MiraCosta College
National Center of Excellence in Bioprocessing
Part No.
N/A
Lot No.
Page 1
Doc No.
STANDARD OPERATING PROCEDURE
BP012
CO No.
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Subject:
of 7
Rev No.
C0004
001
Eff Date
Ion Exchange Column Packing Procedure
25AUG06
1.0
PURPOSE:
1.1
Prepare an ion exchange chromatography column for use.
2.0
SCOPE:
2.1
Describe the appropriate operating instructions to pack an ion-exchange liquid
chromatography column and evaluate packing quality.
3.0
RESPONSIBILITIES:
3.1
It is the responsibility of the course instructor/lab assistant to ensure that this SOP is
performed as described and to update the procedure when necessary.
3.2
It is the responsibility of the students/technicians to follow the SOP as described and to
inform the instructor about any deviations or problems that may occur while performing
the procedure.
4.0
REFERENCES:
4.1
“Column Packing, The Movie” Amersham Biosciences
4.2
ÄktaPrime product manual
4.3
PrimeView software product manual
5.0
DEFINITIONS
5.1
a Front width of peak at 10% height, expressed as time or distance
5.2
b Back width of peak at 10% height, expressed as time or distance
5.3
HETP Height equivalent to a theoretical plate
5.4
h Height of a peak
5.5
L Length of packed column bed
5.6
N Theoretical plate count
5.7
Tris tris(hydroxymethyl)aminomethane
5.8
Ve Elution volume (volume eluted from column from the start of sample application
to the peak maximum)
5.9
w Peak width, expressed as time or distance
5.10 w½ Peak width at half height, expressed as time or distance
5.11 Column Descriptions
5.11.1 Column end pieces. The red end pieces, made of reinforced acetal plastic, hold
the chromatographic and jacket tubes in position. Each end piece houses an
Oring, sealing ring, washer and locking ring. The end piece also carries a tubing
connector through which thermostat fluid enters or leaves the column.
5.11.2 Column bottom piece. The bottom piece screws onto the column end piece .
The gel bed is supported by a 10 μm nylon net ring on top of the plunger. The
bottom piece seals against the chromatographic tube via the O-ring .
5.11.3 Adaptor. The adaptor consists of a top piece that fi ts the column end piece, a
plunger that carries the gel bed protection and support screen, and adjusting
mechanism. The adjusting mechanism allows the plunger to be set in position in
MiraCosta College
National Center of Excellence in Bioprocessing
Part No.
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Doc No.
STANDARD OPERATING PROCEDURE
Lot No.
BP012
CO No.
N/A
Subject:
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Rev No.
C0004
001
Eff Date
Ion Exchange Column Packing Procedure
25AUG06
the chromatographic tube, and forms the mechanism for sealing against the
column wall.
5.11.4 Tubing. XK columns are supplied with fl anged M6-type capillary tubing. The
adaptor tubing is 50 cm long. The bottom piece tubing is 30 cm long. A 5 cm
protection tubing is threaded over the capillary tubing in the inlet of the adaptor
and column bottom piece to prevent squeezing of the capillary tubing during
transport and storage.
6.0
PRECAUTIONS
6.1
Wear safety goggles, gloves, and lab coats when packing a column because pressures
can reach 50 psi.
6.2
The chromatography media is stored and flushed in 70% ethanol. It is harmful to eyes
and mucous membranes.
7.0
MATERIALS and SUPPLIES
7.1
XK16 glass chromatography column (1.6 cm diameter, 20 cm. high ) assembly and
associated components, such as ferrule tubing connectors
7.2
SP Sepharose Fast Flow cation exchange media (mean particle size = 90 m).
7.3
Reagent grade NaCl and acetone
7.4
Equilibration buffer: 0.1M Tris-HCl pH 6 (hereafter called “buffer”)
7.5
20% EtOH (ethanol), ultrapure water (upw)
7.6
Miscellaneous glass and plastic lab ware
8.0
EQUIPMENT
8.1
GE Healthcare ÄktaPrime system
8.2
Laptop with PrimeView software
9.0
PROCEDURE
9.1
Column Packing Procedure
9.1.1 Prepare Media Slurry
9.1.1.1 Equilibrate the chromatography media to the temperature of the
anticipated separations process.
9.1.2 Assemble Column
9.1.2.1 Inspect all components, look for chips in ends of the glass column or
holes in mesh or filters used to support the media.
9.1.2.1.1 You will use a mesh or filter pore size at least two times smaller
than the smallest media particle size.
9.1.2.2 Equilibrate all components to the temperature at which the packing will
be performed.
9.1.2.3 Assemble the bottom end-piece unit, making sure to securely fasten
the tubing inside the end piece and adaptor.
9.1.2.4 Displace the air in the bottom end piece by flushing with 20% EtOH by
using a syringe connected to the tubing. Ethanol will help break the
MiraCosta College
National Center of Excellence in Bioprocessing
Part No.
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Doc No.
STANDARD OPERATING PROCEDURE
BP012
CO No.
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Ion Exchange Column Packing Procedure
25AUG06
surface tension and will not leave a hydrophobic film to entrap air.
Remove the syringe adaptor and replace with a domed nut.
9.1.2.5 Leave a meniscus over the support ring and gently lay the mesh at an
angle so no air is trapped underneath. Snap the top piece on top of the
assembly.
9.1.2.5.1 Reference the picture in §10.1
9.1.2.6 Repeat the steps up until this point for the top adaptor
9.1.2.7 Insert the bottom end piece into the bottom of the column, tighten
securely, and mount to a ring stand.
9.1.2.8 Check that the column is level.
9.1.2.9 Add buffer to a height of about 2 cm at the bottom of the column.
9.1.3 Pour Column
9.1.3.1 Shake the media slurry to ensure homogeneously mixed.
9.1.3.2 Pour the media into the column continuously, holding the column at an
angle and minimizing bubble formation. Fill the column up to the top
of the column until a meniscus of slurried solution is present at the
column top.
9.1.3.3 Mount the column top adaptor and connect to a pump.
9.1.3.3.1 All connections upstream of the column should be made to
ensure no bubbles are introduced.
9.1.3.4 Tighten all connections
9.1.3.5 Connect the bottom outlet to the top of the UV detector and run the
column to check for any leaks or gaps.
9.1.4 Media Consolidation
9.1.4.1 Ideally, the column would be allowed to rest for all the media to settle
out just by gravity. This would take too long, however.
9.1.4.2 The media consolidation can be thought of an accelerated gravity
settling to speed up the process.
9.1.4.3 Pump buffer into the column at a rate of 20cm/hr until the bed
stabilizes.
9.1.4.4 Maintain the packing flow rate for at least three column volumes after a
constant bed height is achieved.
9.1.4.5 Mark the bed height on the column.
9.1.4.6 Loosen the top screw and release the side catch to allow the plastic
thread assembly of the adaptor to be lowered to the media surface.
9.1.4.7 Slide the adaptor down to meet the media surface, expelling any liquid
from the tube at the top as the adaptor is pressed down.
9.1.5 Constant Flow Column Packing
9.1.5.1 Turn the pump rate up to 500 cm/hr.
9.1.5.2 After the level of the column remains stable, pause the pump, loosen
the top adaptor and the connection at the valve, and lower the top
adaptor to again just above the surface of the packed media.
MiraCosta College
National Center of Excellence in Bioprocessing
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Ion Exchange Column Packing Procedure
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9.1.6 Mechanical Compression Column Packing
9.1.6.1 After the media height stabilizes from several more CVs of flow,
perform a final mechanical compression of the packed media.
9.1.6.2 Lower the top adaptor to about 3-mm below the surface of the gel bed
and tighten the adaptor.
9.1.6.2.1 NOTE: Limit mechanical compression to 0.5-cm
9.1.6.3 After packing, the inlet and outlet tubes can be capped and the column
stored until it is needed.
9.2
Column Evaluation Procedure
9.2.1 Prepare Efficiency Test Sample
9.2.1.1 10 mL 0.8 M NaCl and 1% (v/v) acetone in upw
9.2.1.1.1 This volume is sufficient for a class of 20 students
9.2.1.2 The salt solution will be detected by the conductivity sensor and the
acetone will be detected by the absorbance detector.
9.2.2 Apply Efficiency Test Sample
9.2.2.1 Select a sample volume 0.5 – 1.0 % of Cv
9.2.2.2 Inject sample and run buffer through column at normal operational flow
rate for media type.
9.2.2.3 Stop column after obtain both UV and conductivity peaks.
9.2.2.4 Measure Ve, w½, a, b values from plot of peak height vs. time/distance
9.2.3 Calculate Packing Quality Parameters
9.2.3.1 Calculate N, the number of theoretical plates using the formula:
N = 5.54 × (Ve/w½)2
9.2.3.2 Calculate HETP using the formula: HETP = L/N
9.2.3.3 Calculate Hred using the formula: Hred = H/(mean particle size)
9.2.3.4 Calculate As (symmetry factor) using the formula: As = b/a
9.2.4 Evaluate Packing Quality Parameters
9.2.4.1 Compare calculated Hred to values shown in Table:
Hred
2
2-4
3-5
Interpretation
Ideal
Good
High efficiency applications
5-10
Low efficiency applications
MiraCosta College
National Center of Excellence in Bioprocessing
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Ion Exchange Column Packing Procedure
9.2.4.2
9.2.4.3
25AUG06
Compare calculated As to expected values shown in Table:
As
1.0
0.8-1.5
0.8-1.5
Interpretation
Ideal
Good
High resolution applications
0.5-5
Low resolution applications
If Hred and/or As is outside the acceptable range for your application,
then you should repack the column.
MiraCosta College
National Center of Excellence in Bioprocessing
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CO No.
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Ion Exchange Column Packing Procedure
10.0
Doc No.
STANDARD OPERATING PROCEDURE
Lot No.
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ATTACHMENTS
10.1
10.2
XK-16 Schematic and Preparation for Packing
10.1.1 Remove air from the filter and fit the bottom
piece to the XK column to be packed. Tighten
the O-ring with the black adjusting knob.
Close the outlet.
10.1.2 Attach the column that serves as reservoir.
10.1.3 Pour the gel suspension into the column.
10.1.4 Fit the XK adapter to the column and lower it
to the surface of the gel suspension. Tighten
the O-ring with the black adjusting knob.
10.1.5 Connect the inlet tubing from the XK adapter
to the valve (pump).
10.1.6 Your column is now ready to pack.
Flow calculations
10.2.1 From linear flow (cm/hour) to volumetric flow
rate (ml/min)
10.2.2 From volumetric flow rate (ml/min) to linear
flow (cm/hour)
This product was funded by a grant awarded under the President’s High Growth Job Training Initiative as implemented by the U.S.
Department of Labor’s Employment & Training Administration. The information contained in this product was created by a grantee
MiraCosta College
National Center of Excellence in Bioprocessing
Part No.
N/A
Lot No.
Page 7
Doc No.
STANDARD OPERATING PROCEDURE
BP012
CO No.
N/A
Subject:
of 7
Rev No.
C0004
001
Eff Date
Ion Exchange Column Packing Procedure
25AUG06
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