pric ions and selenized Hengar granules. Only pyridine and 2,4,6-trimethylpyridine remained partially unoxidized. Presumably, prolonged or repeated procedures would result in their total destruction. I t is also assumed that the same treatment applied to all the other substances listed in Table I should lead to total oxidation of their carbon content as well. A decided advantage of the sulfuric, nitric, and perchloric acid wet oxidation treatment is that no explosion or ignition occurred during any of the digestions. Furthermore, the use of catalysts (see Table 11) considerably diminished both charring and foaming.
LITERATURE CITED
(2) (3) (4) (5)
(6) (7) (8) (9) (10) (11)
Their Applications in Analysis". G. Frederick Smith Chemical Co., Columbus, Ohio, 1942. G. F. Smith, Anal. Chlm. Acta, 8, 397 (1953). G. F. Smith, Anal. Chlm. Acta, 17, 175 (1957). E. Kahane, "L'Action de I'Acide Perchlorique sur les Matieres Organique", Hermann et Cie., Rue de la Sorbonne. Paris, France, 1934. H. Diehl and 0. F. Smith, "Proceedings of the International Symposium on Microchemistry, Birmingham University, 1958". Pergamon, Oxford, 1960. H. Diehl and G. F. Smith, Talanta, 2, 209 (1959). T. T. Gorsuch, Analyst(London), 84, 135 (1959). T. T. Gorsuch, "The Destruction of Organic Matter", Pergamon, Oxford, 1970. R. E. Mansell, R. P. Tessner, and E. J. Hunemorder, Anal. Chlm. Acta, 51, 323 (1970). G. F. Smith, Analyst(London), 80, 16 (1955). Analytical Methods Committee. Analyst(London), 84, 214 (1959).
RECEIVEDfor review June 26, 1975. Accepted September
(1) G. F. Smith, "Mixed Perchloric, Sulphuric and Phosphoric Acids and
25, 1975.
Evaluation of Combined Applications of Ultrafiltration and Complexation Capacity Techniques to Natural Waters Ralph G. Smith, Jr. Skidaway Institute of Oceanography, P.O. Box 13687, Savannah, Ga. 3 1406
The apparent copper complexation capacities of various molecular weight fractions of dissolved organic matter In estuarine waters were determined by a combination of ultrafiltration and anodic stripping voitammetry techniques. The recovery of total dissolved organic carbon after fractionation varied from 80-90% with a coefficient of variation of f 4 YO.The apparent copper complexation capacity of low molecular weight fractions increased with increasing salinity. The precision of the technique was fa%.
Ultrafiltration techniques have been used in biochemical investigations to separate dissolved organic matter into various molecular weight ranges using a variety of membrane filters. Specifically, the ultrafiltration system developed by Amicon Corporation has been used by several workers ( I , 2 ) for this purpose. Blatt e t al. ( 3 ) conducted studies of the protein retentive capacity of selected membranes. Their results indicate that membranes do not yield absolute quantitative fractionation of the dissolved organics but that their retentive capacity is suitable to separate proteins into reasonably accurate molecular weight ranges. Membrane fractionation techniques have not been used extensively to fractionate dissolved organic matter in natural waters. Most workers employed the use of Sephadex (4-6) for this purpose. Because samples must be preconcentrated for the latter technique, and special equipment is required, it is less attractive than ultrafiltration. The relatively recent application of anodic stripping voltammetry provides a means to evaluate the concentration of specific forms of metals in natural waters. By determining free metal ions and titrating the water with the metal of interest a titration curve is produced from which the complexation capacity of the water sample can be determined (7). Using this technique, Chau et al. (8) have determined the complexation capacity of lake water for copper. The purpose of the work described here was to evaluate the application of a combination of the above techniques to 74
ANALYTICAL CHEMISTRY, VOL. 48,
NO.
1, JANUARY 1976
the study of the distribution of complexing materials in natural waters. Only the complexation capacity of various molecular weight fractions for copper was considered. The main interest was to evaluate the reproducibility of procedures and their preliminary application to an estuarine system.
EXPERIMENTAL Apparatus. An Amicon model 402 Ultrafiltration System was employed using XM-100, XM-50, PM-10, and UM-2 ultrafilters. Anodic stripping was done with a Design Systems model DS-1 Trace Metal Analyzer equipped with an electrolysis cell employing a three-electrode system. Included were a Ag/AgCl reference electrode, a platinum counter electrode and a wax-impregnated graphite electrode as the test electrode. Dissolved organic carbon was determined with a Beckman IR-215B CO2 analyzer. Absorbance spectra were determined with a Beckman DB-G spectrometer. Procedure. The fractionation procedure was begun with the membrane having the highest molecular weight cut-off desired, to avoid membrane clogging. The ultrafiltration cell and membrane was flushed with double distilled water and the filtrate monitored a t 200 mp with an ultraviolet spectrometer until zero absorbance was recorded. A sample of known volume ( ~ 4 0 ml) 0 was then introduced into the cell and the system pressurized with nitrogen. The ultrafiltrate was collected and retained in a preweighed flask. When the sample had been concentrated to approximately 20 ml. the system was depressurized and the concentrate poured into a preweighed flask. The volume of this concentrated fraction was determined by gravimetry assuming a specific gravity of 1.0 and the fraction was saved. The ultrafiltrate from the above step was sequentially fractionated using membranes with the next lower molecular weight cut-off until that having the smallest cut-off desired was used. A t this point, the volume of the ultrafiltrate was determined. An aliquot of each concentrated fraction was diluted to its original concentration using the ultrafiltrate as the diluent. The complexation capacity of the diluted fractions was determined on duplicate 5.0-ml samples after buffering with sodium acetate [Chau et al. ( 9 ) ] .The complexation capacity of given molecular weight fractions was determined by the difference between the observed capacity of the fraction and that of the ultrafiltrate. A 5-min equilibration time was allowed between spikes and electrodeposition was carried out a t -400 mV for 5 min. A linear ramp of 20 mV/sec was used for stripping.
Table I. Observed Flow Rates for Selected Membranes Membrane
M o l wt cut-off
Nitrogen, psi
XM-100 XM-50 PM-10 UM-2
100 000 50 000 1 0 000 1000
5 20 10 50
Flow rate, ml/min
15 10
8 2
Table 11. Reproducibility of Replicate Molecular Weight Fractionations Aliquots
Mol wt range 100 000
50 000-100 000 1 0 000-50 000 1 000-10 000
RESULTS AND DISCUSSION
1000
A major problem associated with the use of the Diaflo ultrafilters is the apparent leaching of organic matter from the membranes. T o elucidate the source of this contamination, an ultraviolet spectrometer was used to monitor the ultrafiltrate while flushing the system with double distilled water. It was observed that the ultrafiltrate had a maximum absorbance at 200
[email protected] and ethanol, used in packing and storing of the membranes, also have a maximum absorbance a t this wavelength and are the apparent source of low molecular weight hydrocarbon contamination. Storage of membranes in a solution of 0.1% sodium azide (NaN3) to prevent bacterial growth minimized contamination problems. T o ensure that leachable carbon was removed from the system before sample introduction, the cell and membrane were flushed with double distilled water and the ultrafiltrate monitored by UV until no detectable absorbance was obtained. Analyses of the ultrafiltrate showed it to be relatively carbon-free at this point. Another important parameter to establish is the minimum nitrogen pressure required with each membrane to attain a constant flow rate. If the pressure is too great, improper fractionation results. Table I shows the observed flow rates of selected membranes at the designated pressures. Large deviations from these flow rates may indicate membrane failure. The recovery of total dissolved carbon from a fractionated sample varies from 80-100% using the technique described. The recovery can be increased to near 100% if the retained fractions are washed from the cell with double distilled water. However, since this may cause structural changes in the dissolved organics, it is an unsuitable approach. The complexation capacity of each molecular weight fraction was determined after the fraction was diluted to its in situ concentration. This dilution was necessary to avoid artifacts of the technique brought about by concentration of the dissolved organics during fractionation. The STATION 1 SalinityO%e
STATION 2 Salinity4%.
Recovery, %
3
2
1
mg Carbon
1.49 0.57 0.30 1.15
1.20 82
1.64 0.89 0.35 0.93
1.36 0.92 0.38 1.43 0.95 89
1.00
84
4 Re1 std - dev, 8
1.46 0.92 0.43 0.95 0.88 82
7.7 21 15 21
14 3.9
choice of the ultrafiltrate as the diluent was to ensure that the complexation capacity obtained for a given fraction is representative of the dissolved organics a t their in situ concentration. To determine the reproducibility of the fractionation process, a large sample was subdivided and treated as described above. The dissolved organic carbon in each fraction was determined by the method of Menzel and Vaccaro (IO).Results indicate a coefficient of variation of 21% or less for each fraction (Table 11).The coefficient of variation of the complexation capacity was less than 8% on 5 replicate samples. APPLICATIONS O F T H E TECHNIQUES Five stations in a Georgia estuary (the Ogeechee River) were sampled in April 1975 and the samples were stored a t 4 O C . The salinity of the samples varied from 0 to 13%. The samples were fractionated and the DOC of each fraction was determined. Most of the carbon in the samples was distributed between the lOOOOO) at the zero salinity station, while a t higher salinities the carbon content was considerably lower. This is probably due to interaction of organic matter with saline waters, perhaps as a coprecipitation with ferric hydroxide. Complexation capacities were determined on each molecular weight fraction and on the unfractionated sample. The results indicate that the molecular weight range designated 100000, (E) 50000-100000,(C)10000-50000,(D) 1000-10000, (E)
