ÄKTA Purifier 10

The ÄKTA purifier is a fully automated liquid chromatography system designed for method development and research applications. The Protein Production laboratories have two machines; an analytical ÄKTA purifier 10 and a preparative ÄKTA purifier 100.

The Table below summarises flow rates and operating pressures for each system and contains links to system descriptions and features:

System
Max flow
Pressure
Features
ÄKTA purifier 10
10 ml/min
25 MPa
Autosampling
ÄKTA purifier 100
100 ml/min
10 MPa
Direct Load
Common features include:
  • easy pump wash for quick solvent exchange without disturbing the column and routine, automated system wash
  • automatic on-line buffer preparation from stock solutions
  • automatic sample application
    • fixed volume loops from 100 µl to 5 ml
    • superloop for 10 ml to 150 ml
    • increased sample throughput using autosampler
  • on-line UV-detection of up to three wavelengths simultaneously
  • on-line monitoring of conductivity
  • on-line monitoring of pH
  • fraction collection options ranging from microtitre plates to large vessels

The purifiers can be regarded as essentially the same for the majority of applications, however, there are some important changes in configuration between the two machines which allow more specialist functions to be performed.

System pathway

ÄKTA Purifier 10 System Pathway

The ÄKTA Purifier 10 at the Technology Facility has been upgraded from a standard system. The standard unit consists of two pumps (A & B) each with buffer selection valves, a mixer unit, in-line filter, injection valve, detectors, outlet selection valve and fraction collector. In addition, the Technology Facility system has column selection valves and autosampling capabilities.

Features

The ÄKTA purifier 10 contains many optional extras which in combination with the low dead volume and high-sensitivity of the system make it a remarkable analytical tool for a range of applications. The system has capacity to receive buffer in either of its two pumps from two different sources in an automated fashion. This allows complex isocratic and linear gradient programs to be performed from a range of buffers in a fully automated fashion. The purifier 10 offers the most variety in sample application, ranging from sample or Superloops, to direct pump loading as well as multiple injections using the peltier cooled autosampler. The autosampler has given this model a high degree of flexibility allowing the user to treat the purifier 10 as a HPLC system or perform 2 Dimensional (2D) chromatography steps with rapid throughput.

The real potential of the purifier 10 can only be exploited when we consider all of the above in combination with the column scouting valves. This gives the system the ability to select automatically any of 7 different columns and thus make method development extremely efficient. An example of this type of use would be in the optimization of a chromatography step. In this case we could screen a series of columns with varying substitution i.e. a hydrophobic interaction test kit, and use the autosampler to load a defined quantity of sample onto each of the resins. The most compatible resin would therefore be rapidly identified.

The purifier 10 has the 10 mm path-length flow cell which gives the instrument 5x greater sensitivity than the purifier 100 system and combined with such features as high-pressure tolerance, low dead volumes, peak fractionation etc. It makes the system ideal for peptide analysis by reversed-phase HPLC. In addition, the software package provides full integration capabilities.

User information

Due to the nature of the pump and to maximize column lifetime it is essential that all buffers are filtered (0.2 µm) and de-gassed before use with the ÄKTA purifier. All facilities for buffer preparation may be found in the Protein Production laboratories if not already available. The purifier 10 is used at room temperature, however, the purifier 100 is housed in a cold cabinet giving this sytem the power to perform FPLC type applications at 4°C.

Suitable samples should be clarified prior to use with the system. This may be achieved by filtering (0.45 or 0.2 µm) or by centrifugation at 10,000 x g for 20 minutes. Sample loading onto the column is system dependent as well as volume dependent.

Important information about sample loops
The sample loops available range from 10 µl to 5 ml, however, the pressure rating of the loops varies with increasing size;

  • Loop 10 µl - Loop 500 µl inc. 25 MPa
  • Loop 1 ml - Loop 2 ml inc. 10 MPa
  • Loop 5 ml 1 MPa

The ÄKTA purifiers are driven by a Windows 2000 software package called UNICORN and are fully networked with the Biology department. The user must first log onto the PC as either themselves or alternatively as Biology in 'workstation' only mode. Next, launch the UNICORN software and then log-on to the package using the supplied password (obtainable from Protein Production staff). The UNICORN software comprises four main areas, a method/results manager, a method editor, system control and an evaluation package. Method construction on the ÄKTA purifier could not be simpler, the system uses a Method Wizard to direct you through the process providing all the necessary options to create complex protocols. Providing you have a basis in either FPLC or HPLC, then we would expect a user to be comfortable with the systems following a half-day training session.

Finally, the Protein Production laboratories would not supply a purification system without the columns to go with it! We have a wide range of columns available and the experience to custom pack resins. For more information on the ÄKTA purifiers and their capabilities, the types of columns which are available or to book a training session on the machine, please contact Jared Cartwright or Mick Miller in the Protein Production laboratories.

ÄKTA Purifier 100