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fv
in fluid
Fd
Figure 4-1
dv
driving force = fv = m (force = ma)
dt
In gravitational potential,
Fd = driving force = mg − mv 2ρ g = m (1 − v2 ρ )g
where g = gravitational acceleration
m = mass of particle
f = frictional coefficient
v2 = partial specific volume of particle
∂V
=
∂ g2 T ρ
1 1 g1
Note that
Therefore,
dv
m (1 − v2 ρ )g − fv = m
dt
dv
When the frictional force balances the driving force, = 0 and the particle acquires its
dt
terminal velocity (vt ) .
m (1 − v2 ρ )g
vt =
f
m(1 − v 2ρ )ω 2 x
vt =
f
where ω ≡ speed of rotation (in radians / sec)
x ≡ distance from the axis of rotation
axis of rotation
meniscus
ω
cup
x
Figure 4-2
vt m (1 − v2ρ )
S= 2 =
ω x f
where S ≡ terminal velocity / unit acceleration
Sedimentation coefficients have units of sec. 10−13 sec is called 1 svedberg (or 1 S).
T. Svedberg pioneered research on sedimentation in an ultracentrifuge.
−13
1 S ≡ 10 sec
Chemistry 24b — Lecture 6 3
(b) As the solution is spun in the ultracentrifuge and macromolecules move down the
centrifugal field, a solution-solvent boundary is generated. The boundary can be
monitored by refractive index, color (absorption) etc.
C0 time 0
plateau region
Concentration time t
solution
solvent
0 x1/2
xm xb
x
meniscus cell bottom
Figure 4-3
(c) By following the boundary with time, the sedimentation coefficient can be
determined.
vt dx1/2 / dt 1 d ln x1/ 2
S = 2 = 2 =
ω x ω x1/2 ω2 dt slope
2.303 d log x1/ 2 logx1/2
=
ω2 dt
2.303
= (slope)
ω2 t
Figure 4-4
Chemistry 24b — Lecture 6 4
buffer + sucrose
Figure 4-5
(b) As the sample is spun in the centrifuge, a band containing macromolecules will
move down the centrifugal field. Also, a sucrose gradient will have developed. The
sucrose gradient ensures that the density of the “solvent” is always greater than the
density of the sedimenting zone. This ensures the stability of the band.
meniscus
ω
sucrose density gradient
species 1 species 2
Figure 4-6
(c) S is determined from the displacement of the band(s) with time in the centrifuge
tube.
Frictional Coefficient
Once S is determined, f can be determined from
m(1− v 2 ρ )
S =
f
Chemistry 24b — Lecture 6 5
Sedimentation-Equilibrium
(1) Consider sedimentation of a homogeneous two-component solution (a solute plus
solvent) in an ultracentrifuge. Because of sedimentation, a concentration gradient is
generated. Diffusion sets in. Since transport by sedimentation and diffusion go in
opposite directions, eventually an equilibrium concentration is generated by
ultracentrifugation. This occurs when
k BT dC
= m (1− v2 ρ ) ω 2 x or
C dx
RT dC
= M (1− v 2 ρ ) ω x or
2
C dx
4.606 RT d log C
M =
ω 2 (1 − v2 ρ ) dx 2
(1 − v 2 ρ(x)) = 0 and vt or S = 0
(c) The various macromolecular species will form bands at points in the salt
gradient where the macromolcules become buoyant; i.e., at x’s where
(1 − v2 ρ(x)) = 0 for the species. Many biological macromolecules have
“buoyant densities” sufficiently different that they can be separated or
resolved by density-gradient centrifugation.
Chemistry 24b — Lecture 6 6
Figure 4-7
Chemistry 24b — Lecture 6 7
Jk (x) Jk (x + ∆x)
x x + ∆x
Figure 4-8
Therefore
d 2 Ck (x,t) ∂Ck (x,t)
D = Fick' s Second Law of Diffusion
dx 2 ∂t
v
2 v ∂Ck ( r ,t)
or D ∇ Ck (r ,t) = in 3- dimensions isotropic medium
∂t
(c) Solution
x2
C(x,t) = α t −1/ 2 exp −
4Dt
where α ≡ a constant
(d) To obtain α, use normalization condition, i.e., conservation of molecules of
that species.
Chemistry 24b — Lecture 6 8
∞
N = number of molecules = ∫ C(x,t)dx
−∞
∞
x2
= α ∫ t −1/ 2 exp − dx = 2 α ( π D)1/2 or
4Dt
−∞
N x2
C(x,t) = exp− Gaussian in x!
2(π Dt)1/2 4Dt
1
(e) Pr obability of finding molecules at position x at time t ≡ P(x,t) ≡
C(x,t)
N
This result can be used to calculate mean-square displacement of the
molecules from the origin
∞
∫x
2 2
x = P(x,t)dx = 2Dt
−∞
2
x = 2Dt
(f)
t =0
t1 t2 > t1 > 0
t2
−x x =0 x
Figure 4-9
Chemistry 24b — Lecture 6 9
How to Measure D?
(a)
Figure 4-10
fluorescence labelled
or spin-labelled
phospholipids
Figure 4-11
Chemistry 24b — Lecture 6 10
x 2 + y 2 = 4Dt = 4 × 10 −8 cm 2 / sec (t )
r2 = 4 × 10−8 cm 2 in 1 sec
2 1/ 2
r = 2 × 10−4 cm in 1 sec
= 2µ (µ = microns)
I0
monochromatic Iscattered
Figure 4-12
Chemistry 24b — Lecture 6 11
Incident light
ν0 ~ 1015 Hz
Figure 4-13