Elution behaviour of some proteins on fresh, acid- or base-treated Sephacryl S-200 HR.
The influence of sodium chloride concentration and the pH of the mobile phase on the distribution coefficient of proteins with different pI values was studied on Sephacryl S-200 HR. The non-size-related behaviour of this gel filtration packing is mainly attributed to small amounts of groups that are negatively charged within the pH range investigated (4.2-10.0). These anionic groups on the packing gave rise to ion-exchange or ion-exclusion interactions depending on the charge characteristics of the protein. Hydrophobic interactions at high ionic strength and intramolecular electrostatic repulsive interactions at low ionic strength were also observed for some proteins. The chemical stability of Sephacryl S-200 HR was studied by comparing the chromatographic results with Sephacryl S-200 HR that had been treated in acidic or basic solutions with those with fresh Sephacryl. After Sephacryl S-200 HR had been stored for 2 weeks in 0.10 M sodium hydroxide the chromatographic results at low ionic strengths clearly showed that groups that are positively charged at pH 4.2 had been formed. However, storage for 2 weeks in 0.01 M hydrochloric acid did not change the chromatographic behaviour of the proteins from that observed when injected on fresh Sephacryl S-200 HR.
Johansson
B L
BL
Department of Quality Control, Pharmacia LKB Biotechnology AB, Uppsala, Sweden.
Gustavsson
J
J
D000180:Acrylic Resins; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002919:Chymotrypsinogen / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D009113:Muramidase / Q000302:isolation & purification; D009211:Myoglobin / Q000302:isolation & purification; D010434:Pepsin A / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D012965:Sodium Chloride; D014168:Transferrin / Q000302:isolation & purification
D000180:Acrylic Resins; D007202:Indicators and Reagents; D009211:Myoglobin; D011506:Proteins; D014168:Transferrin; D012710:Serum Albumin, Bovine; D012965:Sodium Chloride; C023049:sephacryl S 200; D002919:Chymotrypsinogen; D009113:Muramidase; D010434:Pepsin A