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WEDGE® Column Online User's Manual

Introduction

The Wedge column offers you the advantages of scaleable Radial Flow for preparatory chromatography. The revolutionary design of the column allows accurate simulation of large Radial columns, for predictable scale-up as well as scale-down studies. Preparatory scale separations can be carried out using commonly available low pressure media, as well as medium-particle HPLC media. To scale up, simply use a larger Wedge column and increase sample size and flow rate proportionately.

Sepragen is a company of scientists and engineers, experienced in meeting the demands of today’s production environment. We are always interested in new applications for our products and we welcome your questions and comments.

Selecting the Correct Wedge

The choice of wedge is determined by the column packing, solvents, flow rate and other operational parameters. Packing visibility and materials of construction can also have an effect.

Standard Wedge Columns

Standard wedge columns are constructed of acrylic, with Stainless steel frits and EPDM gaskets (Table 1). These columns are designed for applications with temperatures from 4-40o C.

Table 1. Construction materials for Sepragen Wedge Columns.

Column PartMaterial
Column body and end platesAcrylic
frits10 Micron nominal (stainless steel 316L)
GasketsEPDM rubber
FastenersStainless Steel
FittingsPolypropylene
TubingPVC

Custom Stainless Steel and HDPE Columns

Custom-made Stainless steel and HDPE Wedge columns with all stainless steel or HDPE construction are also available.

Working Pressure and Pressure Gauges

Wedge columns are rated for 25 psi, although most applications will run at significantly lower pressure.

A pressure gauge with a gauge guard (to prevent the incursion of fluids into the gauge machinery) is required for proper packing of the Wedge column. It is also recommended for safe operation of the column. Operating the column at pressures in excess of the rated pressure is dangerous and may result in column failure.

Pumps and pumping capacity

Due to the high flow rate capabilities of Wedge columns, a properly chosen pump is critical in deriving the most benefit from your column. Further, for maximum efficiency, columns have to be packed properly for optimal performance. Table 3 indicates the general range of pumps needed for a particular column size. If you need to be more precise, please calculate the flow rate for your application and media as described in chapter 2.

Table 3. Pump capacity needed to pack column

Column Size Pumping Capacity
50 ml 150 mls/min.
100 ml 300 mls/min.
250 ml 750 mls/min.
500 ml 1500 mls/min.
1000 ml 3000 mls/min.

Pumping capacities for running the column may be different than in the above table depending on the maximum flow rate in your chromatographic procedure. It is not advisable to operate these pumps at the bottom or top 15% of their flow rate range.

Packing the Sepragen WEDGE® Column

Preparing the media slurry for packing

Equipment:

  1. Chromatography media, about 115% of total column volume (e.g. 115 mls to pack a 100m ml column);
  2. High ionic strength buffer (the highest ionic strength buffer used in your separation), quantity about 8 to 10 column volumes (c.v.), to be used for packing, called packing buffer.
  3. Beaker with suspended stir-bar and stir plate, beaker size about 4-5 column volumes (for a 100 ml column, beaker size about 500 ml).
Procedure:
If the medium has been stored in alcohol, decant the storage buffer, and wash several times in DI water. Wash the last time with the packing buffer. Pour the medium in the beaker, add additional packing buffer, start stirring gently and make up slurry volume up to about 25% slurry concentration (for 115 mls of media, total slurry volume 460 mls).

Keep stirring gently to keep the slurry in suspension.

Preparing the wedge

Equipment:

  • Variable speed peristaltic pump with forward / reverse flow capability.
  • Fittings, tubing and pressure gauge with guard.
  • Beaker, about 2-3 column volumes (250 ml for a 100 ml column).
Procedure:
Calculate the flow rate at which to pack the wedge.
  • From media specifications, find out the maximum linear velocity recommended for the resin.
  • Determine the volume of your Wedge column.
  • Check the table below to get the correct flow rate. If the exact linear velocity of your medium is in between, you can either work out the flow rate as described later, or take a number in between from the table.
Packing flow rates for various media linear velocities and column sizes.
Column VolumeMedia Linear Velocity
100 cm/hr 200 cm/hr 400 cm/hr 800 cm/hr 1600 cm/hr
Packing flow rate, mls per minute.
50 mls. 8 15 30 61 122
100 mls. 15 30 61 122 243
250 mls. 38 76 152 303 606
500 mls. 76 152 303 606 1212
1000 mls. 152 303 606 1212 2424

To calculate the packing flow rate yourself, follow the steps in this example using your own numbers for linear velocity and column volume. In this example, we will use a resin linear velocity of 150 cm/ hour, and a column volume of 250 mls.

Using these numbers, proceed as follows:

  1. Maximum linear velocity =150 cm/hr. (our assumed linear velocity)
  2. Bed height of column = 11 cm.
  3. Packing flow rate = Max. linear. velocity. / bed ht. = 150/11 = 13.64 column volumes per hour.
  4. For a 250 ml column, the packing flow rate is = 13.64 * 250 = 3409 ml per hour, = 56.8 ml per minute.
It is difficult to precisely set a typical pump to such accuracy, and a flow rate plus or minus 5 to 10 mls. will be sufficient for packing purposes.

Record this flow rate.

Certain media like Poros® (Perseptive Bio-systems), support extremely high linear velocities, and in such cases, your calculations will produce very high packing flow rates. If these are impractical to achieve in the lab, it is sufficient to pack at a flow rate about 20 to 30% greater than the maximum flow rate at which you intend to run your column.

Plumb the wedge, buffer, pressure gauge and pump as shown in figure 1.

Quick Plumbing Checklist

Check to ensure the valves to the packing port and inlet are open, and the outlet valve is shut. Turn on the pump and pump buffer into the column. Use a flow rate of 1/2 c.v./min. This will fill the column with buffer. If you see a few bubbles in the main column cavity, tilt the column from side to side to push the bubbles out through the inlet. (Bubbles elsewhere will be removed in a later step. Do not spend time attempting to remove those bubbles right now).

  • Inlet of pump - In beaker
  • Outlet of pump - to pressure gauge
  • Other side of gauge: to column packing port
  • Inlet of column - In Beaker
  • Outlet of Column - In Beaker
  • Packing port valves - open
  • Column Inlet valve: open
  • Column outlet valve: shut
  • Final Check: Make sure tubes in beaker do not slip out. Tape if necessary
Smartly tap all connections to remove bubbles entrapped within. A few tiny bubbles are not significant. Switch off pump. Adjust flow rate to the packing flow rate. (record this setting on pump dial). Close all column inlets and outlets.

The column is now ready for packing.

Packing the Wedge

Equipment and Material
  • Beaker of slurry (prepared earlier), 2 empty beakers to collect buffer.
  • Column filled with packing buffer, from the previous step.
  • Connector caps (1).
  • Pump with flow rate set to packing flow rate.
Caution: Do not change the pump flow rate at any time during packing.

Method

Move the beaker with slurry close to the pump inlet. Carefully and without letting in air, remove the pump inlet tube from the buffer beaker (previous step), and insert it into the slurry beaker down the side, to the bottom, so as not to interfere with the stirrer. Tape it in place.

Open valves/pinch clamps to set up flow as in figure 2.

Figure the packing time from this formula:

Packing time = Column volume / (slurry concentration * packing rate)

Keep this number handy.

Switch pump on for forward flow. Slurry enters the column, and the packing buffer will exit from column inlet and outlet. Keep a couple of extra empty beakers handy for overflow.

As the medium enters the column, it will begin to build up on the outer and inner frits. (You can see this happening In acrylic columns only. Steel and HDPE columns are not transparent). The density builds towards the middle of the column as it packs. As packing time is nearly over, watch the pressure gauge. Be prepared to turn off the pump as soon as the pressure increases up to the value for the media type being used.

After turning off the pump, quickly close the packing port pinch clamp. Close the inlet and outlet tubing clamps. Keep both inlet and outlet tubing immersed in buffer. Remove the tubing connection at the column packing port. Fill the connector with a little buffer from a squeeze bottle and screw on the connector cap. Wipe off any spilled media or buffer. Put the column back upright. Clean out the tubings of all the packing material. (Run pump in the reverse direction to suck slurry in the tubing back into the slurry beaker. Then, put end of tubing in a beaker of buffer. Pull buffer through to clean out tubing and pressure gauge. Rinse out the gauge guard to clean out any media within.)

Quick Checklist

  • Pump inlet tube in slurry beaker
  • Pump outlet tube to pressure gauge
  • Pressure gauge to packing port
  • Column inlet to beaker immersed in a little buffer, pinch valve open
  • Column outlet to beaker immersed in a little buffer, pinch valve open
  • Packing port pinch valve open
  • No air in tubing

Conditioning the Column

This is the final step before the column is ready to run.

Equipment: Same as before. 6-8 column volumes of equilibration buffer are needed for conditioning.

Setup: Connect pump, tubing as in the figure.

Immerse the pump inlet tubing into a beaker of equilibration buffer, pump outlet to the pressure gauge, and to the column outlet. Be careful not to get air into any of the tubes.

Open pinch clamps on column inlet/outlet tubes.

Turn on pump in reverse direction for a few seconds to pull air, if any, out of column outlet tube. Then forward flow, run the pump at 90% of the packing flow rate for10-15 minutes, or at least 5 column volumes, (whichever is greater) discarding the first couple of column volumes of equilibration buffer, then recirculating. Turn off the pump, wait a minute turn on the pump in reverse direction and re-circulate buffer for the same time. Turn off the pump. The column is now ready to be tested.

Quick Checklist

  • Pump inlet tubing in buffer
  • Pump outlet to pressure gauge
  • Pressure gauge to column outlet
  • Column inlet in buffer beaker
  • Valves: column inlet=open ; column outlet=open

Test Column Packing Efficiency

Prepare a sample of buffer containing 0.5 M NaCl (if a conductivity detector is available), or a 1% acetone solution (if a UV detector is to be used). Apply a sample equal to 2-3% of column volume, at a flow rate 10% of the packing flow rate. Follow through with buffer and calculate the HETP and efficiency from the conductivity or UV trace. Compare to the representative values for the type of media used. Record the data.

Brief instructions on calculating HETP are here. For a detailed discussion on the relevance of HETP, reference to any good basic chromatography text is recommended.

HETP and efficiency are calculated by injecting a tracer spike sample into a packed column and recording the shape of the spike as it comes off the column. The residence time corresponding to the elution volume Ve, and the time period corresponding to the width of the eluting peak Pw at half the height of the peak are then measured off the plot. These numbers, put into the formula in the figure, will give you HETP. See the illustration for more details.

Unpacking Procedure

  • Put the pump inlet tube into a beaker of storage buffer. Equilibrate the column in the storage buffer.
  • Remove the packing port plug.
  • Connect the packing tubing to the packing port. Firmly attach the other end of the tubing into a beaker which will hold at least three CVs.
  • Close the column outlet tube clamp. Open all other clamps.
  • Connect the pump to the column inlet and start the pump at about 0.5 to 1 CV/min. Medium should be seen flowing out of the column packing port. (Be careful that pressure does not increase above 15 psi.)
  • Watch the top of the column. A layer of liquid should form around the top.
  • When the layer covers most of the top, lightly agitate the column to slurry the medium within it.
  • Remove pump inlet tubing from the buffer to allow a little air to be pumped into the column. The air will force slurry out of the column without excessive dilution. It helps to shake the column if possible during this process, to keep the medium slurried.
  • When the column is nearly empty, put the pump inlet tubing back into the buffer and allow a small amount of buffer to be pumped into the column. Agitate the column to slurry the remaining medium, then pump in more air into the column and push out the resin slurry. Repeat this process as necessary to completely remove any medium remaining in the column.
  • If the column has to be very clean, take the column apart and clean the individual parts. The frits should be sonicated at 35 to 40 ° C for about an hour for best results. For instructions on assembly and disassembly, see assembly instructions.

WEDGE® Column Assembly Instructions

Lay out all the parts on a clean tabletop.

Make sure you have all parts. There should be a body, roughly rectangular in shape, two end-plates, two frits, two gaskets, three end caps, one port plug and a sufficient quantity of machine screws. You will also need an allen key to tighten and loosen the screws. The body should have a connector already screwed in, and the end caps should have two similar connectors screwed into them. Here is a list of all the parts:

Body (1) end plate, small (1)
gasket, small (1)
frit, small (1)
end plate, large (1)
gasket, large (1)
frit, large (1)
packing port plugs (1)
packing port caps (1)
inlet/outlet port caps (2)
Machine screws (Sufficient quantity)

Stand the body up on it’s large end. Insert the small frit into its location at the small end of the body so the fine frit is facing inside.

Place the small gasket in the small end-cap.

Put this assembly face-down on the body/frit, and loosely hand-fasten with the screws provided.. Do not tighten the screws yet.

Check to ensure that the gasket is seated correctly , and move the end-cap around as needed to seat the gasket properly. Use the allen key to tighten the screws, alternating each side and gradually increasing the torque. Do not overtighten else the threads in the body may get stripped.

Repeat these steps on the other, larger end. Your wedge should be looking like in figure 4 at this time.

Your wedge column is now ready to be tested for proper assembly.

Testing the assembled wedge

Use one of the end-caps to close off the packing port. Use the supplied tubing to connect the inlet to a pressure gauge and pump, the other end through a tube and pinch clamp to waste.

Pump clean, DI water into the wedge to displace all the air, until the water comes out of the other end. The wedge will have to be tilted to get all the air out. Stop the pump and shut off the outlet tube with the pinch valve provided. Restart the pump very slowly until the pressure reaches 5 psi only. Shut off the pump. Check for leaks, if any. If no leaks are observed, pump up the pressure up to 10 psi, stop and check for leaks again. Repeat in 5 psi increments up to the rated pressure of the wedge, 25 psi. If no leaks have been observed, your wedge is considered ready for use. If leaks were observed at any pressure, read on.

Checking and fixing leaks after assembly

Leakage at end-caps. The most common cause of leaks is improper assembly. If the end-caps are not tightened down uniformly, one side will not sit flat on the seal and body, resulting in a leak. Unscrew the end-cap, and tighten it down uniformly all around, increasing the force applied a little each time. Ensure the gaskets are properly seated.
Leakage at packing ports
  • Port plugs are loosely screwed in. Tighten.
  • Port caps are loose. Tighten.
  • There is insufficient PTFE tape on the threads. Wind at least 4 tight turns and re-assemble
  • Leakage at inlet/outlet ports
  • Port plugs are loosely screwed in. Tighten.
  • Port caps are loose. Tighten.
  • There is insufficient PTFE tape on the threads. Wind at least 3 tight turns and re-assemble.
  • Leakage at body joints Manufacturing defect. Call Sepragen customer service, get an RGA number and return the product. Make sure you pack the wedge properly and write the RGA number prominently on the outside as well as inside the box.
    I don’t know where it’s leaking, but it is wet all over. Condensation on cold surface. Let the wedge and water come to room temperature.



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