipment.
"'I
I fGHR0MIUM WITH
150
TARGET TUBE P H A
-
0
z
8 w
v)
50
-
EXPERIMENTAL
We used a General Electric XRD-3 x-ray instrument for this work. The original heat exchanger and line voltage stabilizer were replaced to make the instrument essentially equivalent to the XRD-5. The integral components of the instrument were: a General Electric S o . 2SPG power supply, a Hamner Model SX-R proportional counter preamplifier, a Hamner Model N302 nonoverload amplifier and pulse height discriminator, a General Electric No. 4SPG counter tube and helium tunnel assembly, a General Electric SPG electronic time register, a General Electric KO,BSPG scaler, a General Electric S o . BSPG rate meter, and a Machlett AEG-50-8 chromium target x-ray tube. Table 1. Comparison of Aluminum Analysis Data from X-Ray Fluorescence and Chemical Methods
Sample No.
A B
C D E
1098
Aluminum, % Chemical X-ray method 4.70 4.68 11.56 11.33 22.98 22.47 4.88 4.85 4.75 4.71 + O . 16 Mean error Relative error +1 .64%
ANALYTICAL CHEMISTRY
X AI
Figure 1 . Plot of counts per second vs. % AI for different target tubes
The procedure followed in setting the pulse height discriminator is that outlined by Miller (2). The electronic components were left on constantly. However, the x-ray source was usually turned off overnight. We found that a 30-minute warm-up period was advisable each morning before checking the pulse height discriminator settings. We have made a modification to the sample chamber of the XRD-3 which should be mentioned because it has certain advantages. The wall of the sample chamber adjacent to the x-ray tube was converted into a cooling panel. Small holes were drilled through the wall forming a water passage around the point of contact of the tube with the sample chamber. Tubing was connected to both ends of this passage. With this arrangement, tap water is circulated through the passageway a t all times so that the only heat to which the sample is exposed is that generated by the x-ray beam. The sample cells we used were made out of nylon by the Instrument Shop in our Research and Development Dept. After two years of operation we have seen no adverse effect of the x-ray beam on the nylon of the sample cell.
The general analytical procedure for determination of aluminum in organoaluminum compounds may be described as follows: prepare a stock solution containing approximately 60% triethylamine and 40% Stoddard solvent (dried over sodium). Place 20 ml. of this solution in a 50-ml. glass-stoppered graduated cylinder. Inject argon into the graduated cylinder through one hole of a two-hole rubber stopper. Purge a syringe with argon and then fill with the organo-aluminum compound to be analyzed, cap with a silicone rubber plug, and weigh. Transfer the sample from the syringe to the graduated cylinder containing the triethylamine solution. Recap the empty syringe and place the glass stopper on the graduated cylinder. Weigh the empty syringe and the graduated cylinder. The size of the sample is determined by the estimated aluminum content in the sample. For pure aluminum triethyl, the best sample size is approximately 4 ml. After thorough mixing, transfer the sample to the x-ray sample cell for analysis. Allow 1 minute after placing the sample cell into the x-ray beam for the helium atmosphere to reach pressure equilibrium. Register counts over a 400-second time period. For such counting intervals, the count level will usually be in excess of 50,000, giving a u value of less than 1.5% resulting from counting error. Total instrument time per sample is approximatcly 8 minutes. Considering calculation time, a sample may be analyzed for aluminum over wide concentration range in approximately 15 minutes. The accuracy of t h e method is shown by the data in Table I. DISCUSSION
The principal factors responsible for the successful application of x-ray fluorescence to the determination of lowlevel aluminum in pyrophoric organic systems were the availability of a chromium x-ray target tube, the modification of the sample chamber to provide sample cooling, and the use of sample cells fabricated out of nylon. Our first work on aluminum analysis was done with a tungsten target tube and without a pulse height discriminator. The background
