Waters AccellPlus QMA and CM Bulk Media - Manual (Page 2)

[ Care and Use ManUal ]
Waters AccellPlus QMA and CM Bulk Media 2
I. calculatIng bulk medIa needs
AccellPlus ion exchange media can be used for batch processes or can
be packed into columns. Columns may be dry- or slurry-packed using
AccellPlus media.
The amount of ion exchange medium and the column size to be used
depends on the amount of sample to be purified. Typically, optimum
performance is achieved when the total sample amount represents
less than 20% of the protein capacity of the AccellPlus medium used
(Tables 2 and 3). Samples exceeding 20% of the column’s maximum
protein binding capacity can be applied with somewhat lower than
optimal resolution of the components contained in the mixture. The
demands of the separation will determine the sample load for amount
of AccellPlus medium used.
Table 2. AccellPlus QMA - Protein Binding Capacity
Measured using Bovine Serum Albumin in 0.02 M Tris/HCl buffer.
pH Mg BSA/gm Accell ® (± 5%)
6.0 122
6.5 180
7.0 180
7.5 171
8.0 105
8.5 62
Protein binding capacity varies considerably based on selected
protein, buffer type, and pH.
Table 3. AccellPlus CM - Protein Binding Capacity
Measured using Cytochrome C in 0.02 M sodium phosphate.
pH Mg Cytochrome C/gm Accell (± 5%)
5.0 157
6.0 171
6.5 172
7.0 155
Note: For best results do no exceed 20% of protein binding capacity.
II. packed column use
Rigid AccellPlus particles greatly simplify column packing compared
to use of many traditional soft gel media. Columns can be easily
packed using either slurry- or dry-column packing techniques.
To pack a column with AccellPlus media:
The bulk density of AccellPlus is 0.5 g/mL. Thus, 1 gm of media will
produce 2 mLs of packed column bed.
AccellPlus media required (grams) = Required column volume (mL)
2
a. Slurry Column Packing
Slurry packing using methanol solvent provides an efficient means
to column packing. We strongly recommend that columns greater
than 5 cm in diameter be slurry packed. Methanol slurry packing
produces more efficient columns providing better peak shapes than
slurry packing using dilute buffer or water eluents. Typically a
3-5:1 v/v methanol:AccellPlus slurry should be used. Pour slurry
directly into the column with the outlet attached to a water aspirator.
Alternatively, the columns can be packed using conventional HPLC
slurry packing techniques. AccellPlus media can withstand pressure of
up to 2000 psi without any problems.
b. Dry Column Packing
To dry pack AccellPlus media into any column follow these steps:
1. Pour the media slowly into the column while either tapping the
side or the base of the column. This will help the media settle
into a densely packed bed.
2. Once the column has been packed, flow mobile phase through
the column for 2-3 minutes at as high a flow rate as the pump
or column pressure limit will allow. This will settle the column
bed slightly. Voids should be filled by stirring the packing at the
column inlet and topping off with a thick slurry media.
3. Adjust the plunger or tap up with media to adjust voids. Repeat
this procedure until a stable column bed is obtained.
c. Column Equilibration
1. Wash the column with 5 column volumes of dilute buffer.
Note: If the column has been packed in methanol, flush with 5
column volumes HPLC-grade water prior to the dilute buffer wash.
2. After the dilute buffer wash, wash the column with 5 column
volumes of concentrated buffer.
3. Wash the column with starting buffer until the pH and ionic
strength of the column eluate match those of the solvent being
pumped onto the column.