Chiral separation of dansyl amino acids by ligand exchange ...

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Electrophoresis 2008, 29, 3941–3948

Shaul Mizrahi1 Dan Rizkov1 Alexander I. Shames2 Ovadia Lev1 1

The Chemistry Institute, The Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel 2 Department of Physics, Ben Gurion University of the Negev, Beer Sheva, Israel

Received February 25, 2008 Revised March 28, 2008 Accepted March 28, 2008

Research Article

Chiral separation of dansyl amino acids by ligand exchange capillary electrochromatography in a low molecular weight organogel Chiral electroseparation is demonstrated, for the first time, by a low molecular weight organogel filled capillary. Five pairs of dansylated amino acids were separated by copper ligand exchange on a trans-(1S,2S)-1,2-bis-(dodecylamido) cyclohexane (1) gel in methanol. Low molecular weight organogels are emerging materials that form stable, fibrillar, thermoreversible and thixotropic gels without covalent bonding of their monomeric building blocks. The dependence of chiral resolution and complex formation stability on the pH*, the ratio between copper and the D-valine selector, as well as other parameters were investigated revealing trends that were unparalleled in previously reports on copper ligand exchange of dansylated amino acids. These observations were explained in view of a simple stacking model of (1) and the difference in axial ligation of the amide carbonyl backbone of the gel to the dansyl D- or L-amino acid:D-valine:copper ternary complexes. Keywords: Capillary electrochromatography / Chiral separation / Ligand exchange / Low molecular weight organogels / Supramolecular DOI 10.1002/elps.200800133

1 Introduction Capillary electrochromatography (CEC) is usually categorized into three main classes; open tubular CEC, packed CEC, and monolith (continuous bed) CEC. Supporters of monolith CEC point out the large loading of functional groups that can be incorporated in polymeric gels in relation to open capillaries while avoiding the difficulties of (silica or another) packing [1]. Our group recently introduced a new form of monolith CEC, which uses a low molecular weight organogel instead of a polymeric or sol–gel filler [2]. Organogels are formed by self-aggregation of small molecules in an organic solvent or water. When heated they form liquid solutions and gelate again upon cooling. Organogels afford versatility in that there is a large variety of organogelators with different functional groups and that they can be formed in virtually any solvent [3]. An additional advantage over polymeric gels is that most of them are reversible. This allows easy capillary formation by injecting a

Correspondence: Professor Ovadia Lev, The Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel E-mail: [email protected] Fax: 1972-2-658-6155

Abbreviations: AA’s, amino acids; Ala, alanine; EPR, electroparamagnetic resonance spectroscopy; Phe, phenylalanine; Ser, serine; Trp, tryptophan; Tyr, tyrosine

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

hot solution of the gelator into the capillary and allowing it to cool and permits removal of the gel and reuse of the capillary. Liquefaction of the gel by mechanical agitation and rapid introduction of the liquefied gel and the analytes to a mass spectrometer was demonstrated. In our previous work we separated a set of dansyl amino acids in an acetonitrile gel of trans-(1S,2S)-1,2-bis-(dodecylamido) cyclohexane (1). The gel is composed of helical twisted ribbons stabilized by amide hydrogen bonding and hydrophobic interactions of the radially oriented alkyl chains. This particular organic gelator was originally introduced and studied by Hanabusa et al. [4] In this paper we describe chiral separations using this interesting matrix. The ligand exchange principle has been used widely as a chiral separation mechanism, first in HPLC [5] and later in capillary electrophoresis (CE) [6]. In this method, a chiral metal (usually Cu21) complex is attached to a stationary phase or added to the run buffer. Resolution between the enantiomers is caused by the difference in their complex formation constants. Since ligand exchange was first applied to CE by Gassmann et al. [7] for separating dansyl amino acids (AA’s), it has been used in virtually all CE techniques. Modified [8] and unmodified amino acids [9], hydroxy acids [10] and pharmaceutical compounds [11] have all been enantiomerically resolved by ligand exchange capillary zone electrophoresis. The same group that opened the door to ligand exchange capillary zone electrophoresis also applied ligand exchange to micellar electrokinetic chromatography, using a pseudo-stationary www.electrophoresis-journal.com

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surfactant phase to improve resolution of the dansyl AA’s [12]. Unmodified AA’s [13] and hydroxy acids [14] were also separated by this method. Amino acids have also been separated using non-aqueous CE [15]. Despite their commercial importance there are very few reports on stationary phases with immobilized ligand exchange selectors. Chiral stationary phases were formed by copolymerization of an amino acid derivative with methacrylamide [16] or chemically bonding it to a silica column [17]. In both cases, Cu(II) is added to the running buffer to form a complex with the incorporated amino acid which serves as the chiral selector. Here we demonstrate enantiomeric separations of dansyl amino acids in a methanol organogel of (1) using a complex of copper and D-valine as the selector. This application demonstrates that a selector can be loaded along with the gel filler to form the solid stationary phase much like conventional polymeric gels derivatized with desired functional groups. Methanol was preferred to acetonitrile due to the higher stability of the methanol gel. The dependence of the separation performance on the running conditions such as pH, copper:valine ratio and buffer composition was studied. Spectroscopic methods were used to elucidate the composition of the complex and its interaction with the gel. The results reveal a novel mechanism for ligand exchange that as yet has never been observed.

2 Materials and methods 2.1 Chemicals Amino acids, dansyl-phenylalanine and cupric acetate monohydrate were purchased from Sigma-Aldrich (St. Louis, MO). Acetic acid was from Frutarom (Haifa, Israel). Ammonium acetate was from Mallinkrodt (Phillipsburg, NJ). Methanol was from J.T. Baker (Deventer, Holland). Trans-(1S,2S)-1,2-bis-(dodecylamido)cyclohexane and dansyl (dns, 5-dimethylaminonaphthalene-1-sulfonyl) amino acids were synthesized according to the procedures previously described [2]. The gels were prepared by dissolving 10 mg of the gelator in 1 mL of run buffer at about 601C and injecting by syringe into the capillary.

measured on a Cary 1E spectrophotometer operated by WinUV software. Electroparamagnetic resonance spectroscopy (EPR) spectra of true and frozen methanol solutions of 1:1.5 copper:valine and the same solution with the addition of dansyl-phenylalanine were obtained using a Bruker EMX220 X-band (n 5 9.4 GHz) EPR spectrometer equipped with an Oxford Instruments ESR 900 continuous flow helium cryostat, an ITC 503S temperature controller and an Agilent 53150A frequency counter. Spectra were recorded at T 5 295 K (room temperature, RT) and 75 K (frozen solutions) with a non-saturating incident microwave power 20 mW and a 100 KHz magnetic field modulation of 0.25 mT amplitude. Temperature dependencies of the EPR spectra for the 1:1.5 copper:valine methanol solutions (both initial and in-gel) were recorded within 298(0.1) rTr335(0.1) K range using Bruker 4121VT temperature accessories. Processing of EPR spectra, determination of spectral parameters and simulation of frozen solutions’ spectra were carried out using Bruker WIN-EPR and SimFonia software.

3 Results 3.1 Separation of dansyl-DL-amino acids We used five pairs of dansyl amino acids as a model set of separands. The separation of dansyl amino acids in a bis(dodecylamido)cyclohexane acetonitrile gel was described in a recent publication [2]. Methanol was preferred because of the greater stability of the gels. The separation in methanol at low pH obeyed a similar trend. At low pH the separation is based on molecule size, hydrophobicity and to a certain extent also on the second pKa corresponding to the protonation of the carboxylic group. A typical electropherogram taken at pH 3.5 is shown in Fig. 1. Plate numbers ranged from 10 000 to 37 000. LOD for the acids ranged from 0.04 to 1 mM. The order of electrophoretic mobilities in the anodic direction was always serine4alanine4 phenylalanine4tryptophan, tyrosine. Serine, small and 20

2.2 Instrumentation A 3D CE system with ChemstationTM software (Agilent Technologies, Santa Clara, CA) was used. Separations were performed at 30 kV with detection at 220 nm. Bare fused silica capillaries (Biotaq, MD) 60 cm long (52 cm effective length) with 100 mm ID were held at 201C. Injection was performed electrophoretically at 30 kV for 10 s. Run buffer contained D-valine, cupric acetate monohydrate and 10 mM ammonium acetate in methanol. pH was adjusted with acetic acid. Samples contained 2 mM dansyl amino acids with an enantiomeric L:D ratio of 2:1. UV spectra were & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

mAU

15

D,L-ser D,L-phe

10

L,D-ala

D-trp

5

L-trp D,L-tyr

0 18

23

28

33

38

time (min)

Figure 1. Separation of dansylated serine (ser), alanine (ala), phenylalanine (phe), tyrosine (tyr) and tryptophan (trp). Run conditions: 30 kV, 52 cm effective length. Buffer: 5 mM copper acetate, 7.5 mM D-valine, 10 mM ammonium acetate, pH 3.5 adjusted with acetic acid. Samples were 2 mM dansyl amino acids (L:D ratio of 2:1) in 1:1 water:acetonitrile. Injection was conducted electrophoretically at 30 kV for 10 s.

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8 Resolution

hydrophilic, elutes first followed by the small and somewhat more hydrophobic alanine, and then follows the larger and even more hydrophobic phenylalanine. The large tyrosine and tryptophan that have a somewhat higher second pKa than phenylalanine (approximately 3.3 for the formers compared with 3.0 for the latter) and therefore have a less significant abundance of the negatively charged species elute last. The samples contained an L:D enantiomeric ratio of 2:1. For all of the acids the D enantiomer eluted first when copper D-valine was used as the selector, with the exception of alanine in which the L enantiomer eluted first. When L-valine was used as the ligand, the elution orders were reversed. It should be noted that at least under the set of conditions that was used for separations in the current study electroosmotic flow could not be observed with neutral probe mesityl oxide.

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6 4 2 0 1:1

1:1.5

1:2

1:2.5

1:3

Cu:valine ratio Figure 3. Influence of the copper:valine ratio on the enantiomeric resolution of the five dansyl amino acids. Run conditions are identical to Fig. 1 with ratio adjusted by the amount of valine. Lines correspond (from top) to trp, ser, ala, phe, and tyr.

3.3 Optical and EPR studies of dansyl AA, valine– copper complexation 3.2 Performance dependence on pH* and Cu:valine ratio Prior work on ligand exchange separation of amino acids has shown the pH of the buffer to have a large influence on the separation [8, 12, 15]. The effect of changing the pH of the acetate buffer in our case for the separation of the five target amino acid pairs is shown in Fig. 2. It is seen that for all acids, the resolution increases as the pH is lowered. The increased resolution at lower pH is rather unusual and we do not know of a literature parallel for the very low optimal pH obtained here. Normally, low pH buffers are avoided in order to increase the competition of the copper binding to the carboxylate and the amide N on the dansyl amino acid [18]. The retention time of all the enantiomers was increased as the pH was lowered. In the absence of electroosmosis, the pH also controls the mobility of the acids. At lower pH they are less charged and hence move slower and have more time to partition with the complex. The retention time for D-serine is shown on the secondary y-axis in Fig. 2 to demonstrate this effect. The ratio of copper to valine, which would provide optimal separation of the enantiomers, was also investigated. The results are shown in Fig. 3. Surprisingly, the optimal ratio turned out to be around 1:1.5 as opposed to the usual 1:2 and higher [8, 12, 15].

Resolution

20

6

15 4

10

2 0

5

time (min)

25

8

0 3.5

3.7

3.9

4.1

pH Figure 2. The effect of pH on the chiral resolution. Run conditions are identical to those of Fig. 1 with pH adjusted with acetic acid. Lines correspond (from top) to trp, ser, ala, phe and tyr. Secondary axis: retention time of D-serine.


In order to understand the underlying reasons for the pH and copper:valine ratio dependencies, we conducted spectroscopic and EPR studies of the valine–copper system with dansylated amino acids. The absorption spectra of various copper compounds and the effect of adding dansylphenylalanine were measured. Figure 4 shows the spectra of copper–valine complexes in ratios of 1:1 (a), 1:1.5 (b) and 1:2 (c) with the addition of successive equivalents of dansylphenylalanine. The 1:1 complex exhibits a blue shift upon adding the dansyl acid with a clear isosbestic point at 635. In the 1:2 complex only a small shift was observed. The 1:1.5 complex showed an initial large shift like those of the 1:1 complex followed by small shifts like those of the 1:2 complex. These observations lead to the conclusion that valine is not exchanged by the dansyl acids. Rather, the dansyl acids coordinate with a 1:1 copper–valine complex. The other ligands in this complex, which are those actually exchanged by the dansyl acids, are likely acetate ions from the buffer. We conducted EPR studies of the 1:1.5 copper:valine complex and the influence of added dansyl-phenylalanine (1 equivalent) on the observed spectra. All other components of the two solutions remained the same. At room temperature both 1:1.5 copper:valine and 1:1.5 copper:valine–dansylphenylalanine solutions show practically the same isotropic hyperfine split (ICu 5 3/2) EPR spectra (Fig. 5A) with the same (within the experimental error) parameters of isotropic spin Hamiltonian (Table 1). In contrast, EPR spectra of frozen solutions recorded at T 5 75 K (Fig. 5B) reveal some differences between the solutions of 1:1.5 copper:valine and 1:1.5 copper:valine–dansyl-phenylalanine complexes. The EPR spectrum of the solution of the 1:1.5 copper:valine complex demonstrates a complicated pattern, which may be described as a superposition of EPR signals belonging to two Cu(II) complexes distinguished by their EPR parameters related to the axial component (both g-factor gjj and hyperfine splitting parameter Ajj —see Table 1). The ratio between the abundances of these complexes may be www.electrophoresis-journal.com

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estimated by spectral simulation as lying between 1:1 and 1:1.5. This supports the hypothesis that there are two predominant species with mono- and di-valine ligation. Although these two complexes differ in their equatorial coordination, these differences are not discernable in the parameters due to the low resolution of the perpendicular component. However, we can see the effect of the different

A

0.35 0.3

ligations on the axial parameters due to the higher resolution and sensitivity of this component. Addition of an equivalent of dansyl-phenylalanine to the complex causes a reduction of the less abundant complex (of the two mentioned above) and reduces the hyperfine splitting parameter Ajj of the more abundant one; see Fig. 5B and Table 1. This may indicate the formation of another, mixed copper:valine:dansyl-phenylalanine complex. Temperature dependencies of EPR spectra line shapes and parameters for the initial and in-gel solutions reveal no significant differences between these samples (Fig. 6). The

more dansyl-phe

A

0.2

T= 295 K

0.15

Normalized EPR Signal Intensity

Abs

0.25

0.1 0.05 0

B

0.4

more dansyl-phe

0.35 0.3 Abs

0.25 0.2

Cu-valine

1.0

0.5 Cu-valine-dns-phe 0.0

-0.5

ν = 9.462 GHz

260

280

300

0.15 0.1

B

0.05

0.3

Normalized EPR Signal Intensity

0

C

more dansyl-phe

0.25 0.2 Abs

320

340

360

380

Magnetic Field (mT)

0.15 0.1 0.05 0 450

550

650

1.5

T = 75 K

Cu-valine

1.0

0.5 Cu-valine-dns-phe

0.0

-0.5

ν = 9.463 GHz

750 200

Wavelength, nm

Figure 4. Absorption spectra of copper–valine 1:1 (A), 1:1.5 (B) and 1:2 (C) complexes with successive addition of equivalents of dansyl-phenylalanine. pH was 3.5, 5 mM copper, dansyl-phe was added in 5 mM increments.

250

300

350

400

450

Magnetic Field (mT)

Figure 5. EPR spectra of 1:1.5 copper–valine complex in methanol without (upper curve) and with (lower curve) an added equivalent of dansyl-phenylalanine at (A) 298 K and (B) 75 K.

Table 1. EPR spectral parameters Sample

T 5 295 K

T 5 75 K

giso70.001

aiso (mT)70.1

gjj 0:001

Ajj (mT)70.1

g?70.001

A? (mT) (estimated)

gav70.005

Cu–valine

2.123

7.6

Cu–valine–dns-phe

2.120

7.7

2.248 2.292 2.249 2.292

17.7 17.0 17.9 16.4

2.055 2.055 2.055 2.055

1.3 1.3 1.3 1.3

2.119 2.134 2.120 2.134



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Normalized EPR Spectra Intensity

Solution

Gel

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Table 2. Enantiomeric resolution of dansyl-AA’s with different acetate concentrations

Step-like changes

0.6

10 mM NH4OAc

20 mM NH4OAc

30 mM NH4OAc

3.40 2.18 1.24 1.08 8.53

0 1.04 0 0 3.33

0 0 0 0 2.17

0.4

Serine Alanine Phenylalanine Tyrosine Tryptophan

0.2 0.0 o

62 C -0.2 -0.4

Separation conditions: 5 mM Cu, 7.5 mM valine, pH* adjusted to 3.5 by acetic acid.

T o

25 C -0.6

305

310

315

305

310

315

3.5 Determination of the binding constants

Magnetic Field (mT) Figure 6. Temperature dependencies of low-field hyperfine components in EPR spectra of copper:valine complex in solution and gel. Temperature increases from the bottom curve to the top one.

only distinguishable feature found was a step-like change in the EPR signal line shape of the in-gel sample approaching the gel melting point (451C). This fact indicates (to some extent) that the gelator does not bind the copper complex strongly and that the EPR line shape is affected by the gelator due to changes in microviscosity at the sol–gel transition.

3.4 Copper:acetate ratio dependence Since the ideal copper:valine ratio was less than 1:2, the remaining ligands in the square coordination plane are probably acetate ions from the buffer. It was therefore interesting to study the effect of changing the acetate concentration in the buffer on the separation. The five dansyl amino acids were separated while changing the acetate concentration and keeping the pH and copper and valine concentrations constant. The resolutions of the acids are shown in Table 2. The binding ability of the acids as determined by their chiral resolution is seriously inhibited by the addition of acetate. The retention times also decreased, for example, from 29.54 min for D-tryptophan at 10 mM acetate to 18.37 at 20 mM and 16.70 at 30 mM, which shows that there is less binding to copper. The increase in ionic strength (I) has a negative effect on the mobility m according to the Onsager equation [19] pﬃﬃ m ¼ m0 CðE; T; Z; m0 Þ I

ð1Þ

where C is a function of m0 the mobility at zero ionic strength, e the dielectric constant, T the absolute temperature and Z the viscosity. Thus, the change in ionic strength cannot explain the decrease in the AA’s retention time. & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Various methods for measuring binding constants of molecular association in electrophoresis using binding isotherms exist. With slight modification, the double reciprocal method as described by Rundlett and Armstrong [20] can be used for ligand exchange as well to determine the different binding constants of L and D enantiomers. Moreover, the analysis of Rundlett and Armstrong allows us to determine the mobility of the dansyl:Cu:valine complex. The equation for the formation of the complex with the dansyl acids is 3n CuVAcn1n þ D Ð CuVDAcn2 þ 2Ac

ð2Þ

where V is valine, Ac is acetate and D is a dansyl amino acid. n is the number of acetates coordinated to the copper in the absence of dansyl AA. The equilibrium constant is K¼

3n ½CuVDAcn2 ½Ac 2 ½CuVAcn1n ½D

ð3Þ

The complex CuVAcn1n is itself in equilibrium with free copper, valine, acetate and other complexes, but for any given set of ratios in the buffer precursors, the amount of this complex will be in a fixed ratio to the amount of total copper, or ½CuVAcn1n ¼ k CT;Cu

ð4Þ

where CT;Cu is the total concentration of all copper species in the system. The acetate is in surplus; therefore, it is reasonable to assume that the acetate concentration (in Eq. (3)) remains constant throughout the experiments so that the equilibrium equation (3) can be rewritten as K0 ¼

3n ½CuVDAcn2 CT;Cu ½D

ð5Þ

K0 [D] gives the fraction of the copper ions that is dansyl coordinated. More importantly, K 0 CT;cu gives the ratio of the complexed dansyl AA to the free dansyl AA. Equation (5) mirrors exactly that used in the standard methods in [20]; hence, the same final form of the mobility equation is reached 1 1 1 1 ¼ 0 þ m mf K ðmb mf Þ CT ;Cu mb mf

ð6Þ


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0 1/(µ−µ µ−µf)

-200

L D

-400 -600 -800 -1000 0

200

400

600

1/[Cu] M-1 Figure 7. Double reciprocal plot for Eq. (6)).

L-

and

D-alanine

(to fit

where m is the observed mobility of the dansyl AA, mb is the mobility of the ligand when bound to the complex and mf is the mobility of the free ligand, which can be measured when no complex is added to the buffer. By plotting (m– mf)1 1 against CT;Cu , K0 can be found as an intercept/slope. An example for dansyl-alanine is shown in Fig. 7. The determined binding constants were 179 M1 for L-alanine and 212 M1 for D-alanine. The mobility of the bound species mb can also be determined from the intercept. The values for L and D, respectively, are 0.00211 and 0.00232 cm2/V min, as compared with 0.00692 for the unbound amino acid. From the Stokes–Einstein relationship, the mobility of the compounds in unrestricted flow is given by q m¼ ð7Þ 3pZD where q is the charge of the particle, Z is the dynamic viscosity of the fluid and D is the particle diameter. The calculated mobilities for the bound complex and free dansylalanine are 0.0172 and 0.0189, respectively (Z 5 0.59 mPa s; ˚ and 9.13 A ˚ , respectively, as determined by D 5 10.0 A modeling in ChemSketch 4.01). The ratio of the expected mobility to the observed mobility can serve as a factor demonstrating the strength of interaction with the gel. For the free acid the factor is 2.74 and for the complex it is 8.13, showing that the complex mobility is influenced to a large extent by interactions with the gel. The use of Eq. (7) is simplistic yet it qualitatively suggests that the complex binds more strongly to the gel than the free acid, though both are relatively mobile and form only weak interactions with the gel.

4 Discussion Given the spectroscopic and thermodynamic data, the dependencies of the separation on the parameters can be better understood. Both the UV and EPR results suggest that the active complex effecting the separation is a Cu:valine 1:1 complex. The copper is distributed between complexes with 0, 1 or 2 valine ligands. The more valine that is added, the more the equilibrium is shifted to complexes with more coordinated valine. Since the lone electron pair on the sulfonamide nitrogen is strongly inducted into the dansyl moiety, it is a weaker ligand than valine which has a & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

free amine. Thus, dansyl amino acids are not expected to replace valine under these conditions and can coordinate only with copper that has fewer than two valine ligands (as indeed observed by the spectrophotometric tests). Such copper complexes have acetate ions from the buffer as the other ligands. The steep drop-off in the resolution of the dansyl acids at ratio 1:2.5 (Fig. 3) likely reflects the point at which the majority of the copper is bound to two valines and can no longer coordinate with the dansyl acids. The ideal ratio of 1:1.5 may be the ratio at which the majority of the copper is bound to one valine ligand. The dependence on the acetate concentration also reflects this suggested type of exchange. As the acetate concentration increased, the dansyl acids were less able to bind to copper as reflected by their reduced resolution and retention times. Were the acids replacing valine in the complex, the acetate concentration should not affect the resolution significantly, and in fact the retention times should increase as indicated by the Onsager equation (1). It must therefore be concluded that the dansyl acids are exchanging acetate ions, and that the higher the acetate concentration, the harder it is for the dansyl acids to replace them. Moreover, the pH dependence can be understood from here as well. The total acetate concentration is divided between acetic acid and the acetate ion. The latter is that which is able to coordinate well with copper. As the pH increases, even if the total acetate concentration remains the same, the concentration of acetate ions increases, and thus there is the same effect as raising the total acetate concentration, i.e. a reduction in resolution and retention time. Similarly, the pH affects the distribution of copper:valine complexes. At higher pH the shift is to complexes with more valine ligands [21]. Thus, increasing the pH has the same effect as increasing the amount of valine. These two effects explain why the ideal pH is very low. The data presented above also shed light on the role of the gel in the separation. It was noted in the thermodynamic analysis that the product of the binding constant K0 and the total copper concentration gives the ratio of the bound to free dansyl acid. At the working concentration of 5 mM Cu, the binding constants of 179 and 212 M1 for L- and D-dansyl-alanine, respectively, give ratios of around 1, or in other words about half of the dansyl acid is bound to the copper. The gel resolves the different unbound acids from each other as demonstrated in our previous work [2]. In addition, the larger interaction factor for the complex suggests an interaction with the gel which slows it down. Chiral recognition usually occurs through a three-point interaction. It has been demonstrated that although it is impossible to achieve the three points within the copper complex itself, a non-chiral stationary phase can provide an additional interaction point and effect the separation [22]. Copper tends to exist as a hexacoordinated or pentacoordinated species, often with solvent molecules at the axial, less strongly bound sites. The carbonyl oxygen of the gel is able to coordinate with copper at the axial position, though this competes with the hydrogen bonding of the carbonyl to www.electrophoresis-journal.com

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A

B

H C 11H 23

N

C

H

H

C 11H 23SIDE CHAIN

N

C 11H 23

N O

H

HN Dns

O

O

C 11H 23

H

SIDE CHAIN

C 11H 23 O

N

O O Cu

C 11H 23

N

H

C 11H 23

N

H

O

N H

H

C 11H 23 HHCC 33 C 11H 23

N

H

O

N

N H

NH2

C11H 23

H

H

NH2 O O

C 11H 23 HH33CC

C11H 23

N

C 11H 23

H

O

C 11H 23

C 11H 23

C 11H 23

N

O C11H 23

N

H

H

Figure 8. Interaction between the ternary copper complex and the gel: (A) with a dansyl acid and the valine side chain away from the gel.

the amide H of the adjacent monomer. Figure 8 shows the expected interaction between the copper complex and the gel. The hydrophobic side chain of the D-valine is attracted to the gel, so that the interacting dansyl acid will have its side chain toward the gel if it is D (Fig. 8A) and away if L (Fig. 8B). Aside from those displayed above, other dansylated amino acids (asparagine, leucine, methionine, threonine, valine, norlecine and norvaline) were separated in this system, and they all eluted with D before L. This can be attributed to their side chains being too big or hydrophilic, and once the D enantiomer binds to the complex the repulsion from the alkyl chains increases the distance between the copper and the carbonyl of the gel preventing the binding which slows down the complex. Since alanine is small and hydrophobic, its D form binds well to the complex and thus elutes after L. Were the copper to bind to the gel with the valine away from the gel as in Fig. 8C, the hydrophobic interactions with the valine ligand and any potential interactions with the dansyl side chain would be lost, and thus this is not a favorable position. It can be concluded from here that the Cu:valine complex moves in and out of the gel, with the axial position being taken by an acetate ion, giving the complex an overall negative charge when not bound to the gel. Such a weak type of interaction is supported by the temperature-dependent EPR studies, which suggest only a weak interaction with the gel.

H

CH3 CH3

O

O N

O Cu O

O

N

Dns

C 11H 23

N O

O

H

HN

O

N

SIDE CHAIN

C 11H 23O

N

O

O

O

O

C 11H 23

H

O

C 11H 23

N O

NH2

H

O Cu

C 11H 23

O H

N

HN Dns

O

O N

O O

H

H

C 11H 23

N

O O

N

H

C 11H 23

N

O

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D

dansyl acid, (B) with an

L

dansyl acid, (C) with a

D

is a novel type of monolithic stationary phase for CEC. The ligand exchange differs from all previously reported chiral LECE and relies on acetate exchange by the different enantiomers rather than on the exchange of the selector ligand. This brings about unexpected chromatographic behavior that was never observed before in LECE. The resolution of the enantiomers depends on the pH, copper:valine ratio and buffer composition in a different way from previously reported LECE. The optimal pH was found to be 3.5. This differs from all previous amino acid separations in which a pH of 5–8 is usually used. The optimal valine:copper ratio is 1.5, much lower than any other reported amino acid:copper ratio used for LECE. The separation efficiencies as determined by plate numbers were on the same order as that reported in optimized polymer gel ligand exchange CEC [16, 17]. The resolution achieved for tryptophan (7.3) is higher than in any other ligand exchange CE known to us. The authors wish to thank the BMBF-Germany—MOS, Israel Water Technology Program and the Ring Foundation for financing this research. The authors have declared no conflict of interest.

6 References 5 Conclusion In conclusion, chiral (electro)separations were performed for the first time in a low molecular weight organogel. This & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[1] Gubitz, G., Schmid, M. G., Electrophoresis 2004, 25, 3981–3996; Preinerstorfer, B., Lammerhofer, M., Electrophoresis 2007, 28, 2527–2565.


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