News

New @ CAT-AgroFood: Asymmetric Flow Field Flow Fractionation (AF4)

Published on
June 5, 2012

CAT-AgroFood has recently invested in Asymmetric Flow Field Flow Fractionation (AF4) equipment. This technique enables separation, fractionation and sizing of (bio)polymers, particles and colloids, without the use of column material. The facility is open for both external users and researchers of Wageningen UR. Experts of the Laboratory of Food Chemistry at Wageningen UR have recently completed a training for operating the new set-up and can now assist clients. They will also use the new equipment themselves to study interactions of proteins and other food components. The CAT-AgroFood AF4 is in full operation and is ready to welcome new users.

The AF4 technique, with the available set of detectors can be used for the separation and characterization of soluble biopolymers (i.e. proteins and carbohydrates), as well as aggregates and particles in the size range of 1 nm to 1 μm.

Surender Dhayal and Peter A. Wierenga of the Laboratory of Food Chemistry of Wageningen University have already successfully used the AF4 to separate monomeric proteins, aggregated proteins, and polysaccharides. The results illustrate how this technique can be used to quickly obtain information on the size-distribution of molecules (or particles, aggregates) in a sample. The results presented below illustrate the possibilities of this technique.

Separation of monomeric proteins

  Method

  • Channel type: Short channel
  • Spacer: 350 W
  • Membrane: 10 kDa Regenerated cellulose
  • Elution buffer: 10 mM Phosphate buffer (pH 7)
  • Sample injected: 20 μg (20 μL of 1 mg/mL concentration)
  • Channel flow: 1 mL/min
  • Cross flow: 3 mL/min for 25 min and then linearly decreased to 0 mL/min

The first experiments focussed on the elution and analysis of monomeric proteins in a range from 14-660 kDa. These proteins are separated at constant cross-flow (see figure 1A). During the elution on the AF4 the detection with MALLS allows the determination the molecular weight. The results show good correlation between the theoretical and experimental Mw (figure 1B).

Figure 1: Elution patterns of monomeric proteins (A), and the relation between elution time and the molar mass of the proteins (B) using determined (Δ) or literature values (O) for the molar mass
Figure 1: Elution patterns of monomeric proteins (A), and the relation between elution time and the molar mass of the proteins (B) using determined (Δ) or literature values (O) for the molar mass
Separation of enzymatically cross-linked protein aggregates
Method
  • Channel type: Short channel
  • Spacer: 350 W
  • Membrane: 10 kDa Regenerated cellulose
  • Elution buffer: 10 mM Phosphate buffer (pH 7)
  • Sample injected: 40 to 100 μg (20 μL of 2 to 5 mg/mL concentration)
  • Channel flow: 1 mL/min
  • Cross flow: 1 mL/min at start and then exponentially decreased to 0.1 with a decay time constant of 2.5 min.
Using Horse-radish peroxidase (HRP) α-lactalbumin was crosslinked for different times, to produce soluble aggregates of different sizes (figure 2A). These were then dialysed to remove monomeric proteins and analysed by AF4 (figure 2B).

Figure 2: (A) Increase in aggregate size (from DLS) during enzymatic cross-linking of α-lactalbumin, and (B) the AF4 elution patterns for the four samples taken during the reaction
Figure 2: (A) Increase in aggregate size (from DLS) during enzymatic cross-linking of α-lactalbumin, and (B) the AF4 elution patterns for the four samples taken during the reaction
For these larger aggregates, the MALLS can be used to determine the Mw as well as the radius of gyration (Rg), as shown in table 1. The results show that the smallest particles tested consist of around 200 α-lactalbumin molecules. During crosslinking the both the Mw as the Rg are increased, but the radius increases more, resulting in a lower density of the final cross-linked aggregate.

Table 1: Properties of cross-linked protein aggregates: Hydrodynamic radius (Rh) obtained from DLS, and radius of gyration (Rg) and molecular weight (Mw) obtained from AF4 separation, and the derived density of the protein aggregates
Crosslinking reaction time [h] z [nm] z [nm] [106 Da] <ρ> [kg/m3]
4 25 27.2 ± 0.6 2.6 ± 0.008 35
7 50 82.9 ± 0.6 22.9 ± 0.18 8.8
10 75 140.1 ± 3.1 89.0 ± 5.8 6.2
15 100 134.8 ± 0.1 79.0 ± 4.8 6.8
Separation of dextran

  Method

  • Channel type: Short channel
  • Spacer: 350 W
  • Membrane: 10 kDa Regenerated cellulose
  • Elution buffer: 10 mM Phosphate buffer (pH 7)
  • Sample injected: 250 μg (25 μL of 10 mg/mL concentration)
  • Channel flow: 1 mL/min
  • Cross flow: 0-5 min: 1mL/min

Dextrans are branched polysaccharides that can vary in molecular weight from several kDa until a few MDa. To test the separation of these polymers by AF4, dextrans with different specified molecular weight were used.

Figure 3: AF4 elution patterns for three dextran samples (D40, D500, D2000), detected with refractive index (dotted lines) and light scattering intensity (solid line)
Figure 3: AF4 elution patterns for three dextran samples (D40, D500, D2000), detected with refractive index (dotted lines) and light scattering intensity (solid line)
In the figure it is clear that the dextrans consist of a heterogeneous mixture of molecules with different molecular weights. The dextran with the lowest has a dominant peak at the short elution time, and the sample with the highest has one dominant peak at the high elution time, while the intermediate sample shows two populations. From the analysis of the different populations, it is seen that the obtained molecular weights of the dominant peaks correspond to the values provided by the supplier (table 2).

Table 2: Properties of the dextran samples
Dextran [kDa] Mw [kDa] z [nm]
40 Peak 1 34 ± 0.8 -
Peak 2 153 ± 7 -
500 Peak 1 229 ± 4 -
Peak 2 607 ± 3 22.8 ± 0.91
2000 Peak 1 - -
Peak 2 2440 ± 10 43.7 ± 0.48

If you are interested in using this facility or if you would like more information, please contact Surender Dhayal or CAT-AgroFood.


A description of the technique and its applications: