RESUMEN
Field-flow fractionation (FFF) is a family of single-phase separative techniques exploited to gently separate and characterize nano- and microsystems in suspension. These techniques cover an extremely wide dynamic range and are able to separate analytes in an interval between a few nm to 100 µm size-wise (over 15 orders of magnitude mass-wise). They are flexible in terms of mobile phase and can separate the analytes in native conditions, preserving their original structures/properties as much as possible. Molecular biology is the branch of biology that studies the molecular basis of biological activity, while biotechnology deals with the technological applications of biology. The areas where biotechnologies are required include industrial, agri-food, environmental, and pharmaceutical. Many species of biological interest belong to the operational range of FFF techniques, and their application to the analysis of such samples has steadily grown in the last 30 years. This work aims to summarize the main features, milestones, and results provided by the application of FFF in the field of molecular biology and biotechnology, with a focus on the years from 2000 to 2022. After a theoretical background overview of FFF and its methodologies, the results are reported based on the nature of the samples analyzed.
Asunto(s)
Biotecnología , Fraccionamiento de Campo-Flujo , Biología Molecular , Alimentos , IndustriasRESUMEN
Asymmetrical flow field-flow fractionation (AF4), a gentle tool for the separation and characterization of particles and macromolecules, has attracted increased interest in recent years owing to its broad dynamic size range and utilization of "open channel" voids in the packing or stationary phase. A steric transition phenomenon in which the sample elution mode change from the normal mode to the steric/hyperlayer mode occurs. Accurate characterization by AF4 requires the absence of steric transition, particularly when the sample has a broad size distribution, because the effect of the combination of different modes is difficult to interpret. In this study, the relative molecular mass (M), radius of gyration (Rg), and conformation of Gastrodia elata polysaccharides (GEPs) were characterized using AF4 coupled with online multi-angle light scattering (MALS) and differential refractive index (dRI) detection (AF4-MALS-dRI). Steric transition was observed during GEP separation by AF4 owing to the broad size distribution of the molecules. This phenomenon would result in the inaccurate characterization of the GEPs in terms of M and Rg because two GEP groups of different sizes may elute together. In this study, the effects of constant and exponentially decaying cross-flow rates, sample mass concentration, and spacer thickness on steric transition were systematically investigated. The results indicated that a high GEP mass concentration (i. e., 0.75 mg/mL) can lead to steric transition. The spacer thickness affected the resolution and retention time of the GEPs and changed the steric transition point (di). An exponentially decaying cross-flow rate not only adjusted the di of the polydisperse GEP samples but also improved the GEP resolution and shortened the analysis time. The influence of steric transition was solved under the following operating conditions: injected GEP mass concentration=0.5 mg/mL; injection volume=50 µL; spacer thickness=350 µm; detector flow rate=1.0 mL/min; and cross-flow rate exponentially decayed from 0.2 to 0.05 mL/min with a half-life of 2 min. Moreover, the influence of GEP origins and ultrasound treatment time on the M and Rg distributions and conformation of GEPs were investigated under the optimized operating conditions. The results showed that the M and Rg distributions of Yunnan and Sichuan GEPs decreased with increasing ultrasound time. When the ultrasound treatment time was 15 min, the Yunnan GEPs had a loosely hyperbranched chain conformation, whereas the Sichuan GEPs had a spherical conformation. When the ultrasound treatment time was increased to 30 or 60 min, the GEPs from both Yunnan and Sichuan had a hyperbranched chain conformation, indicating that ultrasound treatment resulted in GEP degradation. Under the same extraction conditions, GEPs from Yunnan had larger M and Rg values than those from Sichuan. AF4-MALS-dRI showed good repeatability for the characterization of GEPs under the optimized operating conditions. The relative standard deviations of Rg and M were 0.5% and 1.7%, respectively. The data presented in this study can be used as a starting point for in-depth studies on the structural bioactivity of GEPs.
Asunto(s)
Fraccionamiento de Campo-Flujo , Gastrodia , China , Polisacáridos , Fraccionamiento de Campo-Flujo/métodosRESUMEN
Starch occurs naturally in the form of semicrystalline granules, and is composed of two types of carbohydrate molecules, amylose (AM) and amylopectin (AP). Starch granules and starch molecules have sizes in the range of 1-100 µm and 20-250 nm, respectively; these size ranges are among the key factors affecting the functional properties of starch. Asymmetrical flow field-flow fractionation (AF4) is a size-based separation technique. The major difference between AF4 and dynamic light scattering or microscopy techniques is that AF4 enables the separation of particles based on their size; consequently, the elution profile can be converted to the size distribution of the samples. In the last two decades, AF4 systems, when coupled online with multiangle light scattering (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI), have demonstrated to be applicable for the size characterization of starch at the molecular level. Unlike size exclusion chromatography (SEC), AF4 systems use an open channel that does not require a stationary phase or packing materials. Thus, the shear scission of AP molecules during AF4 separation is minimized. The size detection range of a commercial AF4 system ranges from 1 nm to 10 µm, which is smaller than the size range of starch granules. In this study, a home-made AF4 system was developed, and its capability for the size characterization of starch granules extracted from sweet potato, lotus seed, and rice was investigated. The performance of the developed AF4 system was evaluated by running a mixture of polystyrene (PS) with diameter of 2, 6, 12, and 20 µm, respectively. Baseline separation of four PS samples was achieved, and the resolution for 6 µm PS and 12 µm PS was 1.40. The detection limit of the developed AF4 system was higher than that of commercial AF4 systems. Thus, the developed AF4 system is promising for the separation and characterization of starch granules. The effect of the composition of the carrier liquid on the AF4 separation of starch granules was also studied. Moreover, the accuracy of AF4 in terms of size characterization of the starch granules was evaluated by optical microscopy (OM). The results revealed that the type of dispersant and viscosity of the carrier liquid affect the accuracy of size characterization of the starch granules. The size distribution of rice starch granules obtained using a carrier liquid containing 0.01% (w/v) sodium dodecyl sulfate (used as a dispersant), 0.02% (w/v) NaN3 (used as a bactericide), and 0.001% (w/v) hydroxypropylmethylcellulose (used to adjust the viscosity of the carrier liquid) was in agreement with that obtained from OM. Furthermore, a commercial AF4 system coupled with MALS and dRI detectors was employed for the separation and characterization of starch molecules. A molecularly dispersed solution is necessary for the reliable molecular characterization of starch. The effect of the starch dissolution temperature on the AF4 characterization of starch was also investigated. The optimal dissolution temperature for lotus seed and rice starch granules was 75 â, while that for sweet potato starch granules was 78 â; this difference is mainly attributed to the different botanical origins of the granules. The results showed that the ratio of the radius of gyration (Rg) to the hydrodynamic radius (Rh) of rice starch and sweet potato starch is in the range of 0.9-1.1 over the molar mass range of 10 6-108 g/mol. For rice starch, the Rg/Rh ratio is between 1.2 and 1.4. Rice starch has the highest apparent density among the three starches, indicating that rice starch molecules have a dense structure. The results demonstrated that the AF4 system developed in this study is rapid and accurate for the size characterization of starch granules. The developed AF4 system, when combined with commercial AF4 systems coupled online with MALS and dRI detectors, can provide technical support to study the relationship between the size from the nanoscale to the microscale and functional properties of starch.
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Fraccionamiento de Campo-Flujo , Amilosa , Peso Molecular , Refractometría , AlmidónRESUMEN
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is widely used as a crosslinker for fluorescence labeling of protein in the fields of biochemistry and food analysis. Many natural polysaccharides often contain some proteins or peptides that are very low in content but play a vital role in their biological function as well as technical applications. Determination of these low-content proteinaceous matters requires a highly sensitive and selective method. In this study, a methodological approach for investigations of the presence of proteinaceous material over the molar mass distribution (MD) of polysaccharides was developed using gum acacia (GA) as a model polysaccharide. EDC fluorescence-labeling method was modified by changing the pH (7, 9, and 11) of the solution for the analysis of low-content protein in food materials. Fluorescence spectroscopy and asymmetrical flow field-flow fractionation (AF4) were employed for characterizing the labeling efficiency and physiochemical properties of unlabeled and fluorescence-labeled GA. AF4 provided molar mass (M) and the radius of gyration (rG) of arabinogalactan (AG) and arabinogalactan protein complex (AGP) and determined the presence of proteinaceous matter over the MD. The labeling efficiencies of GA at pH 7, 9, and 11 determined by fluorescence spectroscopy were 56.5, 68.4, and 72.0%, respectively, with an increment of 15.5% when pH was increased from 7 to 11. The modified EDC fluorescence-labeling method allows highly sensitive and selective analysis of low-content proteinaceous matters and their distribution in natural polysaccharides.
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Fraccionamiento Químico/métodos , Etildimetilaminopropil Carbodiimida/química , Goma Arábiga/química , Proteínas/química , Albúmina Sérica Bovina/química , Concentración de Iones de Hidrógeno , Sensibilidad y EspecificidadRESUMEN
Liposomes, mainly formed by phospholipids and cholesterol that entrapped different compounds, were separated and characterized using asymmetric flow field-flow fractionation (AF4) coupled with a multi-angle light scattering detector (MALS). AF4 allows the separation of liposomes according to their hydrodynamic size, and the particle size can be estimated directly by their elution time. Besides, different synthesized liposome suspensions of liposomes with different species encapsulated in different places in liposomes were prepared with analytical purposes to be studied. These liposomes were: empty liposomes (e-Ls), magnetoliposomes (MLs) with Fe3O4@AuNPs-C12SH inside the lipid bilayer, and long-wavelength fluorophores encapsulated into the aqueous cavity of liposomes (Ls-LWF). The optimization process of the variables that affect the fractionation has been established. The separation effectiveness has been compared with the results achieved with a photon-correlation spectroscopy analyzer based on dynamic light scattering (DLS) and transmission electron microscopy (TEM), used in self-assembly structures characterization. In all cases, three different classes of liposomes have been obtained; two are commonly appaired in all studied samples, while only a third class is characteristic for each of the liposomes. This mean that the proposed methodology could be used for identifying liposomes according to the encapsulated material.
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Dispersión Dinámica de Luz , Fraccionamiento de Campo-Flujo/métodos , Liposomas/química , Sistemas en Línea , Colesterol/química , Oro/química , Hidrodinámica , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Tamaño de la Partícula , Reología , AguaRESUMEN
Two sets of polystyrene nanoparticles (PSNPs) with comparable core sizes but different carboxyl group densities were made and separated using asymmetric flow field flow fractionation (AF4), capillary electrophoresis (CE), and the off-line hyphenation of both methods. Our results revealed the significant potential of two-dimensional off-line AF4-CE hyphenation to improve the separation and demonstrated for the first time, the applicability of CE to determine the functional group density of nanoparticles (NPs). Compared to the result acquired with conductometric titration, the result obtained with synthesized 100 nm sized PSNPs revealed only a slight deviation of 1.7%. Commercial 100 nm sized PSNPs yielded a deviation of 4.6%. For 60 nm sized PSNPs, a larger deviation of 10.6% between both methods was observed, which is attributed to the lower separation resolution.
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Electroforesis Capilar/métodos , Fraccionamiento de Campo-Flujo/métodos , Nanopartículas/química , Poliestirenos/química , Tamaño de la Partícula , Dodecil Sulfato de Sodio/química , EspectrofotometríaRESUMEN
A method based on asymmetrical flow field-flow fractionation (AF4) coupled with a multi-angle light scattering detector and a differential refractive detector was developed for the separation and characterization of millet starch. In this study, the effects of sample loading, cross-flow rate, half-life, and ionic strength and pH value of the carrier liquid on the AF4 analysis of millet starch were investigated. In addition, the molecular structure of millet starch was determined under the optimum conditions. The optimized operation conditions for the AF4 analysis of millet starch were as follows:injection mass concentration, 0.50 g/L; injection volume, 50 µL; cross-flow rate, 1.2 mL/min; half-life, 3 min; and the carrier liquid, demonized water containing 10 mmol/L pH 7.00 NaNO3 (add 3 mmol/L NaN3). The method developed in this study showed good reproducibility. The relative standard deviations for the radius of gyration (Rg) and molar mass (Mw) were 3.4% and 7.0%, respectively.
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Fraccionamiento de Campo-Flujo , Mijos/química , Almidón/análisis , Peso Molecular , Reproducibilidad de los Resultados , Dispersión de RadiaciónRESUMEN
Asymmetrical flow field flow fractionation (AF4) combined with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to screen allergen protein epitopes. The selected allergen protein (tropomyosin, TM) was enzymatically digested into peptide segments and analyzed via UPLC-QTOF-MS to establish a protein-specific peptide database. The peptide segments were incubated with immunoglobulin E (IgE) for 30 min. During the incubation procedure, the specific peptide segments (with the antigen epitope) combine with IgE while the other peptide segments remain in solution. After incubation, the solution was injected into the AF4 device. The combined peptide segments flowed out of the outlet along with IgE, and the other peptide segments flowed into the waste liquid. The components of outlet were then collected, analyzed by UPLC-QTOF-MS, and the results matched with the spectra of the protein peptides. Eventually the specific peptide segments were identified to detect the antigen epitopes. This study extends the application of AF4 with a preliminary exploration of the detection of an allergen protein epitope, providing a novel research strategy for the screening of allergen epitopes.
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Alérgenos/análisis , Epítopos/análisis , Fraccionamiento de Campo-Flujo , Proteínas/análisis , Cromatografía Líquida de Alta Presión , Espectrometría de Masas en Tándem , Tropomiosina/análisisRESUMEN
Asymmetrical flow field-flow fractionation (AF4) is a kind of moderate separation technology for the analysis of macromolecules, including proteins with a wide range of sizes. In the separation channel, the membrane adsorption and aggregation of proteins affected by the carrier fluid (CF) composition lead to changes in analyte recovery and size distribution, thereby restricting the application of AF4 to biomolecules. Different pH levels (6.2, 7.4, 8.2), several types of cations (Na+, K+, Mg2+) and various ion strengths (0-0.1 mol/L)were studied to demonstrate the influence of carrier fluid composition on the membrane adsorption and aggregation of proteins. The results revealed the following:a) higher ion strength of CF resulted in a greater degree of membrane adsorption and aggregation; b) the zeta potential, determined by the pI of the protein and the pH of the CF, influenced the adsorption and aggregation; c) divalent cations (Mg2+) caused serious adsorption and aggregation. The experimental results can help us achieve better recovery and mitigate aggregate formation by using the optimal CF components in future AF4 studies. Moreover, the findings indicate that AF4 would find extensive application in protein biochemistry assays.
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Fraccionamiento de Campo-Flujo , Ovalbúmina , Adsorción , Cationes , Concentración de Iones de Hidrógeno , Concentración OsmolarRESUMEN
In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations.
Asunto(s)
Glutamato Descarboxilasa/química , Levilactobacillus brevis/enzimología , Dispersión Dinámica de Luz , Estabilidad de Enzimas , Fraccionamiento de Campo-Flujo , Concentración de Iones de Hidrógeno , Levilactobacillus brevis/química , Modelos Moleculares , Multimerización de Proteína , Refractometría , Sales (Química)/química , TemperaturaRESUMEN
Home-made asymmetrical flow field-flow fractionation (AF4) system, online coupled with ultraviolet/visible (UV/Vis) detector was employed for the separation and size characterization of low density lipoprotein (LDL) in egg yolk plasma. At close to natural condition of egg yolk, the effects of cross flow rate, sample loading, and type of membrane on the size distribution of LDL were investigated. Under the optimal operation conditions, AF4-UV/Vis provides the size distribution of LDL. Moreover, the precision of AF4-UV/Vis method proposed in this work for the analysis of LDL in egg yolk plasma was evaluated. The intra-day precisions were 1.3% and 1.9% (n=7) and the inter-day precisions were 2.4% and 2.3% (n=7) for the elution peak height and elution peak area of LDL, respectively. Results reveal that AF4-UV/Vis is a useful tool for the separation and size characterization of LDL in egg yolk plasma.
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Yema de Huevo/química , Fraccionamiento de Campo-Flujo , Lipoproteínas LDL/análisis , PlasmaRESUMEN
Field-flow fractionation (FFF) is a kind of mature separation technologies in the field of bioanalysis, feasible of separating analytes with the differences of certain physical and chemical properties by the combination effects of two orthogonal force fields (flow field and external force field). Asymmetrical flow field-flow fractionation (AF4) is a vital subvariant of FFF, which applying a vertical flow field as the second dimension force field. The separation in AF4 opening channel is carried out by any composition carrier fluid, universally and effectively used in separation of bioparticles and biopolymers due to the non-invasivity feature. Herein, bio-analytes are held in bio-friendly environment and easily sterilized without using degrading carrier fluid which is conducive to maintain natural conformation. In this review, FFF and AF4 principles are briefly described, and some classical and emerging applications and developments in the bioanalytical fields are concisely introduced and tabled. Also, special focus is given to the hyphenation of AF4 with highly specific, sensitive detection technologies.
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Biopolímeros/análisis , Fraccionamiento de Campo-FlujoRESUMEN
Asymmetrical flow field-flow fractionation (AF4) was evaluated as a potential analytical method for detection of a protective antigen (PA), an Anthrax biomarker. The scheme was based on the recognition of altered AF4 retention through the generation of the size-increased Au nanoparticle probes as a result of PA binding, in which a PA-selective peptide was conjugated on the probe surface. In the visible absorption-based AF4 fractograms, the band position shifted to a longer retention time as the PA concentration increased due to the presence of probe bound with PAs. The shift was insignificant when the concentration was relatively low at 84.3pM. To improve sensitivity, two separate probes conjugated with two different peptides able to bind on different PA epitopes were used together. The band shift then became distinguishable even at 84.3pM of PA sample. The formation of larger PA-probe inter-connected species using the dual-probe system was responsible for the enhanced band shift. In parallel, the feasibility of surface-enhanced Raman scattering (SERS) as a potential AF4 detection method was also evaluated. In the off-line SERS fractogram constructed using fractions collected during AF4 separation, a band shift was also observed for the 84.3pM PA sample, and the band intensity was higher when using the dual-probe system. The combination of AF4 and SERS is promising for the detection of PA and will become a potential tool if the reproducibility of SERS measurement is improved.
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Antígenos Bacterianos/análisis , Toxinas Bacterianas/análisis , Oro , Nanopartículas del Metal , Biomarcadores/análisis , Estudios de Factibilidad , Fraccionamiento de Campo-Flujo/métodos , Oligopéptidos/química , Tamaño de la Partícula , Reproducibilidad de los Resultados , Espectrometría RamanRESUMEN
Nanoparticles used as drug delivery systems are of growing interest in the pharmaceutical field. Understanding the behaviour and effects of nanosystems in the human body is dependent on comprehensive characterisation of the systems especially with regard to size and size distribution. Asymmetrical flow field-flow fractionation (AF4) is a promising method for this challenge as this technique enables chromatographic separation of particles and solute molecules according to their respective size. Within this study AF4 was used for the characterisation of human serum albumin (HSA) based nanoparticles. In a first part, the most important aspects of method development like the choice of cross flow rate, focusing and the increase of sample concentration via outlet stream splitting on the sample separation were evaluated. Sample fractionation was controlled by inline-coupling of a dynamic light scattering detector (DLS, Zetasizer) and was confirmed by DLS batch mode measurements. In a second part the applicability of field-flow fractionation for characterisation of the HSA particle formation process by a desolvation method was evaluated. A time dependent particle formation was observed which was controlled by the amount of desolvating agent. Furthermore, field-flow fractionation in combination with in-line dynamic light scattering was used to monitor the increase of particle diameter during PEGylation of the resulting HSA nanoparticles. The separation of nanoparticles from dissolved polyethylene glycol (PEG) could successfully be used for determination of the particles' PEGylation degree.
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Fraccionamiento de Campo-Flujo , Nanopartículas/química , Albúmina Sérica/química , Humanos , Tamaño de la Partícula , Polietilenglicoles/químicaRESUMEN
In field-flow fractionation (FFF), there is the 'steric transition' phenomenon where the sample elution mode changes from the normal to steric/hyperlayer mode. Accurate analysis by FFF requires understanding of the steric transition phenomenon, particularly when the sample has a broad size distribution, for which the effect by combination of different modes may become complicated to interpret. In this study, the steric transition phenomenon in asymmetrical flow FFF (AF4) was studied using polystyrene (PS) latex beads. The retention ratio (R) gradually decreases as the particle size increases (normal mode) and reaches a minimum (Ri) at diameter around 0.5µm, after which R increases with increasing diameter (steric/hyperlayer mode). It was found that the size-based selectivity (Sd) tends to increase as the channel thickness (w) increases. The retention behavior of cyclo-1,3,5-trimethylene-2,4,6-trinitramine (commonly called 'research department explosive' (RDX)) particles in AF4 was investigated by varying experimental parameters including w and flow rates. AF4 showed a good reproducibility in size determination of RDX particles with the relative standard deviation of 4.1%. The reliability of separation obtained by AF4 was evaluated by transmission electron microscopy (TEM).
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Sustancias Explosivas/aislamiento & purificación , Fraccionamiento de Campo-Flujo/métodos , Triazinas/aislamiento & purificación , Algoritmos , Microesferas , Tamaño de la Partícula , Poliestirenos/química , Reproducibilidad de los ResultadosRESUMEN
Dissolved organic matter (DOM) was leached from eight distinct samples of leaves taken from six distinct trees (red maple, bur oak at three times of the year, two sugar maple and two white spruce trees from disparate soil types). Multiple samples were taken over 72-96h of leaching. The size and optical properties of leachates were assessed using asymmetrical flow field-flow fractionation (AF4) coupled to diode-array ultraviolet/visible absorbance and excitation-emission matrix fluorescence detectors (EEM). The fluorescence of unfractionated samples was also analyzed. EEMs were analyzed using parallel factor analysis (PARAFAC) and principal component analysis (PCA) of proportional component loadings. Both the unfractionated and AF4-fractionated leachates had distinct size and optical properties. The 95% confidence ranges for molecular weight distributions were determined as: 210-440Da for spruce, 540-920Da for sugar maple, 630-800Da for spring oak leaves, 930-950Da for senescent oak, 1490-1670 for senescent red maple, and 3430-4270Da for oak leaves that were collected from the ground after spring thaw. In most cases the fluorescence properties of leachates were different for individuals from different soil types and across seasons; however, PCA of PARAFAC loadings revealed that the observed distinctiveness was chiefly species-based. Strong correlations were found between the molecular weight distribution of both unfractionated and fractionated leachates and their principal component loadings (R(2)=0.85 and 0.95, respectively). It is concluded that results support a species-based origin for differences in optical properties.