Spectrophotometric Determination of 5-Ketogluconate MICHAEL SCHRAMM Laboratory o f Microbiological Chemistry, Department o f Biochemistry, H e b r e w University-Hadassah M e d i c a l School, Jerusalem, lsrael
glnconate per ml. It is frozen for storage: suitable dilutions are irrshly prepared before use. 1-Nethyl-1-phenylhydrazinesulfate (Eastman Organic Chemicals, Rochester, N. Y.) is recrystallized by addition of hot alcohol to a hot aqueous solution. Interfering substances present in a commercial sample of 1-methyl-1-phenylhydrazine sulfate can be xmoved readily by shaking the crystals with hot alcohol. The purified product should give a colorless solution, which is freshly prepared in a 2% (w./v,) concentration before use. ;\11 of the following reagents were obtained from commercial soiirces and were prepared as indicated. Perchloric acid, 72Yc (&-./by.). Sodium hydroxide, l2Yc (w./v.). Hydrochloric acid, 3 7 , (w,/v,). Carbonate buffer, p H 10. Dissolve 8.4 grams of sodium bi(,:libonate and 36.0 grams of sodium carbonate in water and adjust the volume to 1 liter. Iodine, 0 . 1 s in 2.57, potassium iodide solution. Stoi,e the solution in a dark bottle out of direct sunlight. Sodium bisulfite, 0.4X. Dissolve 4.2 grams of sodium bisulfite in 100 ml. of ivater. T h e solution is stable for 1 week. Phosphoric acid, 2 . 5 s . Adjust the concentration with water so that 1 volume of phosphoric acid solution and 1 volume of carbonate buffer yield a p H of 3.0 t,o 4.0. Procedure. DEPROTEISIZATIOX. T o remove proteins, perc,hloric.acid reagent is added t o the sample to a final concentration of 12% perrhlorie acid. The solution is then cleared by cwitrifug:ttion. If aldoses are present they are removed as desc.rilwd twloa. I n the absence of aldoses, this step is omitted. OXIDATIVE REhfovAL O F I N T E R F E R I S G ALDOSESBY IODISE. .ildoses give a relatively weak color reaction with the hydrazine reagent. This interference can be eliminated by the prior treatment of the sample with iodine by the method of Fishman and Green ( 2 ) . .i measured aliquot of the deproteinized solution is brought t o pH 7 with sodium hydroxide reagent and diluted t o :I selected volume. A 2.5-ml. aliquot, containing a t least 0.4 pmoles of 5-ketogluconate and not more than 20 pmoles of aldose is placed in a test tube. One milliliter of carbonate buffer is added, foilon-ed by 0.8 ml. of iodine. The solution is immediately shaken, stoppered, and placed in the dark for 30 minutes. Then 0.2 ml. of sodium bisulfite is added and the solution is shaken. If the iodine color does not disappear, more bisulfite is added until the color completely disappears, after which 1 ml. of phosphoric acid reagent is added. After the tubes are shaken to expel carbon dioxide, the pH should be 3 . 0 to 4.0. The solution is then brought to a known volume for assay for 5-ketogluconate content 1,- reaction with hydrazine reagent. REXTIOXWITH HYDRAZINE RE.4GEST. The pH is adjusted to 3.0 to 4.0 with sodium hydroxide or hydrochloric acid. A 2-ml. aliquot, containing 0.05 to 0.5 #mole of 5-ketogluconate is placed with 1 ml. of hydrazine reagent in a test tube covered with :I glass bulb, then heated in a boiling water bath for 40 minutes, and cooled to room temperature.
5-Ketogluconate reacts readily with l-melhj I-l-phenylhydrazine sulfate at 98" C. and at pH 3 to 4 to yield a rose-colored product w-ith an absorption peak at 330 mp. A spectrophotometric micromethod based on this reaction is applicable to the measurement of 5-ketogluconate ( 2 0 . 0 5 pmole) in the presence of aldoses, ketohexoses, and 2-ketogluconate. This reaction is relatively specific and may also be used as a qualitati\e color test.
R E Q U E N T occurrence of 5-ketogluconate in the oxidativr fermentation of glucose mediated by dcetobacter species (3, 6, 8, I S ) , as well as the utility of t,his sugar acid as an int,rrmediate of organic syntheses ( 8 ) , lend interest to the development of rapid and specific methods for its estimation. si. 2ketogluconate is often formed concomitantly tvith 5-ketogluconate (3, 6, 8 ) , a procedure specific for 5-ketogluconate is required. The micromethod described here was developed to permit the measurement' of the formation of 5-ketogluconate by :Icetobacler xylinum suspensions ( 4 ) . Methods proposed for the determination of 5-ketogluconate have been based on copper reduction. The methods described by Militzer (9) and by Stubbs and others ( I S ) are inconvenient, for uze in serial analyses; they also require considerable amounts of substance. h spectrophotometric micromethod recently described by Perlman (11) is applicable to small amounts of material, b u t is subject to interference b y 2-ketogluconate, The ability of 5-ketogluconate to form an osazone has been utilized in this laboratory as the basis of a micromethod for the specific determination of 5-ketogluconate in the presence of 2ketogluconate and aldoses. 1-1IethS.l-1-phenylhydrazine s d fttte, which is known to be much more reactive with ketoses than tvith aldoses, is used as the hydrazine reagent (IO). Because 5lietogluconate possesses a carbonyl group adjacent to a primary alcohol group, it reacts readily x i t h 1-met,hyl-l-phenyl-hydrazine. The reaction proceeds smoothly to completion and yields :i product which follows Beer's Ian and has a strong absorption :it 350 mp. Ketoscs react with 1-methyl-1-phenylhydrazine sulfate: therefore, their presence may be expected to interfere with the assay of 5-ketogluconate. Under the conditions xyhich are proposed here, fructose is a relatively slow reactant, but dihj-droxyacetone reacts readily and thus may interfere seriously with the 5-ketogluconate assay. METHOD
Apparatus. Spectrophotometric measurements were rarricd out with a Beckman Model DU spectrophotometer. Quartz cuvettes with a 1-em. light path \yere used. Reagents. Calcium 5-ketogluconate [ ( C F , H ~ O ; ) ~21/r€IyO] C~, ( 6 , 12) was prepared by Killiani's method ( 5 ) as modified by Barch ( 1 ) . A preparation marketed by General Biochemicals, Inc., Chagrin Falls, Ohio, m-as found to be equally suitable. T o prepare a standard 0.010M solution, dissolve 47.1 mg. of calcium 5-ketogluconate in 4 ml. of 0.LV hydrochloric acid. Add 2 ml. of 0.05M oxalic acid and adjust the pH t o 6 to 7 by careful addition of 0.2N potassium hydroxide (about 2.5 ml.), Centrifuge the solution to separate the calcium oxalate precipitate and transfer the supernate to a 20-ml. volumetric flask. K a s h the calcium oxalate precipitate tm-ice with 4 ml. of water, add the washings to the volumetric flask, and bring the volume to the 20-ml. mark. This stock solution contains 10 pnioles of &keto-
963
SPECTROPHOTOXETRY. The absorbance of the reaction product formed with the hydrazine reagent is measured a t 350 nip against a blank carried through the same procedure. The absorbance shows little change on standing ( < 5y0 after 1 hour). The amount of 5-ketogluconate is directly proportional to the absorbance and may be read from a calibration curve prepared with the 5-ketogluconate standard solution. Treatment with iodine to remove aldoses lowers the absorbance of the product formed from 5-ketogluconate by about 205;. If the procedure has included this step, the calibration curve should be prepared on knovin samples of 5-ketogluconate which have also been carried through this step. If a large excess of 2-ketogluconate is present, the value found for 5-ketogluconate is too high. An appropriate correction is obtained b y independent' determination of 2-ketogluconate with the method of Lanning and Cohen ( 7 ) . Absorbance of the product formed by 2-ketogluconate ivi-ith hydrazine reagent is 4% of that of 5-ketogluconate.
964
ANALYTICAL CHEMISTRY 1 *P
do not seriously interfere with the determination of 5-ketogluconate. Acetate had little effect on the absorbance of 5-ketogluconate. However, the absorbance of the product formed by glucose and 2-ketogluconate was found to be augmented almost twofold by acetate present a t a high concentration (Table 11).
1 .o
.8
Table I. Absorbance of Reaction Products of Various CarbohSdrates with 1-Methyl-1-phenylhydrazine Sulfate
W
v
8
Z .6
, b-, u
0 300
t
I
330
360 390 WAVE LENGTH, Mp
" 420
B 450
Figure 1. Absorption spectra of reaction product of ketogluconates with l-methyl-l-phenylhydrazine sulfate A . 0.4 pmole of 5-ketogluconate B . 0.5 pmole of 2-ketogluconate
QCALITATIVE COLOR TESTFOR 5-KETOGLUCONATE. The reaction product of 5-ketogluconate with hydrazine reagent has a rose color. The color of the product is readily observed if 0.1 pmole of 5-ketogluconate is present. Other reducing substances tested (Table I ) do not yield a rose color even a t considerably higher concentrations. Dihydroxyacetone produces a strong yellow-brown color; therefore, it may interfere in this test.
>
phioles of ComRelative Color pound in Final Molar of RIethoda Compound Reaction Mixture Absorbance b Solution A Dihydroxyacetone 0.5 120.0 Deep yellow 5-Ketogluconic acid 0.2 100.0 Rose Glucuronic acid 0.5 Deep yellow 50.0 Ascorbic acid 0.2 Faint yellow 20.0 D-Ribose 2.0 17.0 Faint yellow n-Xylose 0.2 Faint yellow 17.2 u-Fructose 0.2 Colorless Colorless 8.1 L-Sorbose 0 2 7.3 ~-Sucrose 0.3 5.0 Colorless D-GIUCOSC 2.0 6.1 Colorless Pyruvic acid 0.5 Colorless 4.0 2-Ketogluconic acid 0.5 Colorless 4.1 Gluconic acid Colorless 10.0 0.2 B 2-Ketoeluconic acid 1.8 Colorless 3.0 D-Glucbonic acid 4.0 Colorless 0.4 D-Xylose 4.0 0.4 Colorless D-Ribose 4.0 Colorless 0.4 D-Glucose 6.0 Colorless 0.3 a b
A , without hypoiodide oxidation; B, with hypoiodide oxidation. Absorbance per mole compared t o 5-ketogluconate = 100.
Analytical recovery of 5-Ketogluconate in Biological Mixtures. T o test the method applied t o biological mixtures, known amounts of the 5-ketogluconate standard solution were added to samples of blood serum, urine, and bacteriological culture media. Glucose and gluconate broth of the following compositions (w./v.) were tested: glucose or gluconate 2%; Difco yeast extract 0.5%; Difco beef extract 0.5y0;Difco Bacto peptone 0.5%. I n eveIy case, analytical recoveries of 5-ketogluconate ranged from 95 t o 100%. When 5-ketogluconate was not added to these solutions, apparent 5-ketogluconate concentrations (pmole per milliliter) were as follows: human blood serum, < 0.3; urine and gluconate broth, 0.3 to 0.5; glucose hroth (after osidation by hypoiodide), 0.3 to 0.5.
DISCUSSION
Spectrophotometric Properties of Reaction Product of 5-Ketogluconate with Hydrazine Reagent. As shown in Figure 1, the solution obtained by reacting 1-methyl-1-phenylhydrazine sulfate and 5-ketogluconate under the specified conditions reveals a strong absorption peak a t 345 to 350 mp. With 2-ketogluconate no peak and relatively little absorption occurs in the 350-mp region. The relationship between the amount of 5-ketogluconate and the absorbance follows Beer's law over the range from 0.05 to 0.5 pmole of 5-ketogluconate. Influence of Reaction Time and Hydrazine Reagent Concentration. The dependence of the reaction on l-methyl-l-phenylhydrazine concentration and heating time are illustrated in Figure 2. A reaction time of 40 minutes with 0.6iy0hydrazine reagent (final concentration) is proposed for the quantitative determination of the 5-ketogluconate. Under these conditions small variations in heating time and reagent concentration have little influence on the amount of product formed. Effect of pH. I n the standard conditions, variations of PIT of the reaction mixture in the range from p H 2 to 4 exerted little or no effect on the absorbance. At higher pH values (5 and 6 ) the absorbance of the solution was found to be considerably diminished. Specificity. Absorbances observed under standard conditions with different carbohydrates are listed in Table I. Dihydroxyacetone is seen t o be a serious potential interference. On the other hand, glucose and gluconate as well as 2-ketogluconate, which usually accompany 5-ketogluconate in Acetobacter cultures,
0
PO I
TIME, MINUTES 40 I
60
80
r
1
A
.l
0
.3 .6 .9 1 .P CONCN. OF REAGENT (%, W/V)
1.5
Figure 2. Influence of reaction time and hydrazine
concentration on color yield Mixtures contain 0.2 pmole of 5-ketogluconate A . Absorbance a s function of concentration of 1,-methyl-1-phenylhydrazine sulfate; reaction time, 40 minutes. B . Absorbance a s function of reaction time; concentration of reagent, 0.67% (w./v.).
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V O L U M E 28, NO. 6, J U N E 1 9 5 6 Table 11. Influence of Acetic Acid on -4bsorbance of Product Formed by 2-Ketogluconic Acid or Glucose with 1-Methyl-1-phenylhydrazine Sulfate
cose (cellulose), determined by the method of Schramm and Hestrin ( l a ) ; and 7.0 pmoles of carbon dioxide, determined manometrically.
aMoles
of Compound in Relative .Molar Final Reaction Iletliodn Compound Mixture .Ihsorbance b .?. Acetic acid 400 10.0 2-Ketoduconic acid 0.5 4.1 D-Glucose 2.0 6.1 2-Ketoeluconic acid acetic acid 0.5 400 8.0 D-Glucose acetic acid 2 0 +400 10.0 €3 .icetic acid 400 0 2 1.8 3 0 2-Ketogluconic acid
+
n-OlllcoaP -....
+
+
+
fin
n x
++
2-Ketogluconic acid acetic acid i 8 400 60 D-Glucose f acetic acid 6.0 400 0 3 A , without hypoiodide oxidation: B. with hypoiodide oxidation b Absorhance per mole compared t o Sketogluconate = 100.
Q
The method has been applied to 5-ketogluconate analysis in reaction systems in which suspensions of dried rlcetobacter sylinum cells polymerized glucose to cellulose (4). Analysis of such reaction mixtures by paper chromatography showed that the dried cells converted glucose to 2-ketogluconate and 5-ketogluconate as well as to carbon dioxide and cellulose. A total of 10 pmoles of glucose gave 3.2 pmoles of 2-ketogluconate, determined by Lanning’s method ( 7 ) ; 1.95 pmoles of 5-ketogluconate, determined by the proposed method; 2.0 pmoles of anhydroglu-
ACKNOWLEDGMENT
The author is indebted t o R. 4. Raphael, Queens University, Belfast, Ireland, for suggesting the use of the osazone formation for the determination of 5-ketogluconate, and to Shlomo Hestrin for critical comments. LITERATURE CITED Baroh, W. E., J . Am. Chern. SOC.55, 3653 (1933). Fishrnan. W. €1.. Green. S.. J . B i d . Chem. 215. 527 119551. Frateur, J., Simonart, P., Coulon, T., Antonie van Leeuiienhoek J . M i c r o b h l . Serol. 20, 111 (1954). Hestrin, S.,Schramm, AI., B h c h e m . J . (London) 58, 345 (1954). Killiani, H., Ber. 55, 2817 (1922). Kulka, D., Walker, T. K., Arch. Biochem. and BiophU,s. 50, 169 (1954). Lanning, M. C., Cohen, S.S.,J . BioZ. Chem. 189, 109 (1951). Lockwood, L. B., “Industrial Fermentations,” vol. 11, pp. 1-23, Chemical Publishing Co., New York, 1954. RIilitzer, W. E., J . BWZ. Chem. 154,325 (1944). Neuberg, C., Ber. 35, 95 (1902). Perlman, D., J . Biol. Chem. 215, 353 (1955). Schramm, SI.,Hestrin, S.,Biochem. J . ( L o n d o n ) 56, 163 (19543. Stubbs, J. J., Lockwood, L. B., Roe, E. T . , Tabenkin, B.. W a r d , G. E., Ind. Eng. Chem. 32, 1626 (1940).
RECEIVED for review
J a n u a r y 9, 1956.
.4ccepted March 12, 1956.
Quantitative Spectrographic Determination of Zirconium and Niobium in Uranium Metal J. A. GOLEB Argonne National Laboratory, Lemont,
111.
A spectrographic method enables fast routine analyses of a large number of uranium metal samples containing about j.jq0zirconium and 1.570 niobium. Excitation is by a high voltage condensed spark. The average deviation from the mean was 4~2.8970for zirconium and =t2.1670 for niobium. Comparison of spectrographic and chemical results showed an average deviation of zk3.0170 for zirconium and *3.969’0 for niobium.
source was chosen because of its greater inherent reproducibilit!-, compared to other conventional spectrographic sources, and because of the simplicity of the sampling procedure. The excellent machinability of the alloy permitted a minimum of sample preparation. The “point-to-plane” technique ( 1 ) was used with R Petry stand t o accommodate disks, rods, and bar forms of the alloy, which weighed about 1 kg.
Table I.
I
S T H E course of development work on alloys of uranium with zirconium and niobium, a rapid, accurate method of analysis became imperative. Because chemical methods of determining these alloying constituents were difficult and timeconsuming, it m-as decided to investigate spectrographic procedures. I t was expected that difficulties might occur in a spectrographic procedure because of the complexity of the uranium spectrum (4)with its attendant high background ( 2 ) and the possible interference of uranium with zirconium and niobium lines. However, as the spectra of zirconium and niobium are characterized by a number of prominent lines, some of which might be free of interferences, it appeared worth R-hile to attempt to develop a procedure for the spectrographic determination of zirconium and niobium in the range of about 5.5 and l.570, respectively. The results of this attempt are described. PROCEDURE
A4smooth, machined surface of the alloy was sampled directly by the high voltage, condensed, alternating current spark. This
Chemical Analysis of Spectrographic Standards.
Standard NO. H 366 B H 368 B H 363 B H 361 B H 369 B H 413 B
Zirconium,
Niobium,
%
%
5.10 3.00 3.78 1.78 4.91 5.81
0.52 0.78 0.94 1.53 1.85 1.91
-4 modified Baird spark source with an air-interrupted gap and additional capacitance was employed with the following conditions: Primary. 130 volts, 5.5 amperes Inductance. 4.06 microhenries
Secondary. 25.000 volts Capacitance. 0.005 microfarad
The spectra were photographed on Eastman SA S o . 1 plates, using the Bausch & Lamb large Littrow quartz prism spectrograph. The sample was presparked for 5 seconds and exposed for 15 seconds through an aluminized quartz plate a t the slit, giving a transmittance of about 20%. The image of the electrodes was focused by a 16-cm. quartz lens on the collimator. Standard developing conditions were carefully maintained.
