[ CARE AND USE MANUAL ] WATERS ADVANCED PURIFICATION COLUMNS I. INTRODUCTION CONTENTS I. INTRODUCTION II. USING THE COLUMN III. CARE AND MAINTENANCE

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1 WATERS ADVANCED PURIFICATION COLUMNS I. INTRODUCTION Waters Advanced Purification (AP) series of glass columns are constructed of biocompatible glass and polymeric materials and can be easily used with silica, polymer, or soft gel packings. To optimize flow and ensure uniform sample distribution onto the packed bed, each column incorporates a distributor. A replaceable filter protects the packing from large particulate contaminants. Empty AP glass columns are available in a variety of sizes and utilize the same design to ensure predictable methods transfer among them. AP glass columns are compatible with both analytical and preparative HPLC and FPLC systems. CONTENTS I. INTRODUCTION a. AP Column Accessories II. USING THE COLUMN a. Safety and Solvent Considerations b. Adjusting the Plunger c. Packing the Column d. Installing the Column e. Equilibrating the Column f. Eliminating Voids Inlet tubing Inlet connector assembly Glass column Seal adjustment nut Coarse/fine adjustment slide Inlet cap Inlet filter Fine adjustment knob Plunger III. CARE AND MAINTENANCE a. Troubleshooting b. Replacing Ferrules c. Disassembling and Reassembling the Inlet Connector d. Replacing End Fittings e. Replacing Filters and O-rings f. Storing the Column IV. SCALING UP SEPARATIONS a. Scaling a Seperation b. Adjusting Sample Load and Flow Rate c. Adjusting Gradient Parameters d. Adjusting Gradient Delay Volume V. ORDERING INFORMATION VI. MISCELLANEOUS Outlet connector assembly Outlet filter Outlet tubing Figure 1: Advanced Purification Column Waters Advanced Purification Columns 1

2 a. AP Column Accessories The following accessories are shipped with the AP column: Packing funnel End plugs for column storage Spare Parts Kit consisting of: - 1 inlet O-ring - 1 outlet O-ring - 1 connector tip O-ring - 1 packing funnel O-ring - 2 filter assemblies - 2 ferrules - 10 gripper ring tools - 1 Allen wrench Internal diameter Length (nominal) Maximum operating pressure 10 mm Filter size 20 µm Materials along fluid path: Column Tubing Filter Connectors O-rings Operating temperature 4 C to 50 C Table 1: AP Column Specifications 100, 200, 300, or 600 mm 10 MPa (100 Atm, 1500 psi) Borosilicate glass Tefzel Polyethylene Ryton-4 (polyphenylene sulfide resin) Buna-N II. USING THE COLUMN a. Safety and Solvent Considerations Inherent surface imperfections can weaken glass at high pressures. Maintain lower pressures to prolong column life. The AP glass column has an external plastic jacket which acts as a safety shield. To avoid column hardware damage, never exceed 10 MPa (1500 psi or 100 atm) inside the column. High backpressures may damage resin-based packing materials. Consult your packing material care and use manual for packing material backpressure limits. AP column materials are compatible with most solvents used in HPLC. However: Certain solvents, such as acetone or chlorinated hydrocarbons, may reduce the life expectancy of the O-rings. The transparent plastic jacket may weaken if exposed to certain organic solvents. If the jacket crazes, replace it. b. Adjusting the Plunger Range of movement The packed bed is at maximum volume when the hex at the inlet end of the plunger is flush with the top of the coarse/fine adjustment slide. Do not withdraw the plunger further, or too few threads will be engaged to ensure reliable operation. The slide must be engaged against the locking hex while the column is in use to prevent the plunger from unscrewing under pressure. Inlet Hex is Flush with Top of Slide Inlet Hex Exposed Locking Hex Exposed Threads Exposed Correct Incorrect Incorrect Incorrect Figure 2: Column at Maximum Volume Waters Advanced Purification Columns 2

3 Using the coarse/fine adjustment slide The AP column features a coarse/fine adjustment slide to make plunger adjustment fast and easy. Make sure the slide is in the down position (against the fine adjustment knob) before pressurizing the column, Figure 3 shows: 1. Deactivating the inlet O-ring seal 2. Using the slide for coarse adjustment 3. Using the slide for fine adjustment and locking The slide must engage the locking hex during operation to prevent the inlet plunger from unscrewing under pressure. 4. Activating the O-ring seal Column volumes When you pack the column, you must calculate the column volume from the length of the packed bed you will use in your method. Column volumes are: Column length Column volume 100 mm 8 to 8 ml 200 mm 13 to 16 ml 300 mm 21 to 24 ml 600 mm 45 to 48 ml Short beds To pack a column with a shorter bed, replace the outlet connector with a second inlet connector (see Section V, Ordering Information ). Inlet connectors have a 40 mm range of movement, allowing you to reduce column volume to as little as 3 ml. The minimum bed length for a 100 mm long column with two inlet connectors is 20 mm. Slurry-packing the column Amount of packing material required To determine the amount of packing material required, multiply the column volume, V (ml), by the bulk density of the packing material, d (g/ml): Figure 3: Adjusting the Plunger Downward Amount of packing material required = V x d c. Packing the Column The Waters AP Column is shipped unpacked. The AP Column is compatible with all commonly used silica- and resin-based packing materials. Repack the column when performance deteriorates or when a new method requires a different packing material. When you repack an AP Column, empty the column first (see Emptying the Column ), then proceed to the correct section for slurry-packing or dry-packing the column. Packing Material Waters Protein-Pak HR Waters AccellPlus silica-based packing materials Other packing materials Determining density To determine the density of the packing material: 1. Slurry 1 g of packing material in 10 ml of buffer and allow the material to settle. Bulk Density of the Packing Material 1.2 g/ml 0.5 g/ml Use procedure below to determine density 2. Note the volume of the settled material 3. Calculate density by dividing the weight of the material by the settled volume. Waters Advanced Purification Columns 3

4 Preparing slurry solutions To make slurry solutions, suspend one part of packing material in three parts of degassed solution. The packing funnel can hold 40 ml of slurry. Packing Material Ion-exchange chromatography Reversed-phase chromatography Hydrophobic-interaction chromatography (HIC) Slurry-packing To slurry-pack the column (Figure 4): 1. Unscrew the inlet connector from the inlet cap. 2. Screw the packing funnel into the inlet cap. 3. Clamp the column upright to a rigid support. Drain the outlet tubing to waste. 4. Fill the column with the slurry. If the slurry does not flow into the column, insert a pipette into the tip of the funnel to break the surface tension. 5. When all of the packing material is in the column, plug the outlet tubing. 6. Let the packing material settle. Remove excess solvent from the top of the bed with a pipette. Do not disturb the bed. 7. Remove the funnel, and reattach the inlet connector. 8. Adjust the inlet connector until the plunger contacts the packed bed. Funnel Ensure that the packed bed does not exceed the nominal column length. Solution High concentration buffer Methanol Water or low concentration buffer Dry-Packing the Column Amount of packing material required To determine the amount of packing material required, multiply the column volume, V (ml), by the bulk density of the packing material, d (g/ml): Amount of packing material required = V x d Packing Material Waters AccellPlus silica-based packing materials Other packing materials Waters Protein-Pak HR must be slurry-packed (see Slurry-packing the column ) Determining density To determine the ulk density of the packing material, put 19 g of packing material into a 10 ml graduated cylinder. Tap the graduated cylinder on a flat surface until the material is completely settled. Note the volume and calculate the density. Dry-packing To dry-pack the column: 1. Unscrew the fine adjustment knob from the inlet cap. 2. Screw the packing funnel into the inlet cap. 3. Remove the outlet tubing. 4. Holding the column vertically, gradually fill the column with packing material. While filling, tap the column lightly on a flat surface until the material settles. 5. Remove the funnel and reattach the inlet connector and the outlet tubing. Bulk Density of Packing Material 0.5 g/ml Use procedure below to determine density Inlet cap Slurry 6. Adjust the inlet connector until the inlet plunger contacts the packed bed. Ensure that the packed bed does not exceed the nominal column length. To waste Figure 4: Slurry-Packing the Column Waters Advanced Purification Columns 4

5 Emptying the column To remove packing material from the column: 1. Reverse the direction of flow through the column by connecting the column outlet to the pump. 2. Remove the inlet connector by unscrewing the fine adjustment knob from the inlet cap. 3. Screw the packing funnel into the inlet cap. Position a beaker under the mouth of the funnel. 4. Start flow. Gradually increase the flow until the packed bed begins to move. Continue flow until all packing material washes out of the column. 5. Remove the funnel and clean the column. Make sure the threads are clean. 6. Repack the column (see Slurry-packing the column, or Dry-packing the column, for repacking instructions). d. Installing the Column The AP Column can be installed in: Waters 650 Advanced Protein Purification System Waters 625 system Pharmacia FPLC systems Other high performance liquid chromatography (HPLC) systems (refer to Table 4 for adapters and unions used to connect the AP column to selected systems) e. Equilibrating the Column Before performing a test run, equilibrate the column: 1. Gradually increase the flow in 0.1 ml/min increments until you attain the desired flow rate. 2. While equilibrating the column, check for voids; if present, gradually reduce flow to 0 ml/min, Adjust the inlet connector until the void is eliminated. (If this is unsuccessful, see Eliminating Voids ). 3. Equilibrate the column with 10 to 20 column volumes of mobile phase, or as recommended by the manufacturer of the packing material. f. Eliminating Voids Voids may develop in the column during normal use. To eliminate voids: 1. Stop flow. 2. Disconnect the inlet column tubing from the system to allow displacement of fluid while adjusting the plunger. 3. Deactivate the O-ring seal by turning the seal adjustment nut counterclockwise. 4. Move the slide to the fine (down) position and turn it slowly to eliminate the void. If the fine adjustment knob is all the way down, back if off a few turns. Move the slide to the coarse adjustment position and move the plunger down a short distance. To eliminate the void, return the slide to the fine adjustment position and turn it slowly. Refer to the appropriate system user s guide for specific column installation information. Tightening fittings Finger-tighten plastic fittings. Do not overtighten. Overtightening may damage the threaded parts of the column. Overtightening does not improve performance. End plugs Save the column end plugs for future column storage. Waters Advanced Purification Columns 5

6 III. CARE AND MAINTENANCE a. Troubleshooting Gripper ring Gripper ring tool Symptom Possible Cause Corrective Action Poor resolution Sample overload Reduce sample amount. High backpressure Leakage into plastic shield or out of inlet connector Table 2: Troubleshooting the AP Column b. Replacing Ferrules Each of the four tubing connections on the AP column uses a gripper ring between two washers (to keep the Tefzel tubing from sliding when the column is under pressure) and a ferrule. This section explains how to set the gripper ring and replace a ferrule on the AP column. Installing the Ferrule, gripper ring, and washer assembly To install a new ferrule, gripper ring, and washer (Figure 5): 1. Slide a washer over the end of the tubing. 2. Place a gripper ring in the depression on the gripper ring tool. 3. Insert the end of the tubing into the gripper ring tool as far as it will go (see Figure 5). This seats the gripper ring at the proper distance from the end. Flow rate too high Packed bed void Plugged filter Foreign matter on sealing surfaces O-ring seal is not activated Damaged O-rings 4. Withdraw the tubing from the gripper ring tool and slide another washer over the end of the tubing. Reduce flow rate. Adjust inlet connector to eliminate void. Replace the filter (see Replacing Filters and O-rings ). Clean surfaces and re-tighten. Turn seal adjustment nut counterclockwise to active O-ring. Replace O-rings (see Replacing Filters and O-rings ). 5. Slide the ferrule over the end of the tubing, flared end first. Use l/i6-inch o.d., inch i.d. Tefzel tubing for all connections. Washer Washer Ferrule Figure 5: Using the Gripper Ring Tool c. Disassembling and Reassembling the Inlet Connector To replace the ferrule at the connection between the inlet connector and the column, you must first disassemble the inlet connector assembly. Disassembling the inlet connector To disassemble the inlet connector assembly: 1. Deactivate the O-ring by turning the seal adjustment knob. 2. Move the slide to the coarse adjustment position and unscrew the plunger. 3. Use a screwdriver to remove the O-ring located under the cover. 4. Unscrew the seal adjustment knob, exposing the set screw that holds the core in the proper orientation. Use the Allen wrench from the Spare Parts Kit to remove the set screw. 5. Push the core out of the plunger and unscrew the inlet connector tip. 6. Pull out the tubing. Use a single-edge razor to cut the tubing above the washers. To replace the ferrule, follow the procedure in Replacing Ferrules. Reassembling the inlet connector To reassemble the inlet connector: 1. Insert the end of the tubing and the ferrule into the inlet connector tip. Then slide the core over the tubing and screw it to the tip. Hand-tighten the fitting. 2. Slide the plunger over the core and replace the set screw. The plunger core has three slots and the plastic plunger has two threaded holes. There is only one perfect match. Position the hex of the inlet connector tip to align onwe of the slots with one of the threaded holes. If there is no perfect match, tighten the inlet connector tip a little more. Waters Advanced Purification Columns 6

7 3. Replace the set screw. Do not overtighten the set screw; the plunger core must slide easily to permit the O-ring seal to form. 4. Reassemble the column. d. Replacing End Fittings The end fittings for the AP column are compression screws with gripper rings, washers, and ferrules. Making a new compression screw assembly To make a new compression screw assembly: 1. Use a single-edge razor to make a straight, square cut on the plastic tubing. Make the cut in front of the compression screw. 2. Slide a compression screw fitting and a washer over the end of the cut tubing. 3. Place a gripper ring in the top depression of the tool (see Figure 5). Insert the end of the tubing into the gripper ring tool as for as it will go. This moves the gripper ring to the proper distance from the end of the tubing. 4. Remove the tubing from the tool and slide the other washer and ferrule over the end of the tubing. 5. Insert a union over the end fitting assembly and tighten it to seat the end-fitting. e. Replacing Filters and O-rings The inlet and outlet connector assemblies each contain a filter. Replace filters periodically, depending on: The cleanliness of the sample and solvents The type and particle size of the packing material Distributors for AP filters are inside the filter skirt. Look for the star-like pattern on the distributor surface to ensure the distributor has not become dislodged. The AP column contains three O-rings, one each at the connectors and one at the filter. The outlet connector O-ring does not normally need replacement. Accessing the filters To access the inlet filter: 1. Deactivate the inlet O-ring by turning the seal adjustment knob counterclockwise. 2. Slide the inlet connector adjustment to the coarse adjustment position, and unscrew the plunger from the column. To access the outlet filter, unscrew the outlet connector from the plastic shield and remove from the glass column. Installing the filter To install on inlet filter: 1. Remove the old filter. Pry off the filter with a fingernail (or by inserting a blunt flat tool between the filter and shoulder of the connector tip). 2. Install the new filter as follows: For AP-1 (refer to Figure 6) Ensure that the Tip O-ring is in place on the connector tip. Snap the new filter onto the connector tip. For AP-2 & AP-5 (refer to Figure 7) The Distributor has grooves on one surface and those grooves must face the filter as shown in Figure 7. Each distributor has an insert on the backside which contacts the connector. Should the insert fall out of the Distributor, for consistency, simply re-install it with the circular marks facing the Distributor as shown. Please note that acceptable factory installation of the insert may be such that the circular marks are visible and facing the connector For AP-2 Columns, install the Distributor assembly into the Filter and snap them onto the connector. For AP-5 Columns, install the Distributor assembly into the connector and snap the filter onto the connector. Filter assembly Tip o-ring Connector o-ring Connector (inlet shown) Figure 6: Inlet Connector Tip for AP-1 Column Waters Advanced Purification Columns 7

8 Filter assembly Distributor Connector o-ring Remove all salts from the column before adding an organic solvent. An organic solvent added to a salt can cause precipitation, resulting in increased backpressure. Extended storage in high salt buffers may result in the formation of microcrystals which can lead to high backpressure. (Dissolve microcrystals by slowly introducing a low ionic strength buffer). Insert Connector (inlet shown) Do not allow the mobile phase to freeze. Storage at 4 C is acceptable. Figure 7: Inlet Connector Tip for AP-2 and AP-5 Columns Replacing an inlet connector O-ring To replace the inlet connector O-ring: 1. Deactivate the inlet O-ring by turning the seal adjustment knob. Remove the inlet connector assembly from the column by unscrewing the plunger. 2. Use a small blunt tool, such as a push pin, to gently pry up the O-ring so that you may roll it off the connector with your fingers. Carefully push a new O-ring onto the connector tip. Be sure to select the proper inlet connector O-ring. Do not scratch the O-ring groove. If the groove is scratched, the column will leak. 3. Return the inlet connector assembly to the column. Reactivate the O-ring. Replacing a filter O-ring This small O-ring is located at the edge of the filter skirt. With the filter off, roll the O-ring off the connector tip and replace it with a new one. f. Storing the Column Storage considerations Store a packed glass column according to the recommendations of the manufacturer of the packing material. Note the following: Store the column with end plugs in place to prevent column drying. Use bacteriostatic solutions in appropriate concentrations, for example, 0.2% sodium azide or 30% ethanol. IV. SCALING UP SEPARATIONS a. Scaling a Separation When scaling an analytical separation to a preparative separation, use the following isolation approach: 1. Define the objective by determining the: Complexity of the sample mixture Component to be isolated Required quantity Degree of purity Properties of the components in the mixture 2. Perform crude separations to remove large quantities of extraneous material or to isolate groups or classes of compounds. Simplify a complex sample through coarse separation techniques, such as liquid-liquid or solid-liquid extraction, crystallization, or precipitation. Centrifuge or filter the sample to remove particulates. 3. Develop the separation method. To optimize the separation for compounds of interest, make small-scale injections of the sample mixture, varying the gradient. 4. Perform a loading study. Make progressively larger injections of the sample mixture on a small scale column, such as the AP-Minicolumn, to determine the effect of overload on resolution. This study defines the amount of sample that can be separated in a column of a given volume. 5. Scale up the separation. Translate the small scale parameters to the large scale system values. Adjust the: Sample load Flow rate Gradient volume Waters Advanced Purification Columns 8

9 b. Adjusting Sample Load and Flow Rate When scaling the separation, use analytical and preparative columns that are: The same length Packed with the same particle size material This section describes three equations to adjust the sample load and flow rate. Use Equation l to calculate the sample load and Equation 2 to calculate the flow rate. Alternately, use Equation 3 to calculate a scale factor, which you may use to obtain the preparative value. In all three equations, if you use preparative columns and analytical columns of the same length, both sample mass and flow rate are proportional to the square of the column cross section. In the following equations: p = preparative column a = analytical column m = mass injected I = column length d = column diameter F = flow rate v = column volume sf = scale factor t = times in gradient table V d = delay volume of the instrument Equation 1 To increase sample load in proportion to the volume of the packed bed: Equation 2 To increase flow rate in proportion to the volume of the packed bed: Equation 3 To calculate the scale factor: Multiply the analytical sample mass and/or flow rate by the scale factor to obtain the preparative value. You should obtain similar retention times and peak widths for both the analytical and the preparative column and thus, similar resolution. c. Adjusting Gradient Parameters If you have scaled the flow rate in proportion to the column volume, the gradient time profile should remain constant. However, due to pressure limitations on the preparative instrument or column, you may have to reduce the flow and adjust the gradient time table. Use the following formula to Keep a constant ratio between the gradient volume and column volume Adjust the gradient table d. Adjusting Gradient Delay Volume Before the gradient reaches the inlet of the column, there is an isocratic period caused by the delay volume between the point of mixing of the gradient and the column inlet. The isocratic period exists until the delay volume is purged. In small columns, the delay volume results in a longer isocratic period than in larger preparative columns. To maintain the same separation for analytical and preparative columns, you must delay the gradient for the preparative column by the some number of column volumes. Use the following equation to calculate the additional gradient delay time for the preparative column: Waters Advanced Purification Columns 9

10 V. ORDERING INFORMATION Advanced Purification (AP) Glass Cloumn Accessories and Spare Parts AP Minicolumn Accessories and Spare Parts Advanced Purification Glass Columns for Protein Purifications Waters series of glass columns are constructed of biocompatible glass and polymeric materials and can be easily used with silica, polymer, or soft gel packings. To optimize flow and ensure uniform sample distribution onto the packed bed, each column incorporates a distributor. A replaceable filter protects the packing from large particulate contaminants. Empty AP glass columns are available in a variety of sizes and utilize the same design to ensure predictable methods transfer among them. AP glass columns are compatible with both analytical and preparative HPLC and FPLC systems. Column Dimension Bed Volumn (ml) Flow Rate (ml/min) Pressure Rating (psi/mpa) Part No. 5 x 50 mm psi/10 MPa WAT x 100 mm psi/10 MPa WAT x 100 mm psi/10 MPa WAT x 200 mm psi/10 MPa WAT x 300 mm psi/10 MPa WAT x 600 mm psi/10 MPa WAT x 100 mm psi/6.8 MPa WAT x 200 mm psi/6.8 MPa WAT x 300 mm psi/6.8 MPa WAT x 600 mm psi/6.8 MPa WAT x 100 mm psi/3.4 MPa WAT x 200 mm psi/3.4 MPa WAT x 300 mm psi/3.4 MPa WAT x 600 mm psi/3.4 MPa WAT Description Dimensions of Column Part No. Glass Tube 5 x 50 mm WAT AP Column Accessories and Spare Parts AP-2 Column Accessories and Spare Parts 5 x 100 mm WAT Column Jacket 5 x 50 mm WAT Filters, 10/pkg O-rings, 13/pkg (includes 10 inlet/outlet and 3 funnel) 5 x 100 mm WAT WAT WAT Description Dimensions of Column Part No. Glass tube 10 x 100 mm WAT x 200 mm WAT x 300 mm WAT x 600 mm WAT Plastic shield 10 x 100 mm WAT O-rings, 5/pkg Filters, 10/pkg Replacement tubing (Tefzel) 1/16 inch o.d. x inch i.d. x 10 feet (1.6 mm o.d. x 0.23 mm i.d. x 3 m) 10 x 200 mm WAT x 300 mm WAT x 600 mm WAT WAT WAT WAT Description Dimensions of Column Part No. Glass tube 20 x 100 mm WAT x 200 mm WAT x 300 mm WAT Plastic shield 20 x 100 mm WAT x 200 mm WAT x 300 mm WAT x 600 mm WAT Waters columns feature non-metallic construction and adjustable bed height with easy-to-use coarse and fine adjustments. The AP glass columns are available in a variety of dimensions. O-rings, 5/pkg Filters, 2/pkg Replacement tubing (Tefzel) 1/8 inch o.d. x inch i.d. x 10 feet (3.2 mm o.d. x 1.02 mm i.d. x 3 m) WAT WAT WAT Waters Advanced Purification Columns 10

11 AP-5 Column Accessories and Spare Parts Description Dimensions of Column Part No. Glass Tube 50 x 100 mm WAT x 200 mm WAT x 300 mm WAT Plastic Shield 50 x 100 mm WAT O-rings, 5/pkg Filter, 2/pkg Replacement tubing (Tefzel) 1/8 inch o.d. x inch i.d. x 10 feet) (3.2 mm o.d. x 1.02 mm i.d. x 3 m) 50 x 200 mm WAT x 300 mm WAT x 600 mm WAT WAT WAT WAT Waters Corporation. Waters and The Science of What s Possible are registered trademarks of Waters Corporation. AccellPlus and Protein-Pak are trademarks of Waters Corporation. Tefzel is a registered trademark of E.I. DuPont de Nemours and Company. Pharmacia and FPLC are registered trademarks of Pharmacia LKB. Ryton is a registered trademark of Phillips Chemical Company Waters Corporation. Produced in the U.S.A. July 2016 WAT Rev 6 IH-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: