Chapter 30
Introduction to analytical Separations
An important part of most analyses is dealing with
foreign species that either attenuate the signal from the
analyte or produce a signal that is indistinguishable from
that of the analyte. A substance that effects an analytical
signal is called an interference or an interferent.
Several general methods are used for dealing with
interferences in an analysis; i) masking, ii) chemical or
electrolytic precipitation, iii) distillation, iv) solvent
extraction, v) ion exchange, vi) chromatography, and vii)
electrophoresis.
MASKING
In masking, a reagent is added to the
solution of the sample to immobilize, or
chemically bind, the interferent as a
complex that no longer contributes to or
attenuates the signal from the analyte. A
masking agent must not affect the behavior
of the analyte significantly.
PRECIPITATION AND FILTRATION
Precipitation, in which the analyte or an interferent
is removed from a solution selectively as an
insoluble species, is one of the oldest methods for
dealing with interferences in an analytical
procedure.
SEPARATING SPECIES BY DISTILLATION
Distillation is widely used to separate volatile
analytes from nonvolatile interferents.
SEPARATING SOLUTES BY
EXTRACTION
The extent to which solutes, both
inorganic
and
organic,
distribute
themselves between two immiscible
liquids differs enormously, and these
differences have been used for decades to
accomplish separations of chemical
species.
Principles
The partiton of a solute between two immiscible phases
in an equilibrium phenomenon that is governed by the
distribution law. If the solute species A is allowed to
distribute itself between water and an organic phase, the
resulting equilibrium may be written as
Aaq
Aorg
The ratio of activities for A in the two phases will be
constant and independent of the total quantity of A; that
is, at any given temperature,
 aA org  Aorg
K

 aA aq  Aaq
…continued…
The equilibrium constant K is known as the
distribution constant.
The concentration of A remaining in an aqueous
solution after I extractions with an organic solvent is
given by the equation
i
V
aq


 Ai  
  Ao
 VorgK  Vaq 
where, [A]i is the concentration of A remaining in the
aqueous solution after extracting Vaq mL of the
solution having an original concentration of [A]o with
I portions of the organic solvent, each with a volume
of Vorg.
SEPARATING IONS BY ION EXCHANGE
Ion exchange is a process by which ions held on
a porous, essentially insoluble solid are
exchanged for ions in a solution that is brought
in contact with the solid. The ion-exchange
properties of clays and zeolites have been
recognized and studied since the late nineteenth
century. Synthetic ion-exchange resins were first
produced in 1935 and have since found
widespread application in water softening, water
deionization, solution purification, and ion
separation.
Ion-Exchange Resins
Synthetic ion-exchange resins are high-molecular-weight
polymers that contain large numbers of an ionic
functional group per molecule. Cation-exchange resins
contain acidic groups, whereas anion-exchange resins
have basic groups. Exchangers of the strong-acid type
have sulfonic acid groups ( SO3-H+) attached to the
polymeric matrix. Strong-base anion exchangers contain
quaternary amine [
N(CH3)3+OH-] groups.
xRSO3-H+ + Mx+
(RSO3-)xMx+ + xH+
solid
soln
solid
soln
xRN(CH3)3+OH- + Ax[RN(CH3)3+]xAx- + xOHsolid
soln
solid
soln
Applications of Ion-Exchange Methods
Ion-exchange resins are used to eliminate ions that would
otherwise interfere with an analysis. Another valuable
application of ion-exchange resins involves concentrating
ions from a very dilute solution. Thus, traces of metallic
elements in large volumes of natural waters can be
collected on a cation-exchange column and subsequently
liberated from the resin by treatment with a small volume
of an acidic solution.
The total salt content of a sample can be determined by
titrating the hydrogen ion released as an aliquot of the
sample passes through a cation exchanger in its acidic
form.
Ion-exchange resins are particularly useful for the
chromatographic separation of both inorganic and organic
ionic species.
CHROMATOGRAPHIC
SEPARATION
Chromatography is a widely used
method
for
the
separation,
identification, and determination of the
chemical components in complex
mixtures. No other separation method is
as powerful and generally applicable as
chromatography.
General Description of Chromatography
The term “chromatography” is difficult to define
rigorously because the word has been applied to
several systems and techniques. Common to all
these methods, however, is the use of a stationary
phase and a mobile phase. Components of a
mixture are carried through the stationary phase
by the flow of the mobile phase, and separations
are based on differences in migration rates among
the mobile-phase components.
…continued…
The stationary phase in chromatography is a
phase that is fixed in place either in a column or
on a planar surface.
The mobile phase in chromatography is a phase
that moves over or through the stationary phase,
carrying with it the analyte mixture. The mobile
phase may be a gas, a liquid, or a supercritical
fluid.
Classifying Chromatographic Methods
Chromatographic methods are of two basic types.
In column chromatography, the stationary phase
is held in a narrow tube, and the mobile phase is
forced through the tube under pressure or by
gravity. In planar chromatography, the stationary
phase is supported on a flat plate or in the pores of
a paper. Here the mobile phase moves through the
stationary phase by capillary action or under the
influence of gravity. Column chromatographic
methods can be further subdivided according to
the nature of the mobile phase, specifically liquid,
gas, and supercritical fluid.
Elution in Column Chromatography
Elution is a process in which solutes are
washed through a stationary phase by the
movement of a mobile phase. The mobile phase
that exits the column is called the eluate.
An eluent is a solvent used to carry the
components of a mixture through a stationary
phase.
Chromatograms
If a detector that responds to solute concentration
is placed at the end of the column during elution
and its signal is plotted as a function of time (or
of a volume of added mobile phase), a series of
peaks is obtained, such a plot, called a
chromatogram, is useful for both qualitative and
quantitative analysis. The positions of the peaks
on the time axis can be used to identify the
components of the sample; the areas under the
peaks provide a quantitative measure of the
amount of each species.
Relative Migration Rates of Solutes
The effectiveness of a chromatographic
column in separating two solutes depends
on the relative rates at which the two
species are eluted. These rates are in turn
determined by the rations of the solute
concentrations in each of the two phases.
Distribution Constants
All chromatographic separations are based on
differences in the extent to which solutes are
distributed between the mobile and the
stationary phase. For the solute species A, the
equilibrium involved is described by the
equation
A(mobile)
A(stationary)
The equilibrium constant Kc for this reaction is
called a distribution constant, which is defined
as
 A S cS
Kc 

 A M cM
Retention Times
The dead time tM is the time it takes for an unretained
species to pass through a column.
The retention time tR is the time between injection of a
sample and the appearance of a solute peak at the detector
of a chromatographic column.
The average linear rate of solute migration, , in
centimeters per second is
_
 = L / tR
where L is the length of the column packing. Similarly, the
average linear velocity, u, of the molecules of the mobile
phase is
u = L / tM
Relating Migration Rates to Distribution Constants
_
 = u x fraction of time solute spends in mobile phase
_
moles of solute in mobile phase
 = u 
total moles of solute
_
cMVM
1
  u
 u
cMVM  cSVS
1  cSVS / cMVM
_
1
  u
1  KCVS / VM
The Retention Factor (k)
The retention factor is an important experimental
parameter that is widely used to compare the migration
rates of solutes on columns. For solute A, the retention
factor kA is defined as
KAVS
kA 
VM
where KA is the distribution constant for solute A.
1
  u
1  kA
_
To show how kA can be derived from a chromatogram,
L L
1


tR tM 1  kA
…continued…
This equation rearranges to
tR  tM
kA 
tM
When the retention factor for a solute is much less
than unity, elution occurs so rapidly that accurate
determination of the retention times is difficult.
When the retention factor is larger than perhaps 20
to 30, elution times become inordinately long.
Ideally, separations are performed under conditions
in which the retention factors for the solutes in a
mixture lie in the range between 1 and 5.
The Selectivity Factor
The selectivity factor  of a column for the two
solutes A and B is defined as
 = KB / KA
where KB is the distribution constant for the more
strongly retained species B and KA is the constant for
the less strongly held or more rapidly eluted species A.
According to this definition,  is always grater than
unity.
 = kB / kA
where kB and kA are the capacity factors
 tR  B  tM
 
 tR  A  tM
Quantitative Measures of Column Efficiency
Two related terms are widely used as quantitative
measures of chromatographic column efficiency:
(1) plate height H and (2) plate count or number
of theoretical plates N. The two are related by the
equation
N=L/H
where L is the length (usually in centimeters) of the
column packing. The efficiency of chromatographic
columns increases as the plate count N becomes
greater and as the plate height H becomes smaller.
Determining the Number of Plates in a Column
The number of theoretical plates, N, and the plate
height, H, are widely used in the literature and by
instrument manufactures as measures of column
performance. N can be determined from a
chromatogram. The retention time of a peak tR and
the width of the peak at its base W (in units of
time) are measured. The number of plates can then
be computed by the simple relationship.
N = 16 (tR / W)2
To obtain H, the length of the column L is
measured and N = L / H Equation is applied.
Variables That Influence Plate Heights
It has been found that plate heights can be
decreased, and thus column efficiency increased
by decreasing the particle size of column
packings, by employing thinner layers of film
(where the stationary phase is a liquid adsorbed
on a solid), and by lowering the viscosity of the
mobile phase. Increases in temperature also
reduce band broadening in most cases.
Column Resolution
The resolution Rs of a column provides a quantitative
measure of its ability to separate two analytes. The
resolution is defined as
2 Z
2 tR B   tR A
RS 

WA  WB
WA  WB
A resolution of 1.5 gives an essentially complete
separation of A and B, whereas a resolution of 0.75 does
not. At a resolution of 1.0, zone A contains about 4% B
and zone B contains about 4% A. At a resolution of 1.5,
the overlap is about 0.3%. The resolution for a given
stationary phase can be improved by lengthening the
column and thus increasing the number of plates. An
adverse consequence of the added plates, however, is an
increase in the time required for separating the
components.
Effect of Retention Factor and Selectivity Factor
A useful equation is derived that relates the resolution of
a column to the number of plates it contains as well as to
the retention and selectivity factors of a pair of solutes
on the column. The resolution is given by the equation
RS 
N
4
   1  kB 



    1  kB 
where kB is the retention factor of the slower-moving
species and  is the selectivity factor. This equation can
be rearranged to give the number of plates needed to
realize a given resolution:
2
2
    1  kB 
N  16 R 
 

  -1  kB 
2
S
Applications of Chromatography
Chromatography is a powerful and
versatile tool for separating closely related
chemical species. In addition, it can be
employed for the qualitative identification
and
quantitative
determination
of
separated species.