[ 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 Particle Size Pore Size Qty. Part No. AccellPlus QMA 40 µm 300Å 100 g WAT010742 Anion Exchanger 40 µm 300Å 500 g WAT010741 AccellPlus CM 40 µm 300Å 100 g WAT010740 Cation Exchanger 40 µm 300Å 500 g WAT010739 Waters AccellPlus QMA and CM Bulk Media 1
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. Required column volume (ml) AccellPlus media required (grams) = 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. Waters AccellPlus QMA and CM Bulk Media 2
The AccellPlus column should now be tested (see Packed Column Testing). Use methanol as the mobile phase to run the efficiency and asymmetry test. d. Packed Column Testing Waters recommends that columns be tested prior to use. There are many different ways of doing this, but one of the simplest is to inject a small volume of a 500:1 buffer:acetone solution. This will absorb effectively at 280 nm. The peak should be symmetrical. A typical calculated plate count an AccellPlus IEX slurry packed, 1 x 10 cm column should be greater than 500 plates. Lower observed values would be normal for columns that are packed using the less efficient, dry packing process. N = 16 Rt 2 W III. Bulk Media Use Rt = retention time in minutes W = peak width at baseline in minutes Determine the amount of bulk ion exchange media required for application needs (see Section II, Table 2 or Table 3). Slurry the media with 5 times the media s volume of dilute buffer for 2-3 minutes. The media should be allowed to settle and then the excess buffer drained off. A similar volume of concentrated buffer (0.1 M) should be added, slurried up 2-3 minutes, and then drained off. The same procedure should be repeated with dilute buffer until ph and salt concentration remain at the desired values. IV. Additional Information a. Chemical Compatibility AccellPlus is fully stable in aqueous buffers between ph 2-9. The following reagents are compatible with AccellPlus media for use in cleaning and as anti-microbials. 10% acetic acid (ph 2.5) 2.5 M sodium acetate (ph 8) 20% aqueous propanol, 0.1% trifluoroacetic acid 5 M urea 0.5% Hibitane (chlorohexidine) 5 M urea 1% Triton X-100 0.1% sodium azide 100% methanol 100% acetone 100% ethanol 0.1% sodium azide 70% ethanol 2 M sodium chloride 0.25 M hydrochloric acid (limited) 0.1 M sodium hydroxide (limited) b. Sanitization 70% ethanol, 0.10% sodium azide or 0.5% Hibitane are effective sanitizing agents. c. Regeneration Clean protein solutions, carefully filtering of buffers, and occasional column regeneration will yield 100 or more runs. Occasionally, a reduction in capacity is observed. When this happens, the column should be regenerated by pumping 5 column volumes of 2 M sodium chloride at ph 7.5 for the AccellPlus QMA media and ph 4 for the AccellPlus CM media. More aggressive cleaning solutions may be found in Table 4. Table 4. Suggested Cleaning Solutions Agent Concentration Exposure Sodium Hypochlorite 1000 ppm 2 hours Hydrochloric Acid 0.25 M less than 30 minutes Sodium Hydroxide 0.1 M 3 column volumes pump through in less than 20 minutes d. Short-Term Storage (less than 72 hours) AccellPlus media does not require special storage or treatment for periods less than 72 hours. We recommend, however, that the columns be stored at 4 C. e. Long-Term Storage (more than 72 hours) Although AccellPlus media is not susceptible to microbial attack, an anti-microbial such as Hibitane, sodium azide or 70% ethanol can be added (see Chemical Compatibility). If the medium is to be stored dry, pump the medium out of the column, wash with 10 column volumes of 1 M sodium chloride, 10 column volumes of HPLC-grade water, and 10 column volumes of methanol. Dry the media under vacuum at 80 C. Waters AccellPlus QMA and CM Bulk Media 3
f. Trouble Shooting Problem Cause Remedy Poor Resolution Sample overload Reduce sample amount (see Bulk Media Packing). Flow rate too high Reduce flow rate. Media fouling Reverse flow direction. Flush with suitable cleaning agent (see Chemical Compatibility). Column voiding Check efficiency, and if low, repack or fill column void. Low Flow Rate Media fouling Reverse flow direction. Flush with suitable cleaning agent (see Chemical Compatibility). Frits plugged Reverse flow. Replace connector frits. Low Capacity Salt concentration too high Reduce salt concentration. ph too high (QMA) Lower ph if protein stability is not affected. ph too low (CM) Raise ph if protein stability is not affected. Inappropriate buffer salt Change buffer salt. Waters AccellPlus QMA and CM Bulk Media 4
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