Waters Protein-Pak Epoxy-Activated Affinity Products - Manual (Page 2)

[ Care and Use ManUal ]
Protein-Pak Epoxy-Activated Affinity 2
Figure 1: Epoxy-Activated Affinity Process
c. Description of the Products
Epoxy-activated packings are available in bulk and in Micro-Column
configurations. Bulk material, the Micro-Column, and conditions for
coupling the ligand are described below.
Bulk Material
The bulk packing material is available in 25 and 100 gram amounts.
Larger quantities are available on request. The material is shipped as
a dry white powder and should be stored dry at 4 °C. The bulk density
of the material is about 0.5 g/mL, so about 0.5 grams of material
is required per mL of column volume.The epoxide content is greater
than 100 μmoles/gram.
Micro-Column
The Micro-Column contains 0.5 mL of Protein-Pak Epoxy-Activated
Affiinity packing with 50 to 100 μmoles of available activated sites.
The Micro-Column volume is 3 mL, resulting in a 2.5 mL reservoir.
The outlet has a filter. The top of the bed does not have a filter. The
Micro-Column housing is polypropylene which is stable to all normal
coupling temperatures (4 °C to 45 °C) and pHs (pH 2 to 9.5).
Ligand Coupling
Couple ligands to the epoxy-activated packing by following the
procedures for Coupling Conditions covered in Section II. Unreacted
epoxy groups on the coupled material may interfere with subsequent
use in a chromatographic separation. After the affinity ligand is
immobilized, the remaining epoxy groups can be blocked by reac-
tion with amino compounds of low molecular weight (for example,
glycine, tris buffer, or ethanolamine). The derivatized material is
ideal for use in either batch processes or in packing columns.
II. use of epoxy-ACtIvAted AffInIty pACkIngs
a. Coupling Buffers
Prepare buffers and solvents using the following general guidelines.
– Carbonate, borate, and phosphate buffers are preferred.
Sodium chloride or other non-nucleophillic salts may be
added to achieve a desired ionic strength.
– Do not carry out the coupling in tris, glycerine or other
nucleophillic buffers. These buffers will react with the epoxy
groups.
b. Coupling Conditions
This section outlines general procedures for coupling a ligand to the
material. Table 1 lists general conditions for the covalent binding
of ligands based on different functional groups. The temperature
limitation of the coupling reaction depends on the stability of the
ligrand; generally ligands can be coupled to the support between
4 °C and 55 °C.
CAUTION: Bonded phases on silica are susceptible to hydrolysis at
extreme pHs. When using the material at pH 2 or pH > 9.5, bonded
phase hydrolysis may occur and result in non-specific chromatographic
effects.
Spacer Arm
Epoxide Ligand
Step 1: Ligand Coupling
Step 4: Washing Out Impurities
Step 2: Loading Sample Mixture Step 3: Adsorbing Target Material
Step 5: Eluting Target Material Step 6: Re-Equilibrate and go to Step 2