Waters Protein-Pak HR Ion-Exchange Glass Columns - Manual (Page 2)

[ Care and Use ManUal ]
Protein-Pak HR Ion-Exchanged Glass Columns 2
for general laboratory and VIt ro dIagnostIC
use
a. Protein-Pak HR Series Description
The Protein-Pak HR series packing material support matrix consists
of a spherical, hydrophilic, rigid polymeric gel. The materials
have covalently-bonded, charged functionalities on the polymeric
support, as outlined In Table 1. The packing materials have fully
porous, 1000Å pore-size particles. Biomolecules with up to 10
6
molecular weight penetrate the pores and interact with the bonded
functionalities.
Table 1: Physical Description of Protein-Pak HR Series
Table 2 lists the physical characteristics of the Protein-Pak HR Series
Table 2: Physical Characteristics of Protein-Pak HR Series
b. Theory of Ion Exchange
Ion-exchange chromatography is based on separating ionic
compounds by net charge. Two types of ion exchangers are
available:
Anion Exchangers - Positively-charged supports and negatively-
charged counter ions that bind negatively-charged molecules.
Cation Exchangers - Negatively-charged support and positively-
charged counter ions that bind positively-charged molecules.
A variety of covalently-bonded charged functionalities are available
for both anion and cation exchange. The type of immobilized func-
tionality determines the strength of the ion exchangers.
For example, quaternary amino and sulfonic acid functionalities are
considered strong ion exchangers because these are completely
ionized over a large pH range. Primary amino and carboxymethyl
moieties are considered weak ion exchangers because the degree
of ionization of these is a function of the pH. Weak ion exchangers
are thus limited to a narrower pH range. Both strong and weak ion
exchangers have unique capabilities and advantages as they relate to
individual applications.
Ion-exchange separation involves the following process:
1. The column is first equilibrated with starting buffer.
2. The separation is performed by applying the sample (dissolved in
starting buffer) to the column.
3. Unbound material is washed through the column with starting buffer.
4. Bound, charged molecules have varying association constants for
the ion exchanger due to the differences in their charges and charge
densities. Elution of bound substances is altered by increasing salt
concentration, changing pH, or both.
c. Choosing an Ion-Exchange Column
Choose a starting buffer and its pH to determine the net charge
of amphoteric compounds. Amphoteric compounds can be either
positively or negatively charged, such as proteins. If the net charge
is zero at a particular pH value, it defines the isoelectric point (pl) of
that protein. At this isoelectric point, the protein should not bind to
any ion exchanger.
Because binding takes place due to surface-charge distribution of
the molecule, some proteins bind even at their isoelectric point. At a
particular pH, if the protein of interest is negatively-charged overall
(ph ≤ 7), use an anion-exchange column. If an overall positive
charge (ph≥ 7) resides on the molecule, use a cation-exchange
column. If the charge or pi is unknown, use an anion-exchange
column.
Note: The stability of a particular protein may limit the choice of pH
used and hence the choice of ion exchanger.
Media Type Functionality
DEAE Weak anion exchanger Diethyiaminoethyl
Q Strong anion exchanger Quaternary methylamine
SP Strong cation exchanger Propylsulfonic acid
CM Weak cation exchanger Carboxymethyl
Media Nominal Ligand
Density
Nominal Protein
Binding Capacity*
Nominal Protein
Recovery
DEAE 250 µeg/mL 40 mg/mL >85%
Q 200 µeg/mL 60 mg/mL >85%
SP 225 µeg/mL 40 mg/mL >85%
CM 175 µeg/mL 25 mg/mL >85%
*Conditions:
DEAE and Q: Bovine Serum Albumin 20 mM Tris/HCL pH 8.2
SP and CM:
Cytochrome C 20 mM MES pH 5.0