3.3 Implementation
of HT Column
Chromatography
The step-by-step methodology below describes the implementation of HT column chromatography for testing various buffered
conditions against rotavirus antigens using different multimodal
chromatography (MMC) resins.
1. Fill the Reagents plate (Fig. 1a) with 4 mL of the listed buffers
(Table 2) (see Note 14).
2. The elution plates (ElPlates1–ElPlates4 in Fig. 1a) are 96-well
deep square well plates. Fill the plates with 2 mL of buffers.
These buffers are prepared to return mobile phase conditions
(e.g., pH, buffer concentration and species, salt concentration
and type, additives) that are believed to affect the separation
and from which an optimal condition will be identified. Listed
in Table 3 are the selected buffered conditions used for the
MMC columns.
3. Once all of the plates are in their positions as shown in Fig. 1a.
Place the 96-well microplates (Collection plates) in the hotels.
Fill the tip sanitization trough (Fig. 1a) with 0.5 M sodium
hydroxide.
4. Launch the EVOware software and select the purification script
for 0.6 mL miniature column purification (Appendix 1). The
choice of column size, residence time (see Note 15), and column volume (CV) for each buffered step is decided (Table 4).
This can be selected depending on your requirements.
Table 2
Contents of 48 well deep-well square Reagents plate
Row
Column
1 (Equilibration) 2 (Wash) 3 (Wash) 4 (Strip) 5 (CIP)
6 (Storage)
A(RC1) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
B(RC2) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
C(RC3) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
D(RC4) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
E(RC5) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
F(RC6) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
G(RC7) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
H(RC8) Equilibration
Load
Wash
Strip
1 M NaCl, 0.5 M NaOH 20% EtOH
Each row of the plate (A–H) corresponds to RoboColumn (RC1–RC8)
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Shaleem I. Jacob et al.