Report
Liquid Chromatography as a Measurement Tool for Chiral Interactions
C is well-known as an excellent method for separating and analyzing complex mixtures. However, cleverly designed studies have demonstrated the added breadth of this technique by utilizing LC as a tool for fundamentally measuring interaction chemistry. This paradigm shift results from recognizing that a significant amount of quantitative and qualitative information is contained in a separation. That is, the same interactions that produce a separation in the spatial domain can be measured using that spatial resolution. Many of these measurement studies began with the goal of characterizing the phvsicochemical interactions that drive the senaration process It was soon discovered however that LC can be directly tailored to interaction measurements of practical and nearly five decades since the advent of L C broad
Moira C. Ringo Christine E. Evans University of Michigan
Thermodynamic measurements using LC demonstrate the breadthofthis technique. scope of physicochemical measurements has been realized, including the determination of binding constants, partition coefficients, and diffusional parameters as well as interaction and reaction kinetics (1-3). Indeed, LC inherently incorporates a wide range of chemical interactions, including those involving electrostatic, dipole, and hydrophobic forces. This commonly used separation technique appears to be particularly well suited to the measurement of interaction processes. This measurement perspective is clearly not limited to LC, and all separation techniques may be envisioned as measurement tools (1-4) In this Report we
will focus on thermodynamic measurements of chiral recognition using LC and endeavor to highlight the breadth of this technique as a measurement method. Enantiomers have identical chemical and physical properties in an isotropic environment; however, they often exhibit significant differences in interactions with other chiral species. These enantioselective interactions form the cornerstone for many processes of biological and technological importance and have profound implications for pharmacology, molecular biology, and bioengineering. In many cases, however, the absolute energetics of these interactions are quite small; and the enantiomeric differences of importance are even smaller. Separations methods based on weak interactions are uniquely well suited to the difficult measurement of such important interactions In the following measurements, an enantioselective molecule (selector) is either immobilized on a surface support as a stationary phase or present as an additive to the mobile phase. In all cases, chromatographic figures of merit are determined for these
Analytical Chemistry News & Features, May 1, 1998 315 A
Report
Table 1 . Thermodynamic interaction parameters determined by HPLC. HPLC measurement
Thermodynamic parameter Equilibrium constant for complexation, Kcomp
Chiral selectivity ct Complexation stoichiometry
«
Change in enthalpy upon complexation, AH comp
Change in entropy upon complexation, AS oomp 3
'""•"••ro up
cvi
p e
at
u
, '-* V c o m p
chiral separations and then related to the thermodynamic parameters governing these enantioselective interactions. It is important to note, however, that the measurement regime may extend beyond the realm of separations commonly considered. In fact, the very forces that act to degrade a separation may be of considerable interest in a measurement context Even so, most of the studies highlighted here are within the conventional analytical separation regime, which already provides a rich range of interaction chemistries. Although not exhaustive, the studies cited will demonstrate the broad of measurements that possible using LC and will serve to highllght this exciting new direction in separation science The correlation of separation parameters to the energetics of solution-phase interactions ii possible because LC retention nrocesses are well described by equilibrium thermodynamics (5). Each solute zone proceeds through the column at a rate controlled by competing interactions of the solute with the stationary phase and the solute with the mobile phase. This retention process results in an increase in the solute migration time (£D) relative to the movement to a nonretained species (L) and is often described with the solute capacity factor (k), which is equal to (t -t^/t (A glossary is available on p 321 A.) Allhough mobilephase flow creates an inherently nonequilibrium condition LC retention has been shown to be well modeler] as an equilibrium process This correlation arises because solute retention \< a s ^ ^ e d from movement at tVif» r*pnrpt* ofrfie ynne prrvfilp WTIPTV* nnn
equilibrium effects from mobile-phase are minimized As a result capacitv factor 316 A
Solute capacity factor, k Phase ratio, Solute capacity factor k Solute capacity factor k Solute peak area Temperature dependence of solute capacity factor, k(T) Temperature dependence of so u e capaci y a , l ) snli]tpUranaritv fartor ofP\
measurements in aflowingHPLC system may be directly correlated to the equilibrium thermodynamics of solute interactions with the stationary phase and mobile phase (5). As shown in Table 1, a conssderable range of thermodynamic figures of merit is accessible using LC measurement.
Determining complexation constants Chromatographic studies may be used to advantage in assessing many types of interactions, including enantioselective complexation. When a chiral selector is used as the stationary phase, the primary retention mechanism is complexation with the surface-immobilized chiral selector (Figure la). For 1:1 solute-selector complexation, the equilibrium complexation constant (K ) is related to k by K
[solute-selector] comp [solute][selector]
k ^[selector]
in which [solute], [selector], and [soluteselector] represent the equilibrium concentrations of free solute, free selector, and solute-selector complex, respectively, and § is the volumetric ratio of the stationary and mobile phases. In this measurement regime, the concentration of the chiral selector is always significantly greater than the solute species of interest. Accordingly, K may be determined directly if elector]
m
k k0 + ««Acomp [selector] the enantiomer-selector complexation acts to mediate solute retention; and as a result, Kcomp can be determined by varying tJie equilibrium concentration of the chiral selector (8-12). It is important to note that this formalism assumes that the selector and the stationary phase act independently. Moreover, enantioselective and nonspecific interactions may contribute to the overall retention and directly affect the binding measurement. Nonetheless, measuring complexation constants within the mobile phase has the distinct advantage of simultaneous, multisolute determinations that do not require prior sample purification. When chiral solutes need to be screened this measurement advantage makes the fundamental evaluation of broad classes of interactions truly feasible These measurement advantages have been demonstrated for several structurally related barbiturates with fkyclodextrin (9). In this investigation, the addition of p-cyclodextrin to the mobile phase resulted
in a capacity factor decrease for all solutes caused by complex formation in the mobile phase. By varying the selector concentration, complexation constants for each solute were determined based on the observed capacity factor (Eq. 2). These measurements highlight the unique sensitivity of LC methods, with Kcomp values of < 200 M _1 determined for all lolutes. Even for these modest binding constants, excellent measurement precision of < 5% was sbtained for the esmultaneous Kcomp determinations of methylphenobarbital and hexobarbital enantiomers (9). Concurrent with these measurements, evaluation of k„ indicates that the barbiturateB-cyclodextrin complexes examined in this study have negligible interaction with the C18 stationary phase. This result confirms that enantioselective complexation in the mobile phase is the primary mechanism for chiral interactions of these barbiturates
be a precise and highly adaptable tool for measuring interaction selectivity. Chiral selectivity (a) is an important figure of merit for enantioselective complexation, allowing the direct comparison of the equilibrium complexation constants for each enantiomer with the chiral selector, and is expressed by If
_. _
comp,enan2 comp.enanl
[enanuomer2-selectorlenantiimerlj l>nanriomer1 •selector¥enantiomer21 The ratio of enantiomeric distributions in Eq. 3 can also be directly related to the enantiomeric difference in the change in Gibbs free energy upon complexation (A(AGcomp)) by A(AGc
) = (AGC
(^comp)enana
Selectivity determinations
In addition to complexation constant measurements, LC has been demonstrated to
=
)enan2 -
~RTln a
(4)
This relationship is a key component in discerning the important differences in interaction energy between enantiomers.
Figure 1 . Schematic representation of solute-selector interactions. (a) Using the selector as the stationary phase and (b) using the selector present in the mobile phase and an achiral stationary phase.
Although enantiomers have nearly identical physical and chemical properties, small differences in their interaction energetics with a selector often have a significant impact on molecular-recognition processes ranging from pharmaceutical action to immune response. LC can readily measure solute-selective interactions as low as 1% (a = 1.01)) corresponding to enanttomeric differences in complexation energy changes (A(AG _))of only20J/mol. In the stationary-phase approach, interaction selectivity is assessed by enantiomeric interactions with a surface-bound selector (Figure la). Assuming 1:1 enantiomer-selector complexation, chiral selectivity is directly related to the ratio of enantiomeric capacity factors, a = k2/kl, where k2 > kx. Although LC investigattons often focus on the practical achievement of high-resolution chiral separations, measuring interaction selectivity provides fundamental as well as pragmatic information about enantioselective complexation. The enantioselectivity of an array of chiral selectors has been determined with these equations, including polysaccharides (13,14), proteins (15-18), and crown ethers (19). Although a > 100 has been measured for the interaction of amino acid derivatives with a brush-type stationary phase derived from N- (2-naphthyl) alanine (20), chiral selectivity measurements using this stationary-phase approach more typically range from 1 to 2, which represents a modest A(AG J of < 2 kj/mol. Although the possible impact of selector immobilization must be considered this technique provides a relatively rapid and sensitive means of measuring the selectivity of enantiomeric interactions Selectivity measurements may also be accomplished by incorporating the selector into the mobile phase. In contrast with stationary-phase determinations, however, the interaction selectivity cannot be determined directly as the ratio of solute capacity factors. Because solute retention in this case arises from interaction with the stationary phase as well as a chiral selector in the mobile phase, the selectivity is determined by evaluating A for each enantiomer. This methodology has all of the advantages of liquid-phase measurements mentioned previously but lead to increased imprecision
Analytical Chemistry News & Features, May 1, 1998 3 1 7 A
Report caused by propagation of error in Eq. 2. Nevertheless, stationary- and mobile-phase methods allow the simultaneous measurement of multiple solutes in a nonpurified sample. The power of these methods for determining the selectivity of enantiomeric interactions is perhaps best illustrated by bioanalytical studies of drug-protein interactions. Using a human serum albumin-based stationary phase, chiral selectivity was measured for a series of nonsteroidal antiinflammatory drugs. All the drugs tested showed a high degree of chiral selectivity with human serum albumin, ranging from 1.12 to 3.51 (21). It is interesting to note that the drug that showed the highest degree of chromatographic chiral selectivity with human serum albumin ibuprofen also exhibits a high degree of chiral selectivity in its pharmacological action in humans In another studv enantiomeric interactions with stationary phases of rat- rabbit- and human serum albumin were compared to assess interspeenantioseWtivity taken with a chiral se\ocif\r
t h of r\la\ro a mai/M- rrAc in a n i m a l r\nwei
oloov provide not only
In addition, because the protein is immobilized, a single protein fraction can be used for many measurements, thus eliminating repetitive extractions as well as batch-tobatch inconsistencies. As demonstrated here for common anti-inflammatory drugs, the speed with which large families of drugs can be screened for enantioselective interactions makes LC a powerful method for determining enantiomeric selectivity. Determining complexation stoichiometry It is often presumed that the interactions of interest arise from 1:1 association. Although many solute-selector complexes demonstrate this stoichiometry exclusively, enantioselective complexes may exhibit multiple (e.g., 2:1) or mixed (e.g., 1:1 and 2:1) stoichiometries (12,22-24,, as illustrated in Figure 2. Complexation stoichiometry is an inherent component of the equilibrium complexation constant, and for the general case of m:n solute-selector, complexation is expressed as [solutem-selectorn] comp [soiU);e]m [selector]" As a result, enantiomeric differences in • complexation stoichiometry or the distribution of stoichiometries can have a significant impact on interaction selectivity and all other important figures of merit that arise from Kcomp. One common approach to determining stoichiometry with LC parallels the mobilephase additive technique for determination ofKco . For the general case of m:n stoichiometry, adding selector to the mobile phase induces a change in the solute capacity factor, which is directly related to stoichiometry, K ,k0, and £„, expressed as
consistent with measurement conditions of interest Nonetheless L C has many advan
With this expression, the relationship between the equilibrium selector concentration and 1/k indicates the predominant stoichiometry of the solute-selector complex in the concentration range of the experiment (12,23,25.. This method of assessing stoichiometry is advantageous in that it can be accomplished within the same experiment as the K. and selectiv-
of a pure protein. Serum impurities present when the stationary phase is synthesized are , ,
,.,
,
„
j
Li-
removed by mobile-phase flow and are thereJ:
r
L
•
1
L-
-L.
i
i
,
.
i M .
p
f
x
c
.
, q auquot of mis solute-containi g mobi e ^Jiiaov., (Uiu d o a r L O U I I , a ^J\JL u u i i L/I o u i u i ^ ill
UJJU11 l i l J C d l U l l , U1C SUIUIC SCltAAUI CUlIipiCA
solute. i ne resuiung chromdiogram has a positive peak ttiat represents the solute-selector complex, as well as a negative peak that corresponQS to the decrease in freesolute concentration caused by complexation. i ne area of either of these peaks is directly proportional to the equilibrium concentration of the complex. Stoichiometry is determined based on the change in the equilibrium concentration of the complex when the selector concentration is varied. For example, decreasing the injected concentration of selector by half will yield a corresponding decrease in the peak area by half for a 1:1 complex or approximately one-fourth for a 1:2 complex. As a result, this method provides a relatively unambiguous approach to
1 1 + mKc [selector]" [solute]"1 k k0 + kjmKc [selector]"[solute]"1
fore not a factor in selectivity measurements. 318 A
,
solute-selector complexes that exhibit stoichiometry that can be readily discerned from differences in statistical correlations. The Hummel-Dreyer method provides an interesting analogue to the mobile-phase additive technique. In this stoichiometric determination, the column is equilibrated I n
son but also rapid and effective drug screening for animals and humans alike. Although these methods have significant impact for veterinary and medical applications, the direct comparison of LC selectivity measurements to chiral selectivity in biological systems is often limited by mobile-phase composition. Although the mobile phases used in the studies cited above were buffered to a pH near the physiological range, the high affinity of these drugs for HSA in purely aqueous mobile phases requires adding 6-15% (v/v) organic solvent to achieve reasonable retention times (17 21) This necessity reveals a potentially significant limitation of LC measurement techniques That is the mobile-phase conditions necessary to achieve separation are not always
over more traditional bioanalytical methods such as ennilibriiim dialysis ltr filtratinn wHrh
ity determinations. However, it is limited to
Analytical Chemistry News & Features, May 1, 1998
Figure 2. Illustration of solute* selector stoichiometries.
stoichiometric determination, especially when the availability of selector is limited (22,24). The effectiveness of these techniques is demonstrated in the determination of stoichiometric coefficients for the complexation of chiral terpenes with a- and p-cyclodextrin (12). Using the mobile-phase additive method described previously, enantiomeric capacity factors are measured as a function of selector concentration in the mobile phase. Comparison of terpene complexation with a- and |3-cyclodextrin indicates significant differences in complexation stoichiometry. All terpene-B-cyclodextrin complexes exhibit 1:1 stoichiometry whereas several terpene-a-cyclodextrin complexes clearly indicate 1:2 stoichiometrv Moreover chiral selectivity is observed for the 1:2 complexes onlv This interesting result indicates that enantiomeric differences in the 1:2 complexmajor role in the enantioselectivity of terpene with cc-cvclodextrin (12) Unlike dialysis and filtration methods for assessing complexation, the LC method does not require large differences in size between the solute and selector, which makes it a powerful measurement technique for assessing the complexation interactions of small solutes with small molecules such as cyclodextrins. Moreover, the LC method utilizes interactions to directly assess stoichiometry and, therefore, requires no spectral perturbation to detect these interactions. Determining complexation enthalpy and entropy
Studies have demonstrated the power of the HPLC technique for determining enantiomeric differences in the change in enthalpy [(A(A#comp) = (A// comp )) nan2 (A#comp)en!ml)] and entropy [(A(AScomp) = plexation (11,19,20,26,27.. Although chiral selectivity and A(AG m ) represent an important overall view of enantioselective energetics, a detailed thermodynamic profile requires that the individual roles of enthalpy and entropy be investigated. Whereas A(AH ) assesses enantiomeric differences in the number and strength of interactions with the selector entropic effects from the reordering of solute selec-
ance between these contributions that leads to enantioselectivity. Using this measurement technique, these differences in complexation enthalpy and entropy are also shown to be sensitive to changes in pH and ionic strength. Indeed, variations in pH result in comparable changes for the enthalpic and the entropic components of chiral selectivity. Thus, although pH significantiy affects enantiomeric complexation constants, no enhancement in enantioselective interactions is observed because of enthalpy-entropy , , -AG , -AH AS , compensation. This result further suggests In k = + ln
