Preparative High-performance Size-exclusion Chromatography Research Articles

Differences in fluorescence properties between humic acid and its size fractions separated by preparative HPSEC

We applied three dimensional excitation–emission-matrix (3D-EEM) spectrofluorometry to investigate the fluorescence properties of a humic acid (HA) solution at different pH values and its size-fractions separated by preparative high performance size exclusion chromatography (HPSEC). 3D-EEM fluorescence spectra revealed peak maxima in the bulk HA which were assigned to protein-like, phenol-like and naphthol-like components. Moreover, changes of peak maxima with pH showed that phenol-like and naphthol-like molecular classes were composed of sub-components with different acidities, and, thus, different structures. The 3D-EEM spectra of three size-fractions separated by HPSEC showed similar peak maxima as for the bulk HA, with additional components, such as highly condensed aromatic systems, which had not been visible in the bulk HA. Furthermore, the fluorescence intensity of peak maxima in 3D-EEM spectra of size-fractions appeared invariably greater than that for the bulk HA. This phenomenon as well as the appearance of new maxima in size-fraction spectra is explained by the reduced self-quenching effect in the less complex and conformationally looser size-fractions. In fact, the close mutual proximity of fluorescent humic molecules in the tight supramolecular association of the bulk HA decreased or even inhibited their fluorescence quantum yield. This work shows that, due to self-quenching of humic molecules, fluorescence spectroscopy cannot be used to characterise the molecular complexity of humic matter and, thus, differentiate its environmental origin.

Molecular size fractionation of soil humic acids using preparative high performance size-exclusion chromatography

High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10 mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12–25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size.

Chemical composition and bioactivity properties of size-fractions separated from a vermicompost humic acid

Preparative high performance size-exclusion chromatography (HPSEC) was applied to humic acids (HA) extracted from vermicompost in order to separate humic matter of different molecular dimension and evaluate the relationship between chemical properties of size-fractions (SF) and their effects on plant root growth. Molecular dimensions of components in humic SF was further achieved by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) based on diffusion coefficients ( D), while carbon distribution was evaluated by solid state (CP/MAS) 13C NMR. Seedlings of maize and Arabidopsis were treated with different concentrations of SF to evaluate root growth. Six different SF were obtained and their carbohydrate-like content and alkyl chain length decreased with decreasing molecular size. Progressive reduction of aromatic carbon was also observed with decreasing molecular size of separated fractions. Diffusion-ordered spectroscopy (DOSY) spectra showed that SF were composed of complex mixtures of aliphatic, aromatic and carbohydrates constituents that could be separated on the basis of their diffusion. All SF promoted root growth in Arabidopsis and maize seedlings but the effects differed according to molecular size and plant species. In Arabidopsis seedlings, the bulk HA and its SF revealed a classical large auxin-like exogenous response, i.e.: shortened the principal root axis and induced lateral roots, while the effects in maize corresponded to low auxin-like levels, as suggested by enhanced principal axis length and induction of lateral roots. The reduction of humic heterogeneity obtained in HPSEC separated size-fractions suggested that their physiological influence on root growth and architecture was less an effect of their size than their content of specific bioactive molecules. However, these molecules may be dynamically released from humic superstructures and exert their bioactivity when weaker is the humic conformational stability as that obtained in the separated size-fractions.

Spectroscopic and conformational properties of size-fractions separated from a lignite humic acid

A lignite humic acid (HA) was fractionated by preparative high performance size-exclusion chromatography (HPSEC) in seven different size-fractions. The size-fractions were characterized by cross polarization (CP) magic angle spinning (MAS) 13C NMR spectroscopy and a further analytical HPSEC elution under UV and fluorescence detection. The alkyl hydrophobic components mainly distributed in the largest molecular-size-fraction, whereas the amount of oxidized carbons increased with decreasing size of fractions. Cross polarization time ( T CH) and proton spin-lattice relaxation time in the rotating frame ( T 1 ρ (H)) were measured from variable contact time (VCT) experiments. The bulk HA was characterized by the shortest T CH values and the longest T 1 ρ (H) values which suggested, respectively, one. an aggregation of components in a large conformation that favored a fast H–C cross polarization, and, two. consequent steric hindrances that prevented fast local molecular motions and decreased proton relaxation rates. Conversely, the separated size-fractions showed longer T CH values and shorter T 1 ρ (H) values than the bulk HA, thereby indicating that they were constituted by a larger number of mobile molecular conformations. The UV and fluorescence absorptions were both low in the large size-fractions that mainly contained alkyl carbons, whereas they increased in the olephinic- and aromatic-rich fractions with intermediate molecular-size, and decreased again in the smaller fractions which were predominantly composed by oxidized carbons. These results support the supramolecular structure of humic substances and indicate that the observed variation in conformational distribution in humic association may be used to explain environmental processes with additional precision.

Advanced CPMAS-13C NMR techniques for molecular characterization of size-separated fractions from a soil humic acid

A humic acid extracted from a volcanic soil was subjected to preparative high-performance size-exclusion chromatography (HPSEC) to reduce its molecular complexity and eleven different size fractions were obtained. Cross-polarization magic-angle spinning 13C NMR (CPMAS 13C NMR) analysis performed with variable contact-time (VCT) pulse sequences showed that the largest molecular-size fractions contained aromatic, alkyl, and carbohydrate-like components. The carbohydrate-like content and the alkyl chain length seemed to decrease with decreasing molecular size. Progressive reduction of aromatic carbon atoms was also observed with decreasing molecular size of the separated fractions. Mathematical treatment of the results from VCT experiments enabled cross polarization (T (CH)) and proton spin-lattice relaxation (T(1rho)(H)) times to be related to structural differences among the size fractions. The conformational distribution indicated that the eleven size fractions could be allocated to two main groups. The first group, with larger nominal molecular sizes, was characterized by molecular domains with slower local molecular motion. The second group of size fractions, with smaller nominal molecular sizes, was characterized by a larger number of molecular domains with faster local molecular motion. The T (CH) and (T(1rho)(H)) values suggested that either condensed or strongly associated aromatic systems were predominant in the size fractions with the largest apparent molecular dimensions.

NMR Spectroscopy Study of Freshwater Humic Material in Light of Supramolecular Assembly

The structural similarity-dissimilarity of several humic-type derivatives, separated from a strongly colored freshwater sample by different sorbing solid techniques, tangential-flow ultrafiltration (UF), and large-scale preparative high-performance size-exclusion chromatography (HPSEC), were in detail studied with one-dimensional liquid 1H and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy, especially in light of the native humic-type dissolved organic matter (DOM-HM). The results support the applicability of functional cross-linked poly(vinylpyrrolidone) (PVP) or diethylaminoethyl-cellulose (DEAE) sorbents in concentrating representative integrated wholes of aquatic humic-type material along with a conventional nonionic XAD-8/DAX-8 (polymethyl methacrylates) technique. Apart from the fact that the acidification of the original humic water before a separation procedure seems not to be so destructive to the original structural composition of the DOM-HM as expected, the refinement of aquatic humic solutes, independent of the selected sorbing solid technique, will cause structural changes in the separated humic complexes in comparison with the situation predominating in the original starting material. Tangential-flow ultrafiltration (UF) proved an overpowering reliability to concentrate the aquatic DOM-HM. Most fundamental is the combined outcome of different HPSEC experiments and determined structural functionalities which indicate that almost all original DOM-HM solutes are aggregated mixtures consisting of structurally similar associations possessing various molecular size ranges, which can be separated from their integrated whole as nearly homogeneous and uniform species. This finding permits a reasonable starting point to go on working with more advanced multidimensional NMR techniques in resolving the uncertainty about supramolecular assembly of dissolved humic material. The tested conformity between the obtained molecular NMR descriptors and the corresponding previously collected FT-IR parameters was acceptable thus speaking for the fact that the less sensitive FT-IR spectroscopy can also provide valuable information on the structural and functional properties of heterogeneous humic-type mixtures.

Preliminary Study of Lake Dissolved Organic Matter in Light of Nanoscale Supramolecular Assembly

Large-scale preparative high-performance size-exclusion chromatography (HPSEC) was performed to separate different molecular size fractions in milligram quantities from strongly colored dissolved organic matter (DOM) of a freshwater using a very mild conjugate acid-base pair (10 mM acetic acid-sodium acetate solution at pH 7.0 with an ionic strength of 6 x 10(-5)) as the mobile phase. The homogeneity-uniformity of different molecular size fractions in relation to their combined original mixture was verified by an analytical HPSEC system. In addition to molecular size distribution and basic spectroscopic characteristics, Fourier transform infrared spectroscopy was applied to specify structural features for different size fractions. The results demonstrate clearly that only a very small amount of conjugate organic acid-base pair is required to generate a powerful resolution for a DOM mixture, and very strong treatments with organic acids are not necessarily needed to reach a better SEC resolution. Most essential is the combined outcome of different HPSEC experiments and determined structural functionalities which indicate that almost all original DOM solutes are aggregated mixtures consisting of different associations possessing various molecular size ranges, which can be separated from their integrated whole as nearly homogeneous and uniform species. In summary, the present study strongly speaks for the need to direct the research of natural dissolved and colloidal organic carbon more strongly toward a nanoscale study of supramolecular assemblies. More precise knowledge about the primary, secondary, and tertiary structure of dissolved DOM constituents has its essential function, e.g., for environmental protection and utilization of surface waters.

Reduced heterogeneity of a lignite humic acid by preparative HPSEC following interaction with an organic acid. Characterization of size-separates by Pyr-GC-MS and 1H-NMR spectroscopy.

Preparative high performance size exclusion chromatography (HPSEC) was used to size-fractionate a humic acid (HA) solution with 0.05 M ionic strength before and after having made the humic solution 0.5 x 10(-3) M in acetic acid (AcOH). Size-fractions were characterized by pyrolysis-gas-chromatography/mass spectrometry (Pyr-GC-MS) and 1H NMR spectroscopy. Pyr-GC-MS showed that the AcOH treatment altered the distribution of humic molecular components in the size-fractions. The unsaturated alkyl chains were moved from size-fractions of larger molecular-size into those of lower molecular-sizes. Most of the aromatic moieties, which were found in larger molecular-size fractions for the untreated HA, were spread into fractions of lower molecular size after AcOH addition to HA. Carbohydrates, which were undetectable in anyfraction of the untreated HA, appeared instead in the pyrogram of the lowest molecular-size and most hydrophilic fraction after treatment with AcOH. Our results suggested that AcOH disrupted the weakly bound association of humic supramolecularstructures and HPSEC elution separated size-fractions of different composition without losing humic matter by adsorption on the HPSEC column. The fractions with the largest apparent molecular size were the richest in alkyl chains, thereby suggesting that humic molecules were stabilized into supramolecular associations by multiple weak interactions among apolar groups such as alkyl chains and aromatic moieties. 1H NMR spectra of size-fractions were greatly simplified and more resolved after AcOH treatment. This was attributed to a less complex molecular association in the size separates which provided a larger solubility in the NMR solvent and more favorable relaxation times. Combination of the procedure used here for size-fractionation with NMR and Pyr-GC-MS methods appears to be promising to advance knowledge on the molecular composition of humic substances.

New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane.

We describe a new procedure for isolation of glycoproteins IIb (GPIIb) and IIIa (GPIIIa) from human platelet plasma membrane with high yields (2.7 mg of GPIIb and 3.3 mg of GPIIIa per 100 mg of starting platelet membrane proteins), equivalent to a recovery of 35% and 55% respectively of the total GPIIb and GPIIIa of the membrane. The procedure involves Triton X-100 differential extraction of platelet membranes, SDS solubilization of the 4%-Triton X-100 supernatant, zonal centrifugation in a sucrose density gradient, and preparative high-performance size-exclusion chromatography. The weight percentage of sugar is 15.7% for GPIIb and 12.5% for GPIIIa. Neuraminic acid is present in both glycoproteins, representing 30% and 15% respectively of the total sugar weight of GPIIb and GPIIIa. Mannose, galactose and glucosamine account for 45%, 13% and 28% respectively of the sugars of GPIIIa, whereas galactosamine was not detected. Mannose, galactose, glucosamine and galactosamine represent 17%, 21%, 24% and 10% respectively of the sugar content of GPIIb. The molar percentages of half-cystine and methionine are 4-fold and 2-fold higher respectively in GPIIIa than in GPIIb. From the amino acid and sugar compositions we confirmed the acidic nature of both glycoproteins. The Mr values obtained, 136,500 for GPIIb and 91,500 for GPIIIa, are in very good agreement with those obtained by physical methods. The apparent lack of free thiol groups in both glycoproteins indicates that the tertiary structure of GPIIIa is maintained by 21 intrachain disulphide bonds, and that there are eight intrachain and interchain disulphide groups in GPIIb.

10] Purification of bacterial luciferase by high-performance liquid chromatography

This chapter discusses the purification of bacterial luciferase by high-performance liquid chromatography. Large quantities of bacterial luciferase (Lase) can be rapidly prepared by HPLC using three chromatography procedures, which have their respective counterparts in low-pressure liquid chromatography: high-performance size exclusion chromatography (HPSEC), high-performance anion exchange chromatography (HPAEC), and high-performance hydrophobic interaction chromatography (HPHIC). In particular, HPHIC and its low-pressure counterpart are most useful and provide purifications of Lase that equal or exceed those obtained by affinity chromatography on diphenylpropylamine-Sepharose. The major concern in the HPLC of biological extracts is that the columns will become clogged by particulates, which possibly could ruin a costly HPLC column. Particulates are removed from the solvents by filtration using a membrane filter with a pore size of 0.45 μm or less (Millipore or Nucleopore). The filtered solvents are stored covered to prevent contamination by dust or other debris. The HPLC pumps and columns are secondarily protected from particulates by solvent filters placed on the solvent intake lines. It is recommended that all solvents be filtered on a daily basis to remove any bacteria and microcrystals of salts that form in concentrated buffer solutions. The steps in the purification of luciferase includes: (1) luciferase from photobacterium leiognathi DD17, (2) preparative high-performance size exclusion chromatography (HPSEC), (3) preparative HPAEC, (4) preparative HPHIC, and (5) luciferase from an aldehyde-requiring mutant of vibrio harveyi.

Isolation of human factor VIII:C by preparative high-performance size-exclusion chromatography

Human Factor VIII procoagulant protein (VIII:C) is a plasma protein that participates in the cascade of events leading to blood coagulation. It is absent or defective in patients with hemophilia A. In vivo Factor VIII:C associates with Von Willibrand factor and its multimers to form a high-molecular-weight particle that cna be dissociated into a lower-molecular-weight form in the presence of high concentrations of salt. We have been able to purify rapidly Factor VIII:C on a large scale by sequential high-performance size-exclusion chromatography (HPSEC) under conditions of first low salt and then high salt concentration. Reconstituted commercial Factor VIII:C concentrate was purified by chromatography on a preparative HPSEC column (Toyo Soda, 60 × 2.5 cm, 300ml) in 0.05 M imidazole buffer, (pH 7.0), containing 0.15 M sodium chloride. Factor VIII:C activity was eluted in the void volume in less than 20 min as a high-molecular-weight particle, well separated from low-molecular-weight contaminants. Purification was 20-fold, with a yield of 80%. Up to 4 g of Factor VIII concentrate could be purified at one time in this manner. This material was then concentrated and made 0.35 M in calcium chloride prior to re-chromatography on the same column in a buffer containing 0.30 M calcium chloride. Under these conditions, Factor VIII:C activity was eluted in the inner volume of the column at a position corresponding to a molecular weight of several hundred thousand in less than 1 h. It was well separated from both larger proteins and smaller peptide fragments. Analysis of the preparation with radiolabelled antibody to human Factor VIII:C antigen indicated that at least two molecular weight forms of Factor VIII:C were present.