acetonitrile under identical conditions is 4.3 f 0.5 x lo-*". Thus lanthanum fluoride is appreciably less soluble in 4.OM acetonitrile than in water. The difference in solubility product between the aqueous and partially organic media may be used to explain the shift in potential of the fluoride electrode when used as a reference. The equation for fluoride electrode response a t 25 "C may be written as
E = E,
- 0.0592 log uF-
=
E,*
+ 0.0592 3 1%
ULn
~
-
Since a~~ 3 + and U F - are related through the solubility product constant for lanthanum fluoride,
K.
= ( ~ 1 -)(aF-)' , ~ ~
(3)
the equation for electrode response may be expressed as follows:
E = E , *+
0.059 K. log 3 OF-)
7
~
(4)
This equation separates the E , term normally referred to in describing fluoride electrode response into a membrane solubility term (expressed as 0.0592/3 log K , and E,*,a term representing all other contributions to the "constant" term. In aqueous solution, the potential measured for a given fluoride concentration therefore can be given by
E,,
=
E,," - 0.0592 log
CZF-
0.0592 +_ _ log K , 3
0.0592 +log K , , 3
(6)
where K,,o is the solubility product constant of the lanthanum fluoride membrane in the partially organic solvent mixture. Thus, the shift in potential, JEobs,at a given, constant fluoride concentration on transfer from aqueous to partially nonaqueous solution is given by
(1)
where E is the potential in volts, E, is a constant for a specific electrode system in a specific medium, and uF- is the activity of the fluoride ion. However, the electrode also responds to lanthanum ion, and the potential may be expressed as the following:
E
E,, = E , * - 0.0592 log aF-
(5)
where Ka.wis the solubility product constant for the lanthanum fluoride membrane in water. Likewise, potentials measured in a partially organic medium may be expressed as
For 4.OM acetonitrile, potential changes resulting from changes in LaF3 solubility were determined by substitutand Ks,w = 4.8 X into ing Kh,o = 4.3 X Equation 7. The potential shift predicted on the basis of solubility differences is -40.4 mV. The shift actually observed was -44 mV. Therefore, the shift observed a t the fluoride electrode on addition of acetonitrile may be explained in terms of changes in the solubility of the LaF3 membrane. Since the K , values from which the potential shift was predicted are for freshly precipitated lanthanum fluoride rather than large, well-aged crystals, it is doubtful that any particular significance can be attached to differences of less than f5 mV. The fluoride electrode in a cell without liquid junction has been found less suitable than the calomel electrode with liquid junction as a reference electrode for complexation studies in systems where addition of an organic ligand appreciably alters the properties of the solvent. The relatively large negative shift in potential of the fluoride reference electrode in going from a n aqueous medium 0.100M in acetonitrile to a similar medium 4.OM in acetonitrile can be explained entirely upon the basis of decreased solubility of the lanthanum fluoride electrode membrane in the partially organic medium. Received for review August 20, 1973. Accepted November 7, 1973. This research was supported in part by the United States Department of the Interior Office of Water Resources Research Allotment Grant A-049-Mo. K . M.Stelting gratefully acknowledges support through X.D.E.A. Title IV Fellowship funds and a n American Chemical Society Analytical Division Summer Fellowship sponsored by Carle Instruments, Inc.
Quantitative Spectrometric Determination Specific for Mannose Ralph W. Scott and Jesse Green Forest Products Laboratory, Forest Service. U.S. Department of Agriculture, Madison. Wis. 53705
Specific quantitative methods for single sugars in sugar mixtures generally require a preliminary separation of the sugars or the application of specific enzymes such as glucose oxidase for the measurement of glucose. Photometric methods are of interest because they are sensitive and simple, but their use is often limited by a lack of specificity. Maksimenko et al. ( I ) have recently described a method t h a t deals with the specificity problem in mannose-glucose mixtures by using the different sensitivities of mannose and glucose in the phenol-sulfuric acid reaction. (1) 0. A . Maksimenko. L. A . Zyukova, N . S. Andreev, and R. M . Fedorovich, Zh. A n a / Khim., 26, 2467 (1971).
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In the procedure described here, use is made both of the dehydration of sugars to furans in concentrated sulfuric acid and of a rather specific change in the dehydration of mannose caused by chloride and boric acid. This sensitivity to chloride and to boric acid permits the determination of mannose in plant materials without preliminary separations of sugars.
EXPERIMENTAL Apparatus. Concentrated sulfuric acid and 72% sulfuric acid were dispensed by glass hand-pumped dispensers (5-ml Repipet, Labindustries. 1802 Second St.. Berkeley. Calif. 94710) with caps to fit 9-lb sulfuric acid reagent bottles.
Test solutions were accurately sampled with a spring-loaded syringe set for 0.125 ml a n d fitted with a 0.74-mm Teflon needle (Hamilton Co., P.O. Box 307, Whittier. Calif. 90608). This modification of a 1-ml tuberculin syringe has been described (2). Reaction tubes were 15- X 85-mm culture tubes covered with loose-fitting polypropylene bacteriological caps. T h e tubes closely fitted into a n electrically heated aluminum block with 15- x 50-mm holes. Absorbance was measured with a Beckman DU spectrophotometer. R e a g e n t s . Technical grade concentrated sulfuric acid (about 9670) gave low absorbance blanks between 0.05-0.03 i n t h e range 280 t o 320 n m . If blanks are much higher, it is best t o change to a better lot of acid. T h e only reagent in addition to the HzS04 was a solution containing 1 2 grams of NaCl and 2 grams of H s B 0 3 in 100 ml of water. Purchased sugars were [)-forms except for L-arabinose. Only mannose was recrystallized to a white powder ( 3 ) . 4-0-Methylglucuronic acid was recovered from hydrolyzates of hardwood xylan. P r o c e d u r e for Dissolving Water-Insoluble Polysaccharides. Most carbohydrate polymers dissolve in $270 HzS04 a t room t e m perature. The time for solution may be reduced by heating the sample a t 50 "C for 10- 15 min with some trituration. A suggested procedure for plant cell walls is solution of about 20 mg of 40-80 mesh dry material in 2-5 ml of 72% HzS04 at 50 "C followed by dilution with water to exactly 25 ml. The solution is then ready for the first step in t h e mannose analysis. Lignin from plant material will remain mostly insoluble. It may be allowed to settle out in t h e 25-m1 volume or it may be centrifuged down from a few milliliters of suspension. Amounts of lignin which do not greatly add to absorbance do not adversely affect the absorbance difference due to mannose. P r o c e d u r e for M a n n o s e -4nalysis. Transfer 0.125 ml of sample solution into each of two reaction tubes. T o one tube, a d d the same volume of water and to the other tube, a d d a like volume of the NaCl-HsBOa solution. Add 2.0 ml of concentrated HzS04 t o each tube a n d mix briefly by alternately swirling and tilting the tube. Heat each t u b e in t h e heating block at 70 "C for 30 min; remove each tube and cool by swirling for 15 sec in water a t room temperature. Read absorbances a t 280 n m u.a water reference. The difference between absorbances with and without SaC1H s B 0 3 is directly proportional to mannose concentrations between zero and 250 pg/ml. A reliable standard value is obtained from standard mannose solutions containing 5-6 mg in 25 ml by averaging t h e results of three separate runs. each run having triplicate analyses. Technique. It is advisable to keep tubes free of dust by using loose-fitting caps a t all times, including a drying period in a clean oven after washing. Sulfuric acid can be added to a series of samples at 20-sec intervals with tubes being removed from the heating block at t h e same intervals. The small amount of HC1 released when HzS04 is added to t h e NaCl solution is not a problem in a well-ventilated room. T h e dehydration procedure has been described ( 4 , .5j, S a m p l i n g . T h e amount of sample to be weighed depends upon the percentage of mannose and also upon which carbohydrate monomers are most prevalent in the mixture. It is desirable to maximize the absorbance difference due to mannose without exceeding readable total absorbances due to mannose and other sugars. Mixtures of mannose and glucose varying in mannose content from 2 to 207~require sample sizes varying from 26 to 17 mg. Corresponding amounts of mannose with xylose require s a m ple sizes varying from 17 to 1.3 mg.
R E S U L T S AND D I S C U S S I O N T h e Dehydration Reaction. The usefulness of analytical procedures based upon carbohydrate dehydration in concentrated sulfuric acid is due to the simplicity of reac(2) American Society for Testing and Materials, ASTM Designation D
1915-63 (Reapprov. 1970), 1916 Race Street. Philadelphla. Pa. 19103. ( 3 ) F . J Bates, and Associates, "Polarimetry. Saccharimetry and the Sugars," Nat. Bur. Stand. ( U . S . ) Circ. 440, 1942, p 471 ( 4 ) R . W. Scott, and J Green, Tappi, 55, 1061 (1972). (5) R. W . Scott, W . E. Moore, M . J. Effland. and M . A Millett. A n a / . Eiochem.. 21, 68 ( 1 967)
3Y A
280
//4/5.F'57?,
I' L
".,.
Figure 1. Absorption spectra after dehydration of mannose
in
90% H2S04 without NaCI-H3B03 in solution ( I ) and with NaCIH3B03 in solution ( I / )
tion conditions, the applicability to soluble oligomers without prior complete hydrolysis, the quantitative formation of a few relatively stable furan products, and the characteristic and strong ultraviolet light absorption by these products. Although reagents such as phenol or anthrone may form analytically useful colored products with furans or with intermediates in the dehydration reactions, their use may be less precise, and sometimes even less specific, than a direct ultraviolet absorption measurement. Since the dehydration reaction is dependent upon sugar structure, time, temperature, concentration of sulfuric acid, and reagents such as chloride and boric acid, these factors can be used to steer the dehydration in favorable directions. Major products of dehydration in sulfuric acid are 2-furancarboxaldehyde from pentoses, 5-(hydroxymethyl)-2furancarboxaldehyde from hexoses, and 5-(formyl)-2furancarboxylic acid from hexuronic acids. Although the amounts of these products are not in stoichiometric proportions with the starting sugars, the dehydrations are rapidly completed to give repeatable amounts of furans characteristic of the starting sugars. In Figure 1, curve I is primarily the absorption spectrum of 5-(hydroxymethyl) -2-furancarboxaldehyde formed from mannose in the dehydration mixture without NaC1H3B03. Curve I1 shows the altered absorption spectrum brought about by the dehydration of the same quantity of mannose in the presence of NaCl-H3B03. The chlorideboric acid reagent caused a decreased amount of 5-I:hydroxymethyl)-2-furancarboxaldehyde and an increased amount of an unknown absorber with a maximum absorbance at 278 nm. Tests showed t h a t the alteration in mannose dehydration was primarily due to the chloride; boric acid had a synergistic effect on this change. The slight shift in the 319-nm peak to 325 nm in Figure 1 is most likely due to the conversion of &(hydroxymethyl)-2-furancarboxaldehyde to 5-(chloromethyl)-2-furancarboxaldehyde in the presence of chloride ( 6 ) .A similar shift of absorbance from 322 nm to 325 nm occurs ( 6 ) F. H . Newth, Advan. Carbohyd. Chern., 6,87 (1951)
ANALYTICAL CHEMISTRY, VOL. 46, NO. 4, APRIL 1974
595
The interference by glucuronic acid is due to slow dehydration of glucuronolactone in 90% H2S04 and to an inRelative absorbance creased dehydration rate in the presence of boric acid. It difference a t 280 nm should be possible to greatly reduce interference from glu(by weight; curonic acid by standardizing the procedure without boric Sugar mannose = acid because the increase in absorbance at 280 nm due to Rhamnose $0.58 boric acid is not essential. Galacturonic acid and 4 - 0 +0.42 Fucose methylglucuronic acid do not interfere because they do Glucuronic acid + O .41 not form lactones that retard dehydration in 90% HzSO4. Lyxose + O .23 Although glucose, xylose, and arabinose can be considRibose -0.20 Galactose + O .15 ered as noninterfering sugars, the absorbance which they 4-0-Methylglucuronic -0.13 produce limits the sample size at low percentages of manacid nose. Furthermore, when they are in high percentage they Fructose +o .08 may cause small absorbance differences at 280 nm. These Galacturonic acid -0.07 differences can be estimated from sugar blanks, but an Glucose -0.01 accumulation of errors causes decreasing reliability below Xylose -0.02 Arabinose -0.01 5% mannose. Since mannose dehydration is complete in 20 min and a A negative value results from a decrease in absorbance due to NaC1H3B03, and will cause a low mannose determination. absorbance is then stable, it would be possible to remove tubes from the heating block as a group after a minimum of 20 min and to leave them a t room temperature. Howwhen NaCl is added to 5-(hydroxymethyl)-2-furancarbox- ever, the glucose dehydration rate is decreased by the aldehyde in 90% HzS04. In this case, the peak height is NaCl-H3B03 so that only a t 30 min are the absorbances not decreased. from glucose dehydration approximately equal for the two Interference by Other Sugars. Some sugars which insolutions. The heating period recommended here for the terfere with the determination are listed in Table I. The dehydration was chosen for samples high in glucose. list indicates that cis hydroxyl groups (especially at carMeasurement of Mannose in Sugar Mixtures. The bon atoms 2 and 3) in neutral sugars may cause interferabsorbance difference at 280 nm had a linear response to ence unless there happens to be an intersection of the two mannose concentrations up to 250 pg/ml. Since the priabsorption spectral curves a t about 280 nm as results from mary usefulness of this measurement is for mannose in the dehydration of arabinose. Comparison of recorded absugar mixtures, a few mixtures were prepared and anasorption spectra with and without NaC1-H3B03 permits lyzed (Table 11). Except for the increased relative errors in the estimation of interference and perhaps the choice of a amounts of mannose below 570.the averages of triplicate slightly better wavelength to decrease interference. Absamples show satisfactory estimations for these simple sorbance measurements a t 280 nm are specified here primixtures. marily to decrease interference from glucose, xylose, and The analysis was applied to the materials in Table I11 arabinose. where results are compared to the mannose content mea-
Table I. Interference by Some Sugars
Table 11. Determination of Mannose in Mixtures with Glucose and Xylose (25-ml total volume) Mixture No. Composition
Mg Glucose Mg Xylose Mg Mannose Mg Mannose found Weight percentage mannose found Error in weight percentage found
1
2
20.10 2.12 2.50 2.52 10.2
16.40
2.50 5.00 5.13 21.4
+o. 1
+O. 5
3
4
5
6
20.30 1.25 1.25 1.21 5.3
21.90 2.00 0.50 0.40 1.6
20.30
20.90 2.00
2.00
0.25 0.44 2.0
-0.5
-0.2
0.00 0.00 0.0
+0.9
Table 111. Analysis of Plant Material for Mannose Percentage of mannose anhydridea Source of material
Douglas fir wood Western hemlock wood Ponderosa pine kraft pulp Aspen wood Southern pine kraft pulp Glucomannan (pine) Galactomannan (locust bean gum)
1
13.6 11.9 6.2 2.0 5.4
2
3
Average
14.6 12.0 5.7
13.2 5.8
13.8 12.0 5.9
1.6
2.2
1.9
60.0 49.0
12.2
By chromatography
13.0 13.3 5.7b 1.86
5.6b 61.5 51, Ob
aEach figure under 1, 2 , 3 is an average of three determinations on a single solution. Data calculated from mannose as percentage of total sugar and from a corrected total sugar analysis (4).
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A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 4, A P R I L 1974
sured by paper chromatography ( 7 ) followed by elution and use of t h e Nelson procedure (8). T h e dehydration solutions from t h e galactomannan and from its mannose standard were read at 285 nm rather t h a n at 280 nm to reduce the interference from galactose. From a reading at 280 nm. the apparent mannose content was 10%higher. Application to Certain Total Sugar Analyses. A dehydration procedure can be used for total sugar analysis when one or two sugars dominate t h e carbohydrates in a mixture ( 4 ) . An example is structural tissue of broadleaf plants where the predominating glucose and xylose contents can be measured by comparison to a single glucose standard. Significant amounts of mannose in coniferous wood cause low total sugar estimates (4), b u t the mannose measurement described here can give t h e necessary correction. Precision. Average blank values from water and from NaCl-HsB03 solutions were t h e same: t h u s blank determinations were unnecessary for the analysis. However, (7) J. F. Saeman, W. E. Moore, R. L. Mitchell, and M. A. Millett, Tappi. 37 ( 8 ) , 336 (19541. (8) N . Nelson, J. Bioi. Chem.. 153, 375 (1944).
blanks contained much of the variability of the determination. A standard deviation of 0.012 was found for the absorbance difference between 18 pairs of blanks run during a month. Fifteen sets having three mannose samples in each set had a n average standard deviation of absorbance of 0.010 within a set. The first four lines in Table I11 show the variability between averages of three replicates per solution. These results also include variability in the initial step of dissolving samples in 72% HzS04 and diluting. The relative standard deviations calculated from three averages are 4.576, 1.376, 5.270, and 15.8%. respectively. for the first four lines in Table 111. At the lowest concentration. 2% mannose, the basic variability of the method seen in blank runs becomes large compared to the mannose measurement itself. Received for review August 20. 1973. Accepted October 12, 1973. The Forest Products Laboratory is maintained in cooperation with t h e University of Wisconsin. Mention of trade or proprietary names is for identification purposes only and does not imply endorsement of the product by the Forest Service. U.S.Department of Agriculture.
Application of the Carbon Rod Atomizer to the Determination of Mercury in the Gaseous Products of Oxygen Combustion of Solid Samples Duane Siemer and Ray Woodriff D e p a r t m e n t of Chemistry. Montana State University, Bozeman. Mont. 59715
The analysis of solid samples for fractional part per million levels of mercury is a common problem in analytical laboratories. Several excellent reviews containing the analytical methodology have been published recently (1-3). The most commonly utilized methods a t present are neutron activation analysis or some form of' atomic absorption preceded by wet ashing andjor extraction. The former technique is disadvantageous in many cases in t h a t it is expensive and usually involves sending samples off to central laboratories equipped with high neutron flux sources. Atomic absorption, on the other hand, is relatively inexpensive and can be accomplished rapidly. Atomic absorption analysis for mercury is usually accomplished by adding a reducing agent (usually stannous chloride) to a digestate and sweeping the evolved atomic mercury out of the solution into a cold vapor absorption tube or flame by means of a gas bubbled through the tem. In order to increase the sensitivity and to reduce the error due to the molecular absorption often encountered, some workers have concentrated the elemental mercury in t h e gas streams onto gold surfaces and released the mercury suddenly (by heating the gold substrate) into the atomic absorption cell. Finally, in order to eliminate preashing or extraction steps, some workers have applied the ( 1 ) H . R . Jones, "Mercury Pollurion Control," Noyes Data Corp., Park
Ridge, N . J . . 1971. (2) R. Hartung and B. D. Dinman, "Environmental Mercury Contamination, ' Ann Arbor Science Publishers, Ann Arbor, Mich., 1972. (3) F. M . D'ltri, "The Environmental Mercury Problem," Chemical Rubber Co , Cleveland, Ohio, 1972.
above technique directly to the combustion gases o f s a m ples burned in oxygen ( 4 ) . The approach used in this paper is to burn the samples completely in a modified combustion tube, to collect the evolved mercury on the inner surface of a porous. gold plated, carbon rod atomizer tube, and, finally, to measure the atomic absorption when the tube is heated in the normal fashion.
EXPERIMENTAL A p p a r a t u s . Figure 1 depicts the Combustion apparatus used tor this research. The combustion tube consists ot' a I. tube of about 4-cm radius made of 12-mm diameter \.ycor tubing with a l.i-cm long. 2.0-cm diameter combustion chamber sealed to it. There are oxygen inlets sealed both to the combustion chamber and the "afterburner." T h e combustion apparatus i, wrapped with 20gauge nichrome wire as indicated. The after burner section is loosely filled with a 50-50 v o l ~ v o lmixture of 8-mebh activated alumina and calcium oxide powder. Oxygen f 1 0 ~is controlled and measured by two Gilmont flow gauges and needle valve>. The combustion boat is made of a short section of 1U-mm diameter quartz tubing sealed to a length of 4 - m m quartz rod inserted into a S o . 2 rubber stopper. The combustion gases are passed through a water jacketed cold linger made ot borosilicate glass to condense some of the water evolved if a large numher of sample5 are burned in rapid succession. T h e preparation of' the gold plated atomizer tubes. a description of the filter adaptor and the details of the atomic absorption apparatus used have been described in the paper by Siemer. Lech. and IVoodriff 65). In addition. a deuterium lamp powered by a Beckman power 5upply is utilized for measurement of non( 4 ) V. Liddums and U Ulfvarson. Acta Chem. Scand.. 22, 2150 (1968) ( 5 ) D. Sierner, J. Lech, and R Woodrlff, Appi. Spectrosc in press.
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