Waters AccellPlus QMA and CM Bulk Media User Manual

[ Care and Use ManUal ]
Waters AccellPlus QMA and CM Bulk Media
I. calculatIng bulk medIa needs
II. packed column use
a. Slurry Column Packing
b. Dry Column Packing
c. Column Equilibration d. Packed Column Testing
III. bulk medIa use
IV. addItIonal InformatIon
a. Chemical Compatibility
b. Sanitization
c. Regeneration d. Short-Term Storage (less than 72 hours) e. Long-Term Storage (more than 72 hours) f. Troubleshooting
Waters AccellPlus™ QMA anion exchange and Waters AccellPlus CM cation exchange packing materials are polymer-coated, silica-based media of 37-55 µm particles with a 300Å pore size. AccellPlus media is prepared by a patented copolymerization process that encapsulates the rigid silica base with a hydrophilic bonding layer and a highly stable cross-linked functional layer. AccellPlus media provides excellent recovery and high resolution of bio-molecules. The rigid and non-compressible structure of AccellPlus media makes it well suited for the purification and isolation of proteins, enzymes and immunoglobulins, particularly when preparative or process scale-up is intended.
AccellPlus ion exchange media is available in 100 gm and 500 gm bottles (Table 1). Larger quantities are available on request. The media is shipped as a dry white finely divided powder and can be stored dry.
Table 1. AccellPlus Ion-Exchange Bulk Packings
Particle
Description
AccellPlus QMA
Anion Exchanger
AccellPlus CM
Cation Exchanger
Particle
Size
40
µm 300Å
40
µm 300Å
40
µm 300Å
40
µm 300Å
Pore Size
Qty. Part No.
100 g WAT010742 500 g WAT010741 100 g WAT010740 500 g WAT010739
Waters AccellPlus QMA and CM Bulk Media 1
[ Care and Use ManUal ]
Required column volume (mL)
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
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) =
2
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.
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.
Waters AccellPlus QMA and CM Bulk Media 2
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.
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