UNIT 6 KINETIC METHODS OF ANALYSIS

Kinetic Methods of
Analysis

Structure
6.1

Introduction
Objectives

6.2
6.3
6.4
6.5
6.6

Types of Kinetic Methods

Measurement of Reaction Rates
Mathematical Basis of Kinetic Methods
Catalytic Methods-Enzymatic and Non-enzymatic and Methodsw
Comparison of Graphical Logarithmic Extrapolation Methods and
of Proportional Equations
6.7 Instrumentation
6.8 Applications
6.9 Summary
6.10 Terminal Questions
6.11 Answers

6.1

INTRODUCTION

In this unit, I will introduce you to the kinetic methods of analysis. Modern analytical
chemists make use of a number of methods for analysis based on chemical, physical
and physicochemical changes that are exhibited by substances on chemical reactions.
Kinetic method of analysis is one such important method of analysis and it is most
commonly used nowadays. Every chemical process whatever its nature takes place at
a finite rate tending to an equilibrium (Fig. 6.1). Some reactions are so fast and attain
the equilibrium practically instantaneously. Some of them are slow and take a very
long time to attain equilibrium. There are certain reactions moderate in nature and
amenable to follow the course of reaction with a suitable analytical technique. Fast
reaction techniques are available for studying the very fast (nano and pico second)
reactions.

Reactions with half-times

larger than 10 seconds
are considered slow
whereas those with halftimes smaller than 10
seconds are considered
as fast and reactions with
half-times larger than 2
hours are very slow
reactions.

The most commonly used basic analytical techniques such as precipitation, acid-base
neutralization reactions, redox reactions, complexometry reactions are equilibrium
based kinetic methods with some distinguishable trends. In this unit we will be
studying the kinetics of moderate reactions in the dynamic region before it tends to
attain equilibrium.

Objectives
After studying this unit you would be able to

understand the various methods of classification and types of kinetic reactions.

utilize the different methods for kinetic measurements based on the type of
reaction.

understand how the fast reactions are studied.

make use of rate laws for kinetic studies.

understand how to determine the catalysts, enzymes and substrates by kinetic

methods.

understand the basis of kinetic measurements and the types of instrumentation

used.

visualize the applications of kinetic studies to organic and inorganic species in

various areas of study and make use of them.

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

6.2

TYPES OF KINETIC METHODS

Kinetic methods are classified by a number of criteria. Broadly it can be classified as

catalytic and non-catalytic methods. The catalytic methods are further divided
according to the type of reactions involved. The non-catalytic reactions have been
classified according to whether they are applied to the determination of single species
or of several components in a mixture. Another method is based on the number of
components determined, namely single component and multi-component methods.
The best method of classification of kinetic methods is based on the chemistry of
reactions involved as shown in Table 6.1 for analytical purposes..
Table 6.1: General Classification of Kinetic Methods
Catalytic Methods
Types of
Reactions

Redox and
Chemiluminescence
Complexation

Modified
effects

Activation
Inhibition
Catalytic titration

Non-enzymatic
(Homogeneous)

Enzymatic

Homogeneous
Heterogeneous(immobilized
enzymes)
Modified
effects

Activation
Inhibition

Electrochemical (Heterogeneous)
Non-catalytic methods

Determination of single species

Determination of mixtures (Differential kinetics)

Based on the experimental approaches, it can be broadly classified as mixing methods,

relaxation methods and equilibrium methods. The different approaches, parameters
measured and techniques employed are presented in Table 6.2.
Kinetic methods are
particularly advantageous
when reactions are slow
that it is impractical to
wait until the equilibrium
is reached.

Table 6.2: Classification of experimental approaches for kinetic measurements

Mixing methods: (Methods based on mixing and following of reaction course as the
systems approach equilibrium state)
1.

Batch mixing

2.

Stopped flow mixing

3.

Continuous flow mixing

Relaxation methods: (Systems at equilibrium is suddenly disturbed from the

equilibrium position and observed as it relaxes to equilibrium condition)
1.

Jump techniques (temperature, pressure, electric impulse, photon impulse)

2.

Periodic relaxation techniques ( Ultrasound wave propagation, dielectric)

3.

Spectral relaxation techniques (Fluorescence and phosphorescence)

Equilibrium methods (Techniques that permit extracting rate data without

disturbing the system at equilibrium)
Resonance techniques (Nuclear magnetic resonance (NMR) and Electron spin
resonance (ESR) )

Kinetic Methods of
Analysis

SAQ 1
a)

How can the kinetic methods be classified broadly?

...
...
...
...
...
...
b)

How can the classification for kinetic measurements be done with the help of an
experimental approach?

...
...
...
...
...
...

6.3

MEASUREMENT OF REACTION RATES

Let us consider a simple case that the analyte A decomposes to form the product, P.

A P

(6.1)

The rate of disappearance of analyte and the rate of product formation as a function
of time, t, is pictorially represented in Fig. 6.1. Either the rate of disappearance of
analyte A, or the rate of formation of product P can be used as a measure to follow the
course of reaction by means of a physical, chemical or physicochemical method, say
conductance by conductometry, potential by potentiometry, pH by using a pH meter,
colour by spectrophotometry and so on.
Now let us look at what are the different methods by which kinetic measurements can
be made. In general there are two major types of kinetic methods, namely differential
and integral methods. In the differential method concentration of analytes or products
are computed from the reaction rates by means of differential form of rate expression.
The rates are determined by measuring the slope of the curve relating to analyte or
product concentration to reaction time. In the integral methods, as the name implies,
are based on the integration of the corresponding rate expressions over a finite time.
Each of the above two methods can be carried out by initial rate, fixed time and
variable time methods.

The rate of reaction is

equal to the disappearance
of analyte or the
formation of product and
it is proportional to the
concentration of At at
time t.

The plot of variation of concentration of the reaction process as a function of time

(Fig. 6.1) is representative of any of the differential kinetic equations listed in Table
6.3. In this case the kinetic process is exponential in nature. Kinetic methods are
generally applied to the initial portion of the curve wherein the reaction proceeds only
to 1-3%. This portion of the curve is normally linear and its slope is proportional to
the concentration of the species measured. Kinetic measurements based on this
method is called initial rate method.

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

Fig. 6.1: Change in concentration of analyte [A] and product [P] as a function of time.
Until time te, the analyte and product concentrations are continuously changing.
Then is the equilibrium region

In general in a kinetic reaction, the variation of concentration of product formed is

monitored as a function of time for a series of concentrations of analyte and plotted.
Following this the calibration curve is prepared by plotting the concentration of
product or reaction rate or any other suitable parameter as a function of concentration
to get a straight line with zero or non-zero intercept. From this the concentration of
unknown is obtained.

6.3.1 Differential Methods

6.3.1.1 Initial rate method:
Fig. 6.2 illustrates how the initial rate method is used to determine the
concentration of analyte from the experimental rate measurements. Solid
curves are the plots of experimentally measured product concentration as a
function of time for four standard solutions of analyte (A). Tangents are
drawn to each of the curves at a time near zero (dashed lines). The slopes of
the tangents/reaction rates are then plotted as a function of analyte
concentration giving a straight line. Unknowns are treated in the same way
and their concentrations are determined from the calibration graph.

(a)

(b)

Fig. 6.2: A plot of data for the determination of A by the differential method.
(a) Solid lines are the experimental plots of product concentration as a function
of time for four initial concentrations of A. Dashed lines are tangents to the
curve at t
0. (b) A plot of the slopes obtained from the tangents in (a) as a
function of analyte concentration.

The initial rate is linearly related to concentration whether the initial portion
of the kinetic curve is linear or not, provided that all the measurements are
made at the same time interval for different concentrations. Hence it is also
called as single point method.

Kinetic Methods of
Analysis

The following are the advantages of this method:

1.

Since only a small portion of the plot is utilized for measuring the slope,
a few data points are enough to make the plot and there is no need to
follow the reaction for a long time or to the end. In this way time is
saved and the entire procedure is simplified.

2.

The reaction obeys pseudo zero order kinetics as the change in the
concentration of reactants are very small or negligible.

3.

As the amount of product formed is small, the reverse reaction

virtually does not take place.

4.

Complication arising from possible side reactions is avoided.

5.

The reaction takes place at a faster rate at the start of the reaction and
results better signals.

6.

Useful for reactions whose rate constants are too small to be used in
equilibrium methods.

The shortcoming of the method lies in accurate measurement of the initial

slope. This is overcome by the use of microcomputers or more sophisticated
data handling procedures and numerical analysis which provide the slope of
the curve directly and precisely.
The initial rate method requires the plotting of kinetic curves, certain times it
is preferable to use fixed time or variable time methods which do not require
the plot of kinetic curves.
6.3.1.2 Fixed time method
In this method, the concentration of reaction product is measured at a predetermined time from the start of the reaction (to t1 = t). Since t is kept
constant, the concentration of product is directly proportional to the change in
the concentration of indicator reaction product (P). By plotting P vs.
concentration, you will obtain a straight line with zero intercept as indicated in
Fig. 6.3.

Fig. 6.3: As t is kept constant, plot of P vs concentration will give straight line
with zero intercept.

Normally t is chosen from the start of the reaction. However t can be for
tl t2 or t2 t3 provided it is not too far from the start of the reaction and falls
within the straight line portion of the kinetic curve. t is proportional to
concentration even in the case of reverse reaction and it is practically superior
for pseudo first order reactions.
9

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

6.3.1.3 Variable time method:

In this method the time required for the pre-determined change in the solution
(absorbance) or the formation of product is measured for a series of analyte
concentrations, i.e., P = constant and measured at times, t1, t2, t3, etc. The
application of this method is illustrated in Fig. 6.4.

Fig. 6.4: The plot of reciprocal of time, 1/t vs. concentration

The plot of reciprocal of time, 1/t vs. concentration constitute the calibration
graph (Fig. 6.4). This method is more limited than the fixed time method as
the t does not start from t1 = 0.

6.3.2 Integration Methods

The experimental procedure is similar to that of differential methods, but the values
are integrated over a range and the integrated values are plotted for the tangent, fixed
time and variable time methods.
Integral methods use
integral forms of the firstorder rate equation such
that [A] = [A]0 e-kt.

In the tangent method, the formation of product is integrated over a finite time
interval t = (t1 t2). Log [P] or log[A]o - [P] is plotted as function of time. This
results a series of straight line with positive or negative slopes from which the
calibration graph can be constructed. This is illustrated in Fig. 6.5.

Fig. 6.5: log [P] is plotted as a function of time

10

In the fixed time method (ln[A]) vs. [C]0 for a fixed concentration of A, [A]o
constitute the calibration graph. Plot of 1/t vs. C0 for a series of standards constitute
the calibration graph for the variable-time method.

Kinetic Methods of
Analysis

Integration methods are applied to conditions where the reaction proceeds to a greater
extent.

6.3.3 Induction Period Measurements

This phenomenon was first observed by Landolt in the reaction between iodate and
sulphite in an acid medium and called as Landolt effect.
A+B

Slow

P (product)

k1

(6.2)

fast
P (product) + L (Landolt reagent/catalyst)

k2

Y
... (6.3)

The slow reaction is linked to the fast reaction process through the product (P) of slow
reaction. Since the second reaction is faster than the first, the product can be detected
only when the Landolt reagent/catalyst(L) disappeared completely. The time elapsed
till the appearance of product (P) is a measure of catalyst concentration, which is
directly related to the induction period (t).
The experimental procedure involves the measurement of time elapsed between the
addition of the last reactant (either A or B or catalyst) which starts the slow reaction
and the first appearance of the signal corresponding to the product. Very low
concentrations of catalysts, in the range of microgram and below can be detected by
this method.

6.3.4 Differential Reaction Rate Methods

An important application of kinetic methods is the determination of closely related
species in mixtures such as alkaline earth cations and organic compounds with same
functional groups. Differential reaction rate methods are useful for the determination
of two or more species with a common reagent. Table 6.4 summarises the various
differential rate methods.
For example consider the two species A and B to react with a common excess reagent
R to form products under pseudo-first order conditions,
A+R

B+R

kA

kB

P'

P"

(6.4)
(6.5)

If they are of first order kinetics, [R]o >> [A]o and [R]o >> [B]o the sum of
concentrations of the species at time t is given by
Ct

= [A]t + [B]t = [P] - [P]t = [A]o exp(-kAt) + [B]o exp(-kBt)

... (6.6)

and the sum of concentrations of product is given by

[P]t = [A]o - [A]t + [B]o - [B]t = [A]o [1- exp(-kAt)] + [B]o [1exp(-kBt)]
(6.7)

11

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

It is assumed that no product is formed at the beginning.

If kA > kB then A is reacted before B. Thus if kA/kB is greater than about 500, about
99% of A is consumed before 1% of B is used up. If the measurement is made shortly
after mixing it is possible to determine A with no significant interference from B by
differential determination.
When the ratio of the two rate constants is small, determination of both species is still
possible. Thus by making suitable changes in the reaction conditions such as change
of solvent, temperature, concentration or use of masking agent, so that the k values
differ considerably and make the measurements possible.

6.3.5 Logarithmic Extrapolation Method

Logarithmic extrapolation method is most frequently used in differential kinetic
methods of analysis. For first order reaction, log Ct or log ([P] - [P]t) is plotted as a
function of time (Fig. 6.6) which results a curve. If kA is larger than kB the species A
reacts at a higher rate than B. After time t, [A]t tends to zero and the curve becomes
linear, so that
log Ct = log[P] - [P]t = log[B]o - kBt/2.303

(6.8.)

The extrapolation of the linear portion of the curve that causes the intercept
corresponds to the initial concentration of B, [B]o. The concentration [A]o is obtained
by difference from [P].

Fig. 6.6

Fig. 6.7

Logarithmic extrapolation method for a mixture of species.

For second order reaction, log[A]t or log[B]t is plotted as a function of time (Fig.
6.7). Line 1 corresponds to faster reaction while line 2 corresponds to slower reaction.
The values of [A]o and [B]o can be arrived at from the plot.

6.3.6 Linear Extrapolation Method

For first order kinetics and when kA is larger than kB , [P]t is plotted as a function of
both exp(-kAt) and exp(-kBt) and extrapolated to obtain the required data from them.
Same concentrations of individual components of A and B and in the mixture are
plotted. [A]o and [A]o correspond to the concentration of A by extrapolating the
slower reaction component and faster reaction component respectively. From the
slopes and intercepts, the concentrations of A and B can be computed.

12

For second order kinetic reaction, where [R]o = [A]o + [B]o , the product concentration
P is plotted as a function of ([R]o P)t which on extrapolation yield [A]o as its slope
for the concentration B . The plot of 1/ ([R]o - P) as a function of time and its
intercept on extrapolation yield [B]o directly. The rate constant can be derived from
its slope.

Kinetic Methods of
Analysis

6.3.7 Proportional Equation Method

It is a mathematical approach and widely used in the resolution of closely related
reaction rates in a mixture. Rate constant ratio of 4 is quite enough for this method and
it does not require the prior knowledge of total concentration, Ctot.
In practice, the concentrations Ct are measured at two or more reaction times, t1, t2, .
to formulate two similar equations
[P]

- [P] t1 = [A]o exp(- kAt1) + [B]o exp(- kBt1)

[P] - [P] t

= [A]o exp(- kAt2) + [B]o exp(- kBt2)

... (6.9)
(6.10)

The initial concentrations of A and B are arrived at by solving the simultaneous

equations. The only requirement is that the rate constants must be known earlier.
The general equation for a binary mixture will be in the form of

f1 = k A 1 [A]0 + k B1 [B]0
f 2 = kA 2 [A ]0 + k B2 [B]0

... (6.11)
... (6.12)

where f is a measurable quantity under two different set of conditions and kA 1 ,

k A 2 and k B 1 , k B 2 are empirical proportionality constants obtained under the above set
of conditions.

6.3.8 Activation And Inhibition

The rate of catalysed reactions can either be increased or decreased i.e., activated or
inhibited by the addition of certain substances, called modifiers. If the equilibrium
constant of inhibition reaction is sufficiently large, the rate is equal to the rate of
uncatalysed reaction vc for the stoichiometric ratio of inhibitor I, to the catalyst.
The inhibition reactions are employed in catalytic titrations, wherein the analytical
signal is plotted as a function of volume of titrant added. The curve is rising or falling
depending on whether the monitored species is a reactant or product of the indicator
reaction.

6.3.9 Measurements of Partial Rate Order

Experimentally varying concentrations of analyte are made to react with a large
excess of reactant and the change in concentrations are measured as a function of
time and plotted as in Fig. 6.2. The initial rates for each of the curves are determined
from the tangents of initial straight segment of each curve and plotted against
concentration or log concentration. The log plot of it gives a straight line from which
the partial order of 1/2 is obtained. Plotting [A ]t1 / 2 as a function of time results a set

Initial rates (tangents of

straight segments) are
determined in the case of
differential methods
whereas the value of the
measured property at
different time intervals is
used in the case of
integral methods

of parallel straight lines which confirm the partial order of 1/2 calculated by
differential method. The rate constant can then be readily calculated from the slope of
any one of the lines.
To arrive at the kinetic or rate equation for a given system, it is necessary to determine
the partial reaction orders with respect to different variables influencing the process.
13

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

This can be carried out either by differential methods or by integral methods. As a rule
differential methods are chiefly used for the determination of partial orders, whereas
the integral methods are normally applied to the determination of rate constants.

6.3.10 Non-Catalytic Reactions

As mentioned earlier kinetic methods based on uncatalysed reactions are less
common. Uncatalysed reactions can be rendered useful when selective reagents are
employed in conjunction with sensitive detection methods. For example, the
selectivity of a complexing agent can be controlled by adjusting the pH of the medium
in the determination of metal ions. Sensitivity can be achieved by the use of
spectrophotometric or spectrofluorescence detection to monitor the reagents that form
complexes with large molar absorptivities.
The precision of both catalytic and non-catalytic kinetic methods depend on
experimental conditions such as pH, ionic strength, temperature, solvent medium.
With careful control over these variables, precision of 1-10% RSD can be achieved.
Automation of kinetic methods and computerised data analysis can improve the
relative precision to 1% or less.

6.3.11 Stopped Flow Technique

Slow reaction kinetics generally make use of closed reaction systems whereas the fast
reaction kinetics make use of open systems such as continuous flow analysis.
Measurements of fast reactions generally require special equipment for rapid mixing
of reagents and fast recording of data. Stopped Flow Technique is one of the most
popular and reliable methods for carrying out rapid reactions and used for the study of
reactions with half-lives between micro seconds and a few seconds. In principle, the
streams of sample and reagent(s) are mixed rapidly and the flow of mixed solution is
stopped suddenly. The reaction process is monitored at a position slightly down
stream from the mixing point. The process may be monitored by spectrophotometry or
spectrofluorimetry.

6.3.12 Factors Influencing The Reaction Rate

As pointed out earlier two general types of reactions are associated with kinetic
methods. Slow reactions whose half-lives are 10 seconds and longer and fast reactions
are those with half-lives less than 10 seconds. The rate of slow reactions can be
monitored by direct conventional methods. The reaction is started by adding the
reactants to a suitable vessel and followed by means of performing titrations or
measuring the physical properties such as potential, diffusion current, conductance or
absorbance at pre-selected time intervals.
The reaction rate is largely influenced by a number of parameters such as temperature,
concentration of reactant, solvent medium, pH and ionic strength of the medium. For
this reason, the kinetics of reaction are measured under specified conditions. For slow
reactions with half lives longer than 2 hours, the rate can be adjusted so that their halflives fall within the acceptable range of slow reactions. This is done by (a) selecting a
more favourable temperature (b) changing the reactant concentration (c) changing the
solvent medium (d) adjusting the ionic strength and or pH of the reaction as these
parameters largely affect the reaction rates.

SAQ 2
What is the shortcoming of the initial rate method and how can it be overcome?
...
...

14

6.4

MATHEMATICAL BASIS OF KINETIC METHODS

Kinetic Methods of
Analysis

This section provides a brief account on the mathematical basis of chemical kinetics to
understand the basis of kinetic methods of analysis.

6.4.1 Rate Law for First Order reactions

Let us consider a spontaneous irreversible decomposition of A to form the product P
of Eq. (6.1). whose reaction is first order in A and the rate is given by,

d[A]
= k dt
[A]

(6.13)

when the concentration of [A] = [A]o. Integration of this equation from time zero
(to) to the time t1 when [A] = [A]t gives,

[A] t

( A] 0

t
d[A]
= k dt
0
[A]

(6.14)

Evaluation of integrals gives

ln

[A]t
= kt
[A]o

(6.15)

Finally by taking the exponential of both sides of equation we get,

[A]t
= e kt or [A]t = [A]o e kt
[A]o

... (6.16)

The above integrated form of rate law gives the concentration of A as a function of
initial concentration of A, rate constant k and time t. This relationship is depicted in
Fig. 6.1, both for the reactant and the product.
Now if we monitor the rate of formation of the product P, rather than the
disappearance of analyte A, we have to modify the above equation. If one mole of
analyte form one mole of product, the concentration of A at any time is equal to its
original concentration minus the concentration of product. Thus,
[A]t =

[A]o - [P]t

(6.17)

substituting this for [A]t in the Eq. (6. 16) and on rearrangement we get,
[P]t = [A]o(1-e kt)

(6.18)

Plot of this relationship gives a curve for P, similar to that of A, but mirror image of it
(Fig. 6.1)
A pure exponential variation of concentration of A or P is quite useful, because for
equal lapsed times give equal fractional decrease in reactant concentration or
increase in product concentration. Thus for time interval, t = = 1/k. This on
substitution in Eq. (6.16) gives
[A] = [A]o e k = [A]o e k/k = (1/e) [A]o

... (6.19)

similarly for a period of t = 2 = 2/k

[A]2 = (1/e)2 [A]o

(6.20)

Likewise we get for successive periods (Fig. 6.8).

15

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

Fig. 6.8: Rate curve for a first-order reaction showing that equal elapsed times produce
equal fractional decreases in analyte concentration.

The period = 1/k is referred as natural life time of species A. During time , the
concentration of A decreases to 1/e of its original value and for second and third
period of time 2 and 3 respectively. This is similar to the process we come across
in radioactive decay.

Example 1
A solution of initial concentration of 0.010M on first order reaction showed the rate
constant as 0.0360 s 1. Calculate the concentration of reactant after 18.2 seconds.

Solution
[A]t = [A]oe kt
[A]18.2 = (0.010M)e(-0.0370 )(18.2) = 0.0051 M (Ans.)

6.4.2 Rate Laws for Second Order

Let us now consider a reaction in which one more of analyte A reacts with one more
of reagent R to give a single product P. Let this occur in a single step and also be
irreversible.

k
A + R

(6.21)

If the rate is proportional to the concentration of each of A and R, the rate law is

d[A ]
dt

k [A] [R]

(6.22)

The rate is first order with respect to A as well as R but second order over all.
If we keep the concentration of reactant much larger say 50 to 100 times more than
that of A, the concentration of R changes very little and we get k[R] = constant = k'.
The above equation will then be,

16

d[A ]
dt

= k' [A]

Kinetic Methods of
Analysis

(6. 23)

which is identical to that of the first order reaction. This type of reaction is said to be
pseudo first order with respect to A.

Table 6.3: Rate Equations for irreversible processes

Rate equation

Order

Differential form

Integrated form

Common unit for

rate constant

A single reactant, or reactants at equal concentration

0
1

dx
= k A (a0 x)
dt

dx
= kA
dt
1
dx
= k A (a0 x) 2
dt

3
dx
= k A ( a0 x ) 2
dt

kA =

x
t

2 12
1
[a 0 (a 0 x ) 2 ]
t
1 a0

k A = ln
t
a0 x)
kA =

2
1
1
1

1
t (a 0 x) 2
a0 2
2
kA =
ta0 (a0 x)

kA =

dx
= k A ( a0 x ) 2
dt
dx
= k A (a0 x) (ao - 2x) k = 1 ln a0 x)
A
dt
ta0 a0 2 x)

dx
= k A (a0 x) 3
dt

dx
= k A ( a0 x) n
dt

mol dm 3 s 1
1

mol 2 dm

s 1

s 1
3

dm 2 mol

s 1

dm 3 mol 1 s 1
dm 3 mol 1 s 1

kA =

2ao x x 2
2ta0 / (a0 x) 2

dm 6 mol 2 s 1

kA =

2
1
1
n1

n 1
t (n 1) (a0 x)
a0

dm 3n 3 mol n 1 s 1

Reactants at unequal initial concentrations

2

dx
= k A (a0 x) (bo - x)
dt

kA =

b (a x)
1
ln 0 0

t (a0 b0 )
a0 (b0 x)

b (a x)
1
dx
ln 0 0
= k A (a0 x) (bo - 2 x) k A =

t (2a0 b0 )
dt
a0 (b0 2 x)

dm 3 mol 1 s 1

dm 3 mol 1 s 1

dx
b (a x)
1
= k A (a0 2 x) (bo - x) k =
ln 0 0
A

dt
t (a0 2b0 )
a0 (b0 x)

dm 3 mol 1 s 1

dx
a ( x + x)
1
= k A (a0 x) ( xo + x) k =
ln 0 0
A

dt
t (a0 x0 )
x0 (a0 x)

dm 3 mol 1 s 1

17

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

Reactions are seldom irreversible and we have to take into the reverse reaction
wherever necessary. For such reactions the rate is the difference between the forward
rate and the reverse rate. Thus

Second order and higher

order reactions can be
carried out under pseudo
first order conditions by
the control of
experimental conditions.

d[A ]
dt

k1 [A] [R] - k2 [P]

(6.24)

k1 = second order rate constant for forward reaction, and

k2 = first order rate constant for reverse reaction.

Under circumstances when k2 and/or [P] are relatively small, the rate of reverse
reaction is negligible and only a little error is introduced by the assumption of pseudo
first order behaviour.
In all cases x is the amount of reactant A consumed per unit volume, and except in the
autocatalytic case x = 0 when t = 0

Example 2
The reaction scheme for the compounds A and B with a common reagent is as
follows:
A + R

P + HCl

P + HCl

+ R

The chloride ion concentration was measured as a function of time.

A sample solution of 10 ml containing A and B plus solvent was reacted with an
excess of reagent R. The molar concentrations of chloride ions were observed as a
function time and showed the following data:
Time (sec)
Chloride(sec)

20

50

100

0.000 0.347 0.519 0.577

150

200

0.613 0.644

250

completion

0.670

0.800

Calculate (a) the rate constant kb (b) concentration of A and B in the sample (c) the
time required for 90% completion of reaction of B.

Solution
a)

Plot the data as ln(C0 C) vs. t. Calculate the slope of the straight line portion of
the curve after the break (Slope = -kb)
(Ans. 0.216 min 1)

b)

Extrapolate the straight line portion of the curve to zero time. The numerical
value of the intercept is then equal to ln[B]0 .
(Ans. [B]0 = 0.316M )
The total concentration of A and B is the concentration on completion (i.e.,
0.800M). Hence [A] = 0.800 [B].
(Ans. 0.484M )

c)

If [B]0 = 0.316M, 90% would constitute, 0.284M. Using the expression

ln

Co
= k bt
Co C

and substituting,
2.303 log

0.316
= 0.216 t
0.316 0.284

= 10.6 min

18

(Ans.)

Kinetic Methods of
Analysis

SAQ 3
a)

What is the rate law for second order reaction?

...
...
...
...
...
b)

What do you mean by pseudo first order reaction?

...
...

...
...

6.5

CATALYSED REACTIONS

Many of the reactions are catalysed by inorganic anions and cations and by enzymes
and are widely used for the detection of a variety of biological and biochemical
species. We will now see how the rate laws can be reduced to relatively simple
algebraic relationships for enzyme catalysed reactions and used for analytical
purposes.

6.5.1 Enzyme Catalysed Reactions

Enzymes are high molecular weight molecules and extensively used as analytical
reagents because of their high degree of selectivity to specific species with which they
combine when acting as catalysts. Enzyme catalysed reactions are employed for the
determination of activators, inhibitors, and enzymes itself in addition to the
determination of substrates. Let us consider the reaction,
k
E
Enzyme

k -1

Substrate

k
ES
Enzymesubstrate
complex

Product

E
Enzyme

(6.25)
in which the enzyme E, reacts reversibly to form enzyme substrate complex. This
complex decomposes irreversibly to form the product P and regenerate enzyme. Since
the reaction involves two steps, the rate law for this reaction mechanism assumes one
of the two forms depending on the relative rates of the two reactions. If the second
step is considerably slower than the first step, the second step determines the over all
rate of the reaction and it is termed as rate determining step. If the two steps are
comparable rate, then it assumes a steady state condition. We will now see how these
two processes are useful for analytical studies.

If any chemical reaction

involved more than one
step the slowest step in
the rate determining step
for the formation of the
product

CASE 1: Rate determined by the rate of complex formation

The concentration of S and E at time t for the reaction given above is given by
[S]t =

[S]o - [ES]t and

... (6.26)
19

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

[E]t = [E]0 - [ES]

... (6.27)

Since the second reaction step is slow, equilibrium is achieved rapidly and we can
equate to forward and reverse reaction rates of the first step and obtain

d[E]
d[S]
d[ES]
=
+
dt
dt
dt

(6.28)

At any time t, the rate of forward reaction is proportional to the product of the
concentration of the two reactants ( [E]t [S]t ) and the rate of reverse reaction is then
proportional to the instantaneous concentration of the complex ([ES]t).
k1 [E]t [S]t =

k -1 [ES]t

... (6.29)

On rearranging

k1
[ ES]t
=
= K
k 1
[ E ]t [S]t

(6.30)

When the rate of forward and backward reactions are equal, the system is at chemical
equilibrium. The concentration terms in the above equation are equilibrium
concentrations and K is the equilibrium constant for the first step in the reaction.
Substitution of [S]t and [E]t from Eqs. (6.26) and (6.27) into the Eq. (6.28) and on
rearranging we get the rate expression,
[ES]t = K([S]o - [ES]t) ([E]o - [ES]t)

... (6.31)

In general the catalyst is at low level when the substrate is determined. Further the
enzyme reagents are expensive and the natural level of enzymes are very low. Under
this condition the concentration of substrate is much larger than enzyme-substrate
complex [S]o >> [ES]t.
The Eq. (6.31) then becomes,
[ES]t = K [S]o ([E]o - [ES]t)

... (6.32)

when solved for [ES]t

[ES]t =

K [S]0 [E]o
1+ K [S]0

(6.33)

and the rate of reaction is

d[ P]
k K [S]o [ E]o k 2 [S]o [ E]o
= K 2 [ES]t = 2
=
dt
1 + K [S]o
(1 / K + [S]o

(6.34)

By adjusting the conditions, the above equation can be simplified to determine either
the catalyst or the substrate.

Determination of enzyme
For enzyme determination, the substrate concentration is made large enough so that
[S]o >> 1/K and the denomination in the Eq. (6.34) is approximately equal to [S]o and
the rate is given by
d[P]
= k2 [E]o
dt

(6.35)

Thus the rate of reaction (product formation) is directly proportional to the initial
concentration of enzyme catalyst [E]o and can be used for determining the enzyme
species.

20

Determination of substrate
If the initial concentration of substrate is made small enough so that [S]0 << 1/K the
Eq. (6.34) reduces to

d[P]
= k2 K [S]o [E]o
dt

Kinetic Methods of
Analysis

(6.36)

To determine the substrate concentration, the experiment is conducted at constant

enzyme concentration so that
d[P]
= k ' [S]o
dt

... (6.37)

where k' = k2 K [E]o. Here also the rate reaction is clearly proportional to substrate
concentration. The important point to be noted is that the catalyst is regenerated
continuously (Eq. 6.25). Thus [E]t approximately equal to [E]o.

Case 2: Rate determination by steady-state condition

Steady state approximation is used to arrive the rate law. Here the rate of change in
concentration of ES complex is presumed to be negligible. This assumption leads to
the rate law

d[P]
k2 [E]o [S]t
k [E] [S]
=
= 2 o t
dt
[S]t + (k1 + k2 ) / k1 [S]t + K m

(6.38)

Km = Michaelis constant (Km = (k -1+ k2) / k1) is essentially an equilibrium-like

constant similar to l/K in Case-1.
As it was done earlier the rate law can be simplified so that the reaction rate is
proportional to enzyme or substrate concentration. Thus when the substrate
concentration is large enough ([S]t >> Km) the Eq. (6.38) reduces to

d[P]
= k2 [E ]0
dt

(6.39)

The rate of product formation is directly proportional to the concentration of enzyme.

Further it is independent of substrate concentration and the reaction is said to be
pseudo-first order in substrate.
If the concentration of S is small or Km is relatively large, then [S]t << Km and the Eq.
(6.38) is simplified to
d[P ]
k2
=
dt
Km

[E]0 [S]t = k [S]t

(6.40)

where k' = k2[E]o/Km. The rate of product formation is proportional to the

concentration of substrate and first order in nature. It is also necessary to measure
d[P]/dt at the regions of reactions when [S]t approximately equal to [S]o so that

d[P]
= k [S]0
dt

(6.41)

The analytical applications for the determination of substrate and enzyme using the
rate laws (Eqs. (6.39) and (6.41)) can be depicted. When the substrate concentration is
low (Eq. (6.41)), the region which is linear in substrate concentration is used for the
determination of substrate. For the determination of enzyme, the region of high
substrate concentration is employed (Eq. 6.39) and the rate is independent of substrate
concentration.

21

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

Fig. 6.9: Change in rate of product formation as a function of substrate concentration,

showing the parts of the curve useful for determination of substrate and enzyme.

The limiting rate of the reaction at large value of [S] is often referred to as vmax (vide
Fig. 6.9) and when the substrate concentration at exactly vmax/2 is equal to Michaelis
constant Km.
Treatment to derive rate laws for other catalysts are similar in form to those of enzyme
catalysts. For ease of treatment these expressions can often be reduced to first order
type in nature.

Example 3
Urea of concentration 0.05mM was hydrolysed using urease of concentration of 5.0
micro mole. Using the values k2 = 3 x 104 s 1 and Michaelis constant for urease
2.0mM calculate the initial rate of the reaction .

Solution
Use the Eq. (6.38) and substitute the values

d[P]
k [E ] [S]
= 2 o t
dt
[S]t + K m

d[P]
(3 10 4 ) (5 10 6 ) (0.05 10 3 ) t
=
3.6 10 3 Ms 1 (Ans)
dt
(0.05 10 3 ) + (2 10 3 )
SAQ 4
a)

In an enzyme catalysed reaction how can you determine the rate of complex
formation?

...
...
b)

What is the Michaelis constant with reference to the rate law in an enzyme
catalysed reaction?

...
...

22

6.6

COMPARISON OF GRAPHICAL LOGARITHMIC

EXTRAPOLATION METHODS AND METHOD OF
PROPORTIONAL EQUATIONS

Kinetic Methods of
Analysis

GRAPHICAL LOGARITHMIC EXTRAPOLATION

Advantages
1.

Useful for the determination of mixtures without separation.

2.

No prior knowledge of rate constants are required.

3.

Less prone to error as the linear plot is based on several concentrations

4.

Can be used for the determination of three components in a mixture, provided

the rate constants differ quite considerably.

5.

Selective determination is quite simple with the help of spectrometry.

6.

Temperature is not a crucial variable.

Disadvantages
1.

Rate constants must differ considerably for the mixed components.

2.

Total initial concentration of species must be known.

3.

Faster reacting component must be reacted to about 99%.

4.

A large number of samples are to be drawn for analysis if continuous

monitoring is not possible.

5.

The procedure involved is laborious and time consuming as it requires

continuous monitoring and making plots.

METHOD OF PROPORTIONAL EQUATIONS

Advantages
1.

Ideal for fast reactions and for initial rate methods.

2.

Requires shorter time than the graphical methods.

3.

Prior knowledge of total initial concentrations are not required.

4.

Can be applied to mixtures involving complex kinetics.

5.

Difference in rate constants as small as a value of 4 is sufficient for

multi-component analysis.

6.

Easily adoptable for automation for the determination of more than two
components in a mixture.

Disadvantages
1.

Prior knowledge of rate constants are required.

2.

Not applicable to systems involving synergism.

3.

Though theoretically no limit, not possible to extend beyond four components.

SAQ 5
Fill in the blanks:
i)

In .. method, the advantage is that no prior knowledge

of rate constants are required.

23

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

ii)

6.7

The . of ... is ideal for fast reactions and for

initial rate methods.

INSTRUMENTATION

Kinetic methods of analysis unlike the equilibrium methods do not require the
measurement of absolute values, but rather its variation as a function of time. Strict
control of time, temperature, sample treatment are very important. Use of
microelectronics and micro computers to control the operating parameters and for
data collection and processing and use of novel detecting techniques have led to the
development of kinetic methods and their use in routine analysis, especially the
clinical analysis.
The basic components of kinetic measurements are (i) mixing of sample and reagents
and carrying out the reaction a reaction vessel under thermostated condition (ii)
measurement of concentration changes or any suitable parameter as a function of time
and (iii) data collection and processing. Strict control of temperature is an important
parameter in kinetic studies and hence the reactions are carried out under
thermostated condition.

Fig. 6.10: Schematic diagram of stopped-flow technique

24

The kinetic reactions can be monitored either in a closed system or open system. In
closed systems, mixing operation is done manually or automatically and used for the
study of slow reactions. Open systems are commonly used for the study of fast
reaction kinetics with the help of flow systems or batch systems. The various
parameters measured and the techniques employed are presented in Table 6.2.

Kinetic Methods of
Analysis

In the flow method, after mixing, the required parameter is measured at a point where
steady state condition is ensured. The flow methods comprise of continuous flow,
accelerated flow, segmented flow, pulsed flow and stopped flow. The schematic
diagram of stopped-flow technique is presented in Fig. 6.10.
Other techniques of fast reaction studies are (i) direct relaxation methods such as,
temperature jump, pressure jump, electric field jump, pH jump (ii) periodic
relaxation methods (iii) pulse methods such as flash photolysis and radiolysis.
The detection systems comprise of (a) optical methods such as UV, visible
spectrometry, fluoroscence spectrometry, NMR, ESR (b) Electrochemical methods
like potentiometry, conductometry, polarography, amperometry (c) radiochemical
methods and (d) thermometric methods.

SAQ 6
a)

What are the three basic components of the detection system of kinetic
measurements?

...
...
...
...
...
b)

What does the detection system of kinetic measurements comprise of?

...
...
...
...

6.8

APPLICATIONS

The important areas of applications of kinetic methods are to environmental

chemistry (water, industrial effluents), clinical and pharmaceutical chemistry
(biological fluids and pharmaceuticals), industrial products (alloys, petroleum
derivatives) geochemistry (minerals and rocks), agricultural chemistry (oligoelements
in plants and soils, fertilizers), food analysis (natural and manufactured foods) and
analytical grade reagents (high purity metals, mineral acids and salts).
There are three ways of testing the performance of a kinetic method, namely
application to real samples, spiked real samples and simulated (synthetic) samples.
Real samples are analysed for the analyte of interest. The result of this has to be
compared with an acceptable non-kinetic technique. Thus the kinetic determination of
metal ions are compared with atomic absorption spectrometry, visible absorption
spectrometry or neutron activation analysis.

25

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

When the analyte concentration is too low a known amount of the same sample is
added (spiking) to it and compared with the result obtained by the kinetic method.
Recoveries between 95 and 105% are taken as evidence of usefulness of kinetic
method.
Simulated (synthetic) samples are employed in the analysis of waste waters, sea
waters and nuclear fuels. These should match faithfully the real counterparts (Matrix)
both in analyte content and treatment. Real and simulated samples are more
commonly used for evaluating the efficiency of kinetic methods
Kinetic methods that are used for the determination of both organic and inorganic
species fall into two categories, catalysed and uncatalysed reactions. Catalysed
reactions are more selective and sensitive and most commonly used than uncatalysed
reactions However, uncatalysed reactions are quite useful when high speed automated
measurements are required or when the sensitivity of the detection method is great.
Mn, Fe, Cu, V, C, Bi, Ni, Cr, Hg, Mo, iodide, sulphide, phosphate and cyanide are
the most commonly and frequently assayed in real samples by kinetic methods.

SAQ 7
Mention the important areas of applications of kinetic methods.
...
...
...
...

6.9

SUMMARY

In this unit, you have studied about, how the kinetic methods are classified based on
various parameters. Use of differential and integral methods for the determination of
reaction rates and concentration of analytes by catalytic and non-catalytic methods.
Stopped flow techniques for fast reaction kinetics. You have also learned about
various terms used in kinetic studies and the mathematical approach of kinetic
measurements and application to enzyme catalysed reactions in simplified form. The
basic instrumentation of the techniques has also been described. Typical
applications to organic and inorganic species in different areas of study have also been
illustrated.

6.10 TERMINAL QUESTIONS

1.

Define/Explain the following terms: Order of a reaction, differential method,

integral method, half life.

2.

Distinguish between first order and second order reactions.

3.

Which type of reactions are called fast reactions? How they are measured?

4.

Consider the following reaction

A + 2B

26

a)

What is the overall order of the reaction?

b)

What is the order of reaction with respect A?

c)

What is the order of reaction when B is hundredfold in excess?

5.

For the reaction, A + B

determined.

C + D, the disappearance of A was

Kinetic Methods of
Analysis

The following data were obtained:

Time (min)
0
5
10
15
20
25
30
Concentration 0.150 0.120 0.100 0. 090 0.070 0.060 0.050
of A(mole/l)
a)

What is the order of reaction with respect to A ?

b)

What is the rate constant?

c)

What is the half life for the reaction?

6.

A first order reaction required 10 sec. for 50% conversion to products. How
much time will be required for 99% conversion?

7.

Sucrose is hydrolysed to glucose and fructose in aqueous medium.

C12H22O11

+ H2O -----> C6H12O6 + C6H12O6

What is the order of reaction? If 50% is hydrolysed in 90 minutes how much

time it would require for the reduction of concentration to 75% of initial
concentration?
8.

The rate constant of a pseudo first order reaction is 0.156 s 1. Its rate of
disappearance is 2.80 x10 4 M s 1 after 10 minutes, what will be its initial
concentration?

9.

The following reactions were used for the determination of glucose in blood
glu cos e

C6H12O6 + H2O + O2

oxidase
C6H12O7 + H2O2
Horseradish

Organic dye A + H2O2 peroxidase

Coloured product B
For this 0.5 ml blood serum sample was treated to precipitate the protein,
diluted to 100 ml and filtered. To 0.2 ml of the above, the standard enzyme
containing the organic dye A was added. After 30 minutes sulphuric acid was
added to terminate the reaction and the absorbance measurements of coloured
product B at 540 nm showed 0.125. Standard samples containing 1.0 mg/ml
and 3.0 mg/ml treated exactly in the same way showed absorbance values 0.100
and 0.300 respectively. Calculate the level of glucose in blood in percent mg.

6.11 ANSWERS
Self Assessment Questions
1.

2.

a)

Kinetic methods are classified broadly as catalytic and non-catalytic

methods.

b)

Based on the experimental approaches, kinetic methods can be broadly

classified as mixing methods, relaxation methods and equilibrium
methods.

The shortcoming of the initial rate method lies in accurate measurement of the
initial slope. This is overcome by the use of microcomputers or more
sophisticated data handling procedures and numerical analysis which provide
the slope of the curve directly and precisely.

27

Estimations Based On
Kinetic and Acid-Base
Equilibria Studies

3.

a)

If the rate is proportional to the concentration of each of A and R, the rate

law is

d[A ]
= k [A] [R]
dt

The rate is first order with respect to A as well as R but second order over
all.
b)

In Eq. (6.22), if we keep the concentration of reactant much larger say 50

to 100 times more than that of A, the concentration of R changes very
little and we get
k[R] = constant = k'. The above equation will the be

d[ A]
= k ' [A]
dt

(6. 23)

which is identical to that of the first order reaction. This type of reaction is
said to be pseudo first order with respect to A.
4.

5.

6.

7.

a)

Eq. (6.34)

b)

Km = Michaelis constant (Km = (k -1+ k2) / k1) is essentially an equilibriumlike constant similar to l/K in Case 1 (Refer Eq. 6.38).

a)

graphical logarithmic extrapolation method

b)

method of proportional equations

a)

The basic components of kinetic measurements are (i) mixing of sample

and reagents and carrying out the reaction a reaction vessel under
thermostated condition (ii) measurement of concentration changes or any
suitable parameter as a function of time and (iii) data collection and
processing.

b)

The detection systems comprise of (a) optical methods such as UV,

visible spectrometry, fluoroscence spectrometry, NMR, ESR (b)
Electrochemical methods like potentiometry, conductometry,
polarography, amperometry (c) radiochemical methods and (d)
thermometric methods.

The important areas of applications of kinetic methods are to environmental

chemistry (water, industrial effluents), clinical and pharmaceutical chemistry
(biological fluids and pharmaceuticals), industrial products (alloys, petroleum
derivatives) geochemistry (minerals and rocks), agricultural chemistry
(oligoelements in plants and soils, fertilizers), food analysis (natural and
manufactured foods) and analytical grade reagents (high purity metals, mineral
acids and salts).

Terminal Questions
1.

The order of a reaction is strictly an experimental quantity, and merely provides

information about the way in which the rate depends on concentration.
In the differential method, one deals with the actual rates of reactions as
determined by measuring the slopes of concentration-time curves.
In the integral methods, the experimental procedure is similar to that of
differential methods, but the values are integrated over a range and the
integrated values are plotted for the tangent, fixed time and variable time
methods.

28

The half life of a reaction is the time that it takes for one-half of the original
substance to disappear.
2.

Unit of rate of 1st order reaction is k = sec 1 whereas Unit of rate of 2nd order
reaction is k = M 1 sec1

3.

Kinetic methods are particularly advantageous when reactions are slow that it is
impractical to wait until the equilibrium is reached. Some reactions are so fast
and attain the equilibrium practically instantaneously. Fast reaction techniques
are available for studying the very fast (nano and pico second) reactions.

4.

a)

Three (b) One (c) Pseudo first order.

5.

a)

First order (b) 0.04 M 1 s 1 (c) 17.3 min.

6.

92 sec.

7.

Pseudo first order, 180 min.

8.

8.5 x 10-3M

9.

250 mg. percent.

Kinetic Methods of
Analysis

29