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Data file 29-0211-96 AC
Chromatography systems
ÄKTA ™
pure
ÄKTA pure is a flexible and intuitive chromatography system (Fig 1) for fast purification of proteins, peptides, and nucleic acids from microgram levels to tens of grams of target product. ÄKTA pure is a reliable system where hardware and UNICORN™ system control software are designed to work together with columns and media to meet any purification challenge.
ÄKTA pure is available in two versions: ÄKTA pure 25 designed for a broad range of rearch applications and purification tasks in a multi-ur environment; ÄKTA pure 150 is well suited for optimizing resource utilization and productivity in routine large-scale preparative purification. The system supports a wide range of chromatography techniques and meets the automation requirements needed to deliver the highest purity. The system is configurable and can be upgraded at any time with
a wide range of options to further increa its capabilities depending on your purification needs. ÄKTA pure is the product of over fifty years of
experti in the development of ÄKTA purification systems.
ÄKTA pure offers the following benefits for you:
• Modular system design with a large range of options to allow flexibility in purification of proteins and peptides • Customizable system that is easy to upgrade as your rearch needs develop • Reliable system with components and integrated features bad on the proven design of ÄKTA avant • UNICORN 6 software provides simple, intuitive, and flexible preprogrammed method templates and total system control to simplify your job • Predefined method ttings for all GE Healthcare Life Sciences lab-scale chromatography columns
System overview
ÄKTA pure chromatography system is a highly versatile, modular system with a number of design features to facilitate reliable purification.
The system consists of the ÄKTA pure instrument and UNICORN 6 control software. The system is
modular in
design with all valves, monitors, and columns mounted
GE Healthcare Life Sciences
Fig 1. ÄKTA pure is a flexible chromatography system for the reliable purification of proteins, peptides, and nucleic acids at laboratory scale.
on the forward facing wet side of the system, to allow easy interaction with the instrument modules (Fig 2). Additional components such as valves, monitors, and nsors from the wide range of optional modules can easily be added to the available positions. Multiple rails for attachment of column holders and equipment are located at the front and on the side of the instrument. A buffer tray on the top of the instrument provides a large storage area for vesls and bottles. The instrument control panel shows the system state and allows the possibility to interact with the run (pau/continue) at the touch of a button.
The system weighs only 48 kg in basic configuration and 53 kg when fully equipped with options. The relatively low weight enables easier placement in the lab and the system dimensions allow it to fit conveniently into a standard cold cabinet for work with labile samples.
(A)
Rails for attachment of column holders and accessories (also on Mixer
Pressure Mixer
Pressure
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An in-line filter is mounted on the mixer. The filter is easy to change, and the mixer is also easily changed by snapping it in or out of the mixer holder. The mixer size ud for any given run is always noted in the result file.
Injection valve
The injection valve allows for a variety of sample application techniques using sample loops or Superloop™ sample injection device. The novel valve design eliminates the need for replumbing when changing between various sample
application techniques. A sample loop with a volume of 500 μL is delivered with the system. Sample loops can be filled manually, via a syringe, or with a sample pump; the same sample application options apply to the u of Superloop. Sample loops can also be filled using the system pump.
Moreover, sample can also be applied to the column directly using an optional sample pump or the system pump.
UV monitoring
ÄKTA pure is equipped with either a fixed wavelength UV monitor or a variable multiwavelength UV and visible spectrum monitor.The fixed wavelength (280 nm) UV monitor (U9-L) incorporates LED technology, which is durable and reliable and is ready to u at start-up. Moreover, UV monitor U9-L does not heat the sample. The monitor is available with a 2 mm flow cell as standard (included at delivery) and an optional 5 mm flow cell when higher nsitivity measurements are required.
To determine protein paration at different wavelengths, UV monitor U9-M is designed for multiwavelength detection in the UV and visible spectrum from 190 to 700 nm.
UV monitor U9-M allows monitoring of up to three wavelengths
simultaneously (Fig 3 and 6). For optimized performance when
purifying samples with different protein concentrations, there
are three flow cell path lengths available; 0.5, 2 (included at
delivery), and 10 mm. The flow cell design, together with fiber optic technology, provides a high signal-to-noi ratio without causing any local heating of the UV flow cell. The monitor contains a high-intensity xenon lamp with a long lifetime that requires minimal start-up time. Every time the instrument is switched on, the monitor is automatically calibrated. All U9-M UV cells are calibrated at manufacturing. The UV signal is automatically normalized, which helps when comparing data from different systems.Monitoring with multiple wavelengths can be ud to detect
contaminants, specifically labeled proteins, or target molecules
that do not absorb light at 280 nm. To demonstrate this,
molecular weight standards were monitored at 214, 280, and 340 nm wavelengths. Detection at 214 nm reveals peptide
bonds of all proteins and may be uful if the concentration
and extinction coefficient at 280 nm is low for the target protein.
Ferritin, a multimeric iron-storage protein, showed stronger
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absorbance at 340 nm than the other proteins due to the
high number of ferric ions in the center of the molecule (Fig 3).
Both UV monitor U9-L and UV monitor U9-M can be combined with a cond UV monitor U9-L to give incread application capabilities such as multistep applications or when using small and large flow cells simultaneously to detect both low and high protein concentrations.
Regardless of configuration, ÄKTA pure always comes with two high-performance system pumps, system pressure monitor for column protection, mixer, injection valve, and UV monitor. ÄKTA pure has a wide range of optional modules to allow a large number of possibilities. The system flow path is designed to minimize band-broadening effects, and all wetted materials ud in the flow path are biocompatible and resistant to
commonly ud solvents. The instrument front is designed with empty module positions where optional valves and monitors can be mounted to enable a flexible configuration of the flow path. Examples of two ÄKTA pure system configurations are shown in Figure 2.
UNICORN 6 control software allows a fast and easy start to creating methods and starting runs. UNICORN 6 eliminates the need for programming skills as creation of chromatography methods is done by simple drag-and-drop operations. In addition, the software is modular allowing the addition o
f features such as Column Logbook and Design of Experiments (DoE) functionality for method development. Licensing options for remote access to the system and/or for creating methods or evaluating results give even greater convenience. If preferred, the system can be t up so that it enters “power save mode” after method end, which enables reduction of power consumption by around 80%.
ÄKTA pure system components and available options are described in the following ctions in more detail.
ÄKTA pure standard components
System pump土豆焖面
The two system pumps are bad on the modern technology
developed for ÄKTA avant systems. The robust construction
delivers reproducible flow rates at both low and high back
pressures, allowing short paration times.
Each pump consists of one pair of pump heads, which deliver low-pulsation flow to the mixer. The continuous and accurate flow rates generated enable reproducible isocratic or gradient elution. For ÄKTA pure 25 the system pumps provide a flow rate range of up to 25 mL/min at maximum operating pressure of 20 MPa. For ÄKTApure 150 the flow rate is up to 150 mL/min at maximum operating pressure of 5 MPa. For column packing, ÄKTA pure 25 and 150 can be ud at flow rates up to 50 mL/min and 300 mL/min, respectively. A system pressure monitor is connected to the pumps to continuously measure
system pressure and enable the flow rate to be automatically
adjusted to avoid reaching any defined pressure limit.
Mixer
The mixer enables homogeneous buffer composition during
gradient runs. The choice of mixer chamber size depends on
the flow rate and buffers ud, with a larger mixer volume
required for higher flow rates or difficult-to-mix buffers. Table 1
shows the mixer chamber sizes available for each instrument.
Table 1. Mixer chamber sizes available
System Mixer chamber sizes
AKTA pure 25 Included: 1.4 mL; options: 0.6 and 5 mL AKTA pure 150
Included: 1.4 and 5 mL; option: 15 mL
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Conductivity monitor
The conductivity monitor measures conductivity of buffer and samples for online monitoring of the true gradient. An integrated temperature nsor corrects for variations in
conductivity due to the temperature. The conductivity monitor has a broad reading range and is therefore able to monitor conductivity in all different chromatographic techniques.
ÄKTA pure optional modules for enhanced automation
Sample application options The optional sample pump (Fig 4) is designed to allow automatic sample application directly to a column or indirectly via a sample loop or Superloop. Using the sample pump saves time by eliminating laborious sample application steps and is especially uful when handling large sample volumes. The pump consists of two pump heads and is bad on the same pump principle as the system pumps. Pump purging and air removal can easily be performed automatically. The sample pump is equipped with a pressure nsor for control of the sample flow rate to protect the column while preventing pressure stops and minimizing the time for sample loading. Using the sample pump, samples can be loaded at flow rates of up to 50 mL/min (Sample pump S9) or up to 150 mL/min (Sample pump S9H).
The optional sample inlet valve, V9-IS or V9H-IS, is intended to be ud with the sample pump. Inlet valve allows fast,
automatic loading of up to 7 different samples. The integrated air nsor enables complete sample application without the need to preprogram the sample volume. The valve has ven sample inlet positions plus a dedicated buffer inlet for filling the sample pump with solution before the sample is introduced and for washing out the valve and pump between runs. During sample application, the air nsor detects when sample has been completely loaded so that the method can continue to the ne
xt step without air being introduced into the flow path or column.
Fig 4. ÄKTA pure sample pump.
Fig 3. Gel filtration (GF) with multiwavelength detection (214, 280, and 340 nm) of proteins using ÄKTA pure with UV monitor U9-M. The column ud was Superdex 200 10/300 GL. The peaks obrved on the chromatogram are 1) ferritin (M r 440 000), 2) aldola (M r 158 000), 3) conalbumin (M r 75 000),
4) ovalbumin (M r 44 000), 5) carbonic anhydra (M r 29 000), 6) ribonuclea A (M r 13 700), and 7) aprotinin (M r 6500).
A 214 n m (m A U )
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A 280 n m a n d A 340 n m (m A U )
Volume (mL)
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A 280 nm A 214 nm A 340 nm
Buffer lection
ÄKTA pure can be equipped with two different types of inlet valves that allow lection of buffers and
wash solutions. Valves with multiple inlets enable cleaning reagents to be permanently online, which means that columns and system can be cleaned conveniently at regular intervals.
Inlet lection valve V9-IAB compris two A and two
B inlet positions in a single valve offering a convenient
solution for automation of buffer application and post-run cleaning of columns and system when performing basic
chromatography. Any A inlet can be combined with any B inlet to generate gradients.The inlet automation valves A and B provide up to 2 × 7 inlets. Multiple inlets enable automatic screening of buffer and reagent conditions. Each of the inlet automation valves is equipped with an integrated air nsor, which helps in excluding air from the system. If air is detected, the system can be paud so that the air can be purged before it enters the flow path. Column control A column valve can be connected to the system and ud to control the flow to the column. ÄKTA pure can be equipped with one of two different column valves. Column control valve V9-Cs allows connection of one column and has an integrated bypass function, which enables washing of the system without the need to remove the column. The column control valve also allows rever flow through the column, f
or fast and effective elution of strongly bound proteins, sharper bands, and a concentrated target molecule eluent.
Column: Superdex™ 200 10/300 GL
Sample:
Molecular weight standards for gel filtration Sample volume: 100 μL
Eluent: PBS (10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4)Flow rate: 0.5 mL/min System:
AKTA pure 25
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Fig 6. Column scouting for purification of S-aminotransamina expresd in E. coli . Column lection valve V9-C allowed the connection of five HiTrap HIC columns to ÄKTA pure for this evaluation. UV monitor U9-M was ud for multiwavelength detection. From this scouting, HiTrap Phenyl FF (high sub) 1 mL was lected for u in further scale-up studies.
Fig 5. For incread operational safety, the column lection valve enables continuous measurement of precolumn (Pre-CP) and post-column pressure (Post-CP) during runs. The pressure difference over the packed media bed (Δp) is calculated from the two signals.
Column lection valve, V9-C or V9H-C, also has the integrated bypass and rever-flow functions. Connection of up to five columns for automatic column switching is possible using this valve. Connection of multiple columns minimizes manual intervention and reduces further the risk of introducing air into the column.
The column lection valve has two integrated pressure
nsors: the first nsor measures pressure before the column, enabling protection of the column hardware while the cond measures the pressure after the column. The pressure drop over the column (Δp) is calculated by measuring the difference between the two pressure readings and can be ud to protect the packed media bed (Fig 5).
B u f f e r B (%)
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mL HiTrap Butyl-S FF 1 mL
HiTrap Phenyl FF (high sub) 1 mL
HiTrap Octyl FF 1 mL
HiTrap Phenyl FF (low sub) 1 mL
HiTrap Butyl FF 1 mL
Conductivity (mS/cm)
Conductivity (mS/cm)
Conductivity (mS/cm)
Conductivity (mS/cm)食指戴戒指
System pump pressure 0.6 MPa Column top hardware pressure 0.5 MPa
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Column bottom pressure 0.2 MPa
Columns: Five columns from HiTrap HIC Selection Kit Sample: Supernatant after precipitation with 2 M ammonium
sulfate (AS) at room temperature of extract of E. coli expressing S-aminotransamina (adjusted to 1.5 M AS)
Sample volume: 2 mL Buffer A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0Buffer B: 50 mM sodium phosphate, pH 7.0Flow rate: 1 mL/min UV cell: 10 mm System: AKTA pure 25 equipped with Column lection valve V9-C
and Loop valve V9-L
pH monitoring
An optional pH valve with an integrated pH electrode (not included) enables in-line pH monitoring during the run.
The pH monitor is easily calibrated by injection of calibration buffer directly into the valve with the pH
electrode mounted. A flow restrictor is connected to the pH valve and can be automatically included in the flow path to generate a back pressure that prevents the formation of air bubbles in the UV flow cell. The pH valve is ud to direct the flow to the pH electrode and flow restrictor, or alternatively, to bypass one or both. Bypassing the pH electrode means that it can be stored and kept in place on the valve at all times.
The flexibility of the column lection valve for connection of up to five columns was demonstrated in a column
scouting study using columns for hydrophobic interaction chromatography (HIC). Five columns from HiTrap™ HIC Selection Kit were connected to ÄKTA pure and ud for column scouting for optimization of purification conditions of S-aminotransamina in clarified E. coli extract. UV
monitor U9-M was ud for detection of the protein at two wavelengths. Chromatograms of the five parate HIC runs are shown in Figure 6. Eluted fractions were analyzed using GF and SDS-PAGE (data not shown).
The A 420 signal specifically monitors the target protein. The columns giving the sharpest and most symmetrical peaks at A 420, as well as the highest possible purity, were lected for subquent opti
mization and scale-up experiments. HiTrap Phenyl FF (high sub) 1 mL and HiTrap Butyl FF 1 mL gave the most promising results under the conditions ud, and HiTrap Phenyl FF (high sub) 1 mL was lected for further optimization in this ca.
