[Advances in Chemistry] Aquatic Humic Substances Volume 219 (Influence on Fate and Treatment of Pollutants) || Analysis of Humic Substances Using Flow Field-Flow Fractionation

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<ul><li><p>5 Analysis of Humic Substances Using Flow Field-Flow Fractionation </p><p>Ronald Beckett </p><p>Water Studies Centre, Chisholm Institute of Technology, 900 Dandenong Road, Caulfield East, Victoria 3145, Australia </p><p>James C. Bigelow, Zhang Jue, and J. Calvin Giddings </p><p>Department of Chemistry, University of Utah, Salt Lake City, UT 84112 </p><p>A new method for the determination of molecular-weight distributions of humic substances using flow field-flow fractionation (flow FFF) is outlined. Fairly good agreement between the results obtained by flow FFF and other methods for some humic reference substances was achieved using poly(styrene sulfonate) molecular-weight calibration standards. The molecular-weight distributions obtained for a variety of humic samples were all fairly broad and, in contrast to the data sometimes reported for gel permeation chromatography, did not show any indication of multiple peaks. The number- and weight-average molecular weights can be estimated, and these were shown to vary considerably (Mw from about 4400 to 19,000) for humic acids ex-tracted from different environments. The method is also capable of fractionating a humic sample. However, because it is a very small-scale separation (</p></li><li><p>66 AQUATIC HUMIC SUBSTANCES </p><p>studies being reported in the past decade, either investigating the interaction of pollutants with humic substances or attempting to elucidate the structures of these complex organic compounds. </p><p>Not only do humic substances form metal complexes of varying stability with most trace metals (3-5) but they can also bind and help solubilize nonpolar organic compounds (6-8). In addition, they are adsorbed to many mineral surfaces, and thus they affect the properties of soils, sediments, and suspended particulate matter. In particular, they control the surface charge of aquatic particles (9, 10) and substantially influence their colloid stability (11-13) and adsorption behavior (14-16). Despite much effort and considerable advances, our knowledge of the structure of humic substances is still inadequate. No doubt this lack stems largely from the fact that this very heterogeneous class of materials is defined largely by the method of extraction used to isolate its components. Certainly the introduction of better fractionation schemes is beginning to help simplify the problem of structure determination (17). </p><p>A good illustration of the dearth of reliable information on humic substances is the diversity of views held by scientists in the field on the molecular weight of these compounds. They are very commonly described as having high molecular weight, yet others dispute this claim (18). The molecular weights reported in the literature vary enormously, with values in the range of 500-200,000 being recorded (19). There appear to be several plausible reasons for this observation. Certainly, different workers have used samples extracted from various sources and by various methods. However, the question remains of whether the huge variations reported for different samples are real or artifacts of the methods and conditions used. The introduction of reference samples by the International Humic Substances Society (20) should eventually help to answer this question, and researchers should be encouraged to use such standards as reference materials in their work (21). </p><p>A number of different techniques have been used to determine the molecular weight of humic substances. Each technique is based on a different physical principle and is invariably subject to its own set of limitations and approximations. This variation may be one of the reasons for analytical discrepancies. Another factor that may account for the very high molecular weight sometimes reported is the suggestion that humic substances may aggregate under some solution conditions. Aggregation may be promoted, for example, by high solution concentration, increased ionic strength (particularly in the presence of multivalent cations), or low p H . Wershaw and co-workers (22, 23) have even suggested that humic substances may be capable of forming micellelike agglomerates that can be used to account for properties such as the ability of humic substances to solubilize nonpolar substances (e.g., pesticides, polychlorinated biphenyls [PCBs], polycyclic aromatic hydrocarbons [PAHs]). On the other hand, perhaps degradation reactions may occur under some conditions and lead to lower apparent molecular weights. </p><p>Dow</p><p>nloa</p><p>ded </p><p>by U</p><p>NIV</p><p> OF </p><p>TE</p><p>NN</p><p>ESS</p><p>EE</p><p> KN</p><p>OX</p><p>VIL</p><p>LE</p><p> on </p><p>Sept</p><p>embe</p><p>r 4,</p><p> 201</p><p>4 | h</p><p>ttp://</p><p>pubs</p><p>.acs</p><p>.org</p><p> P</p><p>ublic</p><p>atio</p><p>n D</p><p>ate:</p><p> Dec</p><p>embe</p><p>r 15</p><p>, 198</p><p>8 | d</p><p>oi: 1</p><p>0.10</p><p>21/b</p><p>a-19</p><p>88-0</p><p>219.</p><p>ch00</p><p>5</p><p>In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988. </p></li><li><p>5. BECKETT ET AL. Analysis Using Flow Field-Flow Fractionation 67 </p><p>In this chapter we discuss the development of flow field-flow fractionation (flow F F F ) as a new method for determining the molecular-weight distributions of humic substances. The aim is to describe the experimental methodology, to outline current limitations that need to be overcome, and to contrast this technique with other methods that are currently in use. We also speculate on how the separation capabilities of flow F F F may be used in environmental studies. </p><p>Field-Flow Fractionation Field-flow fractionation refers to a series of separation techniques that are similar in operation to chromatography, but in which the separation of molecules or particles occurs in thin (0.1-0.5-mm) unpacked rectangular channels (24). In normal F F F operation the sample is introduced at one end of the channel; a driving force is then applied across the thin dimension of the channel and causes the sample to migrate toward one wall, the accumulation wall. There the sample particles form a cloud whose mean thickness (/) depends on the velocity induced by the field (U) and their difiusion coefficient (D) as follows (24): </p><p>Thus, the value of / generally depends on the particle or molecular size (or mass). When carrier flow begins, laminar flow is maintained as a consequence of the thin channel cross-section. The carrier flow is greatest in the center of the channel and decreases to zero at the two boundary walls. Indeed, the distribution of flow velocities across the channel is parabolic, as illustrated in Figure 1. As the sample clouds interact with the carrier flow distribution, the particles migrate down the channel with a velocity that depends on the value of I. Sample components wil l thus elute at characteristic retention volumes (V r). It can be shown that Vr and / are related to the key retention parameter, the retention ratio (R), by the relationship (25): </p><p>where V is the channel void volume, is the dimensionless cloud thickness IIand w is the channel thickness. </p><p>Different F F F subtechniques make use of different types of applied fields. The most widely used subtechniques are gravitational or centrifugal sedimentation, thermal gradient, and crossflow methods. Electrical and magnetic fields are less commonly used. Other possibilities exist, including fluid shear induced forces (26). The general strategy of F F F is to measure Vr for the various sample components. Vr is used to calculate R and then or /, </p><p>(2) </p><p>Dow</p><p>nloa</p><p>ded </p><p>by U</p><p>NIV</p><p> OF </p><p>TE</p><p>NN</p><p>ESS</p><p>EE</p><p> KN</p><p>OX</p><p>VIL</p><p>LE</p><p> on </p><p>Sept</p><p>embe</p><p>r 4,</p><p> 201</p><p>4 | h</p><p>ttp://</p><p>pubs</p><p>.acs</p><p>.org</p><p> P</p><p>ublic</p><p>atio</p><p>n D</p><p>ate:</p><p> Dec</p><p>embe</p><p>r 15</p><p>, 198</p><p>8 | d</p><p>oi: 1</p><p>0.10</p><p>21/b</p><p>a-19</p><p>88-0</p><p>219.</p><p>ch00</p><p>5</p><p>In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988. </p></li><li><p>^teu</p><p>T0</p><p>*" </p><p>d^ra</p><p>mf</p><p>th*.</p><p>fl </p><p>FF</p><p>F ch</p><p>ann</p><p>el s</p><p>how</p><p>ing </p><p>the </p><p>sem</p><p>iper</p><p>mea</p><p>ble </p><p>bou</p><p>ndi</p><p>ng </p><p>wal</p><p>ls, </p><p>con</p><p>sist</p><p>ing </p><p>of </p><p>frtt</p><p>s an</p><p>d a </p><p>mem</p><p>bran</p><p>e. I</p><p>nse</p><p>t is </p><p>a cr</p><p>oss </p><p>sect</p><p>ion</p><p> of </p><p>the </p><p>chan</p><p>nel</p><p> illu</p><p>stra</p><p>tin</p><p>g th</p><p>e se</p><p>para</p><p>tion</p><p> mec</p><p>han</p><p>ism</p><p>. </p><p>Dow</p><p>nloa</p><p>ded </p><p>by U</p><p>NIV</p><p> OF </p><p>TE</p><p>NN</p><p>ESS</p><p>EE</p><p> KN</p><p>OX</p><p>VIL</p><p>LE</p><p> on </p><p>Sept</p><p>embe</p><p>r 4,</p><p> 201</p><p>4 | h</p><p>ttp://</p><p>pubs</p><p>.acs</p><p>.org</p><p> P</p><p>ublic</p><p>atio</p><p>n D</p><p>ate:</p><p> Dec</p><p>embe</p><p>r 15</p><p>, 198</p><p>8 | d</p><p>oi: 1</p><p>0.10</p><p>21/b</p><p>a-19</p><p>88-0</p><p>219.</p><p>ch00</p><p>5</p><p>In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988. </p></li><li><p>5. BECKETT ET AL. Analysis Using Flow Field-Flow Fractionation 69 </p><p>which in turn can be related to certain parameters of the particles or molecules (e.g., mass, diffusion coefficient, mobility) by equations derived for the particular field (subtechnique) being used. </p><p>Flow F F F is the subtechnique that uses a crossflow of fluid to drive the molecules to the accumulation wall (Figure 1). This "field" flow is maintained at right angles to and superimposed on the normal channel flow by constructing the channel walls from a porous frit material with semipermeable membranes at the accumulation wall to retain the sample in the channel. In this system the field-induced sample velocity (U) is simply the linear crossflow velocity. Therefore it follows from Equation 1 that (27): </p><p>Uw Vcw2 </p><p>where Vc is the volumetric crossflow rate. Thus, in flow F F F the molecular diffusion coefficient can be estimated </p><p>from the measured elution volume. In addition to its usefulness for characterizing colloidal particles (28, </p><p>29), flow F F F has been shown to be effective for separating proteins (30) and water-soluble polymers (31) with good resolution, as illustrated in Figure 2. The theoretical diameter-based selectivity is unity, a value higher than that for gel permeation chromatography (32). </p><p>Experimental Details The equipment, samples, and method of operation have been described in detail elsewhere (33). In addition to the polysulfone membrane (Millipore PTGC) with a nominal molecular-weight cutoff of 10,000 daltons for globular proteins, which was used as the accumulation wall in the previous study, some experiments were run </p><p>ELUTION VOLUME (mL) </p><p>Figure 2. Flow FFF fractograms showing good resolution in the separation of (A) poly(styrene sulfonate) molecular-weight standards and (B) proteins. </p><p>Dow</p><p>nloa</p><p>ded </p><p>by U</p><p>NIV</p><p> OF </p><p>TE</p><p>NN</p><p>ESS</p><p>EE</p><p> KN</p><p>OX</p><p>VIL</p><p>LE</p><p> on </p><p>Sept</p><p>embe</p><p>r 4,</p><p> 201</p><p>4 | h</p><p>ttp://</p><p>pubs</p><p>.acs</p><p>.org</p><p> P</p><p>ublic</p><p>atio</p><p>n D</p><p>ate:</p><p> Dec</p><p>embe</p><p>r 15</p><p>, 198</p><p>8 | d</p><p>oi: 1</p><p>0.10</p><p>21/b</p><p>a-19</p><p>88-0</p><p>219.</p><p>ch00</p><p>5</p><p>In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988. </p></li><li><p>70 AQUATIC HUMIC SUBSTANCES </p><p>here with a cellulose membrane (Amicon, YC05) with a specified 500-dalton pore size. Most fractograms were collected with conventional UV-visible detectors and chart recorders. Several were recorded with a photodiode array detector (Varian Polychrom 9060) capable of collecting data between 190 and 367 nm. The Suwannee River flvic and humic acids used were the International Humic Substances Society reference stream samples, usually with 40-, injections of 0.25-mg/mL solution buffered at pH 7.9. The carrier solvent in all cases was 0.05 M tris(hydroxy-methyl)aminomethane (TRISMA, Aldrich), 0.0268 H N 0 3 , and 0.00308 M NaN 3, and the pH was 7.9. </p><p>Poly(styrene sulfonate) molecular-weight standards (Polysciences, Inc.) covering the range 4000-100,000 daltons were made up in water (1 g/L), as were some biological test samples of bovine serum albumin, ovalbumin, transfer RNA, and RNase. </p><p>Results and Discussion Fractograms of Humic Substances. Fractograms of the reference </p><p>Suwannee River fulvic and humic acids are shown in Figure 3A. In these plots the ordinate is the absorbanee (arbitrary units) of the sample at 254 nm; it should represent the sample concentration if the molar extinction coefficient remains constant across the molecular-weight distribution. The abscissa is the elution volume, which is related to the dhfusion coefficient through equations 2 and 3. Thus, the abscissa is not linear with respect to molecular weight. </p><p>The fractogram is a smooth single-peaked curve, unlike many of the complex chromatographs based on gel permeation chromatography (GPC). This finding was true of all the samples we have run so far, which have included aquatic, soil, peat, and coal humic substances. The consistency leads us to suspect that the multiple peaks generated by the G P C method are due to anomalous effects, such as adsorption and charge repulsion, involving the sample and gel. </p><p>To investigate whether different molecular-weight components show different U V signals, which may indicate a distinct change in chromophore, some runs were made with a photodiode array detector (Varian) capable of scanning between 190 and 367 nm. An example of the detailed information obtainable from such a fractogram is shown in the three-dimensional plot of Figure 4. However, because of the featureless spectrum of humic substances and the fact that no shift in peak maximum occurs as the molecular weight changes, a rather simple picture is obtained in this case. The diagram does indicate rather clearly the feet that 254 nm (highlighted curve) is a good choice of wavelength for recording fractograms of humic substances. </p><p>Several inherent problems could arise with the flow F F F technique, two of which are associated with the membrane used on the accumulation wall to contain the sample molecules within the channel. These problems are the permeability of the membrane, which may allow some of the sample to leak from the channel, and the possibility of sample adsorption onto the </p><p>Dow</p><p>nloa</p><p>ded </p><p>by U</p><p>NIV</p><p> OF </p><p>TE</p><p>NN</p><p>ESS</p><p>EE</p><p> KN</p><p>OX</p><p>VIL</p><p>LE</p><p> on </p><p>Sept</p><p>embe</p><p>r 4,</p><p> 201</p><p>4 | h</p><p>ttp://</p><p>pubs</p><p>.acs</p><p>.org</p><p> P</p><p>ublic</p><p>atio</p><p>n D</p><p>ate:</p><p> Dec</p><p>embe</p><p>r 15</p><p>, 198</p><p>8 | d</p><p>oi: 1</p><p>0.10</p><p>21/b</p><p>a-19</p><p>88-0</p><p>219.</p><p>ch00</p><p>5</p><p>In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988. </p></li><li><p>5. BECKETT ET AL. Analysis Using Flow Field-Flow Fractionation 71 </p><p>MOLECULAR WEIGHT 1000 10000 20000 </p><p>10 15 20 ELUTION VOLUME (mL) </p><p>3 </p><p>UJ &gt; </p><p>ELUTION VOLUME (mL) 10 15 20 </p><p>0.05 -</p><p>0.04 Fulvate -</p><p>0.03 -</p><p>0.02 " \ -0.0! </p><p>0 </p><p>_Humate \ v </p><p>i </p><p>-</p><p>MOLECULAR WEIGHT </p><p>Figure 3. (A) Flow FFF fractograms of Suwannee River fulvate and humate samples at pH 7.9 with Millipore membrane. (B) Calculated molecular-weight distributions obtained with molecular-weight calibration line for PS S standards </p><p>given in Figure 6. </p><p>membrane. An indication that one or both of these may be occurring to some extent is given in Figure 5, which shows that the peak area of the eluted sample decreases as the stop flow time is increased. (The stop flow time is the time the sample is held without channel flow at the head of the channel, while being forced to the accumulation wall by crossflow. ) Presumably a longer stop flow time gives the sample a greater opportunity to be lost by these two mechanisms. In all other fractograms reported here, a stop flow time of 1.5 min was used to minimize these effects. </p><p>Dow</p><p>nloa</p><p>ded </p><p>by U</p><p>NIV</p><p> OF </p><p>TE</p><p>NN</p><p>ESS</p><p>EE</p><p> KN</p><p>OX</p><p>VIL</p><p>LE</p><p> on </p><p>Sept</p><p>embe</p><p>r 4,</p><p> 201</p><p>4 | h</p><p>ttp://</p><p>pubs</p><p>.acs</p><p>.org</p><p> P</p><p>ublic</p><p>atio</p><p>n D</p><p>ate:</p><p> Dec</p><p>embe</p><p>r 15</p><p>, 198</p><p>8 | d</p><p>oi: 1</p><p>0.10</p><p>21/b</p><p>a-19</p><p>88-0</p><p>219.</p><p>ch00</p><p>5</p><p>In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical...</p></li></ul>

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