High-speed gel-permeation chromatography

To establish optimum operating conditions for high-speed gel-permeation chromatography (GPC), the effects of column packing particle size, solvent flow rate, and column length on the separation efficiency have been investigated by using monodisperse polystyrene samples and polystyrene gel columns (TSK-GEL column, Type-H). Decreasing the particle size of the column packing reduces the time required to obtain a given resolution. Monodisperse polystyrene standards were measured under the optimum operating conditions established (gel particle size 5 μ, column length 2 ft, flow rate 2.5 ml/min). The molecular weight distribution of a polymer mixture was determined in less than 10 min with the same accuracy as by the conventional GPC. Such short analysis time enables one to use GPC for in-plant quality control.     

High-resolution gel-permeation chromatography

The resolution attainable in gel-permeation chromatography (GPC) was investigated by using columns packed with polystyrene gel particles of about 5 μ diameter and mixtures of two monodisperse poly-α-methylstyrene samples studied previously. The resolution of GPC was found comparable to that of the sedimentation velocity method and slightly better than that of precipitation chromatography. Standard polystyrene samples obtained from Pressure Chemical Co. also were measured with the same columns. It was found that weight-average to number-average molecular weight ratios (M?_w/M?_n) of these samples with molecular weight in the range 97,000–411,000 are smaller than 1.006. For samples with molecular weight of 10,000–51,000 and 498,000–860,000, M?_w/M?_n is larger than 1.006, and the width of molecular weight distributions of these samples differed. In particular, molecular weight distributions of samples with molecular weights 19,800 and 51,000 were shown to be bimodal. It is therefore concluded that GPC is useful for samples of very narrow molecular weight distribution if high-resolution columns are used.     

High-speed aqueous gel-permeation chromatography

High-speed gel-permeation chromatography (GPC) of water-soluble polymers has been investigated by using TSK-GEL, type-PW columns packed with small porous particles. Being semirigid, the column packing could be operated under high pressure and, therefore, it was possible to achieve high-speed GPC. A resolution higher than that of ordinary GPC in organic solvent system was attained when the measurement of one sample was completed within 40 min with a 6-ft column set. Samples with a wide range of molecular weights, from oligomers to polymers having a molecular weight greater than 10⁶, could be fractionated. Dextran, polyethylene glycol, polyacrylamide, poly(vinyl alcohol), and poly(vinyl pyrrolidone) were separated according to molecular size with no evidence of adsorption. Investigation of other water soluble polymers is now in progress.     

High-resolution preparative-scale gel-permeation chromatography

Sharp polymer fractions have been obtained in preparative-scale gel-permeation chromatography by use of a column set packed with polystyrene gel particles of about 10 μ diameter. When 0.13 g of standard polystyrene NBS 706 was fractionated per injection, fractions with polydispersities of about 1.02–1.03 were obtained over the molecular weight range 10⁴–10⁶. Effects of a injection volume, solution concentration, and fraction volume on fractionation efficiency were also investigated. Polydispersities of fractions increased appreciably with increasing amounts of polymer injected.     

Analysis of resoles by gel-permeation chromatography

Resoles, the complex, heat-sensitive product mixture from the alkali-catalyzed reactions of phenol with formaldehyde were investigated by gel-permeation chromatography (GPC). The low molecular weight species of these resins which consisted of mono-and dinuclear methylol phenols were resolved into multiple peaks. Model compounds were used to identify the peaks of the specific methylol phenols or methylene etherbridged diphenols. Differences in the refractive index of individual species restricted the quantitative analysis of low molecular weight components in the resole. The effects of sodium and barium hydroxides and hexamethylene-tetramine as catalysts, reaction temperature, and time on the total composition of a resole are demonstrated with the gel-permeation chromatographic spectrum and with the aid of NMR. Formation of a “secondary” resole by methylolation of the bisphenol of formaldehyde was also monitored by GPC.     

Examination of dye-protein interaction by gel-permeation chromatography

The interaction of Cibacron blue F3G-A with two therapeutic proteins, recombinant human growth hormone and recombinant human interferon-alpha2b, has been examined by applying gel-permeation chromatography in combination with the absorption difference spectroscopy. The complexes of these proteins with Cibacron blue F3G-A have been isolated, and their absorbance spectra have been registered. The influence of Cibacron blue F3G-A on the oligomeric state of proteins has been investigated. It was found that Cibacron blue F3G-A promotes the generation of interferon-alpha2b dimers at pH 5.0.     

Steric exclusion and lateral diffusion in gel-permeation chromatography

The peak separation in gel-permeation chromatography (GPC) is attributed to the contributions of the steric exclusion and the lateral diffusion processes. The advantage of using the distribution coefficient K_GPC of the solute molecule in interpreting the GPC separation mechanism is assessed. The physical significance of K_GPC and its relation to measurable GPC parameters are examined in detail. A simple mixing experiment for determining the exclusion effect is described. The results of this experiment, as well as those of the flow rate study, show that the exclusion effect plays the primary role in GPC peak separation. For a column packed with Bio-Rad porous glass of 200 Å designation, the diffusion effect does not contribute significantly to peak separation. However, for the case of a Waters Associates column packed with polystyrene gel of 10⁴ Å designation, both the exclusion and the diffusion effects are shown to be important. A diffusion theory which includes the concept of a restricted diffusion coefficient is proposed to interpret the diffusion effect observed in the polystyrene gel column. The results of the theoretical calculation are found to agree with the observed flow rate dependence of the calibration curve.     

Characterization of diallyl phthalate prepolymers by gel-permeation chromatography

The polydispersity of diallyl phthalate prepolymers was determined by gel-permeation chromatography (GPC). The measurements were carried out in tetrahydrofuran and chloroform on Mexel 2000, Poragel, and Styragel polystyrene gels. The influence of the polymerization conditions, initiator, and type of transfer agent on the molecular weight distribution of the prepolymers is shown. Other correlations of interest, such as molecular weight distribution of Dapon 35 and Dapon 201, are also presented.     

Concentration effects and thermodynamic nonideality in gel chromatography

The dependence of the elution volume V_e on the concentration of injected polymer c in gel chromatography is presented for several systems including poly(styrene) and poly(methyl methacrylate) in a number of pure solvents with SiO₂-based gels. The linear dependence of V_e on c and K_av on c (where K_av is the distribution coefficient) is confirmed in the region of very low concentration. The slopes k of the straight lines increase with increasing relative molecular masses M of the polymer injected and with increasing thermodynamic nonideality of the system (as expressed by the second virial coefficient A₂). The significance of the slope of the GPC calibration curve for meaningful comparison of the concentration effects in various chromatographic systems is pointed out. A recently found correlation between k and (A₂M)γ is confirmed with a γ value of about 0.8. A possible theoretical explanation for the deviation of γ from unity is discussed. Finally, the influence of both the polymer concentration and the thermodynamic quality of the eluent on the resolution index of the chromatographic system is evaluated with the conclusion that thermodynamically poor solvents should be preferred for preparative GPC separations with very high loads.     

Molecular weight determinations by gel-permeation chromatography and viscometry

The hydrodynamic volume concept can be used effectively with gel-permeation chromatographic (GPC) and viscosity data to estimate the molecular weight of a variety of polymers. Agreement is within ±5–10% of the absolute values and thus is satisfactory for many purposes. An iterative computer technique and a method developed by Funt and Hornof for analyzing GPC–viscosity data were found to be equivalent with respect to estimating the molecular weights for the five cases studied. The latter is easily employed but restricted to the case where the sample of interest and the GPC calibration standards have approximately equal Mark-Houwink parameters. Since GPC measurements are commonly performed in thermodynamically good solvents, the general applicability of the method is not impaired. Using the unperturbed dimensions of the polymer chain to estimate the molecular weight of a variety of polymers was not as satisfactory as the above techniques. This approach generally gave biased molecular weight values (consistently low or consistently high). Agreement with the absolute values ranged from 10 to 30%. We therefore believe that either of the techniques based on the hydrodynamic volume concept can be used more effectively to estimate the molecular weight of a series of polymers than the treatment based on the unperturbed dimension.     

Enzymatic hydrolysis of pectic substances by gel-permeation chromatography

Summary A rapid liquid chromatographic (HPLC) method for the determination of pectinolytic activity of enzymes produced by Aspergillus Niger and Rhizopus species is reported.Compared with more conventional methods, HPLC is more reliable and has a much improved maximum sensitivity. The limit of quantitation of galacturonic acid is 0.1g.     

Prehump in the gel-permeation chromatography fractionation of pulp cellulose acetate

GPC chromatograms of cellulose acetate made from wood pulp customarily show a shoulder or small separate peak at the high end of the DP distribution. Material isolated from this “prehump” area is considerably enriched in mannose and xylose, and the size of the prehump in acetates from different pulps correlates with the amount of hemicellulose in the pulp. Prehump is not ordinarily found in cellulose acetate made from linters but was induced by adding mannan at the start of the acetylation, or by prolonging the acetylation beyond the time when the cellulose acetate sulfate triester is first formed. Products of poor solubility, as indicated by increased haze and false viscosity, resulted in both cases. Prehump was reduced in pulp cellulose acetate by many of the steps which give products of improved solubility: mercerization of cellulose prior to acetylation, use of more sulfuric acid catalyst, replacement of part of the acetic acid with methylene chloride during acetylation, and by removal of a small amount of high viscosity or poorly soluble material by fractional precipitation.     

Determination of haptoglobin in human serum by high-performance gel-permeation chromatography

Summary A high-performance gel-permeation chromatographic (GPC) method is described for the determination of serum haptoglobin (Hp), based on the estimation of the haptoglobin-hemoglobin (Hp−Hb) complex. Hemoglobin (Hb) is added to the serum and the resulting Hp−Hb complex is separated by GPC and detected at 415 nm. The Hp−Hb complex separated chromatographically from serum migrated electrophoretically with the globulin fraction. It was found to be stable over a period of one week at 4°C. The total analysis time for a serum sample is approximately 20 min. The minimum detection limit is 30mg/L. Relative standard deviation values were below 1% (peak area). No interferences from bilirubin or from turbidity of samples were observed. The results of the GPC analysis correlate well with those obtained by nephelometric assay (r=0.996).     

Determination of chemical heterogeneity of copolymers by gel-permeation chromatography

Summary The determination of the chemical heterogeneity of copolymers has been carried out by means of gel-permeation chromatography (GPC). Consequently mixtures of different homopolymers with the same relative molecular masses have been fractionated. Although maximum chemical non-uniformity appears, non-uniform effects obtained from the GPC are negligible. It is seen that for copolymers a GPC separation only occurs when the chemical composition of the copolymer changes simultaneously with the degree of polymerisation. We have established that, in a few cases, minute chemical heterogeneity is evident from the GPC curves, but not in the bulk which obeys the kinetics of copolymerisation.     

Polymer characterization by coupling gel-permeation chromatography and automatic viscometry

A method is proposed for use of the universal calibration curve, i.e., the product of molecular weight and intrinsic viscosity versus retention volume, in calculating the molecular weight distribution of a polymer from gel-permeation chromatography (GPC) when the Mark-Houwink relation of the polymer in the solvent used for the GPC is unknown. This is achieved by measuring the viscosity of each fraction with an automatic capillary tube viscometer. Application of this technique to poly(vinyl chloride) and poly(vinyl acetate) proved to be successful.     

Quality control of clinical dextran by rapid gel-permeation chromatography

Summary Clinical dextran can be fractionated by rapid gel-permeation chromatography on Spheron^R with the aid of a high-performance liquid chromatograph. The linear calibration function is obtained by dextran fractions whose molecular weights Mw and Mn are determined by absolute methods (light scattering, membrane osmometry) and end group analysis. The transformation of elution diagrams into a molecular weight distribution is described. Characteristic data for dextran 40 and 70 were obtained from the integral distribution curves. The molecular weight distribution of dextran fractions was found to fit a Schulz-Zimm distribution which is typical for long chain polymers. For quality control purposes clinical dextran can be characterized by its molecular weight distribution with high accuracy within one hour.     

Gas-chromatographic determination of pesticide residues after clean-up by gel-permeation chromatography and mini-silica gel-column chromatography

A new extraction and partition step was introduced into the well established multi-residue method of Specht and Tillkes (DFG method S 19) replacing dichloromethane by ethyl acetate/cyclohexane (1+1). In addition, more simple working conditions were obtained. Results of fortification experiments with organochlorine, organophosphorus and organonitrogen pesticides showed good agreement with those obtained by the formerly published method.