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June 16, 2020

5 frequently asked questions about ÄKTA systems

By Ana Pinto, Support Scientist

Cytiva support scientists answer the five most common questions they receive about our chromatography systems. Read more about how to choose the right columns for ÄKTA start and mixers for ÄKTA pure, and how to carry out lab-scale reversed phase chromatography.

I am looking for advice on columns to use with an ÄKTA start chromatography system. I will be working with protein fragments designed to block enzyme functional sites. The protein fragment sizes will be 9, 35 and 75 kilodalton. Can you advise?

The ÄKTA start is an easy-to-use system for simple applications. It offers a maximum operating pressure of 0.5 MPa and flow range of 0.5 to 5 mL/min – this narrows down the choice of columns that you can use with ÄKTA start.

We recommend gel filtration columns that operate at low pressure. These include HiTrap Desalting and HiPrep Sephacryl (< 0.5MPa).

  • HiTrap desalting columns have a low fractionation range (100 - 10000 Da), so they are used for buffer exchange and group separation.
  • HiPrep Sephacryl columns are low-to-medium pressure operating columns used for macromolecular separation. They cover a wide fractionation range (10 - 1500 kDa).

Since you want to separate protein fragments from 9 to 75 kDa, we recommend the following columns:

I have an ÄKTA pure 150 chromatography system with a 5 mL (standard) mixer chamber. If want to operate a 7 cm diameter column, should I use a 15 mL mixer?

We recommend a 5 mL mixer for flow rates up to 50 mL/min and a 15 mL mixer for flow rates above 50 mL/min. For your situation, we would recommend starting with the 5 mL mixer – you can switch to the 15 mL mixer if you need better gradient performance.

Is there any data on sulfuric acid compatibility with the ÄKTA avant chromatography system?

Unfortunately, there is no data on the compatibility of sulfuric acid with the ÄKTA avant. However, we found that for PEEK and titanium, ÄKTA avant can withstand up to 10% of sulfuric acid.

I am looking for an alternative to 20% ethanol to use as a long-term preservative for a Q-Sepharose FF column. Can I use isopropanol?

We have no long-term study of Q-Sepharose FF in isopropanol, so we cannot recommend it as a storage solution. However, benzyl alcohol is a well-established alternative. You can find storage data for a wide selection of chromatography resins in application note 28-9899-01 AC.

We have an ÄKTA pure 25 chromatography system in our lab. We would like to carry out lab-scale reversed phase chromatography to isolate hydrophobic proteins from natural materials (e.g., nuts, seeds, and fruits). Also, we have an F9-C fraction collector attached to the ÄKTA, and we have some safety concerns when it comes to solvent vapor building up within the unit. Do you have any information or guidelines about using ÄKTA pure systems for lab-scale reversed phase chromatography?

To perform reversed phase chromatography using the ÄKTA pure 25 chromatography system, there are some important guidelines to follow:

  • Organic solvents can weaken PEEK tubing walls more easily than water-based buffers. Take special care when using organic solvents close to pressure limits for prolonged periods. 
  • We recommend replacing the mixer sealing ring with the highly resistant O-ring (Product code 29011326).
  • You can change the pump piston sealing too, but this sealing is dependent on the acetonitrile concentration and pressure used.
  • To extend the life of the pump sealing, use lower acetonitrile concentrations and remove the pump from acetonitrile immediately after each run . 
  • Be aware that organic solvents and high concentrations of organic acids (e.g., acetic acid and formic acid) can also reduce the lifetime of the O-ring and pump piston sealing.
  • Do not fractionate flammable liquids using fraction collector F9-C – this is a fire hazard.
  • Use an outlet valve or fraction collector F9-R (29011362) when running RPC methods or other procedures using organic solvents. 

This information is also included in the handbook for your ÄKTA pure chromatography system.

 

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