Superose™ 6 Increase 5/150 GL

How would I get the best resolution of my column?

For stand alone columns please assemble the monitor to the column outlet.

If the column is connected to a system, connect the column as close as possible to the monitor.

How can I check the functionality of my column?

Leading and tailing peaks as defined according to the following figures:

 

 
Experience shows that the best method of expressing the efficiency of a packed column is in terms of its height equivalent to a theoretical plate (HETP), reduced plate number (h) and its peak asymmetry factor (As). 
Please be aware of that for small columns, less than 10 ml bed volume, the system dead volume has an impact on the column evaluation values.

Can I run two columns in series to increase resolution or capacity?

Gel filtration chromatography
Running columns in series increases the resolution

Affinity chromatography, Hydrophobic interaction, Ion exchange chromatography
Reverse phase chromatography
Running columns in series increases the capacity but may have a bad impact on the resolution
due to increased dead volume.

Please note that back pressure may increase.

How much sample, in mg or ml of protein can I load onto my column?

Affinity Chromatography

• The binding capacity values listed in the selection guide below, are typical for the given species. However, there might be considerable deviations in binding capacity for different immunoglobulins derivated from same species, even if they are of the same subclass.
• Protein binding capacity is protein-to-protein dependent. The given capacities are to be considered as starting values.
• For optimal separation use approximately one fifth of the total binding capacity

Desalting

• In group separation (desalting) the sample volumes can be up to 30% of the column volume.
• Regarding sample volumes for prepacked columns, please refer to Sample preparation for analysis of proteins, peptides and carbohydrates selection guide.

Gel filtration chromatography

• Choose a sample volume of < 0.5% of the column volume for media with average particle size in the range of 10-15 µm and < 5% of the volume for media with average particle size in the range of 30-100 µm.
• In group separation (desalting) the sample volumes can be up to 30% of the column volume.
• Regarding sample volumes for prepacked columns, please refer to selection guide below:

       Sample preparation for analysis of proteins, peptides and carbohydrates selection guide.
       Gel filtration columns and media selection guide and product profile selection guide.

Hydrophobic interaction chromatography

• The binding capacity values in the media and column instructions, there are examples and to be considered as starting values. Protein binding capacity is protein-to-protein dependent.
• For optimal separation use approximately one fifth of the total binding capacity.

Ion exchange chromatography

• The binding capacity values in the Ion exchange columns and media product profile, please refer to Ion exchange columns and media product profile selection guide, there are examples and to be considered as starting values. Protein binding capacity is protein-to-protein dependent.
• For optimal separation use approximately one fifth of the total binding capacity.

Reversed phase chromatography

• The binding capacity values in the media and column instructions, please refer to the related document, there are examples and to be considered as starting values. Protein binding capacity is protein-to-protein dependent.
• For optimal separation use approximately one fifth of the total binding capacity.

The backpressure increases during operation

Find more causes and remedies in the troubleshooting section.

My column contains air, what can I do?

More causes and remedies might be presented in the troubleshooting section.

My column is clogged and/or discoloured. How can I clean my packed column?

Prepacked columns
Please clean the column according to instructions.

Other columns
Please follow the recommendations in the media instructions.

If the issue persists after cleaning you have to replace the column with a new one (prepacked columns) or repack the column with fresh medium.

My column has run dry. Can I reuse the column?

If the column still is free from bacterial growth it might be possible to reuse the column. It could be worth trying.

Remove the air by running liquid slowly from bottom to top of a vertical placed column at a pressure close to maximum column pressure. The size of air bubbles decreases with increased pressure and facilitate the bubbles escape from the column.

If you don't succeed in removing air from the column, the column must be replaced (prepacked columns) or repacked.

My column is leaking buffer, what shall I do?

Leakage around connectors

Leaking tubing

Leakage around end-pieces  ( Not applicable for the pre-packed columns )

How do I store my column?

Prepacked columns
Please store the column according to instructions.

Other columns
Please follow the recommendations in the media instructions.

How should I prepare my sample prior to loading the column?

Please prepare the sample according to instructions and handbooks


Prepacked column instructions

Please refer to relevant Instruction.


Other columns

Please refer to relevant Handbook.

My column has a gap between the packed bed and adaptor. Can I reuse the column?

What chemicals are compatible with my column?

Please see the chemical stability in the instructions for prepacked columns, empty columns and media.

What is the pressure limit of my column?

Prepacked Columns
Please read 
Prepacked chromatography columns for ÄKTAdesign systems

Other Columns