Rates of Reactions

Kuo Kan Liang

RCAS, AS and BST, NTU

Empirical Studies
Reaction rate (progress with time)
measurements:

The progress of the reactions are expressed as
the change of the concentration / pressure of
reactant(s) / product(s) / intermediate(s)

Measured by monitoring physical quantities
proportional to the concentration or pressure. For
example, absorption of radiation.

Spectroscopic Methods
Photon-matter interactions
Plancks constant:

34 J s

h
=
6.626

10
Frequency of light:

34 J s

=
h
/
2
=
1.055

10
Wavelength:
= c/
Angular frequency:
! = 2 = 2c/
Photon energy: E = h = ~! = hc/

Absorption

D*

D + h
D
D+h

D + h

Light scattering

Absorption Spectroscopy
Organic / biological molecules:

UV-Vis (Ultra-violet and visible)

IR (Infrared)

Fundamental principle of absorption spectroscopy:
Beer-Lambert law
The light beam is characterized
by the intensity (energy per unit
area per unit time) at the
frequency of interest:

I0 (!)
I( )

Beam cross-section = A

Molecule concentration: [D]

I(!)

Part of the incident energy

is absorbed by the molecule
I() / I0() < 1
Light path length L
L

Beer-Lambert Law
Absorbance (optical density):

(!) =

log10

I(!)
I0 (!)

Extinction coefficient:

(!)
"(!) =
[D] L

I0 (!)
I( )

Beam cross-section = A

Molecule concentration: [D]

I(!)

Part of the incident energy

is absorbed by the molecule
I() / I0() < 1
Light path length L
L

Beer-Lambert Law
Consider a thin layer of the sample (Thickness = dL)

When one molecule absorbs one photon at frequency , the
energy of the light beam reduces by

Number of incident photon per unit time: I() A/

Number of photons absorbed per unit time:
A dI/. ( dI < 0 )

The probability per unit time that a photon is absorbed when
there is one mole of D in the volume: p()

A dI
A I(!)
= p(!) A [D] dL
~!
~!

I + dI

I
dL

Beer-Lambert Law
Linear dependence of the absorbance on
molecule concentration and light-path:

A dI
p( ) A [D] A I( ) dL
=
)
~
~
1 dI
p( ) A [D] dL
= d(log10 I) =
ln 10 I
ln 10

L
L0 =0

d(log10 I) =

log10
Z

p(!) A [D] L
= (!) =
dL0
ln 10
L0 =0
p(!) A
=
[D] L = "(!) [D] L
ln 10

Exponential dependence of the intensity on

absorbance, molecule concentration, and lightpath:

I(!) = I0 (!) 10

(!)

I
I0

= I0 (!) 10

"(!)[D]L

Composition of 2-Component Rxn

Mixture
The absorbance of the reaction
mixture is measured at two
different frequencies:
(
(!1 ) = "A1 [A] L + "B1 [B] L
(!2 ) = "A2 [A] L + "B2 [B] L

[A] =

(!1 ) /L "B1
(!2 ) /L "B2
"A1 "B1
"A2 "B2

Reaction: A B

1
2

[B] =

"A1 (!1 ) /L
"A2 (!2 ) /L
"A1 "B1
"A2 "B2

Isosbestic Point
If the extinction coefficients of the two components
A and B are the same at a frequency i, that is,
A(i) = B(i) = (i), this frequency i is called the
isosbestic point.

Since for our model reaction we have
[A] + [B] = [A]0 + [B]0 at any time during the reaction,

(!i ) = "(!i ) [A] L + " (!i ) [B] L

= " (!i ) ([A] + [B]) L
= constant

Isosbestic Point
The existence of isosbestic point is the
evidence of having two species in the reaction
mixture in chemical equilibrium without
intermediate.

Definition of Reaction Rate

Reaction rate: rate of change of the amount
(partial pressure, concentration, ... not activity)
of certain reactant or product species.

Rate is always taken as positive

Production rate(s) of product(s), consumption rate(s)
of reactant(s) ...

Rate of finite process:

v = | [J] / t|
Rate of infinitesimal process: v = |d [J] /dt|
(Differential or instantaneous rate)

Stoichiometrically Normalized
Reaction Rates
Consider the species based on which the
reaction rate is defined, and define a rate
which applies to all the species in a reaction

Examples:

A B : -d[A]/dt = +d[B]/dt

(NH2)2CO(aq) + 2 H2O(l) 2 NH4+(aq) + CO32(aq) :
d[NH4+] / dt = 2 d[CO32] / dt = 2 d[(NH2)2CO] / dt

Stoichiometrically normalized rate:

A A + B B C C + D D
d [D] /dt
d [C] /dt
d [B] /dt
d [A] /dt
v
=
=
=
D
C
B
A

Measuring Fast Reactions

Real-time measurements with specialized
instrumentations

Flow methods (continuous flow, stopped flow, )

Time-resolved spectroscopic methods

Relaxation methods
(ways to apply the above methods)

Batch measurements

Quenching methods
(When real-time methods do not have proper
dynamic range)

Flow Method

Stea
dy

flow

S
w ith

veloc
ity v

Distance time ( t = S / v )

Continuous-Flow Method

t = t0 + S / v

t: time since initiation

t0: mixing dead time

Continuous-Flow Method
Disadvantages:

Must be liquid-phase reactions

Require a large amount of reactants

To achieve higher time-resolution for faster
reaction, even more reactants are required

Time resolution: t = S / v
(S = spatial resolution)
Fixed spatial resolution, then
higher v
better time resolution

Stopped-Flow Method

Stopped-Flow Method
Procedure:

Mixing with the flow stopped

Start the flow

Stop the flow

Measurement with time-resolved spectroscopic
methods (CD, FTIR, )

Dead time: td = t0 + S/v

Time-resolution: depends
on the spectroscopic
method

Relaxation Method
Manfred Eigen, Nobel Prize in
Chemistry 1967

If a parameter such as temperature,
pressure, or denaturant concentration
is switched to a new value so rapidly that the
chemical system cannot respond during the
switching process, the subsequent relaxation
towards equilibrium can be monitored.

Protein Folding Kinetics

Protein unfolding can be induced by denaturant,
temperature change, or pH change, etc.

Protein unfolding/refolding kinetics can be
measured by relaxing the unfolding agent
Observed kinetics

Sudden change

Protein + high [D] + solvent

Solvent

Protein + low [D] + solvent

Refolding Kinetics

Observed kinetics

Sudden change

Quenching Method
Chemical reaction is stopped some time after it
is initiated

Advantage: composition of the reaction mixture
can be studied with slow methods in an
off-line manner

Chemical quench flow: species that can quench
the reaction is added into the flow

Freeze quench: temperature is lowered to an
extend that no more reaction will continue

Freeze quench example:

cryo-em
Published October 1, 1984

Downloaded from jcb.rupress.org on February 22, 2011

Nigel Unwin (Cambridge):

open structure of
nicotinic acetylcholine
receptor (nAchR)

Tube formed by nAchR

FIGURE 5 Mature tubes obtained after incubation for 4-5 wk at 17C. Ribbons of paired receptors are visible in a "defective"
region along the length of the tube in b and in the small rounded end regions of the tubes in c and d. Note also the zones of
densest staining on either side of the middle portions of the tubes (see Fig. 2). Bar, 0.1 /~m. x 123,000.

Free quench example:

cryo-em
Nigel Unwin (Cambridge): open structure of nicotinic
acetylcholine receptor (nAchR)

Flash Photolysis (Pump Probe)

The absorption spectra of the reactant(s) is
different from that of the product(s) in a
(photo-induced) reaction

Example: Photo-isomerization of rhodopsin

Polli et al, Nature 467, 440-443 (2010)

Red intensity

Flash Photolysis (Pump Probe)

probe time pump time

r
o
t
c
e
fl
e
r
retro

Sample

Pump pulse
Probe pulse
The sample originally
absorbs the pumping
radiation but not the
probing radiation. After
it absorbs the probe
pulse, however, it will
a b s o r b t h e pro b i n g
radiation

Red intensity

Flash Photolysis (Pump Probe)

probe time pump time

Pump pulse
Probe pulse
Even if the probe pulse
arrives at the same
time as the pump pulse,
the system may not be
able to absorb it, yet.

Red intensity

Flash Photolysis (Pump Probe)

probe time pump time

Pump pulse
Probe pulse
If the probe pu lse
arrives shortly after
the arrival of the pump
pulse, the system may
a b s o r b s it q u ite
strongly, resulting in a
very weak transmitted
probe pulse.

Red intensity

Flash Photolysis (Pump Probe)

probe time pump time

Pump pulse
Probe pulse
If the probe pu lse
arrives much later, the
p u m p e d m o le c u le s
wo u ld have alrea dy
relaxe d (reacte d) so
that the probe is not
absorbe d that much
anymore.

Flash Photolysis (Pump Probe)

1.0

Relative intensity of
transmitted probe pulse

0.8

0.6

0.4

The difference in the

probe beam intensity

is the

induced absorption

0.2

1.

1.5

2.

2.5

3.

Delay time

3.5

4.

4.5

5.

Flash Photolysis (Pump Probe)

Example: Photo-isomerization of rhodopsin

Polli et al, Nature 467, 440-443 (2010)

Empirical Studies of
Reaction Kinetics

Rate Laws / Rate Constants

The differential rate generally depends on the
instantaneous configuration.

Under given physical condition and
configuration, the rate is usually a constant.

A + B P, typically: v / [A]
(power law)

"A

[B]

"B

A, B are not necessarily integers

Example of more complicated rate law:

vmax [S]
Michaelis-Menten law: v =
Not a
Km + [S]

power law

Rate Laws / Rate Constants

If the rate law is a power law, for example,
"A
"B
v = k [A] [B]

the order of the reaction is A + B.

The constant k is the rate constant.

There is no definite relation between the
stoichiometry coefficients { A, B } and the
exponents { A, B }.

The dimension / unit of the rate constant
depends on the rate law

Example: first-order reaction A B, dimension
of k: time1

Determination of Rate Laws

Rate law = fundamental physical model of the
reaction.

Rate law = expressing the instantaneous rate as
a function of time, physical conditions, and
system composition.

Empirical studies: determine function form of
the rate law directly from experimental data.

Isolation method

Initial rate method

Isolation Method
Example: A + B + C + P

Generally
v = k [A]

"A

[B]

"B

[C]

"C

...

Goal: Find k, A, B, and so on.

To find A, prepare B, C, in large excess
[B], [C], can be approximated as constants
"B
"C
"A
[B]0, [C]0, . v = k ([B] [C] . . .) [A]
(pseudo-A-order)

Isolation Method
Measure rate and [A] as functions of time
simultaneously.

Plot the logarithm of rate as function of
logarithm of [A]:

0
ln v = ln kA + "A ln [A]
0
kA : effective rate constant
ln v

A
=
e
slop

ln [A]

Initial Rate Method

Isolation method is often combined with the
initial rate method.

Different terms in a composite rate law are
determined one by one (divide-and-conquer
method).

In the isolation method, the isolation
approximation is worse and worse when more
and more A is consumed.

Initial Rate Method

If, for different initial concentration of A, [A]0,
the progress of the reaction is monitored for a
short time, even [A] can be treated as a
constant. Initial rate v0

Measure v0 for different [A]0, then, like in the
isolation method, we have
ln v0 = ln kA + A ln [A]0
ln v0

A
=
e
slop

ln [A]0

Initial Rate Method

For power law reaction rate law, linear
regression (linear least-square) method can be
used to analyze the experimental data.

Example: Binding of glucose (G) to the enzyme
hexokinase (H). Assume that the rate law is
v = k [H]"H [G]"G ) log v = log (k [H]"H ) + " log [G]
0
G
10 0
10
10
0
0
0
0

Determine k, H, and G according to the

experimental data.

Example

log10([H]0)

log10(v0)

log10([G]0)
-3

-2.81 -2.51 -2.4

-2.87 0.699 0.881 1.19

1.3

-2.52 0.845 1.04

1.36

1.49

1.85

1.98

-2

1.32

1.53

Example

log10([H]0)

log10(v0)

log10([G]0)
-3

-2.81 -2.51 -2.4

-2.87 0.699 0.881 1.19

1.3

-2.52 0.845 1.04

1.36

1.49

1.85

1.98

-2

1.32

1.53

<x> = -2.68, <x2> = 7.24, x2 = 0.0566

Example
0

log10([H]0)

log10(v0)

"H
k [H]0

log10([G]0)
-3

-2.81 -2.51 -2.4

-2.87 0.699 0.881 1.19

1.3

-2.52 0.845 1.04

1.36

1.49

1.85

1.98

-2

1.32

1.53

<x> = -2.68, <x2> = 7.24, x2 = 0.0566

<y>=1.018, <xy>=-2.67, Cov(x,y)=0.0570

G = 1.066, log10k = 3.72

log10k
3.72

Example
0

log10([H]0)

log10(v0)

"H
k [H]0

log10([G]0)
-3

-2.81 -2.51 -2.4

log10k

-2.87 0.699 0.881 1.19

1.3

3.72

-2.52 0.845 1.04

1.36

1.49

4.06

1.85

1.98

-2

1.32

1.53

<x> = -2.68, <x2> = 7.24, x2 = 0.0566

<y>=1.184, <xy>=-3.11, Cov(x,y)=0.0608

G = 1.072, log10k = 4.06

Example
0

log10([H]0)

log10(v0)

"H
k [H]0

log10([G]0)
-3

-2.81 -2.51 -2.4

log10k

-2.87 0.699 0.881 1.19

1.3

3.72

-2.52 0.845 1.04

1.36

1.49

4.06

1.85

1.98

4.6

-2

1.32

1.53

<x> = -2.68, <x2> = 7.24, x2 = 0.0566

<y>=1.670, <xy>=-4.41, Cov(x,y)=0.0619

G = 1.0927, log10k = 4.60

Example
0

log10([H]0)

log10(v0)

"H
k [H]0

G = 1

log10([G]0)
-3

-2.81 -2.51 -2.4

log10k

-2.87 0.699 0.881 1.19

1.3

3.72

-2.52 0.845 1.04

1.36

1.49

4.06

1.85

1.98

4.6

-2
<x>=-2.46,
<x2>=6.20,
x2=0.128

1.32

1.53

H = 1.0169, log10k = 6.63

<y>=4.124,
<xy>=-10.023,
Cov(xy)=0.1299

v0 = k[G]0[H]0, k = 4.26 106 M-1s-1

Integrated Rate Law

From the differential (instantaneous) rate law
we find

1 d [A]
"A
v=
= k [A]
A dt
Often, we may be more concerned about [A](t)

1st-order rate law:
1 d [A]
d [A]
v=
= k [A] ) kA dt =
= d ln[A]
A dt
[A]
Z [A]
Z t
[A]
0
d ln [A] = kA
d ) ln
= kA t
[A]0
[A]0
0

1st-Order Rate Law

Half-life t1/2 : time when [A] = [A]0 / 2.
Let A = 1 and

[A]0 /2
ln 2
ln
= kA t1/2 ) t1/2 =
[A]0
k
For a 1st-order reaction, half-life is
independent of [A]0.

Lifetime (time constant): = 1 / k

When t = , [A] = [A]0 e-1.

Integrated rate law: [A] = [A]0 e-kt = [A]0 e-t/

Meaning of Lifetime
At time t = 0, in one liter of the reaction mixture,
there are [A]0 moles or NA [A]0 molecules inside

At time t, there are NA [A] molecules in the
reaction mixture, where [A] = [A]0 e-kt.

During time t and t + dt, the number of A molecules
which react and disappear is k NA [A] dt. The
time that these molecules remain as A is t.

The average time that each molecule A remain as A,
that is, their lifetime as A, is
R1

kNA [A]0 e
t=0
=
NA [A]0

kt

t dt

1
=
k

1
x=0

xe

1
dx =
k

1st-Order Rate Law

1 1

[A]/[A]0

I
0

g
n
i
s
a
e
r
c
n

1/2

t1/2
1/4
t1/2
0 0

Time

1/8

1/16
0

1st-Order Rate Law

If ln [A] is plotted against t, the result should
be a straight line for 1st-order reaction. If it is
not a straight line, the reaction may not be a
1st-order reaction.
Time / min.
C / M

ln C

6.5

30

699

6.55

6.0

60

622

6.43

120

413

6.02

150

292

5.68

240

152

5.02

360

60

4.09

3.5

480

24

3.18

3.0

ln ( C / M )

t / min.

5.5
5.0
4.5
4.0

100

200

300

400

500

2nd-Order Reaction
Case 1, A P: d[A]/dt = -k[A]2

d [A]
1
= k dt
2 = d [A]
[A]
1
1
)
=
+ kt
[A]
[A]0
Half-life:

[A]0
[A]0 = [A] (1 + kt [A]0 ) ) [A] =
1 + kt [A]0
2
1
1
1
=
+ kt1/2 ) kt1/2 =
) t1/2 =
[A]0
[A]0
[A]0
k [A]0

2nd-Order Reaction
Case 2, A + B P: d[A]/dt = d[B]/dt = -k[A][B]

A
[A]0
x
[A]0 x

B
[B]0
x
[B]0 x

9
! P >
>
=
dx
?
)
= k ([A]0
? >
dt
>
;
?

x) ([B]0

If [A]0 = [B]0, X = [A]0 - x and dX/dt = -kX2.

The function form of the result is the same as
in case 1.

x)

2nd-Order Reaction
1 1

[A]/[A]0

g
n
i
s
a
e
r
c
n

2nd-order
1st-order

0 0

Time

2nd-Order Reaction
Case 2, A + B P: d[A]/dt = d[B]/dt = -k[A][B]

If [A]0 [B]0, [B]-[A]=[B]0-x-[A]0+x=[B]0-[A]0, so
1
[A]

[B]0 [A]0
1
[B] [A]
d [A]
=
=
)
=
[B]
[A] [B]
[A] [B]
[A] [B]

1
d [A] d [A]
) k dt =
[B]0 [A]0
[A]
[B]

1
d [A] d [B]
=
[B]0 [A]0
[A]
[B]

1
[A]
[B]
) kt =
ln
ln
[B]0 [A]0
[A]0
[B]0

k dt