SOP For Ion Exchange Chromatography

1.0 Safety
1.1 Understand the SOP before beginning

1.2 Wear proper safety attire, including: safety goggles, lab coats, and latex gloves.

1.3 The HS50 resin is stored in 0.1M NaOH, use extra caution when working with it.

1.4 Use caution not to damage the fret at the bottom of the column.

1.5 Do not operate above rated column pressure

2.0 Column Packing Procedure

2.1 Slurry resin using pipette as a mixing devise, be careful not to introduce bubbles
into resin slurry.

2.2 Remove top column adaptor

2.3 Use hemostats to close column outlet line

2.4 Once resin is a homogenous solution, carefully pour resin into top inside of
column tube NOTE: additional 0.1N NaOH may be added to slurry container to
slurry all resin.

2.5 Level and align column tube

2.6 Replace column top adaptor

2.7 Allow resin to settle over night

2.8 Remove supernatant from settled resin, leave approximately 5mm liquid on top of
resin bed.

2.9 Determine pump setting to achieve the volumetric flow rate calculated below:
Linear flow rate (LFR): 300 cm/hr
Volumetric flow rate (VFR): Linear flow rate X r2

Use this flow rate for entire packing procedure

2.10 Procure 2 liter beaker of DI water

2.10 Use pump and RODI to purge air from column inlet line and top column adaptor.

2.11 Place column adaptor on column and carefully move adaptor into liquid located
just above the resin bed.

2.12 Secure top adaptor

2.13 Check flow paths and start pump to begin resin bed compression.

2.14 Stop pump when no more bed compression is observed.

2.15 Without disturbing the compressed bed, quickly move the top column adaptor on
top or near the bed.

2.16 Repeat down flow packing procedure until no more bed compression is evident
and the top column adaptor is placed on top of resin bed.

3.0 Equipment Set-Up

3.1 Perform Chart recorder settings as follows:

Left Panel
“int. ext.” up position
“mm/s mm/min” down position
“Record on” down position
“set” up position
“home” up position
“grid” up position

Right Panel short pen (bottom right)

“cal” “var” up position
“pen” down position
“range” 20mV
“zero” up position (N/A for “old” chart recorder)
Chart speed at 5mm/min

UV Control Unit
Setting #2
Range AU

Power on

4.0 Calculations
4.1 Calculate one Column Volume (CV) use the following formula:

r2H

Where : r = radius of column tube , H = height of packed resin in the column tube

4.2 Calculate Volumetric Flow Rate (VFR) use the following formula:

LFR X r2

Where: Linear flow rate (LFR) = 300cm/hr

4.3 Calculate the time it takes to pump one 1 CV through the column use:

CV/VFR
4.4 Determine pump setting to achieve the volumetric flow rate. Use a beaker of DI water
as the pump feed and a graduated cylinder to measure VFR.

4.5 Measure packed bed height and record findings:

4.6 Calculate one column volume (CV) and record findings:

4.7 Calculate the time it takes to pump one 1 CV through the column and record
findings:

5.0 Column Equilibration

1.1 Column Equilibration will use the following solutions 100mM NaCl, 20mM NaCl

1.2 Equilibrate the column as follows by inserting the feed tubing into the
equilibration buffer

1.3 Verify flow path is open and column is set up in down flow mode.

1.4 Start pump at the calculated linear flow rate.

1.5 Maintain linear flow rate throughout entire chromatography run.

1.6 Equilibrate column for approximately 2 column volumes (2 CV’s)

1.7 When column equilibraton is complete, stop pump and discuss the following
questions with lab partners.
Why equilibrate?
 When is the column equilibrated?

6.0 Protein Load

6.1 Verify the column is equilibrated

6.2 Insert pump feed line into Protein Load solution.

6.3 Start pump to begin protein load

6.4 Load approximately 2 CV’s

6.5 Record volume of protein load:

6.6 Once load is completed, stop pump

Describe what you observed during the load:

What is the first absorbance off the column as seen on chart recorder?

What happens to the proteins as they are loaded on to column?

7.0 Post Load Wash

7.1 The Post Load Wash will use the 100mM NaCl, 20 mM NaPO4 buffer.

7.2 Verify the protein load is complete

7.3 Remove feed line from protein load, rinse, and place in equilibration buffer

7.4 Start pump and begin post load wash for approximately 2 CV’s
7.5 When post load wash is complete turn pump off

What is major function of the post load wash?

What do you look for during this step?

When do/can you stop the post load wash?

8.0 First Elution

1.0 The first elution will use the 325 mM NaCl, 20mM NaPO4 solution

1.1 Verify the After post load wash is completed.

1.2 Insert pump feed line into first elution buffer (325mM NaCL,20mM NaPO4)

1.3 Start pump to begin elution for approximately 2CV’s

1.4 When elution is complete, turn off pump

Why perform this elution step?

What did you expect during this step?

What did you observe during this step?

What came off the column as noted by the rise in absorbance?

 When do/can you stop the elution?

9.0 Product Elution

9.1 The product elution will use the 800mM NaCl, 20mM NaPO4 buffer.

9.2 Verify the first elution step is completed

9.3 Insert pump feed line into the elution buffer (800mM NaCl, 20 mM NaPO4)

9.4 Start pump and run elution buffer through column to elute product.

9.5 When product peaks elutes, collect peak in separate container.

9.6 When product elution is complete, stop the pump

When did you start product collection?

How do you know when to expect product elution?

When did you stop product collection?

10.0 Sanitization:

10.1 After Product collection is completed perform column sanitization

10.2 Insert pump feed line into 1N NaOH

10.3 Start pump and run approximately 2 CV’s through column

10.4 When sanitization is complete, stop pump.

Why perform post use sanitization?

Was there absorbance? Why?

When did you stop sanitization?

11.0 Storage

11.1 After sanitization is complete begin column storage.

11.2 Turn off power on chart recorder

11.3 Insert feed line into the 0.1N NaOH

11.4 Run approximately 2CV’s of storage solution through column

11.5 Stop pump when storage is completed

11.6 Use hemostats to clamp tubing

11.7 Remove top column adapter

11.8 Using a pipet, gently slurry the resin (take caution not to hit the bottom fret)

11.9 Place resin back into its storage container

11.10 Rinse column with a small amount of 0.1N NaOH to collect any remaining resin.

11.11 Rinse column with DI water and place top column adapter back into column.

11.12 Store all equipment in its proper place.