Protein column

FIGURE 10.1 I Effect of linear velocity on plate height for proteins. Column SynChropak GPC 500, 250 X 4.6 mm i.d. Mobile phase 0.1 M potassium phosphate, pH 7. (Reprinted from Ref. 4 by courtesy of Marcel Dekker, Inc.)... 

The analyst should be aware that generally, when working with large molecules (particularly proteins), column efficiencies may, under some circumstances, be as little as 10% of the expected value and this must be taken into account when choosing the column and the phase system. 

Figure 18 Very-high-speed gradient anion exchange chromatography of proteins. Column 0.46 x 3.5 cm ZipSep AX, 3 p. Eluent Tris-HCl, pH 8.0, operated on a gradient from 0-0.5 M NaCl. Flow rate 2ml/min. Detection UV absorbance at 280 nm. (1) Ribonuclease A, (2) carbonic anhydrase, (3) conalbumin, (4) bovine serum albumin. (Reproduced from Hatch, R. G., J. Chromatogr. Sci., 31, 469,1993. By permission of Preston Publications, A Division of Preston Industries, Inc.)...

Figure 4.8 Cation-exchange liquid chromatography of basic proteins. Column, Asahipak ES502C eluent, 20 min linear gradient of sodium chloride from 0 to 500 mM in 50 mM sodium phosphate buffer pH 7.0 flow rate, 1 ml min-1 temperature, 30 °C detection, UV 280 nm. Peaks 1, myoglobin from horse skeletal muscle (Mr 17 500, pi 6.8-7.3) 2, ribonuclease from bovine pancreas (Mr 13 700, pi 9.5-9.6) 3, a-chymotrypsinogen A from bovine pancreas (Mr 257 000, pi 9.5) and 4, lysozyme from egg white (Mr 14 300, pi 11.0-11.4). (Reproduced by permission from Asahikasei data)...

Electrophoretic Methods. Little use has been made of electrophoretic techniques for the fractionation of the whey proteins. Column isoelectric focusing has been used to fractionate further the crude immunoglobulin fraction obtained by Smith s procedure (Josephson et al. 1972). Two major peaks, a shoulder, and two minor peaks were obtained, but no attempt was made to identify the components in the peaks. 

The specific amino acid side chains on a-lactalbumin responsible for binding to the metal support are not known however, a-lactalbumin is a metalloprotein. Under physiological conditions, it carries one Ca(II) per molecule hence, there are metal binding sites on the protein. Column-bound a-lactalbumin is eluted by a solution of the free ligand imidazole. A flowchart outlining these procedures is shown in Figure E4.3. 

The use of HPLC for the analysis of bovine milk proteins was introduced by Diosady et al. (59), who compared SE-HPLC on two SynChropac GPC-100 columns in series and RP-HPLC on a lO-jUm-particle-sizc RP-8 column, both with UV detection, for the separation of dialyzed freeze-dried whey proteins. Column temperature was 40°C and 47°C, respectively. Samples were eluted with Tris-buffer pH 6 for SE-HPLC and a linear gradient of two solvents, i.e., 98% 0.5 M KH2P04, pH 2, and 98% isopropanol, each with 2% 2-methoxyethanol for RP-HPLC. They... 

Resolution of the enantiomeric compounds, aromatic amino acids, amino acid derivatives, aromatic sulfoxides, coumarin derivatives, benzoin, and benzoin derivatives have been accomplished on the albumin columns. The a-acid glycoprotein protein column has been used to resolve 50 enantiomeric drugs [288]. The mobile-phase requirements of these sorbents have been reviewed [288]. Although the selectivity of these sorbents are often outstanding, the solute capacity is only 1 nmol per injection. 

RAPID ANALYSIS OF PEPTIDES AND PROTEINS. Columns packed with micropellicular stationary phases can be utilized in many areas of protein chemistry and biotechnology where high analytical speed and resolution are required. Peptide mapping by HPLC is routinely employed for the determination of structure of proteins (2, 3,32). 

Figure 8. Fast analysis of proteins. Column Hy-Tach micropellicular C-18 silica, 30x4.6mm eluent A, 0.1% (v/v) TFA in water, eluent B, 95% (v/v) ACN in water containing 0.1% (v/v) TFA flow rate, 4.0 ml/min. temp., 80°C. Initial column inlet pressure, 260 bars sample, 15 pi of a mixture containing =1 pg each of ribonuclease A (1), cytochrome C (2), lysozyme (3) and P-lactoglobulin A (4). Elution was carried out with a gradient 20 to 50% B in 0.5 min and return to starting conditions in 0.1 min. The dotted line represents gradient profile of ACN. The analysis was carried out with the house built protein analyzer described under Figure 7.

Figure 10. Comparison of micropellicular and porous stationary phases for rapid separation of proteins. Columns Micropellicular, Hy-Tach C-18 silica, 30x4.6mm, (A) and porous, Vydac, 5 im C-4 silica,30x4.6mm, (B). Initial column inlet pressure for the porous column was 105 bars. Experimental conditions were same as described under Figure 8...

The 1-125 protein column (operated in a normal-phase mode) was chosen as the final purification step for two reasons 1) the small (sometimes invisible) amount of residue remaining after the previous purification steps was easily dissolved in 95% acetonitrile 5% water (the initial-conditions solvent for this system) and 2) the peaks collected from this column were in a solution (75%-85% acetonitrile) suitable for peptide stabilization and required no further manipulation (other than capping) prior to storage. The structures of the 11 myotropic cockroach peptides isolated with this method are listed, along with appropriate references, in Table I (leucokinins... 

Fig. 17. High-speed gel permeation separation of the tryptic peptides of human thyro-globulin on dual Waters 1-125 protein columns using a mobile phase comprised of 1 M urea-0.1% HaPO, pH 3.5, at a flow rate of 0.5 ml/min. Reproduced from Hearn era/. (170).

The accurate determination of the adsorption isotherm parameters of the two enantiomers on a CSP is of fundamental importance to do computer-assisted optimization to scale up the process. Such determinations are usually done with an analytical column and the most traditional method to determine the parameters and saturation capacity is by frontal analysis (see section 3.4.2). The aim of paper III was to investigate the adsorption behavior and the chiral capacity of the newly developed Kromasil CHI-TBB column using a typical model compound. Many of the previous studies from the group have been made on low-capacity protein columns which has revealed interesting information about the separation mechanism [103, 110, 111], For this reason a column really aimed for preparative chiral separations was chosen for investigation [134], As solute the enantiomers of 2-phenylbutyric acid was chosen. 

To test the binding of biotin-bearing Rh-PE-labeled, TATp-bearing liposomes before and after incubation at lowered pH values, the corresponding samples are kept for 3 h at pH 7.4 or 5.0 and then applied onto the Immobilized NeutrAvidin protein column (rrr Note 6). 

Prepare Immobilized NeutrAvidin protein column well ahead so that it would stabilize as per manufacturer s (Pierce) manual for at least 30 min of application of samples. 

Figure 16.5 Affinity chromatography of peroxidase (after A. Borchert, P.O. Larsson and K. Mosbach, J. Chromatogr., 244, 49 (1982)]. Conditions sample, 4.1 ml of solution with 1 pg ml of protein column, 5cm x 5 mm i.d. stationary phase, concanavalin A on LiChrospher Si 1000, 10pm mobile phase, 0.05M sodium acetate, 0.5 M sodium chloride, 1 mM calcium chloride, 1 mM manganese chloride, pH 5.1 t shows pulse of 4.1 ml of 25 mM a-methyl-o-glycoside detector, solid line, UV, 280 nm broken line, visible, 405nm. Peaks = nonretained proteins this peak contains less than 2% peroxidase 2 = peroxidase.

Porous silica Zorbax PSM Li Chrospher Li Chrospher Diol Synchropak GPC Chromegapore Sphere sil p-Bondagel E Protein Column TSK Type SW... 

Insulin Bondagel E-12S, Toya-Soda G2000 SW and G3000 SW and Waters 1-125 protein column Various buffers At 280 nm 112... 

Chemically modified silica gel. Waters also sells Protein Columns 1... 

In some ways, analyses of these profiles are disappointing since they indicate that the adsorption portion of the elution profile does not provide a great deal of information concerning the adsorption kinetics. However, analysis of these frontal profiles has allowed us to examine protein-protein exchange processes that occur as the protein solution passes through the column. This has been accomplished by collecting and analyzing fractions by HPLC (Waters Associates 1-250 protein column). 

Immobilised protein columns. A further group of chiral stationary phases has been developed by immobilising naturally occurring asymmetrical peptides, e.g. bovine serum albumin, ai-acid glycoproteins, ovomucoid and... 

Values of AT as a function of flow rate were measured for the SEC separation of 12 proteins (12), using both denaturing and nondenaturing conditions, and columns of two different pore diameters (1S- and 30-nm nominal pore diameters). Comparison of these experimental data with the above model for SEC allows values of p to be derived for each protein/column combination, for either native or denatured conformations of the protein. ... 

FIGURE 5.17 Effect of ACN concentration in the eluent on the separation of four proteins. Column, 39 cm (effective length 29 cm) x 50 p.m i.d., fused-silica capillary with porous methacrylic monolith having tertiary amino functions mobile phase, ACN (%, v/v) in 60 mM aqueous sodium phosphate, pH 2.5 applied voltage, —25 kV detection, 214 nm sample (1) ribonuclease A, (2) insuhn, (3) a-lactalbumin, and (4) myoglobin. (Reprinted from Zhang, S., et al., J. Chromatogr. A, 887,465-477, 2000. With permission from Elsevier.)... 

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