Purification and analysis of hematoporphyrin and hematoporphyrin derivative by gel exclusion and reverse-phase chromatography

A ternary-solvent HPLC method for analysis of the components of the tumor-localizing product HPD is described. This HPLC system was used to assess the utility of a new method, based on gel exclusion chromatography, for purification of hematoporphyrin.     

Aqueous gel permeation chromatography of polyelectrolytes and salt exclusion effect

The dependence of the elution volume of NaCl samples is discussed as a function of the volume and concentration injected and of the ionic strength of the eluent. The same is done with two fractions of polystyrene sulphonic acid in the sodium form. It is concluded that the exclusion depends directly on the screening parameter proportional to $\text{μ}{}^{\mathrm{<mtext>?</mtext><mtext>1</mtext><mtext>2</mtext>}}$ for NaCl and that a steric parameter has to be added with polyelectrolytes, depending on the molecular weight and on the ionic strength of the eluent. The behaviour is directly correlated to the pore size distribution in the gel.     

Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography.

A gel permeation chromatographic method for quantifying neutral oligosaccharide fractions from human milk has been developed. Oligosaccharides from monofucosyllactoses to trifucosyllacto-N-hexaoses were separated according to size on a Fractogel TSK HW 40 (S) column. Refractive index detection of monofucosyllactoses to difucosyllacto-N-tetraoses yielded a constant mass response factor of ca. 1 relative to glucose. After the addition of glucose as an internal standard, oligosaccharides were isolated from human milk by ethanol precipitation or two ultrafiltration procedures. The oligosaccharide concentrations found by the ultrafiltration procedures were significantly lower (significance level 0.05) than those determined by the ethanol precipitation procedure.     

Study of protein association and micelle formation in surfactants by microcolumn exclusion chromatography

The application of microcolumn exclusion chromatography to the determination of equilibrium and kinetic association constants for proteins is described. Taking phospholipase A₂ as an example, the relationship between association and enzymatic activity was investigated. A procedure for determination of the critical concentration of micelle formation (CCM) in surfactants was developed.     

Molecular mass ranges of coal tar pitch fractions by mass spectrometry and size‐exclusion chromatography

A coal tar pitch was fractionated by solvent solubility into heptane‐solubles, heptane‐insoluble/toluene‐solubles (asphaltenes), and toluene‐insolubles (preasphaltenes). The aim of the work was to compare the mass ranges of the different fractions by several different techniques. Thermogravimetric analysis, size‐exclusion chromatography (SEC) and UV‐fluorescence spectroscopy showed distinct differences between the three fractions in terms of volatility, molecular size ranges and the aromatic chromophore sizes present. The mass spectrometric methods used were gas chromatography/mass spectrometry (GC/MS), pyrolysis/GC/MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICRMS) and laser desorption time‐of‐flight mass spectrometry (LD‐TOFMS). The first three techniques gave good mass spectra only for the heptane‐soluble fraction. Only LDMS gave signals from the toluene‐insolubles, indicating that the molecules were too involatile for GC and too complex to pyrolyze into small molecules during pyrolysis/GC/MS. ESI‐FTICRMS gave no signal for toluene‐insolubles probably because the fraction was insoluble in the methanol or acetonitrile, water and formic acid mixture used as solvent to the ESI source. LDMS was able to generate ions from each of the fractions. Fractionation of complex samples is necessary to separate smaller molecules to allow the use of higher laser fluences for the larger molecules and suppress the formation of ionized molecular clusters. The upper mass limit of the pitch was determined as between 5000 and 10 000 u. The pitch asphaltenes showed a peak of maximum intensity in the LDMS spectra at around m/z 400, in broad agreement with the estimate from SEC. The mass ranges of the toluene‐insoluble fraction found by LDMS and SEC (400–10 000 u with maximum intensity around 2000 u by LDMS and 100–9320 u with maximum intensity around 740 u by SEC) are higher than those for the asphaltene fraction (200–4000 u with maximum intensity around 400 u by LDMS and 100–2680 u with maximum intensity around 286 u by SEC) and greater than values considered appropriate for petroleum asphaltenes (300–1200 u with maximum intensity near 700 u). Copyright © 2009 John Wiley & Sons, Ltd.     

Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis

Capillary zone electrophoresis (CZE) was used for characterisation of soil humic acid (HA) fractions obtained by coupling size-exclusion chromatography with polyacrylamide gel electrophoresis, on the basis of their molecular size and electrophoretic mobility. CZE was conducted using several low alkaline buffers as background electrolyte (BGE): 50 mM carbonate, pH 9.0; 50 mM phosphate, pH 8.5; 50 mM borate, pH 8.3; 50 mM Tris-borate+1 mM EDTA+7 M urea+0.1% sodium dodecyl sulphate (SDS), pH 8.3. Independently of BGE conditions, the effective electrophoretic mobility of HA fractions were in good agreement with their molecular size. The better resolution of HA were obtained in Tris-borate-EDTA buffer with urea and SDS. This results indicated that CZE, mostly with BGE-contained disaggregating agents, is useful for separating HAs in fractions with different molecular sizes.     

Determination of cows' milk in goats' milk and cheese by capillary electrophoresis of the whey protein fractions.

The use of capillary zone electrophoresis to determine the adulteration of cows' milk in goats' milk products is described. The detection and quantification of cows' milk was based on the presence of the specific whey proteins: the relative calibration curve is reported. The peaks of interest were well resolved by using sodium borate at pH 9.2 as background electrolyte in methyl-silanized capillaries. The minimum amount detectable of cows' milk was 2% in milk mixtures and 4% in cheeses. Restrictions due to genetic variability and possible heat treatments, on only one of the two types of milk employed, are taken into account. Qualitative analysis of goat-ewe-cow and goat-ewe samples are also reported.     

Speciation of iron in breast milk and infant formulas whey by size exclusion chromatography-high performance liquid chromatography and electrothermal atomic absorption spectrometry

Speciation of iron in milk was carried out by high performance liquid chromatography (HPLC) and electrothermal atomic absorption spectrometry (ETAAS). Milk whey was obtained and low molecular weight protein separation was performed by size exclusion chromatography (SEC) with a TSK Gel SW glass guard (Waters) pre-column and a TSK-Gel G2000 glass (Toso Haas) column. After studying water as a possible mobile phase, this mobile phase was carefully selected in order to avoid alterations of the sample and to make subsequent iron determination in the protein fractions easier by ETAAS. The proposed method is sensitive (limit of detection [LOD] and LOQ 1.4 and 4.7 μg l⁻¹, respectively) and precise (relative standard deviation [RSD]<10%). Iron is principally found in the proteins of 3 and 76 kDa in breast milk, and it is irregularly distributed in infant formulas.     

A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy

A simple and economical method to isolate whey protein from fresh raw milk is developed by serial defatting, casein eliminating, lactose removing, and separating by gel filtration chromatography. Four major whey components, including immunoglobulin (Ig), bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and -lactalbumin (-Lac), and a non-protein of low molecular mass (1.7 kDa) but strong absorbance at 280 nm, are detected simultaneously. The small non-protein molecule is rich in aromatic amino acids and thiol groups as supported by the structural characterization with near infrared Fourier transform Raman spectroscopy (FT-Raman). FT-Raman results show that the secondary structure of Ig is dominated by anti-parallel β-pleated sheet; BSA is mainly in -helix; both β-form and unordered structure are important in β-Lg; while -Lac is mostly in -helix coupling with random coil. Differences in the Raman profile for each whey component reflect their intrinsic compositional differences and distinct spatial arrangement. The S–S linkages diverging around 510–540 cm⁻¹ indicate that the conformation of disulfide bonds in each whey components is different, which may be responsible for their diversified behaviors in solubility, rheological and functional properties.     

Preliminary studies on selenium-containing proteins in Brassica juncea by size exclusion chromatography and fast protein liquid chromatography coupled to ICP-MS

An approach for screening and resolving selenium-containing plant proteins was developed based on the combination of sample preparation and multi-dimensional liquid chromatography coupled to ICP-MS. Different protein extraction protocols were investigated. A 24 h dodecylsulfate-mediated protein extraction in a sonication bath followed by acetone precipitation was found to be optimal. The use of different protein precipitate solubilizing agents (sodium dodecyl sulfate media and Tris-HCl buffer) demonstrates possible fractionation of the selenium-containing proteins. Selenium-containing protein screening and fractionation were carried out by means of SEC-ICP-MS. High molecular weight selenium containing proteins were solubilized with a sodium dodecyl sulfate-containing buffer, whereas the low molecular weight compounds were released into a Tris-HCl buffer. Size exclusion chromatography-fast protein liquid chromatography coupled to ICP-MS allowed separation and detection of several selenium-containing proteins in Se-supplemented wild type Brassica juncea plant, a fast growing selenium accumulator.     

Characterization of low-density polyethylene by gel permeation chromatography—II. Study on the Drott method using fractions

Polyethylenes of different structures were fractionated and the fractions characterized by light scattering, gel permeation chromatography and viscometry. Intrinsic viscosities were measured in solvents of different thermodynamical quality including a θ-solvent (diphenyl at 118° for low-density polyethylene and at 130° for high-density polyethylene and ethylene-butene-1 copolymer). The results were used for examining two aspects of the Drott iterative procedure: (a) the relationship between thermodynamical quality of the solvent and depression in the intrinsic viscosity due to branching; and (b) analytical form of expression relating the so-called g-factor to the number of long-chain branches. The ratio of intrinsic viscosities of branched and linear species at a given weight-average molecular weight has been clearly proved to be solvent independent, and the equation relating the g-factor to the number of branches for polymer monodisperse with respect to molecular weights appears to be a fair representation of long-chain branching in low-density polyethylene. For the polymers examined, the branching frequency λ is not independent of molecular weight.     

Comments on exclusion of polymer chains from small pores and its relation to gel permeation chromatography.

Some criticisms of our theoretical treatment of the partial exclusion of flexible-chain polymers in solution from cavities of macromolecular size and its application to gel permeation chromatography are examined. In other discussion, it is confirmed by simple reasoning that the identification, explicit or implicit in various studies, of the mean projection of a polymer molecule onto a line as a characteristic dimension governing the extent of permeation of simple pores does not depend on specific molecular models. Our previous calculation of permeation by certain random-flight branched-chain species is shown to lead, incidentally, to the mean projection for these structures. From relations between the mean projection and the hydrodynamic volume of a molecule, it appears that the product of intrinsic viscosity and molecular weight is not a common calibration factor for elution of all molecular species from a gel chromatographic column, but theory and experience do support the validity of this correlation among solutes with similar molecular architecture.     

Study of chromium-containing proteins in subcellular fractions of rat liver by enriched stable isotopic tracer technique and gel filtration chromatography

Ten male Wistar rats were intravenously injected with a single approximately physiological dose of enriched stable isotopic Cr-50 tracer solution (200 ng (50)Cr(3+)/100 g body wt). The fundamental distribution patterns of the chromium-containing proteins in the nucleic, mitochondrial, lysosomal, microsomal, and cytosolic subcellular fractions of the rat liver were investigated by means of Sephadex G-100 gel chromatography combined with neutron activation analysis via (50)Cr (n, gamma) (51)Cr reaction. In total, nine kinds of Cr-containing proteins were found in the five subcellular fractions, whose relative molecular masses were 96.6+/-6.2, 68.2+/-1.4, 57.9+/-4.7, 36.6+/-1.2, 24.2+/-1.8, 14.0+/-1.5, 8.8+/-0.6, 6.9+/-0.4, and 4.2+/-0.4 kDa. Approximately 64.5% of Cr proteins accumulated in the cytosolic fraction. The second enriched part was the nucleic fraction; about 12.2% Cr proteins were stored in this section. The 4.2-kDa molecular mass might contain the so-called low molecular weight chromium-containing substance; however, in this research, it was only observed in the mitochondria, lysosome, and microsome. In the mitochondrial fraction, most of the Cr proteins were present as relatively low molecular weight substances: about 56% of chromium-containing proteins had molecular masses < or =6.9 kDa. Nevertheless, more than 69% of Cr-containing proteins were observed with molecular masses > or =57.9 kDa in the liver cytosolic fraction.     

Characterization of large water-soluble macromolecules by size exclusion chromatography

A method for characterizing very large, water-soluble polymers by size exclusion chromotography (SEC) has been developed. Sephacryl S1000 packing material, a precision syringe pump, and an eluent pressure detector have been utilized to produce highly accurate chromatograms of polymers having molecular hydrodynamic diameters up to 250 nm. Previous SEC analysis has been limited to polymers having hydrodynamic diameters of less than 120 nm.     

Characterisation of modified whey protein in milk ingredients by liquid chromatography coupled to electrospray ionisation mass spectrometry

Whey proteins are an important ingredient in the food industry. We have investigated the protein composition of commercial whey samples by liquid chromatography coupled to electrospray ionisation mass spectrometry on a time-of-flight instrument. We found that industrial whey protein contains a multitude of different modifications, ranging from almost native proteins through different degrees of glycosylation and oxidation up to almost completely oxidised forms. The information obtained allows characterisation of the influence of industrial processing on protein modifications and classification of whey protein-based ingredients or milk powders from different suppliers.