Improved molecular weight analysis of streptococcal hyaluronic acid by size exclusion chromatography

Summary Four size exclusion chromatography (SEC) calibration techniques were tested for use in the molecular weight characterisation of Streptococcal Hyaluronic Acid (HA). An exponential equation, evaluated using the Hamielec method, was superior to the customary peak position method. It provided the most accurate estimates of the weight average molecular weight, Mw. The calibration was valid for HA in the range 800 – 2500 kDa, and permitted the calculation of both polydispersity and molecular weight distributions for HA from Streptococcal fermentations. This exponential calibration approach should have application in the characterisation of other large biopolymers, particularly where pore size of available SEC media is limiting.     

Determination of molecular weight of heparin by size exclusion chromatography with universal calibration

The molecular weight (MW) of heparin can be accurately determined by size exclusion chromatography using "universal calibration." A universal calibration curve was constructed for Superose 12 with standard pullulan samples and verified using characterized ficoll fractions. This calibration yielded the correct MW of heparin as determined by light scattering, when the ionic strength of the mobile phase was maintained over 1.0M. Sodium poly(styrenesulfonate) samples were not suitable standards because of adsorption at high salt concentration and repulsion from the packing material at low ionic strength. The extraordinarily high charge density of heparin leads to the need for high salt concentration to screen such repulsions.     

Absolute molecular weight and molecular weight distribution of guar by size exclusion chromatography and low-angle laser light scattering

A procedure to determine the absolute weight-average molecular weight (M_w) and molecular weight distribution (MWD) of guar by aqueous size exclusion chromatography coupled with low angle laser scattering is described. It is shown that for a rigorously purified sample of guar solution the values for M_w and MWD are 2·2×10⁶ and 1·9 respectively. The effect of sample preparation and purification on these molecular parameters are discussed. Limitations and challenges in the aqueous size exclusion chromatography of complex water soluble polymers such as guar are also explored.     

Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array

The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters.     

Calibration mixture for estimation of peptide molecular weight by exclusion chromatography.

A procedure is described for the rapid preparation of a partial cyanogen bromide digest of horse heart cytochrome c. This digest serves as a calibration mixture for estimation of peptide molecular weight in the range 500 to 20,000 by exelusion chromatography in denaturing solvents. The elution profile of the calibration mixture from Sephadex G-50 equilibrated and developed with 10% formic acid is described by the relationship log M = 4.79 ? 0.42 ($\text{V}{}_{\mathrm{<mtext>o</mtext>}}\text{V}{}_{\mathrm{<mtext>o</mtext>}}$).     

Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography

Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.     

Separation of both fibrous and globular proteins on the basis of molecular weight using high-performance size exclusion chromatography

A high-performance size exclusion liquid chromatographic system has been used to separate proteins with different shapes solely on the basis of their molecular weights. After the effects of ionic and hydrophobic interactions with the stationary phase have been overcome, protein elution is normally governed by their effective size in solution. Conditions are described under which proteins, with isoelectric points within the normal operating pH range of the columns, are eluted independent of their Stokes' radii. Even fibrous proteins with axial ratios of 50 elute according to their known molecular weights over the range 2000–2,000,000.     

Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography-multiangle laser light scattering

The feasibility of size exclusion chromotography (SEC)-multiangle laser-light scattering as a technique to investigate aggregation and degradation of glycosylated and nonglycosylated proteins, and antibodies under various conditions such as addition of detergent, changes in pH, and variation of protein concentration and heat stress temperature was examined. Separation of proteins and their aggregates was performed using SEC-high-performance liquid chromatography. Detection of analytes was carried out with on-line UV, refractive index, and multiangle laser light-scattering detectors. Quantification and molecular weight determination were performed using commercial software. Aggregation and degradation were examined under various conditions and quantitative results are presented for bovine serum albumin, choriogonadotropin, glyceraldehyde-3-phosphate dehydrogenase, Herceptin, and ReoPro. This method can simultaneously determine both the quantities and the molecular weights of macromolecules from a single injection. The determination of molecular weight is absolute which avoids misleading results caused by molecular shape or interactions with the column matrix. This technique is valuable not only for assessing the extent of aggregation but also for effectively monitoring molecule degradation as evidenced by molecular weight reduction and change in monomer amount.     

Measurements of protein-protein interactions by size exclusion chromatography

A method is presented for determining second virial coefficients (B(2)) of protein solutions from retention time measurements in size exclusion chromatography. We determine B(2) by analyzing the concentration dependence of the chromatographic partition coefficient. We show the ability of this method to track the evolution of B(2) from positive to negative values in lysozyme and bovine serum albumin solutions. Our size exclusion chromatography results agree quantitatively with data obtained by light scattering.     

Molecular weight distribution of carrageenans by size exclusion chromatography and low angle laser light scattering

A procedure to determine the absolute weight average molecular weight and molecular weight distribution of carrageenans by high pressure aqueous size exclusion chromatography coupled with low angle laser light scattering is described. Experimental parameters are successively discussed, particular attention being focused on the absence of shear degradation during elution. The distribution curves were highly reproducible in time and weight average molecular weights integrated along the chromatogram were in good agreement with static light scattering results. A large difference in the molecular weight range between native (food-grade) and acidic degraded carrageenan samples was observed. Weight average molecular weights were found to be in good correlation with viscosity values, for degraded as well as undegraded products. It is also shown that the method described can help people using carrageenans in pharmacological studies by providing information on the real molecular weight distribution of the products they are employing.     

Characterization of loaded liposomes by size exclusion chromatography

This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes.     

Determination of the molecular weight of microbial polysaccharides using high performance exclusion chromatography

An automatic method of determining the molecular weight parameters (Mw, Mn) of microbial polysaccharides such as dextran, pullulan was developed based on the use of high performance size-exclusion chromatography on the two types of columns: Zorbax PSM 60 + 300 + 1000 and SynChropack GPC 100 + 500 + 1000. The Mw and Mn values were determined for a number of domestic and foreign dextran preparations. Changes in the molecular weight of pullulan and hydroxyethylstarch resulted from acid and enzymatic hydrolysis were estimated.     

Protein-sodium dodecyl sulfate complexes: determination of molecular weight, size and shape by controlled pore glass chromatography

The peak position vs log molecular weight curves of protein-SDS complexes chromatographed on controlled pore glass of narrow pore size distribution is linear over a molecular weight range of 17,000–385,000. A glass with a pore size of approximately 500 Å allows the inclusion of all complexes in this range. Peak position curves on glasses with broad pore distributions show decreased resolution and deviate from linearity at low elution coefficients.Exclusion size analysis of the elution coefficients of individual complexes from different columns with pore diameters ranging from 197 to 650 Å gives from 120 to 423 Å as their longest dimension. Assuming constant hydration and SDS-to-protein ration, the found dimension suggests the shape of a football, rather than a sphere or rigid rod.     

Liposome retention in size exclusion chromatography

Background  

Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void.     

Refolding of denatured ribonuclease observed by size exclusion chromatography

The unfolding and refolding of pancreatic ribonuclease have been observed by absorbance, fluorescence, and size exclusion chromatographic measurements in solutions of guanidinium chloride continuously maintained at pH 6.0 and 4 degrees C. The spectral measurements were fitted with a minimal number of kinetic phases while the chromatographic measurements were simulated from an explicit mechanism. All of the measurements are consistent with a minimal mechanism involving seven components. The folded components include the native protein and two transiently stable intermediates each having the same hydrodynamic volume. The intermediate having all native peptide isomers has an unfolding midpoint in 3.8 M denaturant while the intermediate having one nonnative peptide isomer has an unfolding midpoint in 1.3 M denaturant. The unfolded protein is distributed among four components having the same hydrodynamic volume but differing peptide isomers. At equilibrium, 10% of the denatured protein has all native isomers, 60% has one nonnative isomer, 5% has a different nonnative isomer, and 25% has both nonnative isomers. In low denaturant concentrations, the dominant component with one nonnative isomer can refold to transiently populate the compact intermediate with the same nonnative isomer.     

Monitoring xanthan quality during fermentation by size exclusion chromatography

Summary Xanthan concentration and molecular weight distribution are determined by size exclusion chromatography in the fermentation medium after dilution and cell removal by centrifugation. The analysis is rapid enough for process control. During a batch fermentation, the average molecular weight is found to be in the range of 7.2–9.3·10⁶ g/mole and to run through a maximum.     

Aluminium speciation in soil solutions as studied by size exclusion chromatography

Size exclusion chromatography was used for the fractionation of the aqueous extracts taken from different soil horizons (LOf, Oh, Ah, 15 and 35 cm). The aluminium content in the fractions was determined by graphite furnace atomic absorption spectrometry. In the fractions obtained from the LOf, Oh and Ah horizons, a great part of the total aluminium was bound to organic molecules. Over 90% of the aluminium in mineral soil solutions (15 and 35 cm depth) was of low molecular weight or associated with those species.