Scaling size exclusion chromatography

Instrumentation. A Pharmacia BioPilot Column Chromatography system was used to perform large-scale size exclusion chromatography (SEC) with an 11.3 x 90 cm BioProcess column packed with Sephacryl S-200 HR gel. High performance size exclusion (HPSEC) and ion exchange chromatography (HPIEC) were conducted with Pharmacia Superose 6 and 12 (HR 10/30) and Mono-Q (HR 5/5) columns respectively, equipped with Beckman model 520 system controller and Beckman model HOB HPLC pumps. 

Nielen, M. W. R, Polymer Analysis by Micro-Scale Size-Exclusion Chromatography/MALDI Time-of-Flight Mass Spectrometry with a Robotic Interface, Anal. Chem., 70, 1563, 1998. 

Nielen MWF. Polymer analysis by micro-scale size exclusion chromatography MALDl time-of-flight mass spectrometry with a robotic interface. Anal Chem 1998 70 1563-8. 

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

This reversed-phase chromatography method was successfully used in a production-scale system to purify recombinant insulin. The insulin purified by reversed-phase chromatography has a biological potency equal to that obtained from a conventional system employing ion-exchange and size-exclusion chromatographies (14). The reversed-phase separation was, however, followed by a size-exclusion step to remove the acetonitrile eluent from the final product (12,14). 

Toyopearl HW size exclusion chromatography resins are macroporous packings for bioprocessing chromatography. They are applicable for process-scale... 

Styragel columns are compatible with most solvents commonly used in size exclusion chromatography. Exceptions are found on both sides of the polarity scale the use of standard general-purpose Styragel columns with aliphatic hydrocarbons or with alcohols (except hexafluoroisopropanol) and water is generally not recommended. However, it is possible to pack columns in special solvents for special-purpose applications. The interested user should contact Waters for additional information. 

Analysis of the MHR by high-performance size-exclusion chromatography (HPSEC) revealed three distinct populations to be present, which were isolated on a preparative scale. The composition of the populations differed mainly in the relative proportion of rhamnose, xylose and the amount of methyl esters and acetyl groups, although the general characteristics were rather similar (Table 2). However, degradation studies with RGase showed that, in contrast... 

IEC is also important in large-scale manufacturing of gene vectors such as plasmid DNA. In one process flow scheme, IEC and size exclusion chromatography (SEC) were used sequentially to purify the plasmid after lysis... 

Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), was used for the separation and fractionation of macromolecules on an analytical and preparative scale [17]. The separation occurs predominantly by the hydrodynamic volume of the macromolecules in solution, however, in some cases the polarity of the molecules can also influence the retention times. Like HPLC, the SEC technique is generally very reproducible with regard to its elution times (typically < 1 h) and hence can be used for automated synthesis. But because the cost for an automated SEC system is high, it must be considered as a serial separation technique. In addition, larger scale separations > 100 mg, usually require repetitive injection of small aliquots. 

Fig. 19 VCD spectra of the size-separated AuNPs protected with (R)-BINAS with the fraction 1-3 samples obtained using size exclusion chromatography and of free (R)-BINAS in CD2C12 solution. The spectra of the nanoparticles were scaled by a factor of 4. Adapted with permission from [156], Copyright (2010) American Chemical Society...

In order to calculate the molecular weight M) or molecular-weight distribution (MWD) of the polymer, the dependence of the Soret coefficient on M must be known. Because is virtually independent of M, at least for random coil polymers, the dependence of retention on M reduces to the dependence of D on M. The separation of molecular-weight components by D (or hydrodynamic volume, which scales directly with D) is a feature that thermal FFF shares with size-exclusion chromatography (SEC). In the latter technique, the dependence of retention on D forms the basis for universal calibration, as D scales directly with the product [rjjM, where [17] is the intrinsic viscosity. Thus, a single calibration plot prepared in terms of log([i7]M) versus retention volume (F,) can be used to measure M for different polymer compositions, provided an independent measure of [17] is available. In thermal FFF, a single calibration plot can only be used for multiple polymers when the values of for each polymer-solvent system of interest are known. However, a single calibration plot can be used with multiple channels. In... 

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