Enhancement of Retention by Ion-Pair Formation in Liquid
`Chromatography with Nonpolar Stationary Phases
`
`Csaba Horvath,‘ Wayne Melander, Imre h‘lolnar.1 and Petra Molnar
`
`Chemical Engineering Group, Department of Engineering and Applied Science, Yale University, New Haven, Connecticut 06520
`
`In Ion-pair reversed-phase chromatography, the retention ot
`ionized analytes on a nonpolar banded stationary phase is
`enhanced by the presence oi a “hydrophobic" counterion
`(hetaeron) In the mobile phase. Either Ion-pair tormation In
`the mobile phase with relatively strong retention oi the complex
`or the conversion at the stationary phase Into an ion-exchanger
`may explain the phenomenon. Analysis of the pertinent
`equilibrla shows that the observed hyperbolic or parabolic
`dependence at the capacity factors on the hetaeron con-
`centration cannot shed light on the mechanism. The ex-
`perimental data obtained tor the retention oi catecholamlnes
`by using CFC 1,, alkyl sulfates and other similar hetaerons In
`a wide concentration range. however. could be mechanisticaly
`Interpreted from the chain length dependence oi the pa-
`rameters tor the relationship between the capacity factors and
`hetaeron concentration. Although the results clearly dem-
`onstrate that In the system Investigated, Ion-pair formation
`governs retention, ion-exchange mechanism can be operative
`under certain conditions. Changes In retention upon addition
`at salt to the eluent are treated both theoretically and ex-
`perimentally. The ettect of organic solvents on the behavIOr
`oi the chromatographic system Is discussed in view of the
`proposed theory.
`
`According to the popular notion, the selectivity in chro-
`matographic separations is determined by the differences in
`the equilibrium distribution of eluite molecules, i.e. analytes,
`between the stationary and mobile phases. Quite frequently,
`however, secondary equilibria between the eluites and certain
`species present in the eluent can drastically affect retention
`(1}. In a recent paper (2) we analyzed the effect of protonic
`equilibria in the mobile phase on retention in liquid chro-
`matography with nonpolar stationary phases. The selectivity
`of the chromatographic system for ionogenic eluites was shown
`to be greatly influenced by their dissociation constant and the
`hydrogen ion concentration in the eluent because the binding
`of protons increases and decreases the retention of weak acids
`and bases, respectively.
`Recent work has demonstrated that retention of charged
`eluites on nonpolar bonded stationary phases can be aug-
`mented by the presence of suitable counterions. which have
`a substantial hydrophobic moiety, in the mobile phase (3-5}.
`This technique is often referred to as “soap" or “ion-pair
`reversed-phase" chromatography. The counterions used in
`the mobile phase belong in the group of detergents such as
`alkyl sulfonates and sulfates or tetraalkylammonium com-
`pounds, and ion—pair formation between the eluite and
`counterion is assumed to be responsible for the increase in
`retention (6).
`This approach is particularly interesting because the use
`of nonpolar bonded stationary phases for liquid chromato-
`graphic separations has a wide currency (7}.
`In most cases
`
`'Present address, K. G. Dr. Herbert Knauer, Hegauer Weg 38,
`Berlin 37', West Germany.
`
`octadecyl-silica is the stationary phase and hydro-organic
`mixtures with methanol or acetonitrile as well as neat aqueous
`or organic solvents are used as eluents. The technique is
`simple and can be used for the separation of a wide variety
`of substances. For this reason it is the most popular method
`in high performance liquid chromatography. The mechanism
`of retention is believed to be the same as involved in the
`
`so-called hydrophobic effect (8) and we recently adapted the
`solvophobic theory (9) to treat the interaction of eluites with
`the hydrocarbonaceous functions of bonded phases in a
`rigorous thermodynamic fashion (2, I 0). We have shown that
`the magnitude of retention is governed by the effect of the
`solvent on these species and their adducts.
`The increasing popularity of using an ion-pair forming agent
`in the mobile phase to increase the retention of oppositely
`charged eluites prompted us to investigate the fundamental
`aspects of this technique. Our treatment, however, is quite
`general and applicable to a variety of cases where the retention
`of an eluite on nonpolar bonded phases is enhanced by
`complex formation with a component of the eluent. For sake
`of convenience we propose to call the complexing agent
`hetaeron, a term derived from the Greek work for companion
`(Efatpoul'. Thus “hetaeric” chromatography would denote a
`technique in which a certain concentration of a complexing
`agent is intentionally maintained in the mobile phase in order
`to affect the selectivity of the chromatographic system by
`secondary equilibria. The name should be restricted to
`situations where the eluite-hetaeron complex is formed in the
`mobile phase and distributed between the two phases. Of
`course, secondary equilibria may change the properties of the
`stationary phase. Indeed, it was recently suggested {11) that
`in ion-pair reversed-phase chromatography the stationary
`phase acts as a dynamically coated ion exchanger because of
`the adsorption of the detergent. ions. By using extensive
`experimental data and applying the solvophobic theory for
`the interpretation of the results, we intend to demonstrate
`in this article that in the situations examined the mechanism
`
`of the chromatographic process entails ion-pair formation in
`the mobile phase and binding of the neutral complex to the
`stationary phase.
`
`THEORY
`
`Phenomenological Treatment. In order to shed light on
`the relationship between the capacity factor, which is a
`convenient measure of retention, and the equilibrium con-
`stants. which govern the retention on nonpolar bonded
`stationary phases in the presence of various concentrations
`of a complexing agent (hetaeron) in the mobile phase, the
`process first will be treated phenomenologically.
`Figure 1 illustrates the various equilibria which are involved
`in the chromatographic process. The eluite, E, whose retention
`is of interest, can interact with the hetaeron, H, to form a
`complex. EH, which is bound to the hydrocarbonaceous ligand,
`L, of the stationary phase to form LEH. Alternatively, the
`hetaeron may bind first to the stationary phase and then form
`LEH. In addition the species E and H can individually form
`the adducts LE and LH with the ligands of the stationary
`phase. It is assumed that binding of the eluite and the heteron
`2030
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`
`

`

`K2
`——-—>
`
`E + H
`
`———9
`
`EH
`
`E + H
`
`K2
`
`+ L
`
`K1
`
`+ l.
`
`KL,
`
`+ L
`
`K3
`
`K6
`—-—-—-)
`
`K5
`
`LE + H
`
`(__._..._
`
`LEH
`
`E + LH
`
`Flame 1. Schematic Illustration of the equlllbria Involved in the
`chromatographic process with nonpolar bonded stationary phases and
`a complexing agent In the mobile phase. The meaning of the symbols
`is: E. eluite; H. complexing agent (hetaeron); L, hydrocarbonaceous
`ligand bound to the support: K 1 to K5 are the corresponding equilibrium
`constants
`
`to the stationary phase ligands takes place independently.
`The equilibrium constants are expressed by the following
`set of equations in which the species concentrations in the
`mobile and stationary phase are denoted by the subscripts
`m and s, respectively. The concentration of L is defined as
`the accessible ligand concentration in the stationary phase.
`
`K1 = [LElsMElmlLls
`
`K2 = [EHlm flE]m[Hlm
`
`K3 = [LHls/[HlmlLls
`
`K4 = [LEHls/[EHlmlLls
`
`K5 = [LEHLMLHldElm
`
`K6 = [LEHlsMLElslHlm
`
`(1)
`
`(2)
`
`(3)
`
`(4)
`
`(5)
`
`(6}
`
`The capacity factor of the eluite, k, is defined in the usual
`way as
`
`k = MILEHls + [LEllelElm + [EHlml
`
`(7)
`
`where o is the phase ratio, i.e. the ratio of the volume of
`stationary phase to the volume of the mobile phase in the
`column.
`In chromatographic practice when the column is equili-
`brated with the eluent the hetaeron concentration in the
`mobile phase is constant.
`If [E] << [H]m, only a negligible
`fraction of the hetaeron is in the form of a complex so that
`the hetaeron concentration can be considered invariant and
`we can write
`
`The assumption that complex formation occurs with the
`hetaeron already bound to the stationary phase yields, by the
`combination of Equations 1—3, 5 and 7-9 the following ex-
`pression for the capacity factor.
`
`k = ¢[L1{K1+ KaKslHlml + K2[H]}(1 + KalHll
`(11}
`
`A third possible combination uses Equations 1—3 and 6-9 and
`yields
`
`k = ¢[Ll(Ki + KinHlml + K2[H1J(1 + K3[H])
`
`(12)
`
`Equation 12 would imply that the eluite first binds to the
`stationary phase and then forms a complex with the hetaeron.
`Equations 10—12 all express the dependence of the capacity
`factor on the hetaeron concentration and have the general
`form
`
`’6 = [kc + B[H])f(1 + Klellil + K3[Hl)
`
`(13)
`
`where k.) is the capacity factor of the eluite in the absence of
`hetaeron, K2 is the association constant for the eluite and the
`hetaeron, K3 is the binding constant of the hetaeron to the
`stationary phase and B is the product of the two equilibrium
`constants as shown in Equations 10—12. A plot of h vs. [H]
`according to Equation 13 yields a parabola provided 1 ,1 K3[H]
`< lng.
`If either K2[H] <4. 1 or K3[H] (< 1, Equation 13 can be
`written as
`
`k = (a. + B[H]}f(1 + P[H])
`
`(14)
`
`where P can be either Kg or K3. Equation 14 is the equation
`of a rectangular hyperbole, for the dependence of the capacity
`factor on the hetaeron concentration. Both parabolic and
`hyperbolic dependence of the capacity factor on the hetaeron
`concentration have been observed in ion-pair reversed phase
`chromatography (5, 6).
`When the complex is an ion-pair. both the eluite and the
`hetaeron have to be fully ionized for the above treatment to
`be valid. Whereas the hetaeron is usually a strong electrolyte
`in ion-pair chromatography, the eluites are often weak bases
`or acids and therefore the pH of the eluent can have an
`influence on the retention. The correspOnding protonic
`equilibria can readily be incorporated into the above model
`as will be shown by the example of a weakly basic eluite.
`The protonation of the neutral eluite, E", is characterized
`by its acid dissociation constant, Ka, related to the equilibrium
`
`E0 + H" 2 EH+
`
`(15)
`
`[H]m E [H]
`
`Both forms, E” and EH+, can bind to the ligands of the
`stationary phase according to the following equilibria
`
`(8)
`
`Since the extent of binding of the eluite by the stationary
`phase is expected to be small and the total ligand concen-
`tration [Ll-1- is conserved, we may write that
`
`EEl + L z LE‘J
`
`and
`
`[LlT = [L15 + [w]; E [L]
`
`(9)
`
`EH“ + L : LEH”
`
`(16)
`
`(17)
`
`There are several ways to evaluate from Equations 1—6 a
`combination of the equilibrium constants which govern the
`chromatographic process.
`If we assume that the eluite is
`bound by the stationary phase as its complex with the he-
`taeron, which is formed in the mobile phase, then the
`combination of Equations 1—4 and 7—9 yields the following
`expression for the capacity factor
`
`k = ¢[L](K1+ K2K4lHllftl + K2[H]){1 + K3[H])
`(10}
`
`2296 I ANALYTICAL CHEMISTRY, VOL. 49, N0. 14, DECEMBER 19??
`
`The equilibrium constants corresponding to Equations 16 and
`17 are denoted by K10 and K1, respectively.
`In this case. however, only the protonated eluite molecules
`can form an ion-pair [HEH'] with the hetaeron—counterion and
`bind as a complex, [LHEH], to the stationary phase. Con-
`sequently, mass balance yields the following expression for
`the capacity factor
`
`
`[LHEH], + [LEL + [LEHWS
`lit-ti
`
`[Elm + [EH+]m + [HEHlm
`
`(18)
`
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`

`Following the previous approach and using Equations 1-4
`and 15—17 to substitute the equilibrium constants into
`Equation 18. we obtain for the capacity factor the expression
`
`Xi‘Ka
`
`_ _
`(K. + [H1 + KzKaHn
`k _ “up + K. + KalHDfl + KaiHJ)
`
`[H1
`
`(19)
`
`Equation 19 can also be written in a form similar to that of
`Equation 13, but in this case the magnitude of the parameters
`would also be dependent on the acid dissociation constant of
`the eluite and the hydrogen ion concentration in the mobile
`phase.
`Enhancement Factor. Experimental data show that in
`ion-pair reversed phase chromatography the dependence of
`the capacity factor on the detergent concentration often
`follows hyperbolic behavior (6} such as represented by
`Equation 14. According to this expression the two limiting
`values of the capacity factor are kg and BKP at zero and at
`sufficiently high hetaeron concentrations, respectively. The
`ratio of the two quantities gives the highest possible am-
`plification of the capacity factor due to the presence of the
`hetaeron. It is termed the enhancement. factor, a, and given
`by
`
`I? = 3/380?
`
`(20)
`
`where he is the capacity factor of an eluite in the absence of
`hetaeron, P is either the stability constant of the eluite—
`hetaeron complex or the equilibrium constant for the binding
`of the hetaeron to the stationary phase and the physical
`meaning of B also depends on the particular mechanism which
`governs eluite retention in the presence of the hetaeron. We
`shall see later how Ti can be used for both the elucidation of
`mechanism and the practical selection of a hetaeron.
`Mechanistic Implications of the Solvophobic Theory.
`In view of the preceding section, on a closer examination of
`the process, the retention of the eluite in ion-pair chroma-
`tography on nonpolar bonded phases can occur either by
`“dynamic ion-exchange“, i.e., ion-pair formation takes place
`between the eluite and the hetaeron bound to the stationary
`phase, or by ion-pair formation in the mobile phase and
`binding of the complex to the nonpolar stationary phase.
`Since a phenomenological approach cannot distinguish be-
`tween the two cases on the basis of the dependence of the
`capacity factor on the hetaeron concentration, we shall use
`the solvophobic theory to estimate the relative magnitude of
`the equilibrium constants on the basis of the molecular
`properties of the hetaeron and eluites.
`The solvophobic theory was developed to describe the effect
`of solvent on chemical phenomena (9) and has successfully
`accounted for inter alia, the solubility of small nonelectrolytes
`in water and other solvents (i2) and the effect of solvent
`variation on reaction rates for several different chemical
`reactions (13). It is not restricted to water as the solvent and
`expresses the energetics of the solvent effect in terms of, at
`least in principle, measurable properties of the solute and
`solvent, unlike other theories for the hydrophobic effect.
`We have recently adapted this approach to quantitatively
`treat the effect of eluite and eluent properties on chroma-
`tographic retention using polar solvents, especially water, and
`a nonpolar stationary phase (2, 10). In our model, we assumed
`that the chromatographic process entails a reversible asso-
`ciation of a solute, S, with the hydrocarbonaceous ligand, L,
`of the stationary phase to form a complex SL. The logarithm
`of the corresponding equilibrium constant for a nonionized
`solute, K10, was expressed for fixed column and eluent
`properties at a given temperature by Equation 47 in Ref. (10}
`
`which for our purpose can be written in a simplified form as
`
`1nK,”%a—b+cAA
`
`{21}
`
`where a, b. and c can be regarded as constants dependent upon
`solvent and column properties: (7 also depends on the eluite
`properties such as dipole moment, polarizability and molecular
`volume. AA is the difference between the molecular surface
`
`area of the complex, Ast, and those of the eluite, A3, and the
`hydrocarbonaceous ligand, AL, so that
`
`AAZASL—AS—AL
`
`(22)
`
`For the capacity factor of an ionized solute, the following
`simplified expression can be derived from Equation 20g of Ref.
`(2)
`
`ln K12: o' + b' flZ} + c’AA
`
`[23)
`
`where a”, b’, and c’are again solvent and column dependent
`parameters. The effect of charge on the solute molecule is
`represented by the function 1'(Z) which goes approximately
`as the absolute value of the product of charges on the ion and
`its counterion.
`
`The simplified expressions in Equations 21 and 23 allow
`us to make some qualitative and semiquantitative statements
`regarding the constants lumped together in the enhancement
`factor as far as the mechanism of ion-pair reversed-phase
`chromatography is concerned.
`It is recalled that the pa-
`rameters k0, B, and P in Equation 20 are directly related to
`the equilibrium constants defined in Equations 1-5.
`As an is the capacity factor of the eluite in the absence of
`hetaeron, in view of Equations 1, 10, and 14 we may write that
`
`11‘! ko= inlelLlKil = 0' + b' M) + 9AA
`
`{24)
`
`Obviously the value of Jet. is independent of any kind of
`ion-pair formation. 0n the other hand, the meaning of the
`constants B and P in Equation 20 is dependent on the actual
`mechanism of the chromatographic process.
`The energy of any electrostatic interaction and hence the
`logarithm of the corresponding equilibrium constant depends
`upon the product of the charges on the interacting species.
`Thus, for the ion-pair formation in the mobile phase the
`equilibrium constant, K2, in Equation 2 can be expressed by
`
`in K: = f'(ZEZH} + const.
`
`(25)
`
`where ZE and Za are the charges on the eluite and hetaeron,
`respectively. Similarly, in the case of dynamic ion-exchange,
`represented by Equation 5, the equilibrium constant, K5, for
`the interaction between the eluite and the hetaeron bound
`
`to the stationary phase can be expressed by
`
`In K5 = f”(ZEZH) + const.
`
`(26)
`
`The other equilibrium constants of interest, K3 and K4,
`correspond to the binding of the hetaeron and the complex
`to the stationary phase ligands, as shown by Equations 3 and
`4, respectively. According to the solvophobic theory, both
`equilibrium constants can be expressed as a function of the
`decrease in the molecular surface area upon binding of the
`species to the stationary phase. Thus, by using Equation 23,
`we can write for the equilibrium constant representing the
`binding of the charged hetaeron, that
`
`In K3 = a’ + b' f(ZH) + c’AA,
`
`(27)
`
`On the other hand, the equilibrium constant for the binding
`of the neutral ion-pair can be expressed from Equation 21 as
`
`In K4: [1 _ b + CAAq
`
`(28)
`
`CUREVAC EX2030
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`
`

`

`
`
`
`
`Table II. Relationship between Eluite Properties and the
`Parameters of Equation 14 as Predicted for the Two
`Limiting Mechanisms with Ion-Pair Formation
`
`Parameter
`
`mobile phase
`
`stationary phase
`
`Ion—pair formation occurs in
`
`Table I. Relationship between Hetaeron Properties and
`the Parameters of Equation 14 as Predicted for the Two
`Limiting Mechanisms in Ion-Pair
`Reversed-Phase Chromatography
`
`Parameter
`it
`BD
`
`P
`
`Ion-pair formation occurs in the
`
`mobile phase
`
`.
`.
`.
`hydrophobic surface
`area
`(carbon number)
`charge type
`(P 2 K3}
`
`stationary phase
`.
`.
`.
`hydrophobic surface
`area
`(carbon number}
`hydrophobic surface
`{carbon number)
`area
`
`1— charge type
`(P = K.)
`charge type
`hydrophobic surface
`BikoP
`area
`(Carbon number)
`
`According to the previous discussion the meanings of 3A,,
`and AA, in Equations 27 and 28 are different and given by
`
`AA3=AHL_AH_AL
`
`and
`
`AquAHEL-AL—AHE
`
`(29)
`
`(30}
`
`where A,- is the molecular surface area of the species i denoted
`by the subscripts.
`We can express the enhancement factor, n, by two different
`combinations of the equilibrium constants. In the first case,
`assuming dynamic ion exchange we obtain from Equations
`11, 14, 20, 24, and 26 that
`
`in n = ln(K51K,)= const. +
`
`f"(ZEZH) — f(zE) _ C(ALEH “ AEL — ALE) {31)
`
`In the second case, when ion-pair formation in the mobile
`phase dominates, the enhancement factor can be expressed
`by using a similar combination of the pertinent equations as
`
`In n = ln (3'4le] = const. -
`
`leE) + CiAHEL “ AHE ‘l' AB ‘ ALE)
`
`{32)
`
`From the results presented earlier [2) we know that
`
`AEL_AE_AL°‘AE
`
`(33)
`
`Since this relationship is expected to hold for all species under
`investigation we can write for the last term in Equation 32
`that
`
`AHEL _ AHE + As _ ALE ‘1 (ARE _ As)
`
`(34)
`
`In other words the last term in Equation 32 depends upon
`the difference in the surface area of the complex and eluite.
`If the complex is formed to maximize the electrostatic effect,
`this difference is very nearly the surface area of the hetaeron,
`AH, alone. Hence, if the retention proceeds primarily by the
`formation of ion-pairs in the mobile phase, the enhancement
`factor will depend upon the surface area of the hetaeron as
`
`log n 0‘ AH
`
`(35)
`
`Consequently, in the case of normal alkyl sulfates or sulfonates
`log :7 is expected to be proportional to the carbon number of
`the hetaeron.
`On the other hand, no such a dependence is expected in
`the “ion-exchange“ mechanism. Indeed, the relevant hetaeron
`property, as seen from Equation 30, is a function of its charge,
`flZHZE), only. In view of these relationships, the analysis of
`
`2293 O ANALYTICAL CHEMISTRY, VOL. 49. NO. 14. DECEMBER 1977
`
`kn
`
`.8
`
`P
`BikflP
`
`charge and
`hydrophobic
`surface area
`charge and
`hydrophobic
`surface area
`...
`charge and
`hydrophobic
`surface area
`
`charge and
`hydrophobic
`surface area
`charge and
`hydrophobic
`surface area
`charge (P: K2)
`charge
`
`experimental data obtained with hetaerons containing the
`same ionic groups but different alkyl chain lengths can shed
`light on the actual mechanism of the process. The dependence
`of the enhancement factor on the properties of the hetaeron
`and eluite is shown in Tables I and II, which summarize the
`conclusions of this approach.
`EXPERIMENTAL
`
`A Model 501 (Perkin-Elmer, Norwalk, Conn.) high pressure
`liquid chromatograph was used with a Model 7010 sampling valve
`{Rheodyne. Berkeley, Calif), a Model FS 770 (Schoeffel,
`Westwood. N.J.) variable wavelength detector at 254 nm, and a
`Perkin-Elmer Model R-56 recorder were used. Partisil 1025 ODS
`(Whatman. Clifton, NJ.) columns packed with 10-pm octadec-
`yl—silica containing about 5% lwfw) carbon were used in the study
`of hetaeron behavior. The chromatogram in Figure 2 was obtained
`with a LiChrosorb RP-18 column (Rainin, Boston, Mass.) packed
`with 5mm octadecyl-silica. All columns were 25GI mm long and
`had 6.4 mm o.d. and 4.6 mm id. Most experiments were carried
`out by isocratic elution using neat aqueous 5 x 10'2 M phosphate
`buffer, pH 2.5, and the hetaerons were also dissolved in this buffer.
`In most cases the flow rate and the column temperature were 2
`mL 1 min and 40 °C, respectively. Some experiments were carried
`out with hetaerons dissolved in mixtures of methanol and the
`above mentioned phosphate buffer as the eluent.
`Catecholamine derivatives were obtained from Aldrich
`(Milwaukee, Wis.) or Schwartszann {Orangeburg, NY.) and
`reagent grade H3P04 and KH2P04 were supplied by Fisher
`(Pittsburgh, Pa). The alkyl sulfates and hexylsulfonate used were
`Eastman products (Rochester, N.Y.), whereas the alkyl phosphates
`were gifts from Hooker Chemicals Corp. (Buffalo, N.Y.).
`rI‘he
`perfluorated carboxylic acids were purchased from Aldrich.
`Methanol was “distilled in glass" from Burdick and Jackson
`(Muskegon, Mich).
`Retention times were measured from the distance between the
`injection point and the peak maxima on the chromatogram. The
`mobile phase hold-up times were measured as described previously
`(2) and the capacity factors have been calculated in the usual way
`(14).
`The analysis of the data was performed on a PDP 1] f 10
`minicomputer equipped with a RXOI floppy disc, a VT55 display
`unit, and a Decwriter. The computer program used for parameter
`estimation by the least squares method was written in BASIC
`language.
`The symbols used in this study for the sample components are
`as follows: DOPA, 3,4-dihydroxyphenylethylarnine (dopamine);
`EP, 1-[3,4-dihydroxyphenyl)-2-(methylamino]ethanol
`(epi—
`nephrine, adrenaline); OP. 1-(4-hydroxyphenyll-2-aminoethane
`{octopamine); NE, 2-amino—l—[3,4—dihydroxyphenyl]ethanol
`[norepinephrine, noradrenaline); DOS, 3.4-dihydroxyphenylserine.
`RESULTS AND DISCUSSION
`
`A typical chromatogram in Figure 2 illustrates the sepa-
`ration of certain catecholamines on octadecyl—silica in the
`absence and in the presence of n-octylsulfate in the neat
`aqueous phosphate buffer used as the eluent. As the chemical
`nature and concentration of the alkyl sulfate have great
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`cr
`
`E
`
`.
`
`l
`
`|—I—l—I_._._.|
`o
`4
`a
`12
`16
`MINUTES
`
`Flame 2. Chromatograms illustrating the effect of ion-pair formation
`with n-octylsulfate in the eluent on the separation of catecholamines
`by reversed-phase chromatography. Column. 5 am LiChrosorb RP18;
`flow rate. 2.0 mLimin; temp, r0 °G: inlet pressure. 2200 psi. Eluents:
`A. 5 X 10'2 M phosphate in water, pH 2.2; B. 5 X 10" M phosphate
`and 3 X 10‘3 M octylsulfate in water, pH 2.2
`
`
`
`
`O
`
`20
`
`4O
`
`60
`
`80
`
`I00
`
`BUTYLSULFATE [m M]
`Figure 3. Dependence of the capacity factor of protonated catechol
`amine derivatives on the concentration of n-butylsulfate in the eiuent
`Column. 10 um PartisilODS: flow rate. 2 0 ran’min: temp. 40 °C: inlet
`pressure. 400 psi; eluent. 5 X 102 M phosphate in water. pH 2. 55.
`containing various concentrations of the hetaeron
`
`influence on the retention of the eluites, experiments were
`carried out in a wide range of conditions in order to shed light
`on the chromatographic process in View of the preceding
`theoretical anaysis. In addition to alkylsulfates of different
`chain lengths, hexylsulfonate as well as butyl- and amyl-
`phosphates were also employed.
`Equation 13 predicts that the observed capacity factor will
`initially increase with increasing hetaeron concentration
`followed by a monotonic decrease at high hetaeron concen-
`trations. However, if over the experimentally accessible range
`of hetaeron concentrations, either the binding of hetaeron to
`
`
`
`O
`
`20
`4O
`HEXYLSULFATE [mm]
`
`60
`
`
`
`
`
`
`Figure 4. Dependence of the capacity iactor of charged catecholamine
`derivatives on the concentration of n-hexvlsultete In the neat aqueous
`mobile phase. Conditions are given in Figure 3
`
`0
`
`2O
`4O
`60
`OCTYLSULFATE [m M]
`Figure 5. Dependence of the capacity factor of charged catechalamlne
`derivatives on the concentration ol‘ n-octylsultate in the neat aqueous
`mobile phase. Conditions are given In Figure 3
`
`the stationary phase, Kng], or the extent of ion-pair formation
`in the mobile phase, K2W], is negligible. the capacity factor
`can be expressed by Equation 14. In this case the capacity
`factor first rises and eventually becomes practically inde-
`pendent of the hetaeron concentration. Therefore, if Equation
`14 holds over the experimental range of hetaeron concen-
`tration, a plot of h vs. [H] yields a rectangular hyperbole.
`The capacity factor of four catecholamines and the amino
`acid, 3,4—dihydroxyphenylserine, as a function of the con-
`centration of various n-alkyl sulfates in the eluent is shown
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`
`
`
`
`
`
`DECYLSULFATE [mM]
`Figure 8. Dependence of the capacity factor of charged catecholamine
`derivatives on the concentration 01' n-decylsuliate in the neat aqueous
`mobile phase. Conditions are given in Figure 3
`
`in Figures 3-6. The alkyl chain of the hetaerons ranges from
`butyl to decyl groups and the upper limit of the concentration
`range was usually determined by the solubility of the hetaeron
`in the neat aqueous eluent.
`Inspection of the data shows that with butyl-, hexy -, and
`decylsulfates the capacity factor in most cases rises with
`increasing hetaeron concentration to a constant value from
`which it does not decline significantly. 0n the other hand
`when decylsulfate is used, the capacity factor increases to a
`maximum from which it rapidly decreases with further in-
`crease in the hetaeron concentration. Thus, the qualitative
`predictions are supported by the data as is also illustrated by
`the dependence of the capacity factor of adrenaline on the
`concentration of the hetaerons in Figure 7.
`In order to test the validity of Equations 13 and 14. the data
`shown in Figures 3—6 were analyzed by a least-square fit. The
`data obtained using butyl-, hexyl-, and octylsulfates did not
`converge by using Equation 13 but they did fit Equation 14.
`On the other hand, the data obtained using decylsulfate could
`be well fitted to Equation 13, whereas the fit to Equation 14,
`was very poor.
`The quality of the fit can be illustrated by a generalized
`plot of the data. Equation 14 is divided by kn, the expression
`can be rearranged to obtain
`
`(Miran) — [1/nil + PiHlll = P[Hlf(1 + PIHD (36)
`
`The RHS of Equation 36 represents a normalized capacity
`factor corrected for the effect of unconjugated eluite binding.
`When it is plotted against. the normalized hetaeron con-
`centration. P[H], a rectangular hyperbole should be obtained.
`Such a plot of data obtained with butylsulfate, hexylsulfate,
`and decylsulfate as hetaerons and DOPA as the eluite is shown
`in Figure 8.
`In the case of other acidic hetaerons such as
`
`2300 I ANALYTICAL CHEMISTRY. VOL. 49. NO. 14, DECEMBER 1977
`
`'5'
`
`DECYL
`
`
`
`CQPRCITYFACTOR
`
`O
`
`20
`
`4O
`
`60
`
`ALKYLSULFATE [mM]
`Figure 'i'. Plots ol' the capacity tector oi adrenaline vs. the hetaeron
`concentration for various ri-alkylsufiates. Conditions are given in Figure
`3
`
`
`
` 0 DECYLSULFATE
`
`
`l0
`
`0.2
`
`i
`
`0
`
`K HEXYLSULFATE
`' BUTYLSULFATE
`
`Figure 3. Normalized plot of the capacity factor data obtained for
`dopamine using three difierent n-alkyl sulfates as the hetaerons. The
`theoretical curve calculated from Equation 41 Is given by the solid line.
`The data were obtained under conditions described in Figure 3
`
`hexylsulfonate, butyl- and amylphosphates in neat aqueous
`eluents, the relationship between the capacity factors mea—
`sured with these eluites and the hetaeron concentration was
`found to conform well to Equation 13.
`We noted in the theoretical section that the dependence
`of the capacity factor on the hetaeron concentration alone does
`not shed light on the actual mechanism of the process.
`However, as one changes from one hetaeron to another. both
`representing the same type of compounds, predictions of the
`effect on the capacity factor can be made by recourse to
`solvophobic theory as shown in Tables I and II. If ion-pair
`formation occurs in the mobile phase, the enhancement factor
`will depend strongly on the hydrophobic area of the hetaeron
`CU REVAC EX2030
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`I00—
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`50-—
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`I
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`ru 0
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`FACTOR.7} 5 r
`
`ENHANCEMENT
`
`
`
`4
`
`6
`
`3
`
`IO
`
`CARBON NUMBER OF THE HETrf-lEROlNlJ'il|=
`
`Figure 9. The dependence of the enhancement factor of catecholamine
`derivatives on the carbon number of n-alkyl sulfates as the hetaerons.
`The straight line obtained by least squares analysis fits the expression,
`log n = 0.225 ($0.031?) NC, where n is the enhancement factor and
`N6 is the carbon number. The intercept is zero within experimental
`error
`
`and the charge of eluite but. it. will be independent of the size
`of the eluite. 0n the other hand, if ion—pair formation occurs
`on the stationary phase, i.e., in the case of the ion-exchange
`mechanism, the enhancement factor will depend on the charge
`of the hetaeron and the charge and size of the eluite.
`The predictions based on the solvophobic treatment of
`chromatographic retention support the concept that in our
`experiments soap chromatography proceeds through the
`formation of ion—pairs in the mobile phase followed by ad—
`sorption onto the nonpolar stationary phase. The logarithm
`of the enhancement factor evaluated with the least-squares
`parameters of Equation 14 is linear in the carbon number of
`the hetaeron and largely independent of the size of the eluite
`as illustrated in Figure 9. This is predicted for the ion-pairing
`mechanism according to Equation 35, whereas no dependence
`on the hetaeron size but a dependence on the molecular area
`of the eluite is expected for the ion-exchange mechanism. The
`constant P of Equation 14 should be equal to K2 and inde-
`pendent of hetaeron size in the ion-pair mechanism but will
`depend upon the charge of both eluite and hetaeron. It will
`also be dependent upon the charge type since the distance
`of closest approach of the two ions is implicitly included in
`the function HZEZH} in Equation 25 and 26.
`The mean values of the pertinent parameters as obtained
`by least squares analysis are shown in Table III. The standard
`deviation of the values is also indicated. The capacity factor
`of the eluite in the absence of hetaeron, k0, is not shown
`because it is not a property of the hetaeron. It is seen from
`the data in Table III that the enhancement factors are fairly
`independent of the eluite, but they strongly depend and, in
`fact, increase exponentially with the carbon number of the
`hetaeron in agreement with the data shown in Figure 9. The
`only exception to this rule is