Handbook of Size Exclusion Chromatography and Related ...

the importance of secondary retention mechanisms for several commercial GFC
columns. As discussed, after derivatization with a hydrophilic bonded phase,
silica-based packings exhibit residual and accessible silanol groups that dissociate
within the usable pH range as a function of the pretreatment of the base silica. It
was found that the pH of a solution of TSKgel G3000SW packing material in
0.5 M NaCl was slightly below 5 and that the number of dissociated silanol groups
reached 0.013 meq/mL packing material at pH 8 (100). As a consequence, a basic
solute, such as arginine, or a protein, such as lysozyme, is retained longer than
expected because of interaction with the negatively charged silanol groups; acid
proteins or small acids, such as citric acid, are repelled from the surface and elute
earlier than expected based on their size. This is illustrated in Table 8, in which the
distribution coefficients for citric acid and arginine are listed for various
commercial columns as a function of the ionic strength of a pH 7.05 phosphate
buffer (33). Normal SEC behavior for citric acid and arginine, that is, elution from
Table 8 KD Values for Citrate, Arginine, and Phenylethanol as a Function of Ionic
Strength for Commercial Silica-Based Gel Filtration Columns a
Solute and
ionic strength
TSKgel
G3000SW
LiChrosorb
Diol
SynChropak
GPC 100
TSKgel
G2000SW
Waters
I-125
Citrate
m ¼ 0:026 0.66 0.54 0.46 0.43 0.39
0.12 0.89 0.81 0.76 0.75 0.72
0.24 0.92 0.95 0.89 0.84 0.79
2.40 0.94 0.99 0.91 0.88 0.88
Arginine
m ¼ 0:026 1.30 1.53 1.35 1.57 1.70
0.12 1.05 1.15 1.06 1.06 1.23
0.24 1.02 1.05 1.01 1.02 1.16
0.60 1.00 0.99 — 0.99 1.08
2.40 0.98 1.07 0.98 0.98 1.00
Phenylethanol
m ¼ 0:026 1.47 2.49 1.44 1.95 1.83
0.12 1.50 2.56 1.49 2.02 1.88
0.24 1.53 2.64 1.53 2.10 1.88
0.60 1.61 2.93 1.63 2.30 2.03
1.20 1.81 3.52 1.81 2.71 2.29
2.40 2.35 5.31 2.35 4.01 3.03
a The distribution coefficient KD (or KSEC) is defined by VE ¼ Vi þ KDVP, in which VE is the solute
retention volume, Vi the interparticle or interstitial volume, and VP the pore volume. Mobile phase:
pH 7.05 phosphate buffer of indicated ionic strength.
Source: Ref. 33.
© 2004 by Marcel Dekker, Inc.