Eclipse AF4 and accessories (Wyatt Technology Corporation)
The Eclipse is the control unit for Asymmetric Flow Field-Flow Fractionation (AF4). This technique belongs to the chromatographic methods enabling separation of the biomolecules or the particles based on the hydrodynamic behaviour. The sample is separated in the empty channel by a combination of laminar and perpendicular flow. Thanks to the adjustment of the separation force and the choice of both the separation profiles and the separation channels, the AF4 technique enables the separation of particles in the range from 1 nm to 1000 nm. The sample injection to the separation channel is automated (Agilent Technologies, Inc.) with a 100 μl injection loop. The separation takes place at a constant temperature in the adjustable range of 4 °C to 90 °C. The Analytical channel with a capacity of about 100 μg of the sample, the Semi-preparative channel with a capacity of about 1 mg of sample and the Hollow fiber channel (HF5) are available. Due to the small volume of the HF5 channel, the separation is processed in a small sample volume, high sensitivity and short analysis time. The separation is followed by a series of detectors: The Agilent 1200 Series Diode Array and Multiple Wavelength Detector UV-VIS DAD detector (Agilent Technologies, Inc.) with a flow cell allowing online determination of absorbance of up to 8 wavelengths ranging from 190 nm to 900 nm; The Agilent InfinityLab LC 1260 Series Infinity II Fluorescence Detector (Agilent Technologies, Inc.) for online measurements of multiple excitation or emission wavelengs. DAWN® HELEOS® (Wyatt Technology Corporation) Multi-Angle Light Scattering (MALS) detector measuring the angular dependence of the intensity of the scattered light to determine the particle size expressed as gyration radius (range 10 nm to 500 nm), molecular weight (200 Da to 1 GDa), and a second virial coefficient (describing the level of the interaction between the particles and the solvent). The sensitivity of the detector, depending on the nature of the sample, is about 0.2 μg/ml. The ambient detector has 18 angles between 15° and 160° and He laser with a wavelength of 658 nm. The 149° detector is equipped with a dynamic light scattering module allowing online measurement of the hydrodynamic radius (in the range of 1 nm to 300 nm). The Optilab® T-rEX ™ is an online detector for determining the differential refractive index (dRI) between the eluting sample and the reference liquid (pure mobile phase) and the absolute refractive index (aRI). As a light source, there is an LED with a wavelength of 660 nm. The AF4 allows the collection of individual fractions through a septum into tempered vessels using a 1260 Infinity II Analytical-Scale Fraction Collector (Agilent Technologies, Inc.).