Column Calibration Research Articles

NEW APPROACH TO MODEL FITTING IN MULTI-DETECTOR GPC

The main limitation of multi-detector GPC arises from the nature of detector sensitivities in the tails of a polymer distribution. In the low molecular weight tail of this distribution, molecular weight-sensitive detectors (such as a capillary viscometer or a static laser light-scattering photometer) have low sensitivity while concentration detectors (e.g., differential refractometer) have high sensitivity. This situation is reversed in the high molecular weight tail. These imbalances in sensitivity raise the question of how best to obtain an estimate of column calibration curves. The question is central to the successful application of the multi-detector GPC technique. For example, the accuracy and precision with which structural information for polymers with broad molecular weight distribution, especially with long-chain branches, can be obtained depends critically on the accurate estimation of such calibration curves in the tails. Traditionally, calibration curves are fit to the logarithm of the ratios of detector responses. However, the logarithm of a ratio will not give meaningful values in the regions where at least one of the responses is near zero. Thus, low detector sensitivity in the tails requires that a calibration curve be fit only to the heart of the peak, where all detectors have good response. The optimized curve is then extrapolated to the regions in the tails that were excluded from the fit. This data truncation has two consequences that limit the accuracy and precision of the multi-detector GPC technique. Truncation eliminates potentially useful responses with which to constrain the calibration curves, and the resulting curves can be sensitive to the choice of the fitting region. We describe a new data analysis method for multi-detector GPC where the complete chromatographic profile obtained from one detector is compared, in a least-squares sense, to a model that is a function of responses from the other detector. This formulation of least-squares avoids the use of logarithms, ratios, and eliminates the need for extrapolation. The approach allows the inclusion of regions in the least-squares fit that contain low detector's signal, e.g., near baseline responses that fluctuate about zero from either, or both, detectors. We apply this approach to obtain column calibration curves with each of two molecular weight-sensitive detectors, coupled to a GPC system. Such calibration curves are the necessary intermediate steps in determining the polymer's molecular weight and intrinsic viscosity distributions. If suitable calibration standards are available, we further show how the polymer's intrinsic viscosity law can be obtained directly from dual-detector responses without requiring - or depending on - a sample-dependent calibration curve.

Determination of Molecular Size by Size‐Exclusion Chromatography (Gel Filtration)

Size-exclusion or gel filtration chromatography is one of the most popular methods for determining the sizes of proteins. Proteins in solution, or other macromolecules, are applied to a column with a defined support medium. The behavior of the protein depends on its size and that of the pores in the medium. If the protein is small relative to the pore size, it will partition into the medium and emerge from the column after larger proteins. Besides a protein's size, this technique can also be used for protein purification, analysis of purity, and study of interactions between proteins. In this unit protocols are provided for size-exclusion high-performance liquid chromatography (SE-HPLC) and for conventional gel filtration, including calibration of columns (in terms of the Stokes radius) using protein standards.

Comparison of the fractionation of a coal tar pitch by solvent solubility and by planar chromatography

Two methods of fractionation of a coal tar pitch, one based on solvent solubility separation and the other on preparative thin layer chromatography were compared for effectiveness of the separation. The fractions were characterised using size exclusion chromatography (SEC) and UV-fluorescence spectroscopy (UV-F). Fractionation by planar chromatography was found to give a better separation and to use less solvent than the solvent separation method. Both SEC and UV-F clearly showed less overlap in fractions separated by planar chromatography, compared to solvent fractionation. The cleaner separation of the heaviest fraction is particularly relevant for the mass spectroscopic investigation of large molecular mass materials, where the presence of lighter materials was observed to suppress high-mass signal. The close agreement observed between SEC and UV-F is consistent with previously reported correlations for coal tar pitches. However, the relevant SEC work was performed using pyridine as the mobile phase. We have found that 10%–15% of coal tar pitches cannot be dissolved in pyridine whilst total dissolution of coal tar pitch may be achieved using NMP. The calibration of analytical columns operated with NMP would be expected to lead to more accurate evaluations of molecular masses in these complex mixtures.

Estimation of sediment–water exchange of solutes in Lake Veluwe, The Netherlands

Two methods for the determination of sediment–water exchange of dissolved components –(a) laboratory column experiments and (b) calibration of a model for vertical transport of dissolved components in pore water– are discussed. The three parameters of the transport model –the effective dispersion coefficient at the sediment–water interface, the attenuation constant of dispersion in the sediment and the advective flow rate– were highly correlated. Estimates for the dispersion coefficient at the sediment–water interface in the shallow Lake Veluwe, The Netherlands, showed a large variability, but were always one to two orders of magnitude larger than the molecular diffusion coefficient. Dispersive transport dominated advective transport at the sampled sites. The dispersion coefficient at the sediment–water interface in shallow lakes can be enhanced by several mechanisms, such as pressure gradients caused by wind stress. Exclusion of this turbulent dispersion, as in laboratory column experiments with intact sediment cores, can therefore result in an underestimation of the real flux. For reactive compounds the introduced error may be partly compensated by adjustment of the concentration gradient due to reaction during transport and incubation.

Characterization of Methocel™: Correlation of static light‐scattering data to GPC molar mass data based on pullulan standards

Using low-angle laser light-scattering (LALLS) and multiangle laser light-scattering (MALLS) techniques, absolute molar mass averages were determined for a wide variety of polymers in the Methocel™ product line produced by The Dow Chemical Company. These data were correlated to GPC molar mass averages obtained from column calibration using pullulan standards. It was determined that the pullulan equivalent molar mass averages overestimated the values determined by light scattering by a factor as high as 3.2. Mark-Houwink parameters were calculated based upon values for the pullulan standards and application of the universal calibration concept. Sample preparation issues and chromatographic conditions that impact data quality are also presented. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2197–2210, 1998

The functional relationship of the column calibration factor in thermal diffusion column measurement

An approximate formulation of the column calibration factor (CCF), , at any given temperature is derived for thermal diffusion columns (TDC) from the Navier-Stokes equation in cylindrical coordinates. The values of are then used to estimate the experimental thermal diffusion factor of a neon gas mixture with its natural isotopic abundances from measured in four columns of different column geometries. The molecular force parameters of the molecule are estimated from the intercepts and slopes of the linear variations of and the collision integral against and respectively. The excellent agreement of the estimated force parameters with the available literature values and the comparison of the temperature variation of by the CCF method with the existing experimental and theoretical ones establish the fact that the derived relationship of with the column geometries is more than adequate.

Flow microcalorimeter auto-calibration for the analysis of immobilized enzyme kinetics

A new calibration procedure of a flow microcalorimeter (FMC) for the direct investigation of immobilized enzyme kinetics was developed. The procedure is based on the use of a flow-through microcalorimetric column packed with the immobilized enzyme adapted to the differential reactor system with infinite flow recirculation. The true reaction rate is determined from the substrate consumption as calculated from the decrease in thermometric response. The response is transformed to the substrate concentration based on a microcalorimeter column calibration using known substrate concentrations. The method was experimentally verified using the invertase immobilized by biospecific binding on concanavalin A - cellulose conjugates. From the measurement of the sucrose hydrolysis in the FMC, all kinetic parameters of immobilized invertase, V max, K m, and K i were estimated. Results were compared to those obtained by the simultaneous spectrophotometric determination of the reactant concentrations. Both techniques gave equivalent results of reactant concentrations and kinetic parameters.

Production of Antisera Using Fusion Proteins

AbstractThe use of fusion proteins for the production of antisera allows specific areas of proteins to be targeted as epitopes and facilitates the purification of the antisera. This unit first describes the use of standard molecular biological techniques to construct a fusion‐protein expression plasmid by inserting a region of cDNA into a pGEX vector. Next, E. coli are transformed with the plasmid and induced to generate fusion protein. Also described is the purification of the soluble fusion protein, which is necessary for immunization and other subsequent procedures. This is accomplished by taking advantage of the GST fusion tag's affinity for glutathione. The purified fusion protein is then used to immunize animals, and antisera from these animals are then purified using affinity columns. Support protocols describe the construction and calibration of affinity columns for purifying antibodies using soluble fusion proteins, the use of insoluble fusion proteins for animal immunization, and preparation of affinity columns for purifying antibodies using insoluble fusion proteins.

Polydispersity Study of Narrow Polystyrene Standards Using Light Scattering

Manufacturers of narrow polystyrene standards for use as column calibrants with size exclusion chromatography (SEC) often label the corresponding polydispersity (Mw/Mn) as < ∼1.05. These values appear to reflect more the errors associated with the measurement than the true narrow polydispersity. Ideally, we would measure polydispersity by a light scattering measurement of Mw and a membrane osmometry measurement of Mw. The ratio of such measurements would be expected to have an error on the order of 5%- a number comparable to the difference of the measured polydispersity and unity Multi-angle light scattering (MALS) allows the absolute determination of the rms radius independent of the separation technique and any other detector. Using this information and following the method of Shortt,1 column band broadening effects may be corrected and a far more precise determination of polydispersity made. A variety of standards from several manufacturers were measured, and the polydispersity of each was determined to be far smaller than stated, further confirming their suitability for column calibration purposes.

Absolute Molecular Weight Distribution of Low-Molecular-Weight Heparins by Size-Exclusion Chromatography with Multiangle Laser Light Scattering Detection

The absolute molecular weight (Mr) distribution of seven low-molecular-weight (LMW) heparin products was determined by size-exclusion chromatography (SEC) coupled with multiangle laser light scattering (MALLS) detection. The SEC/MALLS technique does not rely on relativeMrstandards for column calibration and yields absoluteMrestimates directly from the angular dependence of scattered light intensity as a function of concentration, as formulated by light scattering theory. The SEC/MALLS method we describe is rapid, precise, and accurate. In 1 h it yields results from triplicate injections that agree well with the manufacturers’ own independent analyses and that exhibit coefficients of variation of ∼1%. By eliminating the requirement for finite quantities of highly purified, well-characterizedMrstandards derived from heparin, the present procedure represents a clear improvement over relative methods ofMrdetermination. Thus, it is concluded that the SEC/MALLS method is ideally suited to routine quality control of commercial LMW-heparin products.

Molecular Force Parameters through Thermal Diffusion to Ensure the Model Independency of Column Calibration Factor

Using Ar 36 –Ar 40 as calibrating gas, the simultaneous determination of thermal diffusion factors α T 's at two available experimental temperatures as well as the prediction of pressure dependence of ln q e at an intermediate temperature is possible for some isotopic inert gas mixtures in terms of column calibration factor F s (CCF) of a given column. The variation of α T with \bar T is then used to estimate the force parameters of the molecules only to ensure the reliability of the proposed temperature dependence of α T . The experimental α T 's due to Maxwell and Slieker shape factors are also calculated for comparison with those by the CCF method as well as by theoretical ones based on elastic collisions among the molecules. It seems that α T 's by CCF method play an important role in determining the force parameters of molecules indicating thereby the model independency of F s as a sensitive α T measuring parameter.

Estimation of Column Calibration Factor and Force Parameters to Predict Temperature Dependence of Thermal Diffusion Factor of Some Simple Molecules

The column calibration factor (CCF) F s of a column (Roos and Rutherford 1969) has been accurately estimated to yield the thermal diffusion factor α T 's of Kr 80 –Kr 86 , Xe 129 –Xe 136 , CO 28 –CO 29 , CH 4 16 –CH 4 17 and N 2 28 –N 2 29 respectively at two different experimental temperatures. The probable temperature dependence of α T 's are used to obtain the force parameters ε i j / k and σ i j of the respective molecules. Again, the α T 's as obtained from lnq max and F s are compared with the experimental α T 's by the existing method involved with column shape factors as well as theoretical α T 's based on elastic and inelastic collisions among the molecules. The comparison of the estimated force parameters with the reported ones finally suggests the technique of predicting α T 's as a function of temperature is extremely useful.

Application of Triple-Detector Size Exclusion Chromatography (On-Line Differential Refractometer, Viscometer and Light Scattering Detectors) for the Characterization of Brominated Polystyrene

Abstract A brominated polystyrene sample was used as a probe to explore the increased capabilities of size exclusion chromatography (SEC) with on-line light scattering and viscosity detectors—triple-detector SEC (TriSEC). Conventional SEC using only a concentration detector is not capable of providing accurate analysis for these types of polymers. Several flowrates and sample concentrations were used to study their effects on the precision of the various TriSEC-determined molecular parameters. Our study clearly shows the strength and weakness of each of the three detectors, and their interesting synergism in the TriSEC configuration. Overall, the molecular weight distribution results are better determined by light scattering compared to those determined by viscometer using the universal calibration approach. The viscometer, however, gives the important intrinsic viscosity distribution results. These intrinsic viscosity results can be considered for polymer quality control applications, because of their excellent precision and independence from SEC column calibration. The determination of the Mark-Houwink (intrinsic viscosity versus molecular weight) plot for any polymer conformational and structural studies is best done by using all detector signals from TriSEC.

HPLC, a practical user's guide

PREFACE. I HPLC PRIMER. 1 Advantages and Disadvantages of HPLC. 1.1 How It Works. 1.1.1 A Separation Model of the Column. 1.1.2 Basic Hardware: A Quick, First Look. 1.1.3 Use of Solvent Gradients. 1.1.4 Ranges of Compounds. 1.2 Other Ways to Make My Separation. 1.2.1 FPLC-Fast Protein Liquid Chromatography. 1.2.2 LC-Traditional Liquid Chromatography. 1.2.3 GLC-Gas Liquid Chromatography. 1.2.4 SFC-Supercritical Fluid Chromatography. 1.2.5 TLC-Thin Layer Chromatography. 1.2.6 EP-Electrophoresis. 1.2.7 CZE-Capillary Zone Electrophoresis. 2 Selecting an HPLC System. 2.1 Characteristic Systems. 2.1.1 Finding a Fit: Detectors and Data Processing. 2.1.2 System Models: Gradient Versus Isocratic. 2.1.3 Vendor Selection. 2.1.4 Brand Names and Clones. 2.1.5 Hardware-Service-Support. 2.2 System Cost Estimates. 2.2.1 Type I System-QC Isocratic (Cost: $10-15,000). 2.2.2 Type II System-Research Gradient (Cost: $20-25,000). 2.2.3 Type III System-Automated Clinical (Cost: $25-35,000). 2.2.4 Type IV System-Automated Methods (Cost: $30-50,000). 2.3 Columns. 2.3.1 Sizes: Analytical and Preparative. 2.3.2 Separating Modes: Selecting Only What You Need. 2.3.3 Tips on Column Use. 3 Running Your Chromatograph. 3.1 Set-up and Start-up. 3.1.1 Hardware Plumbing 101: Tubing and Fittings. 3.1.2 Connecting Components. 3.1.3 Solvent Clean-up. 3.1.4 Water Purity Test. 3.1.5 Start-up System Flushing. 3.1.6 Column Preparation and Equilibration. 3.2 Sample Preparation and Column Calibration. 3.2.1 Sample Clean-up. 3.2.2 Plate Counts. 3.3 Your First Chromatogram. 3.3.1 Reproducible Injection Techniques. 3.3.2 Simple Scouting for a Mobile Phase. 3.3.3 Examining the Chromatogram. 3.3.4 Basic Calculations of Results. II HPLC OPTIMIZATION. 4 Separation Models. 4.1 Partition. 4.1.1 Separation Parameters. 4.1.2 Efficiency Factor. 4.1.3 Separation (Chemistry) Factor. 4.2 Ion Exchange Chromatography. 4.3 Size Exclusion Chromatography. 4.4 Affinity Chromatography. 5 Column Preparation. 5.1 Column Variations. 5.2 Packing Materials and Hardware. 5.3 Column Selection. 6 Column Aging, Diagnosis, and Healing. 6.1 Packing Degrading-Bonded-Phase Loss. 6.2 Dissolved Packing Material-End Voids. 6.3 Bound Material. 6.4 Pressure Increases. 6.5 Column Channeling-Center-Voids. 6.6 Normal Phase, Ion Exchange, and Size Columns. 6.7 Zirconium and Polymer Columns. 7 Partition Chromatography Modifications. 7.1 Reverse-Phase and Hybrid Silica. 7.1.1 Ionization Suppression. 7.1.2 Ion Pairing. 7.1.3 Organic Modifiers. 7.1.4 Chelation. 7.2 Acidic Phase Silica. 7.3 Reverse-Phase Zirconium. 7.4 Partition Mode Selection. 8 Nonpartition Chromatography. 8.1 Ion Exchange. 8.1.1 Cationic:Weak and Strong. 8.1.2 Anionic:Weak and Strong. 8.2 Size Exclusion. 8.2.1 Organic Soluble Samples. 8.2.2 Hydrophilic Protein Separation. 8.3 Affinity Chromatography. 8.3.1 Column Packing Modification. 8.3.2 Chelation and Optically Active Columns. 9 Hardware Specifics. 9.1 System Protection. 9.1.1 Filters, Guard Columns, and Saturation Columns. 9.1.2 Inert Surfaces and Connections. 9.2 Pumping. 9.2.1 High- and Low-Pressure Mixing Controllers. 9.2.2 Checking Gradient Performance. 9.3 Injectors and Autosamplers. 9.4 Detectors. 9.4.1 Mass Dependent Detectors. 9.4.2 Absorptive Detectors. 9.4.3 Specific Detectors. 9.5 Fraction Collectors. 9.6 Data Collection and Processing. 10 Troubleshooting and Optimization. 10.1 Hardware and Tools-System Pacification. 10.2 Reverse Order Diagnosis. 10.3 Introduction to Data Acquisition. 10.4 Solvent Conservation. III HPLC UTILIZATION. 11 Preparative Chromatography. 11.1 Analytical Preparative. 11.2 Semipreparative. 11.3 True Preparative. 12 Sample Preparation and Methods Development. 12.1 Sample Preparation. 12.1.1 Deproteination. 12.1.2 Extraction and Concentration. 12.1.3 SFE (Cartridge Column) Preparations. 12.1.4 Extracting Encapsulated Compounds. 12.1.5 SFE Trace Enrichment and Windowing. 12.1.6 Derivatives. 12.2 Methods Development. 12.2.1 Standards Development. 12.2.2 Samples Development. 12.3 Gradient Development. 13 Application Logics: Separations Overview. 13.1 Fat-Soluble Vitamins, Steroid, and Lipids. 13.2 Water-Soluble Vitamins, Carbohydrates, and Acids. 13.3 Nucleomics. 13.4 Proteomics. 13.5 Clinical and Forensic Drug Monitoring. 13.6 Pharmaceutical Drug Development. 13.7 Environmental and Reaction Monitoring. 13.8 Application Trends. 14 Automation. 14.1 Analog-to-Digital Interfacing. 14.2 Digital Information Exchange. 14.3 HPLC System Control and Automation. 14.4 Data Collection and Interpretation. 14.4.1 Preinjection Baseline Setting. 14.4.2 Peak Detection and Integration. 14.4.3 Quantitation: Internal/External Standards. 14.5 Automated Methods Development. 14.5.1 Automated Isocratic Development. 14.5.2 Hinge Point Gradient Development. 14.6 Data Exportation to the Real World. 14.6.1 Word Processors: .ASC, .DOC, .RTF, .WS, .WP Formats. 14.6.2 Spread Sheets: .DIF, .WK, .XLS Formats. 14.6.3 Databases: .DB2 Format. 14.6.4 Graphics: .PCX, .TIFF, .JPG Formats. 14.6.5 Chromatographic Files: Metafiles and NetCDF. 15 Recent Advances in LC/MS Separations. 15.1 A LC/MS Primer. 15.1.1 Quadrupole MS and Mass Selection. 15.1.2 Other Types of MS Analyzers for LC/MS. 15.1.3 LC/MS Interfaces. 15.1.4 LC/MS Computer Control and Data Processing. 15.2 Microflow Chromatography. 15.3 Ultrafast HPLC Systems. 15.4 Chip HPLC Systems. 15.5 Standardized LC/MS in Drug Design. 16 New Directions in HPLC. 16.1 Temperature-Controlled Chromatography. 16.2 Ultrafast Chromatography. 16.3 Monolith Capillary Columns. 16.4 Micro-Parallel HPLC Systems. 16.5 Two-Dimensional HPLC Systems. 16.6 The Portable LC/MS. APPENDICES. APPENDIX A Personal Separations Guide. APPENDIX B FAQs for HPLC Systems and Columns. APPENDIX C Tables of Solvents and Volatile Buffers. APPENDIX D Glossary of HPLC Terms. APPENDIX E HPLC Troubleshooting Quick Reference. APPENDIX F HPLC Laboratory Experiments. Laboratory 1: System Start-up and Column Quality Control. Laboratory 2: Sample Preparation and Methods Development. Laboratory 3: Column and Solvent Switching and Pacification. Appendix G Selected Reference List. INDEX.

Calibration of Size-Exclusion Chromatography: Use of a Double Gaussian Distribution Function to Describe Pore Sizes

A method for the calibration of size-exclusion chromatographic columns is proposed that takes into account the nonlinear dependence of the Stokes radius Rs upon the partition coefficient KD. The method is based on the assumption that the pore size distribution of a molecular sieve column can be described by the sum of two Gaussian distributions and has been successfully tested with low-pressure chromatography gels (Sephacryl and Superose) and HPLC gels (TSK SW) over a wide range of protein sizes. An application of this method is described, in which aggregation states of the membrane protein prostaglandin H2 synthase solubilized in nonionic detergents are estimated.

Calibration of gel-permeation columns in the high-molecular-mass range: fixed human thrombocytes for the estimation of interstitial volume and the haemocyanin of the Vineyard snail Helix pomatia as a molecular mass calibration substance

Human thrombocytes, fixed with formaldehyde, are shown to be a universal indicator of the interstitial volume m gel-permeation chromatography (size-exclusion chromatography) for gels of all molecular mass separation ranges. The fixed thrombocytes are simple to prepare and to handle. Furthermore, native or intramolecularly cross-linked haemocyanin of the Roman snail Helix pomatia is shown to be well suited as a molecular mass calibration substance. We propose 8.8 · 10 6 as its molecular mass for calibration. This haemocyanin can therefore be added to the list of globular protein molecular mass calibration substances in the range of very high molecular masses.

Thermal diffusion factors of hydrogenic trace mixtures with helium by column calibration factor

Using column calibration factor (CCF)F s for a given column, the temperature dependence of experimental thermal diffusion factors αT of hydrogenic trace mixtures in helium are accurately determined. This study, however, observes the inelastic collision effect in these trace mixtures when αT by our CCF method are compared with those by the existing methods and theoretical ones respectively.

Calculation of retention in reversed-phase liquid chromatography: IV. ChromDream software for the selection of initial conditions and for simulating chromatographic behaviour

A computer-aided method to assist the chromatographer in the determination of initial conditions in reversed-phase HPLC has been developed. ChromDream software predicts the optimum methanol, acetonitrile or tetrahydrofuran concentration in a mobile phase on the basis of the structural formulae of the compounds. The effects of the concentration of an organic solvent in water on retention and chromatograms of compound mixtures are simulated. Reversed-phase column parameters responsible for selectivity and retention are calculated. Volume and energy increments for more than 100 volume fragments and more than 30 bond dipoles have been derived. An approach for the selection of reference compounds for column calibration is proposed.

Size-exclusion chromatography in an analytical perspective

Modern media for size-exclusion chromatography combine maximum selectivity with high plate count to resolve solutes differing in molecular mass by as little as 20%. Separations of solutes differing in molecular mass by only 10% will be very hard to accomplish even for rod-shaped molecules. The reduced column lengths and porosity of small-particle-sized media unfortunately reduces the inherent resolving power to the same order as traditional columns but with a substantial reduction in analysis time. The use of well-calibrated columns for obtaining estimates of molecular mass yields a precision close to that of absolute methods. The simplicity and accuracy of the integral calibration method using a sample of broad molecular mass distribution makes this procedure very suitable for manual calibration of size-exclusion columns.

Study of size distribution and stability of liposomes by high performance gel exclusion chromatography

Applications of high performance liquid chromatography on a gel exclusion column (HPLC-GEC) for the characterization of liposomes are presented following a review of the conventional uses of gel exclusion chromatography (GEC). The practical aspects of HPLC-GEC concerning the choice of the gel and the column operation are examined. A procedure for column calibration with vesicles is proposed, in order to obtain the selectivity curve of the TSK-G6000 PW gel; this appears to be the most convenient gel for sizing of liposomes. The mechanism of elution is discussed as a function of the particle size and composition. Methods involving this type of column and different detection modes (light scattering, refractive index, fluorescence and radioactivity) or their combination, were investigated in detail either to analyse the polydispersity of liposomes or their size stability in terms of aggregation and/or fusion.