field flow fractionation (FFF) Flashcards
what is fff
is the family of technique suitable for separation of large analyte and colloid particles
how is separation occurs in FFF
- FFF has no separation column, has separation channel without stationary phase.
- Mobile phase is carrier liquid
what is the principle of separation
we obtain separation of analyte depending on diffusion coefficient and size of the analytes
what does retention depends on
- crossflow velocity and diffusion coefficient of analyte
what happens at the steady state of analyte between crossflow and diffusion
they are at equilibrium
what cause the distance between 2 analyte
it depends on the balance between crossflow and diffusion transport
what does retention/ distance of analyte depends on
-for small analyte they have higher diffusion coefficient so they will travel long distance than large analyte with lower diffusion coefficient and they will be far from ultrafiltration membrane
- for large analyte they have lower diffusion coefficient and they are close to the ultrafiltration membrane and they will travel small distance than small analyte
how does velocity affect separation of analytes
velocity increase as we move away from the wall of the channels (ultrafiltration membrane ).
so means velocity increase with increase of diffusion coefficient which decrease retention time
what does retention time depends on in the fff
tr is determined by Qc / Qout ratio
- when Qc increase with constant Qout retention time increase and
- when Qout increase with constant Qc retention time decrease.
- depends on height of the channel, increase with Tr
- diffusion coefficient increase with decrease of Tr
- radius/ size of analyte increase with retention time
note: more detail in the paper note
what cause zone broadening in AF4
- diffusion: long residing in the channel can cause broader peakes
- unwanted interaction of analyte with accumulation wall
what are FFF technique
- symmetrical flow FFF: which give low migration velocity, long retention time, low flow rate which result in broadening of peaks and analyte getting stuck in the channels
- asymmetrical flow FFF (AF4): use of (rectangular channel) and different channels has only one permeable walls. which results in high speed and high resolution power
- asymmetrical flow FFF (trapezoidal channel): using rectangular channel they will be zone broadening because migration time decrease when approaching the channel outlet causing sample zone to broaden. but using trapezoidal channel: this allow us to narrow the channel outlet which will decrease the volume to ward the channel outlet causing migration velocity to be kept resulting in decreasing sample zone to broaden
how to solve the above problem caused by symmetrical flow FFF
by introducing HPLC pump
what happens when light interact with matter
it cause charges to polarize
what cause charges to easy shift within the molecule
this depends on polarization of the molecule
what are light scattering detector
- MALS ( multiangle light scattering )
-dynamic light scattering
what are two type of light scattering
isotropic light scattering and anisotropic light scattering
differentiate between isotropic light scattering and anisotropic light scattering
- isotropic light scattering:
- no angular dependent on scattered light because when the light enter the light volume will be distributed equal.
- size d of object being analyzed is much small than wavelength λ.
d < 15-20nm, used on small protein/ samll particle
- anisotropic light scattering:
* angular dependent on the scatted light because light volume is not distributed equally.
* size d is small or equal to the wavelength. d < 7000nm. large molecule/particle
what we know using MALS
angle, rayleig ratio, concentration, reflective index and wavelength
what we don’t know using MALS
molecular weight, Rg ( radius of ratio), A2 ( virial coefficient )
can we determine Rg from the isotropic and anisotropic MALS
isotropic: as we don’t have any slope we don’t obtain any information about the form factor so we can not determine Rg/ radius of their ratio.
anisotropic: because we have slope we can determine radius of their ratio.
what can we determine molecular weight using using isotropic and anisotropic MALS
yes, because when w consider two limits which are concentration and angle approaches to zero.
see the equation in note paper
how can we separate large molecule and small molecule
under mass signal as Y-axis and elution time as X-axis. small particle will be obtained at lower retention/elution time and large particle will be obtained at large elution time
why is the drms noisy on first peak
because its near isotropic scattering means we have no angular dependence in scattered light so diameter of rms can’t be determined
how is dn affected in the diagram
dn (hydrodynamic diameter) is obtained from elution time so it increase with elution time
what is X and Y axis of diagram with two signal (what are they)
X-axis is elution time and Y-axis is diameter.
two signal are MALS and UV
how does analyte size affect light scattering signal
light scattering is very sensitive to the size of analyte even tho the concentration will be high.
- so with short elution time, means analyte are small and light cattering will be low
how does molecular mass affect the elution time
Mm increase witth elution time
how to avoid overloading
inject small amount to avoid noise signals
how can we increase resolution when we have constant cross flow
we can use exponentially decaying cross flow from ..ml/min (choosing cross flow that can give us maximum resolution of the 2 peaks) with half-time of ..min.
what is the limitation
when we increase half-time a lot we can loose resolution again
