Size-exclusion chromatography conditions

Fig. 3. Overview of puriftcation sequence for the nonrecombinant tissue plasminogen activator (t-PA) which also contains urokinase plasminogen activator (u-PA). Serum-free culture conditional media is from normal human ceU line. The temperature for aU. steps, except for size-exclusion chromatography...

This chapter makes no distinction between gel-permeation chromatography (GPC) and size exclusion chromatography (SEC). We make mention of specific analysis conditions wherever possible. We have attempted to include a variety of conditions but by no means should this chapter be considered a comprehensive review of conditions for analyzing polyacrylates. We have drawn extensively from our own experience in selecting examples. 

As mentioned above, two experimental methods were examined as a source of kinetic samples Method A Agitated Glass Ampoule and Method B Static Mixer. These are described in turn in the following paragraphs. Analysis of samples was done using high temperature size exclusion chromatography (SEC) under conditions previously described (9.101. 

Mourey, T. H. and Bryan, T. G., Size-exclusion chromatography using mixed-bed columns with dimethylformamide at near-ambient conditions comparison of pStyragel HT Linear and PL Gel mixed-bed columns, /. Liquid Chromatogr., 14, 719, 1991. 

Polymerization conditions temperature 35 °C time 18.5 h solvent CH2C12, 100 mL total volume b TON turnover number, E ethylene, P propylene, H 1-hexene c Determined by size exclusion chromatography with polystyrene standard "Temperature 25 °C... 

Fig. 12. Separation of styrene oligomers by reversed-phase (left) and size-exclusion chromatography (right) (Reprinted with permission from [121]. Copyright 1996 American Chemical Society). Conditions (left) column, molded poly(styrene-co-divinylbenzene) monolith, 50 mm x 8 mm i.d., mobile phase, linear gradient from 60 to 30% water in tetrahydrofuran within 20 min, flow rate 1 ml/min, injection volume 20 pi UV detection, 254 nm (right) series of four 300 mm x 7.5 mm i.d. PL Gel columns (100 A, 500 A, 105 A, and Mixed C), mobile phase tetrahydrofuran, flow rate, 1 ml/min injection volume 100 pi, toluene added as a flow marker, UV detection, 254 nm temperature 25 °C,peak numbers correspond to the number of styrene units in the oligomers...

Buchacher et al. [43] discussed the continuous separation of protein polymers from monomers by continuous annular size exclusion chromatography. The P-CAC used for the experiments was a laboratory P-CAC type 3 as described in Table 1. The results were compared to conventional batch column chromatography in regard to resolution, recovery, fouling, and productivity. The protein used in the studies was an IgG preparation rich in aggregates. Under the conditions used, the polymers could be separated from the monomers, although no baseline separation could be achieved in either the continuous or the batch mode. The... 

Size-exclusion chromatography combined with RP-HPLC-MS was employed for the separation of pyranoanthocyanins from red wine. Wine samples (10 ml) were acidified with 3 M HC1 to pH 1 then sodium bisulphite was added at a concentration of 400 g/1. After 15 min reaction time the treated wine was loaded into a gel column (200 X 15 mm i.d.). Pigments were eluted with 95 per cent ethanol followed with 100 per cent methanol. The various fractions were acidified to pH 1, concentrated and redissolved in water. HPLC-DAD was carried out in an ODS column (150 X 4.6 mm i.d. particle size 5 /nn) at 35°C. Solvents were 0.1 per cent aqueous TFA (A) and ACN (B). The gradient started with 10 per cent B for 5 min to 15 per cent B for 15 min isocratic for 5 min to 18 per cent B for 5 min to 35 per cent B for 20 min. The flow rate was 0.5 ml/min and analytes were detected at 520 nm. MS conditions were sheath and auxiliary gas were a mixture of nitrogen and... 

Several affinity screening methodologies that include MS-based readout and work under protein-excess conditions have been developed in the past decade [1]. Some examples include affinity selection/mass spectrometry (ASMS Abbott Labs [10]), size exclusion chromatography with LC-ESI-MS (see Chapter 2 and 3 [11-19]), the use of coupled or non-coupled pulsed ultra-filtration/mass spectrometry (summarized in this chapter [11, 20-23]), restricted access phase chromatography (see Chapter 5 [24, 25]), capillary electrophoresis [26, 27], target shift mass spectrometry [28], and multitarget affinity/specificity screening (MASS, see Chapter 10 [29, 30]). 

The present experimental approach is based on the chromatographic advantages provided by the diol or glycerol derivatives of porous silica stationary phases available for use in HPLC. These have recently become available for estimating the molecular size of polyelectrolytes using aqueous size exclusion chromatography. The conditions for reproducible polyelectrolyte size measurements, and their possible purturbations have been summarized by Barth (8). 

Several variants of separation methods based on dialysis, ultrafiltration, and size exclusion chromatography have been developed that work under equilibrium conditions. Size exclusion chromatography especially has become the method of choice for binding measurements. The Hummel-Dreyer method, the vacancy peak method, and frontal analysis are variants that also apply to capillary electrophoresis. In comparison to chromatographic methods, capillary electrophoresis is faster, needs only minimal amounts of substances, and contains no stationary phase that may absorb parts of the equilibrium mixture or must be pre-equilibrated. 

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