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3
Equilibrium Types in HPLC
O U T L I N E
H3C CN H
H
General Comments H
O O H
H
H O
H H H
In Section 1.1 it was indicated that HPLC O O H H
separation is based on the equilibrium estab- H
O
H
lished between the molecules from the mobile H
H O
H
H O H
phase and those present in the stationary phase, O H H H O H
H O O
and that different types of equilibria are used for O O
H H
achieving separation in HPLC. This chapter H H H H
H H H H H
presents a more detailed description of types O
O O O O O
of equilibria acting in HPLC separation. Each Si Si Si Si Si
equilibrium is dependent on the analyte reten- O O O O O O
O O O O O
tion, which is commonly described in chroma-
tography by the capacity factor k. The formal Water immobilized on a polar surface
Distribution Coefficient i1 i2
the distribution constant Ki for a given system shape and tailing. The reverse will happen for
appears to depend only on temperature. The cases when at the apex of a peak K > K ideal,
graph representing ci,A as a function of ci,B at and the peak will show deviation from Gaussian
a given temperature is called an isotherm and shape and fronting.
has the equation:
ci;A Ki ci;B (3.1.22) Evaluation of Capacity Factor k from
Liquid-Liquid Distribution Constants
The isotherm is a straight line when Ki is a true
Different studies on liquid-liquid extraction
constant. In this case, at a constant temperature,
(see, e.g., [1]) showed that the partition
the equilibrium process maintains a constant
constants Ki,A and Ki,B for a compound i in two
ratio between ci,A and ci,B during the separation.
systems, (a) solvent A/water and (b) solvent
However, in some cases Ki varies with ci,A and
B/water, are related by the expression:
ci,B, and the isotherm is no longer a straight line.
When ci,A represents the concentration of log Ki;A a log Ki;B b (3.1.24)
species i in the stationary phase, and ci,B the
where a and b are constants that are character-
concentration of i in the mobile phase, rel.
istic for the two solvents that are utilized and
3.1.22 is identical with rel. 2.1.11 (where species
can be obtained using best regression fit for
X is now indicated as i). As shown in Section 2.1,
a large number of compounds. Relation
the retention time t0 R (i) is given by expression
(3.1.24) gives excellent results for systems that
2.1.15, which can be written in the form:
are somewhat similar such as octanol/water
t0R Ki J t0 (3.1.23) and hexanol/water. This type of correlation
becomes weaker for systems that are very
Relation 3.1.23 indicates that for a chromato- different. Further studies [2e4] showed that
graphic separation where Ki is a true constant, this type of correlation can be extended from
the concentrations of ci,A and ci,B do not affect liquid-liquid extraction data to values for the
Ki and the retention time tR (i) is not affected capacity factor ki for chromatographic systems.
by the concentration of the analyte. However, The correlation between Ki,A and ki (for a specific
when Ki varies with the concentration of the stationary phase) is similar to rel. 3.1.24 and has
analyte, the result will be that the retention the following expression:
time will also be modified. This is going to
log ki a log Ki;A b (3.1.25)
happen across the chromatographic peak where
the analyte concentration starts at zero before The partition constant Ki,A that is of particular
the peak reaches a maximum at the peak apex, interest in HPLC is the partition constant for
and then decreases again to zero. This change octanol and water, typically indicated as Kow
in the retention time is not large, but in some (or Pow). For this constant, extended information
cases it can be large enough to modify the shape is available in the literature regarding its values,
of the peak from the Gaussian shape to peaks measurement, and applications [4e6]. Also,
showing fronting or tails. For example, it can since Kow describes the partition of a compound
be assumed that for a given analyte at the begin- i between a polar solvent (water) and a solvent
ning of the peak K Kideal (and does not depend with a large hydrophobic moiety (octanol), its
on the concentration), but at the apex of a peak values are expected to correlate well with the
K < Kideal. In this case the retention time tR for capacity factor for reversed phase liquid chro-
the apex will be slightly shorter than ideal and matography that uses a C8 or C18 stationary
the peak will show deviation from Gaussian phase. This assumption was proven correct for
3.2. ADSORPTION EQUILIBRIUM 91
2 FIGURE 3.1.3 Dependence of log ki vs. log
Kow for 72 mono and disubstituted aromatic
compounds with ki values obtained for a C18
stationary phase with water/methanol 50/50
1.5 (v/v) as a mobile phase [2].
y = 0.5199 x - 0.3311
R2 = 0.9355
1
log k
0.5
0
-0.5 0 0.5 1 1.5 2 2.5 3 3.5
-0.5
log K ow
numerous compounds, and the following rela- solid stationary phase. An equilibrium is
tion is valid. assumed to be established between the mole-
log ki a log Ki;ow b (3.1.26) cules of the solute (analyte) dissolved in the
mobile phase and those adsorbed on the
surface of stationary phase. Similar to the case
Depending on the stationary phase and the
of partition equilibrium, a small amount of
mobile phase utilized, the coefficients a and
solute is assumed to participate in this equilib-
b are obtained as best regression fit. As an
rium. The stationary phase can be practically
example, the dependence of log ki versus log
considered covered by a monolayer of mole-
Kow for 72 mono and disubstituted aromatic
cules of either solute i or mobile phase A. The
compounds, with ki values obtained for a C18
distribution of i between mobile phase and
stationary phase using water/methanol 50/50
stationary phase can be expressed by the
(v/v) as a mobile phase [2], is described by a linear
displacement equilibrium:
expression of the form 3.1.26. Such a line, with a
0.5199 and b 0.3311, is shown in Figure 3.1.3. As imo n Ast % ist n Amo (3.2.1)
shown in this figure, dependence has R2 0.9355
indicating a very good correlation. where the solute i can displace on the stationary
phase n molecules of adsorbed phase A and
where the index st is used for the stationary
3.2. ADSORPTION EQUILIBRIUM
phase and mo for the mobile phase A. This
type of process is exemplified by activated char-
Liquid-Solid Equilibrium coal that adsorbs organic molecules. In HPLC
In adsorption HPLC, the molecular species the adsorption can be exemplified by a silica
of the solutes from the mobile phase have the dry surface (covered with a monolayer of
capability to be adsorbed on the surface of the water), which adsorbs a monolayer of a solvent
92 3. EQUILIBRIUM TYPES IN HPLC
Si Si Si Si Si Si Si Si Si Si
O O O O O O O O O O O O
O O O O O O O O O O
Molecules of mobile phase adsorbed on silica Replacement of mobile phase with the analyte
FIGURE 3.2.1 Schematic diagram of the replacement from a monolayer of three molecules of solvent with a molecule of
analyte.
(e.g., CH2Cl2) followed by the replacement of n in isocratic conditions). However, from a solvent
solvent molecules by a molecule of an analyte. mixture (e.g., for solvent A with the composition
Figure 3.2.1 shows a schematic picture of this %A and for solvent B with the composition
process with nitrobenzene as the analyte (100e%A), it can be assumed that only one of
molecule. the solvents (e.g., A) is adsorbed on the surface
The process described by rel 3.2.1 can be of the stationary phase and is replaced by the
characterized by a thermodynamic distribution analyte in an equilibrium of the type 3.2.1. The
constant K.i given by the ratio (a indicates activity of solvent A in the mobile phase will
activities): be indicated by aA,mo and on the stationary
phase by aA,st. Not all these requirements are
K i ai;st =ai;mo aA;mo =aA;st n (3.2.2)
always fulfilled, and therefore the assumption
that Ki is a constant should be considered only
Several assumptions are necessary to obtain
an approximation [7].
a constant value for Ki (for a given tempera-
From the equality of chemical potentials for
ture). These assumptions include the
the components of the two sides of the equilib-
following: (1) the surface is uniform; that is,
rium 3.2.1, the thermodynamic distribution
all adsorption sites are equivalent, (2) the
constant can be written as follows (see
adsorbed molecules do not interact such that
rel. 3.1.4):
Ki will be independent of the molecules
concentration, (3) all adsorptions take place K i expm0 i;mo n m0 A;st m0 i;st
by the same mechanism, and (4) only a mono-
layer of molecules is formed even at maximum n m0 A;mo =RT (3.2.3)
adsorption.
In practice, the mobile phase usually consists In rel. 3.2.3 the typical notation m for standard
of a mixture of two or even more miscible chemical potentials was utilized. The two values
solvents (the composition of this mixture is for the standard chemical potentials in the
kept constant when the separation is performed mobile phase m0i,mo and (n m0A,mo) are usually
3.2. ADSORPTION EQUILIBRIUM 93
equal and cancel each other. This leads to the the constant for the equilibrium 3.2.1 can be
relation: written in the form:
K i expn m0A;st m0i;st =RT (3.2.4) Ki ci;st =ci;mo xA;mo =xA;st n (3.2.10)
The concentration of solvent A in the mobile where ci,st, for example, indicates the molar
phase is not affected by the adsorption process; concentration of the solute i on the stationary
therefore the equilibrium can be considered phase, and xA,mo indicates the molar fraction of
only for the solute i between the stationary solvent A in the mobile phase. In rel. 3.2.10, it
and mobile phases: can be considered that xA,st 1 when the entire
surface of the stationary phase is covered with A
imo % ist (3.2.5) molecules. By replacing rel. 3.2.6 in rel. 3.2.10
This equilibrium is characterized as shown in followed by multiplication with J, it can be
Section 3.1 by an equilibrium constant: seen that log ki can be written in the form:
0
Ki ci;st =ci;mo (3.2.6) log ki log ki A n logxA;mo (3.2.11)
where ci,st is the number of moles of i per gram In rel. 3.2.11, ki(A) indicates the capacity
of adsorbent and ci,mo is the molar concentration factor of the solute i in pure solvent A as a mobile
of i in the mobile phase. If the quantity of phase. Indeed, for the molar fraction xA,mo 1 its
adsorbed mobile phase per unit weight of logarithm value is zero and log ki log ki(A). For
adsorbent is indicated as CA, the relation lower concentrations (molar fraction < 1) of A in
between Ki and K0 i will be the following: the mobile phase, the logarithm is negative and
0 the capacity factor increases. Relation 3.2.11 is
Ki K i CA (3.2.7)
also known as the Soczewinski equation. It is
With rel. 3.2.7 and the assumption gi,st / gi,mo 1 common to express in practice this expression
and gA,st 1, the expression for Ki becomes: in the form:
0
Ki expn m0A;st m0i;st =RT CA (3.2.8) log ki z log ki A n log fA;mo (3.2.12)
thermodynamic variables of the system by an the sample is in contact with the stationary
expression similar to rel. 3.1.12, such that: phase and another part is in contact with mole-
cules already adsorbed. Also, when different
0
ln K DH0 T DS0 =RT (3.2.13) molecules in a mixture are competing for the
stationary phase surface, the adsorption of one
where DH 0 and DS0 are the standard enthalpy component may depend on the adsorption of
and entropy changes for the transfer of the ana- another component. Isotherms of different
lyte from the mobile to the stationary phase shapes deviating from linear have been reported
expressed by the equilibrium 3.2.5. This indi- in the literature (see, e.g., [8,9]).
cates that the vant Hoff equation is equally Assuming for simplicity in rel. 3.2.10 that
applicable regardless of partition or the adsorp- n 1 and that the molar fraction of component
tion mechanism. i in solution is xi,mo, the molar fraction of the
solvent in solution will be xA,mo 1 xi,mo.
Also, assuming that the molar fraction of the
Peak Shape in Adsorption adsorbed compound i is xi,st, the molar fraction
Chromatography of the adsorbed solvent is xA,st 1 xi,st. In
Similar to the case of partition chromatog- this case, rel 3.2.10 can be written in the form:
raphy, the graph representing ci,st as a function xi;st 1 xi;mo
of ci,mo at a given temperature is called an Ki (3.2.14)
xi;mo 1 xi;st
isotherm. The isotherm is a straight line when
K0 i is a true constant at a constant temperature. Since typically the value for the molar frac-
However, in some cases K0 i varies with ci,mo tion xi,mo is very low, 1 e xi,mo z1 and rel.
and the isotherm is no longer a straight line. 3.2.14 can be rearranged to give:
This can be seen, for example, at large loads of
Ki xi;mo
the column when, because of the limited surface xi;st (3.2.15)
capacity of the stationary phase, some part of 1 Ki xi;mo
0.25 0.70
K = 1.6 0.60
0.20
K = 1.6
0.50
0.15
0.40
K = 0.8
K = 0.8
xi,st
xi,st
0.10 0.30
K = 0.2 0.20
0.05 K = 0.2
0.10
0.00 0.00
0 0.05 0.1 0.15 0.2 0 0.2 0.4 0.6 0.8 1
xi,mo xi,mo
FIGURE 3.2.2 Hypothetic graphics for Langmuir isotherm Ki 0.2, 0.8, and 1.6 for two concentration ranges.
3.3. EQUILIBRIA INVOLVING IONS 95
Relation 3.2.14 is known as the Langmuir connected through ionic interactions with the
isotherm. The representation of this isotherm stationary counterionic groups bonded to the
for hypothetic systems with Ki 0.2, 0.8 and stationary phase. Ion-exchange chromatog-
1.6 on two concentration ranges for xi,mo (0 to raphy is subdivided into cation exchange
0.2, and 0.2 to 0.99) are shown in Figure 3.2.2. chromatography and anion-exchange chroma-
From Figure 3.2.2 it can be seen that at low tography. In cation-exchange chromatography,
concentrations for the analyte i and for lower the ions M in solution are exchanged with
Ki values, the dependence of the molar fraction the cations C that were initially retained by
of the adsorbed material practically depends stationary ionic groups of the type R-X. In
linearly on the concentration in the mobile anion exchange the anionic species B in solu-
phase. This is not the case at higher concentra- tion are exchanged with the anions A that
tions or higher Ki values when the amount of were initially retained by the stationary coun-
adsorbed material is higher. Not all adsorption terionic groups of the type R-Y. In this way,
systems are well described by a simple Lang- the electroneutrality of the system is always
muir isotherm, and other formulas have been maintained (the ions in solution also have coun-
developed for a better description of the adsorp- terions). A special type of ion exchange may
tion process [10]. For a system where depen- also take place on zwitterionic stationary
dence between ci,st and ci,mo is linear, the value phases (ZIC phases containing, for example,
of K0 i is a true constant and is not affected by a sulfonic and an amino group in the bonded
the increase in concentration of the analyte. phase) when the mobile phase is a totally
Since the retention time in a chromatogram is aqueous buffer. In this case, only one group is
related to the equilibrium, the retention time t0 R involved in the ion-exchange process, and the
(i) for component i is given by expression 2.1.15, phase acts either as cation- or as anion-exchange
which can be written in the form: material.
ion-exchange behavior of simple, dilute strong and 3.3.6, the equilibrium constants 3.3.2 can be
electrolyte solutions. However, when dealing expressed in the form:
with more complex systems, such as weak elec-
KM;C Kres;M =Kres;C (3.3.7)
trolyte solutions or solutions with high solute
concentrations exceeding the resin capacity,
For a multicharge ion, the equilibrium taking
the simplifying assumptions are not always
place between the solution and the resin can be
valid. For example, the uptake of ions may
written in the form:
exceed the total capacity of the resin in case of
high concentrations. z resX C Mz % resXz z Mz z C
For a given ion M in solution and a resin (3.3.8)
in C form, the exchange equilibrium is the and the equilibrium constant is:
following: z
Mz res C res
KM;C (3.3.9)
resX C M % resX M C (3.3.1) Mz mo C mo
The constant for this equilibrium can be Based on rel. 3.3.2 or 3.3.9, it can be seen that
written using previous assumptions as follows: the concentration of an ion in solution and the
value of the equilibrium constant are important
M res C res factors in the retention of an ion from solution.
KM;C (3.3.2)
M mo C mo In practice, the ion-exchange column must be
initially conditioned and made in the form C.
where the index res indicates resin and mo indi- The ion selected as C is frequently H, and in
cates mobile phase. The exchange constant KM,C this case rel 3.3.9 will become:
indicates the degree to which an ion M is z
Mz res H res
preferred in the exchange process, compared KM;H (3.3.10)
to the ion C. Larger constants for KM,C indicate
z
M mo H mo
higher affinity for the resin of species M. The
When a solution containing the ions M
exchange constant can be expressed as a func-
having a relatively high Kres,M is passed through
tion of a distribution constant between the resin
the resin, the ions will attach to the resin and H
and the solution. The equilibrium described by
ions will be released in solution. The exchange
rel. 3.3.2 can be viewed as equivalent with two
of H ions with M ions can continue until the
independent equilibria:
equilibrium described by rel. 3.3.10 is reached.
C mo % resX C (3.3.3) The change of the column into H form
before the separation can be accomplished by
M mo % resX M (3.3.4) slowly flowing through it a solution of an inor-
ganic acid. For example, a solution of 0.1 N
which are described by the constants: HCl is passed through the stationary phase
resin. This changes the resin into H form. Rela-
C res
Kres;C (3.3.5) tion 3.3.9 (or 3.3.10) is applicable only to
C mo describe an equilibrium, and this is not the
M res case during the column conditioning when the
Kres;M (3.3.6) concentration [Mz]mo 0 and [H]mo is high.
M mo
However, rel. 3.3.10 shows that [H]res will
Each solute has a distribution constant (reten- become as high as possible when [H]mo is
tion constant) for a specific resin. Using rel. 3.3.5 high and since [Mz]mo is practically zero,
3.3. EQUILIBRIA INVOLVING IONS 97
[Mz]res will also tend to zero After condi- described by the equilibrium constant KLM
tioning, the resin is thoroughly washed with given by the expression:
water to eliminate the remaining HCl traces.
LMmo
The same discussion for the equilibrium on KLM (3.3.15)
a cation-exchange column can be easily applied L mo M mo
for an anion-exchange column. In this case,
the concentration of M in the resin is given by
a column in the form A- exchanges a base B- in
the expression:
the following equilibrium:
resY A B % resY B A (3.3.11) LMmo
M res Kres;M (3.3.16)
L mo KLM
This equilibrium is governed by the constant:
Expression 3.3.16 shows that a higher concen-
B res A res
KB;A (3.3.12) tration of ligand or a high complexation
B mo A mo constant diminishes the amount of species M
retained in the column. However, complexation
For any anion, a retention constant can be can be used to favor retention on the resin. For
defined with the formula: example, specific ions form negatively charged
B res complexes. Assuming that an ion M2 forms
Kres;B (3.3.13) with a ligand L four combinations ML, ML2,
B mo
ML 2
3 , and ML4 , the negatively charged
Frequently, in anion-exchange chromatog- complexes and the ligand can be retained on
raphy A is OH, but other anions such as Cl an anion-exchange resin, while the positive
are also common. ions and the neutral molecules are not retained.
In a mixture of ions in which only some have the
complexing capability with the formation of
Equilibrium in the Presence negatively charged compounds, an anion
of a Complexing Reagent exchanger can be used for separating the
desired species. In this case, considering the
The use of various complexing agents in the constant Kcomp describing the equilibrium:
solution interacting with an ion-exchange resin
is another procedure used in ion-exchange 4 L M2 % ML4 2 (3.3.17)
HPLC for obtaining separations. The equilib- and the equilibrium constant Kres,ML4, the distri-
rium between the complexing agent (ligand) bution constant between the mobile phase and
in solution and the ions to be exchanged the resin for ML2
4 species, the concentration of
reduces the concentration of the free ions avail- ML24 in the resin can be estimated using the
able for the exchange process. In this case two expression:
simultaneous equilibria take place in the mobile
phase: ML4 2 res Kres;ML4 Kcomp L 4 M2 (3.3.18)
resX C M % resX M C and
Each complex ion has its specific distribution
L M % LM constants in the resin. The constant depends on
(3.3.14) factors such as bond strength, hydrophobic
interactions, and steric hindrance. The adsorp-
Assuming that the reaction with the ligand is tion of complex ions on resins is a more compli-
present only in the mobile phase and is cated process because in addition to the
98 3. EQUILIBRIUM TYPES IN HPLC
a molecule is the volume of a hypothetical hard for HPLC in Section 2.1 as having expression
sphere that diffuses with the same speed as the 2.1.14 will be given in SEC by the formula:
particle under examination. For SEC, the hydro- Vpores
dynamic volume is therefore the true parameter JSEC (3.4.3)
Vinter
related to the separation.
Using rel 3.4.3, the expressions for capacity
Partition Equilibrium between factor kSEC will be given by the formula:
Interstitial Mobile Phase and Pore
Mobile Phase Vpores
kSEC KSEC (3.4.4)
Vinter
The separation in SEC is done on a porous
stationary phase. The small molecules from the The direct proportion between kSEC and JSEC
sample moving with the mobile phase penetrate expressed by rel 3.4.4 has, however, a problem.
the pores of the stationary phase and have The value of JSEC may increase by an increase
a long path through the column and therefore in the number of pores and not only by the
long retention times. Very large molecules increase in the (average) pore volume. If the
cannot enter the pores at all and elute without pores are numerous but keep a small average
retention (total exclusion). The macromolecules volume, these pores cannot contribute to the
of medium size enter only some larger pores retention of larger macromolecular analytes. In
and are only partly retained, eluting faster this case, rel. 3.4.4 is no longer valid, and the
than small molecules and slower than the very proportionality remains valid only for a specific
large ones, thus achieving separation. range of molecular sizes.
Similar to any HPLC process, the retention The partition constant KSEC in rel. 3.4.4 repre-
process in SEC of a given molecular species sents the ratio of the concentrations of the
can be formally characterized by the reduced macromolecule analytes in the pore (cpore) and
retention volume V0 R given by rel. 2.1.16. From the concentration in the interstitial volume
rel. 2.1.16 the following expression can be imme- (cinter):
diately obtained:
KSEC cpore =cinter (3.4.5)
VR V0 K Vst (3.4.1)
For understanding the variation of KSEC, the
In SEC the partition process takes place as
elution process can be viewed as the movement
a migration of the analytes between the intersti-
of a sample zone of a solution containing the
tial volume of the column filled with the
macromolecular analytes (a nonzero concentra-
packing and the pores of the packing. In expres-
tion of analyte) along the column packed with
sion 3.4.1 the dead volume V0 can be indicated
porous particles and filled with the mobile
for this process as the interstitial volume Vinter,
phase (with the analyte at zero concentration,
and the volume Vst can be considered as equal
c 0). The initial concentration of macromole-
to the volume of the pores of the packing Vpores.
cules within the pores is also zero. The concen-
Therefore, the general equation 3.4.1 can be
tration gradient between the interstitial
written for SEC separation in the form:
volume outside the pores during the sample
VR Vinter KSEC Vpores (3.4.2) zone (cinter > 0) and within the pore (at cpore
0) pulls macromolecules into the pores
Using the same assumptions as for rel. 3.4.2, because of the tendency to equalize the chemical
the phase ratio that was described in general potentials in the interstitial volume and in the
3.4. EQUILIBRIUM IN SIZE-EXCLUSION PROCESSES 101
Expanded
macromolecule
FIGURE 3.4.1 Schematic representation of the partition of a coiled macromolecule between interstitial space and the pore
of stationary phase (expansion of the molecule is associated with increase in entropy and shrinking is associated with
decrease in entropy).
pore volume. Macromolecules outside of the However, in the ideal case of no interaction
pores are expanded. When pulled into the with column packing, the value for DH0 in rel.
pore in order to equalize concentrations outside 3.4.6 is zero, and the separation mechanism is
and inside the pore, the macromolecules are controlled exclusively by the entropy of the
squeezed and their conformational entropy process. Therefore the expression for a pure
decreases. The macromolecules inside the pore size-xclusion process described by the constant
are contracted and lose part of their conforma- Kpure is given by the formula:
tional entropy. Some macromolecules cannot
enter completely the pore volume because the Kpure expDS0 =R (3.4.7)
loss in entropy would exceed the pulling force Size exclusion of macromolecular analytes
inside the pore. A schematic representation of in the absence of any energetic interactions
the partition process in SEC separation is illus- with the stationary phase is therefore an
trated in Figure 3.4.1. This process indicates entropy-controlled process. Such a process is
that the values of KSEC are situated within the usually indicated as an entropic partition
interval [0, 1]. If KSEC 0, the sample fraction [15]. The loss of entropy when the molecules
will elute in the void volume (total exclusion), are trapped inside the stationary phase causes
and when KSEC 1 the sample fraction elutes DS0 to have negative values [16]. Expression
in the total column volume (Vinter. Vpore). 3.4.7 indicates that temperature should not
However, this process implies an ideal mecha- significantly influence the exclusion processes.
nism of SEC where the analytes do not exhibit This was proven experimentally in several
any attractive or repulsive interaction with eluents that are good solvents for polymers
column packing except for the effects caused [17]. The change in entropy is more significant
by the imperviousness of the pore walls. In for larger molecules (of polymers) and less
some instances, values KSEC > 1 are seen, indi- important for smaller ones. This can be under-
cating that other interactions take place between stood by starting with the following expres-
the analyte and the stationary phase. The sion for entropy:
expression for KSEC using the general formula
2.1.12 is the following: S kB ln U (3.4.8)
KSEC exp DH 0 T DS0 =RT (3.4.6) where kB is the Botzmann constant and U is the
number of possible (equally probable) micromo-
where e DH 0 is the standard enthalpy and DS0 is lecular states. The number of ways in which the
the entropy change for the transfer of the ana- individual molecules can occupy the space
lyte from the mobile to the stationary phase. within the pore of a stationary phase is
102 3. EQUILIBRIUM TYPES IN HPLC
VR mL
for large and small molecules in solution, the 7.5
large molecules will have a smaller S value in 7
the stationary phase, and therefore a considerable 6.5
loss of entropy. At the same time, the small mole-
6
cules will have only a minor loss of entropy. The
result is that during the adsorption process, the 5.5
-6 -5 -4 -3 -2 -1 0
large molecules will have a larger (in absolute
value) negative DS0. From rel. 3.4.7, it can be S cal
0
HX % H X (3.5.4)
The Influence of pH on Partition
where the equilibrium constant Ka is defined by
the well-known formula In a simple partition equilibrium between
two liquid nonmiscible solvents, the distribu-
tion coefficient (Di) and the distribution constant
H X (Ki) are identical for apolar or polar solutes that
Ka (3.5.5)
HX have no acid or base functional groups in their
104 3. EQUILIBRIUM TYPES IN HPLC
100 CH3 O
1 C C
C OH
H 3C H H
80 N H+ 3
1 2 3
CH 3 O
60
% Con tent
2 C C _
C O
H 3C H H
40 +
NH 3
20 CH3 O
3 C C _
C O
0 H 3C H H
NH2
0 2 4 6 8 10 12 14
pH
FIGURE 3.5.1 Variation in the proportion of different forms of valine in a solution at different pH values.
P
molecule that would lead to some dissociation cHX;o cHX;o
in the aqueous or partially aqueous medium. DHX P (3.5.10)
cHX;w cHX;w cX ;w
When the solutes contain ionizable functional
groups such as -OH, -SH, -COOH, -SO3H, Expressions 3.5.9 and 3.5.10 show that higher
-NH2, and others, it is possible for those solutes KHX or DHX indicate a higher concentration of
to have interactions into aqueous phase with the analyte in the organic phase. This remains
proton or hydroxide ions leading to ionized valid when the organic phase (o) is the
species. In such cases, the two distribution stationary phase (e.g., in reversed phase chro-
parameters (Di and Ki) are not identical, and matography). For these cases, a higher KHX or
the one that truly describes the partition process DHX is related to a higher capacity factor k
is the distribution coefficient Di. In Figure 3.5.2 based on rel. 2.1.13 and 3.1.20, respectively,
a schematic example is shown, which describes and therefore to longer retention times in the
the partition process for a molecular weak acid, chromatographic process.
denoted by HX. Since in the case of ionizable groups the
Similar to the case of solvents is the one distribution process is controlled by the
where the organic phase role is taken by an
organic stationary phase, and the aqueous
phase is a mobile phase with a certain water HX Organic
content (indicated as w). In the case of the acids s olvent (A = o)
KHX
HX, the distribution constant K (see rel. 3.1.7) Aqueous
and distribution coefficient D (see rel. 3.1.18) H + X-
+ phas e (B = w)
HX
are given by the following relations: Ka
cHX;o
KHX (3.5.9) FIGURE 3.5.2 Representation of the partition process of
cHX;w a weak acid HX.
3.5. THE INFLUENCE OF PH ON RETENTION EQUILIBRIA 105
distribution coefficient D and not by the A weak-base-type compound Y can be in-
distribution constant K, it is important to volved in an acid/base equilibrium as shown
analyze the variation of D for several partic- in the following:
ular cases. In the case of an acid HX, the
Y HOH % YH HO (3.5.13)
concentration of the dissociated form in rel.
3.5.10 can be calculated from the dissociation
This equilibrium is characterized by a basicity
constant (Ka) as:
constant Kb (Kb Kw / Ka). The same calcula-
cHX;w tions as applied for the acids distribution lead
cX ;w Ka (3.5.11)
H w to the following dependence of DY on pH:
Kw
By substituting cX ;w given by rel. 3.5.11 and DY KY (3.5.14)
KHX given by rel. 3.5.16 in expression 3.5.10, Kw Kb ,10pH
the relation between KHX and DHX can be (where Kw is the ionic product of water (1014 at
written as follows: 25 C). The plot of this dependence is shown in
Figure 3.5.4 for a base with Kb 104.64
c 10pH
DHX HX;o KHX (pKb 4.64).
1 Ka 10pH The formulas 3.5.12 and 3.5.14 (as well as the
cHX;w 1 Ka
H graphs from Figures 3.5.3 and 3.5.4) indicate
(3.5.12) that for weak acidic or basic analytes, the pH
plays a major role in the distribution of the ana-
The dependence given by rel. 3.5.12 is illus- lyte. Even if the distribution constant is highly
trated in Figure 3.5.3 for a compound with favorable for the organic phase (large K values),
Ka 10e4.54, (pKa 4.54). At low pH values, since the parameter that truly describes the distri-
DHX z KHX. At high pH values, DHX z 0; bution process is D, its value can be very small if
and at pH pKa which is the inflection point the pH of the aqueous phase is not selected prop-
of the sigmoid curve of dependence, DHX z erly. This effect can also be successfully used in
0.5 KHX. a separation process. For example, the separation
1 1
0.8 0.8
DHX / KHX
0.6 0.6
DY / KY
0.4 0.4
0.2 0.2
0 0
0 pKa 7 14 0 7
pKw -pKb 14
pH pH
FIGURE 3.5.3 The dependence of DHX/KHX as a function FIGURE 3.5.4 The dependence of DY/KY as a function
of pH for an acid HX (pKa 4.54) of pH for a basic compound Y (pKb 4.64).
106 3. EQUILIBRIUM TYPES IN HPLC
of a number of acids with different pKa values can The graph showing the variation of DHXY /
be easily separated using a gradient elution with KHXY as a function of pH is given in Figure 3.5.5
the mobile phase changing its pH value. for a compound with pKa 2.47 and pKb 4.55
Compounds with amphoteric character (phenylalanine).
contain both acidic and basic functional groups Taking into account that Ka,Kw << 1014,
(for example, amino acids). If such a compound Kw 1014 and Kb >> Kw, the limit value of D
is indicated as HXY and participates in a distri- for the acidic pH domain is given by the
bution process, but in the mobile phase (partly expression:
aqueous) is also part of two acidebase equi-
libria, these equilibria and the corresponding pH/0 Kw
DHXY KHXY (3.5.23)
Ka and Kb constants can be written as follows: Kb
HXY % XY H (3.5.15) In the example shown in the graph from
c XY;w ,cH ;w Figure 3.5.5, Kw << Kb and the value for D is
Ka (3.5.16) much lower than that for K (practically zero).
cHXY;w
For pH / 14 and taking into account that
HXY HOH % HXYH OH (3.5.17) Ka >> Kw, the limit value of DHXY for basic pH
domain is given by the expression:
cHXYH ;w ,cHO ;w pH/14 Kw
Kb (3.5.18) DHXY KHXY (3.5.24)
cHXY;w Ka
The distribution of the neutral species For the example shown in the graph from
between the two phases (o and w) is character- Figure 3.5.5, Kw << Ka, and the value for D is
ized by partition constant (KHXY) and distribu- again much lower than that for K (practically
tion coefficient (DHXY): zero).
cHXY;o The maximum value for the ratio D/K can be
KHXY (3.5.19) obtained from the condition vDHXY =vH 0,
cHXY;w
cHXY;o
DHXY (3.5.20)
c XY;w cHXYH ;w cHXY;w
D max
Combining rel. 3.5.16, 3.5.18, and 3.5.19 with
DHXY / KHXY
log D ow
where {ci,o} i 1,2,.n represent all the forms of -0.5
the compound i present in octanol and {ci,w} i -1
1,2,.n represent all the forms of compound i -1.5
present in water. (Note: The terms partition and -2
distribution are used interchangeably, and the
-2.5
difference must result from the meaning of
the parameter). The general discussion on the -3
0 2 4 6 8 10 12 14
dependence of distribution coefficient D on pH
given in this section is equally applicable to pH
the distribution coefficient Dow, which depends
FIGURE 3.5.7 Variation of log Dow with pH for butyric
on the water pH for ionizable compounds. For
acid. log Kow 0.98 (max Dow Kow)
compounds having no ionizable groups, Kow
Dow . Variation of Dow with the pH can be
obtained using computing programs (e.g., regarding their octanol/water distribution, the
MarvinSketch 5.4.0.1). values for Dow should be considered at a specific
Since the structure of ionizable compounds pH. For amino acids, peptides, and proteins that
depends on pH, the distribution coefficient are present in zwitterionic form, the value for
Dow is also pH dependent. For example, in an log Dow at the isoelectric point (pI) is important
acidic pH, an acid may be very little or not for their characterization (the isoelectric point
ionized at all. In this case, at pH values where (pI) is the pH value at which the molecule
the compound is not ionized Dow Kow, while carries no electrical charge). For molecules
for other pH values they are different and with multiple zwitterionic points, log Dow at
Dow < Kow . This is exemplified for butyric acid
in Figure 3.5.7, where at low pH (pH < 3) the -2
maximum value for log Dow is 0.98 and Kow
Dow . For some amphoteric compounds, even in -2.2
a pH where a neutral form would be expected,
-2.4
the compound is present in zitterionic form,
and always Dow < Kow . Amino acids are this -2.6
log Dow
FIGURE 3.6.1 Chemical formulas for retinol (Vitamin A) and cholecalciferol (Vitamin D3).
(larger absolute value) indicates a stronger two forms of compound i, the result for ln ki
interaction [28]. is the following:
Ki1 Ki2 K1;2
ln ki ln ln J (3.6.5)
Nonlinear Dependence of the Capacity 1 K1;2
Factor on 1/T
A replacement of the expressions for indi-
When a compound i can be present during vidual equilibrium constants in rel. 3.6.5
the separation in several forms i1, i2, i3, and leads to a complicated formula, and for
so on, that are in equilibrium and account for simplicity a power series can be used to express
the total level of i, the capacity factor can be ln ki. In such cases, the following formula can be
expressed by formula 3.1.21, where the distri- used:
bution coefficient Di replaces the simple equi-
librium constant Ki. In such cases, the linear 1 1
dependence of ln k on 1/T is not necessarily ln ki a b c 2 . ln J (3.6.6)
T T
obeyed. The simple expression of the form
3.6.2 cannot be written (without including The number of terms in expression 3.6.6
approximations). Replacing in rel. 3.1.21 the can be larger, but a polynomial form limited to
expression 3.1.19 for Di for the simple case of (1/T2) is sometimes sufficient to describe the
Retinol 1.4
1.8 94% CH3 OH Calciferol
70%CH3OH
1.6 1.2
1.4 1.0
96%CH3 OH 75%CH3 OH
1.2
0.8
ln k
ln k
1.0
98%CH3OH 0.6
0.8 80%CH3 OH
0.4
0.6
0.4 0.2
3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.00 3.05 3.10 3.15 3.20 3.25 3.30
1/T*1000 1/T*1000
FIGURE 3.6.2 Vant Hoff plots for retinol and cholecalciferol separated on a C18 stationary phase and mobile phase
containing water and methanol in different proportions [26].
3.6. THE INFLUENCE OF TEMPERATURE ON RETENTION EQUILIBRIA 111
TABLE 3.6.1 Evaluation of thermodynamic parameters DH 0 and DS0 for the separation of retinol and calciferol on
a C18 column [26].
H3C O
O CH3
H H
N N
N N
O H3C O
H3 C HO NH
OH H 3C
O CH3 O O CH3
O
H 3C
2 Evaluation of Enthalpy-Entropy
1.8 Compensation from vant Hoff Plots
vincamine
1.6 In the retention process, a higher interaction
1.4 of the retained molecule with the stationary
phase is associated with a more negative
1.2
enthalpy of the interaction. However, this
ln k
[32] Miyabe K, Guiochon G. Thermodynamic interpretation problems associated with the analysis of vant Hoff
of retention equilibrium in reversed-phase liquid and Arrhenius data. J. Phys. Chem. B 1976;80:2335e41.
chromatography based on enthalpy-entropy compen- [34] Krug RR, Hunter WG, Grieger RA. Enthalpy-
sation. Anal. Chem. 2002;74:5982e92. entropy compensation.2. Separation of the chemical
[33] Krug RR, Hunter WG, Grieger RA. Enthalpy-entropy from statistical effect. J. Phys. Chem. B 1976;80:
compensation.1. Some fundamental statistical 2341e51.