Fractionation range

Fig. 6. Fractionation ranges of commercially available gel-filtration matrices (D) small, ( f- ) medium, and ( . i) large (31).

This indicates that the target efficiency of the fiber is increased by the proximity of other fibers. The value of Ka averaged 4.5 for values of the void fraction ranging from 0.90 to 0.99. Extending use of the equation to values of , lower than 0.90 may result in large errors. 

Sephadex type Grade Dry bead diameter (/urn) Fractionation range peptides and proteins (g/mol) Fractionation range dextrans (g/mol) Swelling factor (ml/g dry Sephadex) Maximum operating pressure" (cm H,0) Permeability Ko Maximum linear velocity" (cm/hr) Swelling time (h) ... 

Bead Composition (% agarose) Size exclusion pore dimension° (nm) Fractionation range Maximum Maximum operating linear velocity (cm/hr) ... 

Type Bead size djo (/xm) Size exclusion pore dimension (nm) Exclusion limit protein (M,) Fractionation range protein (/V ,) pH stability (long term/ short term)... 

Bead size dso (/im) Size exclusion Exclusion Fractionation range pH stability... 

II. INTRODUCTION OF PolyHYDROXYETHYL ASPARTAMIDE AND THE EFFECT OF CHAOTROPES ON THE FRACTIONATION RANGE... 

A SEC material should be hydrophilic if it is to be used for biological applications. One such material, introduced by PolyLC in 1990 (8), is silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) the trade name is PolyHYDROXYETHYL Aspartamide (PolyHEA). This material was evaluated for SEC of polypeptides by P.C. Andrews (University of Michigan) and worked well for the purpose (Fig. 8.1). Because formic acid is a good solvent for polypeptides, Dr. Andrews tried a mobile phase of 50 mM formic acid. The result was a dramatic shift to a lower fractionation range for both Vq and V, (Fig. 8.2) to the point that V, was defined by the elution position of water. 

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

Controlling for these forces requires variation in the amount of salt, organic solvent, and the pFI of the mobile phase. It is impractical to perform such experiments with 50 mM formic acid an alternative additive must be used that maintains its chaotropic properties independent of salt content or pFI. Fortunately, mobile phases containing 50 mM hexafluoro-2-propanol (HFIP) afford a fractionation range comparable to that of the formic acid (Fig. 8.6), permitting the effects of these variables to be studied systematically. 

IV. FRACTIONATION RANGE WITH PolyHEA AS A FUNCTION OF PORE DIAMETER... 

Another important parameter for column selection is the proper choice of sorbent porosity. The pore size of the sorbent determines the fractionation range of the column. The best way of doing this is by looking at the calibration curves of the columns, which are normally documented by the column vendor (cf. Fig. 9.3 for PSS SDV column calibration curves and PSS SDV fractionation ranges) (7). 

If two columns with different porosity are used in a column combination, the efficiency of the separation will not change much, but the fractionation range will be increased immensely. In this case, longer chromatography times allow a better separation of broader samples (or samples with high and low molar mass components). 

The linear column (PSS SDV 5 /mm linear) has a wider molar mass fractionation range while keeping the analysis time roughly the same. Therefore the slope of the calibration curve is much steeper and the resolution will be poorer in this case. The second column with a single pore size (PSS SDV 5 /mm 1000 A) separates only below 50,000 Da, but does this very efficiently in the same time. 

TABLE 16.3 Producers (Pharmacia Biotech) Specification of Fractionation Ranges of Cross-Linked Allyl Dextran/N,N -Methylene Bisacrylamide Copolymer-Based Sephacryl Gels for Dextrans... 

TABLE 16.4 Producers (Merck) Specification of Fractionation Ranges for Polyethyiene Giycoi Dimethylacryiate-Based Fractogei HW Geis... 

TABLE 16.8 Producers (Bio-Rad) Specification of Fractionation Ranges of Polyacrylamide-Based Bio-Gel P Gels... 

In the table,. Ys.min and. Y2.max give the mole fraction range over which the parameters a and a apply. 

Samples were fractionated by size on stainless steel columns packed with TSK-GEL ToyoPearl gel filtration medium, (Supelco Inc., Bellefonte, PA) in 50 mM NH4AC, pH 5.2. For HW-55 S (fractionation range for dextrans 1000-200,000 daltons) and HW-50 S (fractionation range for dextrans 500-20,000 daltons), the column was 10 x 500 mm and the flow rate was 1 ml/min. For HW-40 S (fractionation range for dextrans 100-7000 daltons),... 

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