Anal. Chem. 1981. 53. 1715-1717
T l Y l I ..conds)
Figure 5. Comparative, superimpoJed paaks BlusWating the effect of straight and coiled reactors on practical dispersion wimout chemical effect: Row rate, 0.42 mLlmin; reactw. 30 cm362 cm (2.5 cm o.d., coiledt30 cm [422 cm total length]; skaight reactw length. 422 cm. Curve A, coiled reactor [ D = 9.371. Curve E. straight reactor [ D = 12.941.
Table I. Comparative Values for Practical Dispersion and Time for Return to Base Line for Straight and Coiled Reactors (Flow Rate, 0.16 mC/min; Reactor, Tygon tubing, 180 cm length, 0.5 mm i.d.; pH 5.05) straight reactor
coiled reactor 3.4 mm 22.3 mm iden idc'
1715
for return to base line, th. as a result of increased radial diffusion. When a chemical reaction is operative its effect can be characterized by change in D- as described above. Table I shows that Deb., (chemical contribution to practical dispersion) is larger for straight reactors as a result of an increase in mixing with the flowing stream (background reactant) since axial diffusion is greater. The same is true for the increase of D,,, when the diameter of the coil reactor is increased. We hope this paper adds additional insight to earlier helpful descriptions of dispersion in flow injection analysis and points to the need for further work to lead to a more thorough understanding of parameters and concepts describing the transient signal observed in unsegmented, continuous-flow systems. LITERATURE C I T E D (1) ROfi6ka. J.: Mnsen. E. H. A w l . CMm. Acta 1975, 78, 145-157. (2) Tljssen. R. AMI. Chlm. Acts 1080. 114. 71-89. (3) Van Der Berg. J. H. M.; DeslJa. R. S.: E g M k . H. G. M. Aml. CMI. Acta 1980. 114. 91-104. (4) Reijn. J. M.; Van Der unden.W. E.: P m . H. AMI. C M . Acta 1910. 114. 105-118. (5) Vandersllce. J. T.: StewaBn. K. K.; QeWy Rossnfeld, A,: Higgs. D. J. rabnra mi. 28. 11-18, (6) Ruii&a. J.: Hansen. E. H. AMI. C h h . Acts 1978, 99. 37-76. (7) Motlola. H. A.: brim. A. AMI. Chlm. Acta 1978. 100, 167-180. (8) Fansen. E. H.; RuiiEka. J. J. C h m . Educ. 1979. 56. 677-680. (9) Cam, C. 0. J. mysbl. i988, 185. 501-519.
Dispersion without Chemical Effect 26 7 219 230
C. C. Painton Horacio A. Mottola*
tbasr
D
4.36
4.08
4.17
Dispersion with Chemical Effect S
166
Dchem
7.44 3.08
tbas,
D
153 6.24 2.16
154
Department of Chemistry Oklahoma State University Stillwater, Oklahoma 74078
6.69 2.52
idc = internal diameter of coil. for the injection of sample transported without imposed chemical reaction adding to changes to concentration levels of the monitored species. Figure 5 confirms this effect coiled reactors produce sharper peaks (smaller D ) and shorter time
RFCEIVEDfor review March 6,1981. Accepted June 2,1981. This work is being supported by a grant from the National Science Foundation (CHE-7923956). This paper was presented a t the 1980 Southeast/Southwest Regional Meeing of the American Chemical Society, New Orleans, LA, Dec 12. 1980.
Carbon- 13 Enriched Nuclear Magnetic Resonance Method for the Determination of Hydroxyl Functionality in Humic Substances Sic We wish to report a method for the characterization of hydroxyl functional groups in humic substances. Treatment of humic and fulvic acids with W e n r i c h e d methylating reagents followed by 13CNMR spectroscopy permits one to identify and estimate the relative abundances of the various OH groups. Humic substances, especially fulvic and humic acids, are present in soil,water, and coal ( I , 2). They are active chelating agents (3-5),adsorb hydrophobic compounds, and promote plant growth. In natural waten, they affectaquatic organisms, form toxic haloforms upon chlorination, transport insecticides, and remobilize metal ions from sediments. They appear to interfere with the flotation process in copper miniig and c a w engineering problems in coal gasification. T h e activity of these polymeric humic substances resides in their hydroxyl functionality, which is composed of carboxyl, phenolic, hydroxyl, and saccharide groups. Amino nitrogen may also be present in some humates. Hydrogen bonding may 0003-270018110353-1715t0 1.2510
affect their state of aggregation and may also influence their chemical behavior (6. i?.Despite many years of m a r c h , little is known about the chemical structure of these ubiquitous compounds or the exact nature of oxygen functionality. Direct spectral measurements reveal very little information. Recent IT NMR studia @,9)show four broad bands, which indicate aliphatic, hydroxyl, aromatic, and carboxyl regions. One can make rough estimates of relative abundances of these four regions from the CPIMAS (cross polarization/magic angle spinning) NMR spectra (9). In order to enhance and separate the NMR signals of the hydroxyl functions from the broad featureless spectra of humic substances, we have methylated humic and fulvic acids with "C-enriched reagents. This derivatiration labels hydroxyl groups. eliminates hydrogen bonding and facilitates signal assignment in the 13C NMR spectrum. Estimation of the relative ahundance of functional group types can also be inferred from integral absorptions. A variety of model com0 1981 Amwkan UWkA Soclay
1716
ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
2bo
lbo
ibo p,n
(re.a:i"e
to
L
I 5'0
7s)
I
I
B
spectrum of soil fulvic acid in D,O. Scale in parts per million relative to Me3Si.
Figure 1. 13C NMR
pounds was used to establish chemical shifts and calculate percentage errors in the integration of peak areas.
EXPERIMENTAL SECTION Diazomethane (0.01mol) was prepared from N-rneth~l-~~C-Nnitroso-p-toluenesulfonamideand its ether solution was distilled into a solution of 50 mg of substrate in 25 mL of DMF in an ice bath. The solution was allowed to warm to room temperature and stirred overnight. The DMF was removed under vacuum and the residue redissolved in dry DMF (distilled over molecular mol) sieves). To this solution was added 0.25 g of NaH (5 X and 0.1 mL of I3CH3I(5 X lo4 mol) under nitrogen. The mixture was allowed to stir overnight. The excess NaH was decomposed with water and concentrated HC1 was added until the solution was neutral to litmus. The solution was then dried under vacuum and the methylated residue dissolved in CDC13for NMR analysis. Complete methylation of OH functionality was confirmed by the absence of OH bands in the IR spectrum. As a control, the same reaction was carried out without humate substrate; the product showed no I3C-NMRbands downfield from 40 ppm (relative to Me3Si). Reagents. N-Methyl-13C-N-nitroso-p-toluenesulfonamide and I3C iodomethane (90% or 60% of '%) were obtained from Merck, Sharp and Dohme (Canada, Ltd., Montreal, Quebec). Flavonoid compounds were obtained from Aldrich Co. The Mollisol humic acid was obtained from M. Schnitzer, Canada Agriculture, Ottawa, Canada. Its analytical properties are as follows: 56.4% C, 5.5% H, 4.1% N, 1.1% S,and 32.9% 0;the functional groups (mequiv/g) are COOH 4.5,phenolic OH 2.1,and OCH3 1.0. Fulvic acid from the Biscayne FL aquifer was provided by E. M. Thurman, USGS, Denver, CO, who isolated it from its source (IO). Its analytical properties are as follows: 55.44% C, 4.17% H, 1.77% N, 35.39% 0,1.06% S, 0.43% ash, 7.1 mequiv/g COOH, 2.5 f 1.5% mequiv/g phenolic. NMR Spectra. The 13C NMR spectra were recorded on a Bruker Instruments WH-90 Fourier transform NMR Spectrometer operating at a frequency of 22.62 MHz in the 'H broad band decoupler mode. Spectra of the same compounds were also recorded on a Bruker Instruments WM-250spectrometer operating at a frequency of 62.90 MHz (to ascertain whether any significant structural differences were observed between the spectra of the two spectrometers). Both instruments gave comparable spectra. All samples were measured in CDC13 solution, which served as the internal '% lock. The spectra were obtained by using 8K data points over a 6000 Hz spectral width with quadrature detection. Humic samples were subjected to delay periods between pulses from 1 to 40 s with no apparent changes in band absorptions in the OCH, region (40-70 ppm relative to Me3Si). Chemical shifts were assigned relative to internal Me3Si. The areas were computer generated with a program which assumed Laurentian line shapes. RESULTS AND DISCUSSION Figure 1 shows the spectrum of a typical underivatized fulvic acid in D20 accumulated after lo8 scans by the NMR spectrometer. It is characterized by four broad bands at 40 ppm, 79 ppm, 129 ppm, and 172 ppm (relative to Measi). A CP/MAS spectrum of the same compound shows somewhat enhanced peaks a t the same chemical shifts. Figure 2 shows
C 2
34
I 1
l , , , l , l . l , l , , , , , , , , , j
60
50
4c
ppm (relative to WS)
Flgure 2. 'C NMR spectra of 13Cpermethylatedhumic and fukic acids and reference compounds: (A) Mollisol soil humic acid; (B) Biscayne aquifer fulvic acid; (C) mixture of reference compounds: (1) pentamethylquercetin, (2) diglyme, (3) methyl benzoate, (4) methyl pentanoate.
Table I. 13CNMR Chemical Shift Assignments for Methylated Compoundsa chemical shift, functionality PPm compd 51.3 1 aliphatic COOCH, aromatic COOCH, 52.3 2, 3 aryl OCH, 55.7-56.3 4 55.6 3 58.9 5 ROCH,CH,OCH, carbohydrate OCH, 58.9-60.8 6 R,CHOCH, 57.5 ref 11 61.0 5 (eno1)OCH; 45.6 7 R,NCH, a All spectra taken in CDC1,; shifts in parts per million relative to Me,Si. Compounds: 1,methyl hexanoate; 2, methyl benzoate; 3, methyl 2,6-dimethoxy-4-hydroxybenzoate; 4,pentamethylquercetin; 5, diglyme; 6, 2,3,4,6tetra-0-methylglucopyranose; 7,methylpiperidone.
the 13CNMR spectra of a 13C-permethylated humic acid and a 13C-permethylatedfulvic acid in the 40-70 ppm region. The spectrum of a known mixture of methylated model compounds is also displayed in Figure 2 as a reference. See Table I for chemical shifts of model compounds. Intense bands for the methoxyl groups appear in these spectra, as well as a band for a R2NCH3group a t 45.6 ppm. When the 40-65 ppm portion of these spectra are displayed on an enlarged scale, these bands can be separated into discrete clusters. Aliphatic and aromatic methyl esters can be separately assigned a t 51.2 and 52.5 ppm, respectively. Another narrow group of peaks a t 55-56 ppm represent methyl aryl ethers. Aliphatic methyl ethers range from 57 to 61 ppm;
ANALYTICAL CHEMISTRY, VOL.
Table 11. Estimated Abundance of Hydroxyl Groups as Percentages of Total Hydroxyl Functionality 70of total OCH, chemical shift -area of spectrum of methoxyl Fulvic humic band, P P ~ assignments acida acidb 51.2
52.5 55-56 57.5 58-60
61-62 45.6'
aliphatic carboxyl aromatic carboxyl phenol aliphatic OH aliphatic or carbohydrate OH aliphatic or carbohydrate OH amino nitrogen
' Biscayne aquifer fullvic acid.
' As a percent of the total OCH,
15 24 12 28
9 20 14 9 38
10
10
20'
44c
11
Mollisol humic acid. peak area.
derivatives of ROCH2CH20CH3structures or carbohydrate methyl ethers show resonances between 59 and 60 ppm, while unoxygenated alkyl or c ycloalkyl methyl ethers appear in the 57-58 ppm region (11). These assignments are made on the basis of comparisons to model compounds and mixtures of model compounds which were measured under identical conditions to the spectra for the humates. The chemical shifb measured in our laboratory were consistent with literature values (12-15). The relative abundarices of the various functional groups were determined by integration of the peak areas. Enhancement due to nuclear overhauser effects (NOE) for the OCH3absorption bands was uniform, as determined by pulse delay experiments. The estimated abundance of the hydroxyl functionality in humic and fulvic acid is shown in Table 11. From a study of peak areas of model Compounds, we found that the estimates of relative abundance could be in error by as much as 15%. An analysis of the spectrum of permethylated Mollisol (Black Chernozem) humic acid reveals some interesting features. A narrow cluster of four lines at 51.3 ppm represents four different aliphatic carboxyl groups, while a broader band characterized by two sharp peaks indicates a variety of aromatic carboxyl functions. The aromatic carboxyl groups exceed the aliphatic groups by more than a 2 1 ratio. At 56 ppm, a band for phenolic groups shows an intensity of about 48% of the total COOH intensity; this is in good agreement with a reported value of 44% based on titration experiments. Intense absorptions in the 57-61 ppm region reflect aliphatic or carbohydrate hydroxyl groups, comprising slightly more than half (57%) of the total hydroxyl functionality. A dialkyl amine functionality is indicated by a peak at 45.6 ppm; it has 44% of the area of the total hydroxyl bands. The Mollisol humic acid sample contains 4.1% N. The aquatic fulvic acid reveals features quite different from the humic acid. The ratio of aromatic to aliphatic carboxyl is less than 21; the aromatic carboxyl band is much narrower than the corresponding band for humic acid, suggesting a small variety of structural types. There appears to be very little phenolic character; less than one-third of the total carboxyl content is phenolic. This is consistent with the reported titration values, although the latter are subject to considerable error. The dialkyl amine content is 20% of the total hydroxyl content; the fulvic acid contains 1.7% nitrogen. While the total aliphatic-carbohydrate contents of the humic and fulvic acid are about the same, they are qualitatively quite different. The humic acid appears to have more carbohydrate-like groups, while the fulvic acid has two uplfield peaks which cannot be assigned. However, this appears to be the first
53, NO. 11, SEPTEMBER 1981
1717
direct measure of aliphatic hydroxyl functionality in humic substances; previous estimates have been based on difference measurements (2). In summary, we have shown that the 13CNMR spectra of 13C-enriched permethylated humates can unambiguously identify aliphatic and aromatic carboxyl groups, phenolic groups, aliphatic hydroxyl groups, and probably amino nitrogen. When fully exploited, this method will become a much more sensitive probe of chemical structure. For example, preliminary experiments with coupled H-13C NMR have revealed hyperfine structure related to the immediate proton environment of the OCH3groups; other experiments with mild methylating agents selectively enhance specific functional group absorbances. Finally, the compilation of the spectra of a representative group of humates and fulvates should establish a basis for a rational classification of these substances and help predict their physicochemical interactions with their environment.
ACKNOWLEDGMENT The authors wish to thank Dale McKay, Colorado State University NMR Center (funded by NSF Grant CHE. 7818581) for the CP/MAS spectrum of soil fulvic acid; Kenner Christiansen, University of Arizona, Chemistry Department, for invaluable technical help with the 13C NMR spectra; Michael Barfield, University of Arizona, for interpretation of the spectra, and Morris Schnitzer, Canada Agriculture, Ottawa, Ontario, for the sample of Mollisol humic acid. LITERATURE CITED (1) Steelink, C. J. Chem. Educ. 1977, 54, 599. (2) Schnitzer, M.; Khan, S. U. "Humic Substances in the Environment"; Marcel Dekker: New York, 1972. (3) Bresnahan, W. T.; Grant, C. L.; Weiser, J. H. Anal. Chem. 1978, 50, 1675. (4) Reuter, J. H.; Perdue, E. M. Geochim. Cosmochim. Acta 1977, 47, 325.
(5) Buffle, J.; Greter, F. L.; Haerdl, W. Anal. Chem. 1977, 49, 216. (6) Wershaw, R. L.; Pinckney, D. J.; Booker, S. E. J. Res. U . S . Geol. Surv. 1972. 5. 565. (7) Wershaw,
571.
d. L.;
Plnckney, D. H. J. Res. U . S . Geol. Surv. 1972, 5,
(8) Wilson, M. A.; Jones, A. J.; Wllliimson, B. Nature (London) 1978, 276,
487. (9) Hatcher, P. G.; Breger, I. A.; Mattingly, M. A. Nature (London) 1980, 285, 560. (IO) MacCarthy, P.; Peterson, M. J.; Malcolm, R. L.; Thurman, E. M. Anal. Chem. 1979, 51, 2041. (11) Blunt, J. W.; Munro, M. G. H. Org. Magn. Reson. 1980, 73, 26. (12) Scott, K. N. J. Am. Chem. SOC. 1972, 94, 8564. (13)Sundholm, E. G. Tetrahedron 1977, 33, 991. (14) Ludemann, H. D. Blochem. Biohys. Res. Commun. 1973, 52, 1162. (15) Blunt, J. W.; Munro, M. H.; Paterson, A. J. Aust. J. Chem. 1976, 29, 115.
Michael A. Mikita Cornelius Steelink* Department of Chemistry University of Arizona Tucson, Arizona 85721
Robert L. Wershaw U.S. Geological Survey Denver, Colorado 80225 RECEIVED for review October 6,1980. Resubmitted February 2,1981. Accepted May 29,1981. This paper was presented before the 180th National Meeting of the American Chemical Society, Las Vegas, NV, Aug 24-29, 1980. The work was supported in part by the Office of Water Research and Technology, A-094, U.S. Department of Interior, Washington, D.C., as authorized by the Water Research and Development Act of 1978, and in part by the U.S. Environmental Protection Agency, Office of Research and Development, EPA No. CR807363.
