Protein losses with retention time

Table II. Protein Losses with Retention Time for Cytochrome C at 85 C.

The methacrylic backbone structure makes the spherical Toyopearl particles rigid, which in turn allows linear pressure flow curves up to nearly 120 psi (<10 bar), as seen in Fig. 4.45. Toyopearl HW resins are highly resistant to chemical and microbial attack and are stable over a wide pH range (pH 2-12 for operation, and from pH 1 to 13 for routine cleaning and sanitization). Toyopearl HW resins are compatible with solvents such as methanol, ethanol, acetone, isopropanol, -propanol, and chloroform. Toyopearl HW media have been used with harsh denaturants such as guanidine chloride, sodium dodecyl sulfate, and urea with no loss of efficiency or resolution (40). Studies in which Toyopearl HW media were exposed to 50% trifluoroacetic acid at 40°C for 4 weeks revealed no change in the retention of various proteins. Similarly, the repeated exposure of Toyopearl HW-55S to 0.1 N NaOH did not change retention times or efficiencies for marker compounds (41). 

FIGURE 7.4 Separation of a standard protein mixture on a Fractogel EMD BioSEC-column (600-16 mm) after incubation with 30% acetonitrile. The sample contained BSA ( ), ovalbumin ( ), and cytochrome c (A) (sample volume 500 ftl flow rate 1.0 ml/min). No significant shifts of the retention times and no loss of the resolution were observed even after 900 hr of exposure. 

These high performance size exclusion separations of alpha-chymotrypsin SI and myosin light chains compare favorably with those achieved by ion exchange chromatography but require only a fraction of the time to accomplish. Furthermore, the very short retention times allow for separation of these labile proteins at room temperature, whereas operation at 0-40 C would otherwise be mandatory to avoid the loss of enzymatic activity. 

The flow rate of the mobile phase is an important parameter which determines the elution time. In the dipeptide separation, the optimum flow rate producing the best peak resolution is present in all spiral disks. In the four-spiral disk systems (columns III and IV), which retain a large volume of the stationary phase, the application of a high flow rate of up to 10 mL/min is possible to reduce the separation time, without substantial loss of peak resolution. In the separation of proteins with PEG-phosphate, the low flow rate produced the best peak resolution with higher retention of the stationary phase. 

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