Introduction

AMBERCHROM™ chromatographic resins are macroporous, polymeric resins for adsorption and reversed phase liquid chromatography. They are designed for laboratory and process scale purification of proteins, peptides, nucleic acids, antibiotics, and small molecular weight pharmaceuticals. Amberchrom™ CG161, CG300 and CG1000 are based on polystyrene-divinylbenzene polymers, and Amberchrom™ CG71 is based on acrylic polymers. All of these are in slurry form in quantities ranging from 25ml to 50 liter containers (Figure 1).

This brochure addresses the optimum procedures for packing these resins in lab-scale columns. For assistance in packing larger process-scale columns, or for any additional information, please contact Rohm and Haas. Amberchrom™ resins are supported by MRS (material regulatory support) documents, support you use in regulated applications and can be customized for your specific application.

Preparation for Packing

GENERAL CONSIDERATIONS FOR PACKING AMBERCHROM™ CHROMATOGRAPHIC RESINS

The best results will be achieved by packing Amberchrom™ resins by the application of gentle pressure from 0.5 to 10 bar (7 to 150 psi). Rohm and Haas does not recommend packing Amberchrom™ by simple gravitational settling.

Amberchrom™ resins are supplied in a slurry form in an aqueous (20%) ethanol solution. For best packing results, it is recommended to use 10-20% organic solvent (ethanol, methanol, isopropanol, acetonitrile, etc.) solution with 0.1% acid (TFA, sulfuric or acetic) as a packing buffer. Do not use an oxidizing acid such as nitric acid! Nitric acid can react violently with polymers.

REMOVAL OF FINES

When working with any resin, good chromatographic practice requires the removal of fines because these could block frits/filters and increase the pressure drop across the column. The following decantation process may be required to remove fines prior to packing the column.

1. Resuspend the resin in its original container by shaking it.
2. Pour the resin into a suitable vessel for defining. The length of time required for settling depends on the geometry of the vessel, the amount of dilution, type of buffer, and the particle size of the resin.
3. Decant or suction off the supernatant from the settled resin.
4. Add fresh buffer and resuspend the resin with gentle overhead stirring. Do not use a magnetic stirrer or sonication since these methods will generate fines.
5. Allow the resin to settle again; decant as above.
6. Repeat the above steps until fines appearance is minimal. Depending on the particle size of the resin, this may require from 1 to 24 hours.

SLURRY PREPARATION

The slurry concentration is defined as the volume of the settled gel divided by the total volume of the slurry. Rohm and Haas recommends a slurry concentration of 50-65% for optimal column packing results. The slurry concentration can be adjusted as follows:

1. Re-suspend the resin in its original container by using gentle agitation.
2. Pour an aliquot of the re-suspended resin into a graduated cylinder to measure the total slurry volume as shown in Figure 2.
3. Pour the aliquot into a glass column equipped with a vacuum source as shown in Figure 3.
4. Attach the bottom of the column to a suitable vacuum source as shown in Figure 3.
5. Apply sufficient vacuum to remove the excess liquid. When the liquid has been removed, calculate the dried resin volume. See Figure 3. Calculate the resin slurry: (dry resin volume/total slurry volume) x 100 = slurry concentration.
6. A slurry concentration of 50-65% is recommended for packing in standard columns. If needed, adjust the remaining resin slurry by either decantation/suction or addition of fresh mobile phase.
7. Once the total slurry concentration is determined, calculate the desired packed column volume. All polymeric resins will compact slightly when packed under pressure, and an excess of resin is typically required to pack a tight resin bed. Use 1.1X resin volume to pack the calculated column volume.

EQUIPMENT REQUIRED FOR PACKING
(Figure 4a and Figure 4b)

The following equipment is required to successfully pack Amberchrom™ resins:

pump– - (required for flow packing only) - The pump must be capable of delivering at least 2-3 times the operational flow rate desired. Pulseless flow pumps are recommended. The pump hardware must be compatible with the packing and chromatographic solvent.

pressure gauge - A pressure gauge is recommended for packing and operating the column. A gauge should be selected so that the target packing pressure is in the midrange of the gauge display.

column–- The column should be compatible with the packing and chromatographic solvent. Rohm and Haas does not recommend acrylic columns because of solvent incompatibilities. The column must be capable of radial or axial compression, and should have an adjustable flow adapter. Fixed head columns are not recommended due to resin shrinkage and expansion. Flow-packed columns, such as those manufactured by Millipore (Vantage and Moduline) and E. Merck have been used successfully with Amberchrom™ resins. Dynamic axial compression columns, such as those manufactured by TechniKrom and ProChrom, have also been used successfully with Amberchrom™ resins.

bed support - –A stainless steel or nylon mesh is preferable for a bed support. A disc support can be used; however, it will add to overall pressure drop of the column. The size of mesh support which can be used is as follows:

• "s" grade - 10 to 15mm
• "m" grade - 30 to 40mm
• "c" grade - 50 to 70mm

reservoir–- A packing reservoir may be used to pack the column, depending on the volume requirements of the resin slurry. If the volume of the resin slurry is greater than the volume of the column, a packing reservoir should be used. This will ensure that the column can be packed in one operation. Although not recommended, multiple pours of the resin slurry into the column can be performed.

Packing Procedures

PACKING METHOD - FLOW PACKED COLUMNS

We recommend a packing flow rate which is at least 2-3 times the nominal operating flow rate for lab scale columns. However, pilot and process scale columns have been packed successfully at less than two times the nominal operating flow rate. For assistance or information about packing larger columns, please contact Rohm and Haas at one of the numbers on the back page of this manual.

Table 1
Recommended Packing and Operational Flow Rates for Amberchrom™ Resins

1. Set up the column as shown in Figure 6a. The total volume of the column should be sufficient to allow the entire resin slurry to be poured in one operation
2. Wet the bottom frit or screen in the column with packing solvent, using a squirt bottle or reverse flow. Plug the outlet of the column and leave 1-2 cm of buffer in the bottom of the column (Figure 5).
3. Resuspend the resin slurry to assure homogeneity (Figure 6).
4. Pour the resin slurry slowly down the inside of the column to prevent air entrapment (Figure 7).
5. After the resin slurry has been fully transferred to the column, rinse the inside of the column using a squirt bottle containing the packing solvent (Figure 8).
6. Immediately place the flow adapter into the column (Figures 9 and 10). The adapter should go into the buffer without contacting the resin slurry. There should be no trapped air between the flow adapter and the buffer.
7. Open the column outlet, and start the pump.
8. Slowly ramp up to the target flowrate. This prevents hydraulic shock to the forming bed, and therefore prevents uneven packing of the column. The flowrate can be ramped up in several incremental changes. These increments will be determined by the size of the column and target flowrate. Some examples are listed below:
   
   
   

   Column size (cm ID x cm)	Media type	Target flow rate (ml/min)	Increment (ml/min)	Hold time (min)
   1.0 x 10	CG71 c	15	5	0.5
   2.2 x 30	CG161 m	40	10	1
   7.0 x 100	CG1000 s	200	40	2

   
   
   
9. After the bed has fully consolidated, shut off the pump, and close the column outlet.
10. If the column was packed without a reservoir, skip to Step #11. If a reservoir was used to pack the column, the entire bed should reside in the lower column section. Using a pipette or siphon tube, draw off all the supernatant from the upper reservoir, if necessary. Then remove the upper reservoir and the coupling ring. Carefully place the flow adapter into the column, and on top of the consolidated resin bed. Take care to avoid introduction of air into the column.
11. Carefully loosen the flow adapter seal and lower the adapter onto the resin bed. Take care not to disturb the resin bed when moving the flow adapter. The flow adapter should now rest gently on the resin bed. (Notice where the flow adapter rests in Figure 11.)
12. Open the column outlet, and start the pump as described in Step #8.
13. The resin bed should now be compressed further. After the bed has stabilized, shut off the pump, and leave the column outlet open. Adjust the flow adapter down onto the bed. The adapter should be pushed onto the bed until resistance is such that the adapter will not go any further. Be careful not to get any resin around the sealing ring. Close the column outlet.
14. The column is now ready for equilibration.

Packing Method - Dynamic Axial Compression Columns (DAC)

1. Set up the column as shown in Figure 4b. The total volume of the column should be sufficient to allow the entire resin slurry to be poured in one operation.
2. Wet the bottom frit or screen in the column with packing solvent, using a squirt bottle or reverse flow. Plug the outlet of the column and leave 1-2 cm of buffer in the bottom of the column.
3. Resuspend the resin slurry to assure homogeneity (Figure 12).
4. Pour the resin slurry slowly down the inside of the column to prevent air entrapment (Figure 13).
5. After the resin slurry has been fully transferred to the column, rinse the inside of the column using a squirt bottle containing the packing solvent.
6. Refer to the column manufacturer's instructions to complete the column packing operation. A pressure setting of 8-10 bar is recommended when packing a DAC column. Figure 14 shows the packing piston in place above the column prior to packing, and Figure 15 shows the piston lowered as the column is packed.
       

Column Equilibration

Amberchrom™ resins must be equilibrated after packing to ensure optimum column performance. This equilibration needs to be performed only on fresh resin. Normal clean-in-place (CIP) procedures can be used on the resin afterwards.

1. Connect the column outlet to a suitable waste container. Do not connect the column to the detector!
2. Wash the column with 8 - 10 column volumes (CV) of 100% acetonitrile, acetone, or propanol at a maximum linear velocity of 150 cm/h.
3. Wash the column with 5 CV of 45% acetonitrile/55% water, 50% acetone/50% water, or 50% propanol/50% water at a maximum linear velocity of 40 cm/h.
4. The column can now be equilibrated for an efficiency evaluation.

Column Operation

EVALUATION

Once the column equilibration is completed, re-equilibrate the column with 3-5 CV of mobile phase for the efficiency evaluation. The mobile phases used for this evaluation procedure are:

• 25% acetonitrile/75% water for CG 1000s
• 35% acetonitrile/65% water for CG71 and CG300
• 45% acetonitrile/55% water for CG161

A solution of either methyl paraben (1mg/ml), sodium chloride (5%) or acetone (5%) can be used to determine the asymmetry and efficiency of the packed column at a linear velocity of 40 cm/hr. The volume of the test solution loaded onto the column should be 0.5% to 1% of the column volume. It is also important to match the sample composition with the mobile phase, as shown in Figure 16. The asymmetry values obtained for the column should be between 0.8 to 1.6. The minimum efficiencies for Amberchrom™ columns are listed in Table 2.

Table 2
Minimum Column Efficiencies for Amberchrom™ Media

Chromatographic Separation

Equilibrate the resin with 3-5 CV of the mobile phase used for the separation. Separations on Amberchrom™ resins can be performed under isocratic or gradient conditions using organic solvents such as propanol, ethanol, methanol, or acetonitrile. Ion pairing reagents such as trifluoroacetic acid (TFA) can also be used to improve separations. If the column is to be used in a gradient separation, it is recommended that a blank run be performed prior to actually attempting a separation. Check the column after the blank run, and adjust the column flow adapter if necessary.

Although Amberchrom™ resins exhibit low swelling characteristics (2-6%), it is recommended to maintain a minimum level of organic solvent (2-3%) in the loading, elution, and cleaning steps. Column performance may be reduced due to bed shrinkage after use in 100% aqueous conditions.

Column Maintenance

CLEANING

Amberchrom™ resins can be cleaned in the column or removed from the column and treated in bulk. Fouled resin can be cleaned by treating the resin with 0.5M NaOH (it is recommended to maintain a minimum level of organic solvent (2-3%) in the loading, elution, and cleaning steps) for 12-18 hours and rinsing with organic/water solution. Alkaline solutions are useful for removing protein aggregates and other fouling contaminants. The column can then be cleaned with 100% solvent by increasing the solvent concentration gradually in 10% steps or by means of a linear gradient at 4% solvent per minute.

Caution: Under no circumstances should nitric acid be used to clean Amberchrom™ resins!

STORAGE

Store the column or used bulk resin in 20% propanol, methanol, ethanol, or acetonitrile, preferably at 4°C to 25°C.

Column Unpacking

Amberchrom™ resins are durable enough to be packed and unpacked many times. To unpack the resin, place a container large enough to contain the unpacked resin below the column. Remove the column bottom end fitting, and begin flow through the column (Figure 17a and 17b). Collect the resin slurry in the container.

An alternative method for unpacking the column can be employed by removing the top flow adapter from the column. Pour a sufficient amount of mobile phase into the column, and re-slurry the column using gentle overhead agitation. The resuspended resin can then be siphoned into the appropriate storage container.

Questions

Please contact Rohm and Haas for additional information about Amberchrom™ chromatographic resins.