field flow fractionation (FFF)

Question 1

what is fff

Answer 1

is the family of technique suitable for separation of large analyte and colloid particles

Question 2

how is separation occurs in FFF

Answer 2

• FFF has no separation column, has separation channel without stationary phase.
• Mobile phase is carrier liquid

Question 3

what is the principle of separation

Answer 3

we obtain separation of analyte depending on diffusion coefficient and size of the analytes

Question 4

what does retention depends on

Answer 4

• crossflow velocity and diffusion coefficient of analyte

Question 5

what happens at the steady state of analyte between crossflow and diffusion

Answer 5

they are at equilibrium

Question 6

what cause the distance between 2 analyte

Answer 6

it depends on the balance between crossflow and diffusion transport

Question 7

what does retention/ distance of analyte depends on

Answer 7

-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

Question 8

how does velocity affect separation of analytes

Answer 8

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

Question 9

what does retention time depends on in the fff

Answer 9

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

Question 10

what cause zone broadening in AF4

Answer 10

• diffusion: long residing in the channel can cause broader peakes
• unwanted interaction of analyte with accumulation wall

Question 11

what are FFF technique

Answer 11

• 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

Question 12

how to solve the above problem caused by symmetrical flow FFF

Answer 12

by introducing HPLC pump

Question 13

what happens when light interact with matter

Answer 13

it cause charges to polarize

Question 14

what cause charges to easy shift within the molecule

Answer 14

this depends on polarization of the molecule

Question 15

what are light scattering detector

Answer 15

• MALS ( multiangle light scattering )
  -dynamic light scattering

Question 16

what are two type of light scattering

Answer 16

isotropic light scattering and anisotropic light scattering

Question 17

differentiate between isotropic light scattering and anisotropic light scattering

Answer 17

• 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

Question 18

what we know using MALS

Answer 18

angle, rayleig ratio, concentration, reflective index and wavelength

Question 19

what we don’t know using MALS

Answer 19

molecular weight, Rg ( radius of ratio), A2 ( virial coefficient )

Question 20

can we determine Rg from the isotropic and anisotropic MALS

Answer 20

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.

Question 21

what can we determine molecular weight using using isotropic and anisotropic MALS

Answer 21

yes, because when w consider two limits which are concentration and angle approaches to zero.

see the equation in note paper

Question 22

how can we separate large molecule and small molecule

Answer 22

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

Question 23

why is the drms noisy on first peak

Answer 23

because its near isotropic scattering means we have no angular dependence in scattered light so diameter of rms can’t be determined

Question 24

how is dn affected in the diagram

Answer 24

dn (hydrodynamic diameter) is obtained from elution time so it increase with elution time

Question 25

what is X and Y axis of diagram with two signal (what are they)

Answer 25

X-axis is elution time and Y-axis is diameter.
two signal are MALS and UV

Question 26

how does analyte size affect light scattering signal

Answer 26

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

Question 27

how does molecular mass affect the elution time

Answer 27

Mm increase witth elution time

Question 28

how to avoid overloading

Answer 28

inject small amount to avoid noise signals

Question 29

how can we increase resolution when we have constant cross flow

Answer 29

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.

Question 30

what is the limitation

Answer 30

when we increase half-time a lot we can loose resolution again