Separation of very hydrophobic analytes by micellar electrokinetic chromatography. III....

Carolin Huhn1

Michael Pütz2

Ute Pyell1

1Department of Chemistry,University of Marburg,Marburg, Germany

2Bundeskriminalamt – FederalCriminal Police Office,Forensic Science Institute,KT 34 – Toxicology,Wiesbaden, Germany

Received August 23, 2007Revised November 6, 2007Accepted November 6, 2007

Research Article

Separation of very hydrophobic analytes bymicellar electrokinetic chromatography.III. Characterization and optimization of thecomposition of the separation electrolyteusing carbon number equivalents

Four coefficients are suggested to characterize the separation properties of separation elec-trolytes in MEKC. These coefficients are the electrophoretic mobility of the pseudosta-tionary phase (PSP), the electroosmotic mobility, and two parameters which can beobtained via the Martin equation using the retention data of the members of a suitable ho-mologous series. These four coefficients are used for the characterization of separationelectrolytes applicable to the separation of very hydrophobic analytes (log POW = 3–4) withSDS as PSP and to quantitatively describe the influence of the buffer additives ACN andurea for the fine-tuning of retention factors and the additive calcium chloride for the fine-tuning of the width of the migration time window. Together with carbon number equiva-lents (NC

*) as analyte descriptors the suggested coefficients provide a tool for the fast opti-mization of the composition of the separation electrolyte. The proposed method optimiza-tion scheme is applied to the separation of ingredients of essential oils. Predicted resolu-tions are compared to experimental results.

Keywords:

Essential oils / Micellar electrokinetic chromatography / Migration time win-dow DOI 10.1002/elps.200700628

Electrophoresis 2008, 29, 783–795 783

1 Introduction

The separation of very hydrophobic solutes using MEKCwith conventional surfactant is still a difficult task if notimpossible in many cases. MEKC is based on an aqueousseparation electrolyte, so that solubility problems withhydrophobic analytes can often not be avoided. In addition,without appropriate modifiers the retention factors are toohigh, so that hydrophobic analytes are transported virtuallyexclusively within the micellar phase. Separation and quan-tification are then impossible. The following solutions tothese problems have been published:

(i) Most often, organic solvents are used to increase thesolubility of the analytes in the separation electrolyte and toreduce retention factors. Among these organic modifiers areACN [1–4], methanol, isopropanol, N,N-DMF [4–7], form-amide [8], and urea [7, 9]. Bütehorn and Pyell [10] demon-strated that a combination of the buffer additives urea andACN can have advantages over the use of a single modifierreducing largely retention factors of very hydrophobic ana-lytes (dansylated amines) without compromising efficiencywhen employing SDS as pseudostationary phase (PSP). (ii)The addition of a CD to the separation electrolyte containinga conventional surfactant induces a secondary equilibriuminfluencing the separation process and reducing largelyapparent retention factors. The use of CD-MEKC (CD-mod-ified MEKC) has found widespread application for theseparation of hydrophobic solutes and for enantioselectiveseparations [7, 11–17]. (iii) Several new PSPs were shown toenable the separation of hydrophobic analytes in EKC.Examples are polymeric PSPs employed with high volumefraction of an organic solvent in the separation electrolyte[18–23]. (iv) Another approach is the use of mixed micelles,

Correspondence: Professor Ute Pyell, Department of Chemistry,University of Marburg, Hans-Meerwein-Strasse, D-35032 Mar-burg, GermanyE-mail: [email protected]: 149-6421-2822124

Abbreviations: PSP, pseudostationary phase; SF, separation fac-tor; SN, separation number

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

784 C. Huhn et al. Electrophoresis 2008, 29, 783–795

where separation characteristics can be fine-tuned via themolar fractions of the two surfactants, and in addition,higher volume fractions of organic modifiers can beemployed [7, 24, 25].

The use of analyte descriptors similar to the analytedescriptor carbon number equivalent (N�C) was first proposedin 1984 by Jandera [26] for HPLC. He introduced the lipo-philicity index nce, which is the carbon number equivalent ofan analyte for a purely aqueous mobile phase. In order todescribe and to predict retention factors at different volumefractions of an organic modifier in the mobile phase, a sec-ond descriptor the polarity index qi was introduced which is ameasure of polar interactions. In order to determine nce andqi, it is necessary to measure k dependent on the volumefraction of the organic modifier for the solute and for thehomologous calibration series.

In a previous paper investigating the separation ofhydrophobic analytes by MEKC with conventional surfactant[27] the separation of aromatic ethers in essential oils of for-ensic interest containing safrole as main constituent isdescribed (safrole is precursor for the clandestine synthesisof (3,4-methylenedioxymethamphetamine) [27, 28]. MEKChas been chosen for the separation of these analytes becausethis technique provides good selectivity and a high peak ca-pacity and can handle large differences in the concentrationsof main and minor components. The empirically optimizedseparation electrolyte consisted of 7.5 mmol/L borate,60 mmol/L SDS, pH 9.2 with the additives ACN (20% v/v),urea (4 mol/L), and CaCl2 (0.5 mmol/L). This paper alsoaddresses the optimization of the composition of the sampleinjection solution. In the second part of this series [29] weinvestigated the use of carbon number equivalents N�C asanalyte descriptors. It was shown that for the analytes selectedtaking the homologous series of alkyl phenones as referencecarbon number equivalents are quasi-independent of thecomposition of the separation electrolyte with RSD �4% for awide parameter range varying the concentration of urea, thevolume fraction of ACN, and the concentration of CaCl2.

In this third part of the series we want to gain furtherinsight into the influence of these three additives on param-eters affecting the resolution of a peak pair.

Associated with the determination of the migration ve-locity of the micellar phase according to the method pre-sented by Bushey and Jorgenson [30, 31] retention data areobtained for the members of a homologous series. Thesedata permit direct access to the two parameters a and bresulting from plotting log k against the number of methyl-ene groups Nc in the alkyl chain (Martin equation). We pro-pose to use these parameters (a and b) together with theelectroosmotic mobility meo and the electrophoretic mobilityof the micelles mMC as a set of coefficients to characterize theproperties of a separation electrolyte. It will be investigatedwhether together with carbon number equivalents N�C asanalyte descriptors modeling of separations is possible.Employing N�C in place of the retention factor k permits todefine a new function f N�c

� �which proved to be particularly

useful for rapid resolution optimization concerning theseparation of very hydrophobic analytes (log POW = 3–4) withconventional surfactants.

2 Theoretical considerations

2.1 Resolution

The large number of structurally related analytes containedin sassafras oils requires a fine-tuning of the separationelectrolyte composition to yield adequate resolution for allanalytes of interest for quantitative analysis. According to theequation derived by Terabe et al. [32] (Eq. 1) the resolution Rs

of two adjacent peaks (neutral analytes) can be optimizedwith regard to efficiency, selectivity, retention, and ratio ofphase velocities.

Rs ¼ffiffiffiffiNp

4

� �a� 1a

� �k2

1þ k2

� � 1� t0

tMC

1þ k1t0

tMC

0

B@

1

CA (1)

where N is the plate number, a is the selectivity factor, k1, k2

are the retention factors of Analytes 1 and 2, respectively, t0

and tMC are the migration times of the aqueous and themicellar phases, respectively. The efficiency (described by theplate number) is generally high in MEKC and will not bediscussed further. In this study we concentrate on the opti-mization of the resolution via selectivity fine-tuning (givenby the selectivity factor a in the second term), retention factoroptimization, and widening of the migration time windowquantified by the migration time ratio tMC/t0 [33].

2.2 Determination of the migration time of the

micelles

The successful separation of hydrophobic analytes in MEKCwith SDS as micellar phase affords additives which are ableto reduce the retention factors of these analytes to a greatextent. However, under these conditions, the use of a markerfor the determination of tMC is no longer reliable [34, 35].Therefore, Bushey and Jorgenson [30, 31] published aniteration procedure for the determination of the migrationtime of the micellar phase based on the retention factors ofthe members of a homologous series. This method is basedon the constant contribution of each methylene unit to thefree energy change DG associated with the transfer of asolute from the aqueous phase to the PSP. The logarithms ofthe retention factors for the members of a homologous seriesare thus linearly related to the number of carbon atoms Nc inthe alkyl chain, yielding the well-known Martin equation (Eq.2).

log k ¼ aþ bNc (2)

It should be emphasized that in the case of the homologousseries of alkylphenones Nc corresponds to the number of


Electrophoresis 2008, 29, 783–795 CE and CEC 785

methylene units in the alkyl chain. For the first step of theiteration procedure the migration time of the solute with thehighest carbon number is taken as an estimate of tMC. Withthis value the retention factors are calculated for all othermembers of the homologous series. With these data aregression line is calculated. The extrapolation of the regres-sion line to the member of the homologous series with thehighest carbon number is then used to calculate a new esti-mate for tMC. The iteration is continued until the change intMC is lower than a predefined threshold value.

2.3 Carbon numbers and carbon number equivalents

During the iteration procedure for the estimation of tMC, theslope b and the intercept a of Eq. (2) are simultaneously cal-culated. It is also possible to calculate “interpolated” carbonnumbers for neutral analytes in a similar way as it is done forretention indices in GC. However, in order to simplify fur-ther calculations we did not transfer retention data intoretention indices but used carbon number equivalents N�C.In a previous publication [29], it was shown that for the ana-lytes selected taking the homologous series of alkyl phe-nones as reference N�C is quasi-independent of the composi-tion of the separation electrolyte regarding type and con-centration of the modifier. Other publications [36, 37]showed the independence of retention indices from the con-centration of the PSP and the temperature. Carbon numberequivalents are calculated from retention data for the analyteand for the members of a homologous series (taken as refer-ence) obtained in the same run or in subsequent runs.

N�c ¼1b

logtR � t0

t0 1� tR

tMC

� �

2

664

3

775� a

8>><

>>:

9>>=

>>;(3)

where N�C is the carbon number equivalent, a and b are theintercept and slope of the regression line plotting log k of themembers of a homologous series against the number ofmethylene groups in the alkyl chain, and tR is the retentiontime of the analyte. Taking alkyl phenones as referencesolutes, N�C for a number of sassafras oil constituents werecalculated for different compositions of the separation elec-trolyte (see Section 3) [29]. The resulting mean values andRSDs are given in Table 1. While for the major part of solutesRSD �4%, the RSDs for piperonal and the internal standardIS are significantly higher, reflecting the higher uncertaintyin the calculation of retention factors for analytes migratingclose to t0 or tMC [38]. As it is known that in MEKC retentionindices are independent of the total surfactant concentrationand the temperature [36, 37], these parameters are notinvestigated in the present study.

Based on N�C it is possible to calculate the retention factorfor the solute of interest in a given separation electrolyte,provided the parameters a and b (Eq. 2) are known. Theseparameters have to be determined for each separation elec-

Table 1. N�C calculated according to Eq. (3) for all separationelectrolytes listed in Table 2

�x SD RSD

Piperonal 0.91 0.20 22.32IS 1.75 0.13 7.45Eugenol 2.84 0.11 3.81cis-Isoeugenol 3.13 0.14 4.51trans-Isoeugenol 3.29 0.12 3.76Methyleugenol 3.56 0.14 3.83Safrole 3.86 0.14 3.71Myristicin 4.01 0.15 3.69Asaron 4.11 0.16 3.89Isosafrole 4.18 0.17 4.03Anethol 4.31 0.14 3.34

�x mean value; data taken from ref. [29].

trolyte or can be deduced from interpolation formula. Also,the selectivity factor a for a pair of solutes can be calculatedusing the carbon number equivalents N�1 and N�2 of these twocompounds.

log k ¼ aþ bN�c (4)

a ¼ k2

k1¼ 10aþbN�2

10aþbN�1¼ 10 aþbN�2ð Þ� aþbN�1ð Þ ¼ 10b N�2�N�1ð Þ (5)

Equation (5) shows the interdependence of the slope b of theMartin equation (the logarithm of the methylene selectivity)and the selectivity coefficient a. Thus b can be used to quan-tify selectivity differences induced by various additives de-pendent on their concentration in the separation electrolyte.

The last two factors in Eq. (1) have been defined by Ter-abe et al. [32] as f kð Þ: if k1 and k2 are replaced by the meanretention factor �k (�k = (k1 1 k2)/2), the last two factors can bedefined as f �k

� �. If k1 and k2 are replaced by expressions fol-

lowing from Eq. (4) and a is replaced by Eq. (5), the resolu-tion of two neutral solutes can be described as a function ofthe number of theoretical plates N, the parameters a and bdetermined for this electrolyte with the members of a speci-fied series of homologous, the carbon number equivalentsN�1 and N�2 of the solutes of interest, and the migration timesof the aqueous and the micellar phase t0 and tMC, respectively(Eq. 6).

Rs ¼ffiffiffiffiNp

410b N�2�N�1ð Þ � 1

10b N�2�N�1ð Þ10aþbN�2

1þ 10aþbN�2

1� t0

tMC

1þ 10aþbN�1t0

tMC

(6)

If N�1 and N�2 are replaced by the mean carbon numberequivalent N�c of the two solutes to be separated we candefine the function f N�c

� �analogously to f �k

� �. In the normal

elution mode with a velocity of the EOF higher than the(oppositely directed) velocity of the micelles, this functionshows a maximum, whereas in the restricted/reversed elu-



tion mode [39] with the absolute velocity of the micellarphase higher than that of the EOF, a pole can be found.Similar to the definition of negative electrophoretic mobili-ties a negative migration time of the micelles can be definedin case of the restricted/reversed elution mode, indicatingthat the micelles migrate towards the anode.

Foley [40] showed by differentiating the function f �k� �

with regard to �k (keeping all other parameters constant) thatmaximum resolution is obtained in the normal elution modefor �kmax ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffitMC=t0

p. Accordingly, we can calculate N�c;max at

the maximum of the function f N�c� �

at fixed a, b, t0, and tMC.

N�c;max ¼1b

log

ffiffiffiffiffiffiffitMC

t0

r� �� a

� �(7)

For the restricted or reversed elution mode f �k� �

and f N�c� �

have a pole. It is known that in this case f �k� �

is reachinginfinity for �k ¼ �tMC=t0. Accordingly N�c;pol for f N�c

� �reach-

ing infinity can be calculated at fixed a, b, t0, and tMC:

N�c;pol ¼1b

log � tMC

t0

� �� a

� �(8)

The characterization of the separation properties of differentseparation electrolytes is possible using these maxima orpoles of the function f N�c

� �. Highest resolution with respect

to the function f N�c� �

can be expected for those separationelectrolytes having N�c;max or N�c;pol similar to the carbonnumber equivalents of the analytes to be separated. However,variations in a are not taken into account with this simplifiedapproach. Therefore, it can have advantages to predict theresolution for the solutes of interest (at predefined platenumber N) using carbon number equivalents as analytedescriptors and tabulated values for a, b, tMC, and t0 asseparation electrolyte coefficients (Eq. 6).

2.4 Relationship to octanol–water partitioning

coefficients

Regarding the biphasic systems octanol/water or micellarphase/aqueous phase, a congeneric behavior (differentslopes for different analyte classes) can be observed for thefunctions log POW = f(log k) or log k = f(log POW), if SDS isthe PSP [34, 41–44]. The same congeneric behavior wasobserved for retention indices [34, 45, 46]. This congenericbehavior is induced by the differences in the type of interac-tion between the solute and the two “solvents” involved. Themajor difference in case of SDS as PSP can be ascribed todifferences in the hydrogen-bond basicity of SDS relative tothe hydrogen-bond basicity of wet octanol. For SDS asmicellar phase, three analyte classes are observed: neutralnonpolar analytes like aromatic hydrocarbons, alkyl ben-zenes, and halogenated benzenes and more polar analyteswith the possibility to establish hydrogen bonds, divided intotwo classes: strong hydrogen bond acceptors (including alkylphenones, anilines, and aromatic ethers) and hydrogen bonddonators (e.g., phenols) [47].

The calculation of carbon number equivalents fromlog POW is possible, if the analytes belong to the same class ofcompounds as the compounds which were employed todetermine the parameters of the function log POW = f(Nc). Inref. [29], c = 1.229 and d = 0.502 were obtained via linearregression of log POW plotted against Nc for alkyl phenones,for log POW = c 1 d6Nc, with log POW taken from(www.vcclab.org/lab/alogps, 23.10.2006). With these param-eters, carbon number equivalents can be obtained directlyfrom octanol–water partitioning coefficients.

Consequently, all equations presented in Section 2.3 canbe transformed into equations in which N�C is replaced by((log POW 2 c)/d).

tR ¼ t01þ 10aþblog POW�c

d

1þ t0

tMC10aþb

(9)

Rs ¼ffiffiffiffiNp

410b

log POW;2�cd � log POW;1�c

d

� �� 1

10blog POW;2�c

d � log POW;1�cd

� � �

� 10aþblog POW;2�c

d

1þ 10aþblog POW;2�c

d

1� t0

tMC

1þ 10aþblog POW;1�c

dt0

tMC

(10)

where log POW;1 and log POW;2 are the logarithm of the octa-nol–water partitioning coefficients of Analytes 1 and 2,respectively. If log POW ¼ log ðPOW;1 þ POW;2Þ=2

� �, the

last two factors in Eq. (10) can be defined as f ðlog POWÞ. Themaximum of this function (for the normal elution mode)and the pole of this function (for the reversed/restricted elu-tion mode) are given in Eqs. (11) and (12).

log POW;max ¼db

log

ffiffiffiffiffiffiffitMC

t0

r� �� a

� �þ c (11)

log POW;pol ¼db

log � tMC

t0

� �� a

� �þ c (12)

With Eq. (10), resolutions can be predicted for the solutes ofinterest (at predefined plate number N) directly from log POW

values and tabulated values for a, b, tMC, and t0.

3 Materials and methods

3.1 Chemicals

Methanol, NaH2PO4, eugenol, safrole, thymol, piperonal,and asaron were from Fluka (Buchs, Switzerland); urea andSDS were from Roth (Karlsruhe, Germany); disodium tetra-borate, methyl 4-cyanobenzoate, CaCl2, isosafrole, aceto-phenone, propiophenone, butyrophenone, and valero-phenone were from Merck (Darmstadt, Germany); ACNfrom J. T. Baker (Deventer, The Netherlands); methyleu-



genol, trans-anethol, hexanophenone, heptanophenone,octanophenone, decanophenone, and dodecanophenonewere from Aldrich (St. Louis, USA); myristicin was fromSigma (Steinheim, Germany). Structures of the analytesstudied are depicted in Fig. 1.

Figure 1. Structures of constituents in sassafras essential oils,2 = internal standard.

3.2 Buffer and sample preparation

Analytes were dissolved in methanol at a concentration of10 mmol/L. These standard solutions were mixed withdiluted separation electrolyte (1:1). The resulting analyteconcentrations in the sample solution are given in the figurelegends. All separation electrolytes contained 7.5 mmol/Lsodium borate (1.875 mmol/L Na2B4O7) and 60 mmol/LSDS, pH 9.2. Three buffer additives were used, ACN, urea,and CaCl2. The concentrations of these additives in theseparation electrolytes tested are summarized in Table 2.

3.3 Instruments

A Beckman P/ACE 5510 equipped with a DAD(labs = 240 nm) was used. Sample injection was done at34.5 mbar (0.5 psi) for 3 s. Fused-silica capillaries from Poly-

micro Technologies LLC (Phoenix, AZ, USA) were used withan inner diameter of 50 mm and an outer diameter of363 mm. The length was set to 20/27 cm. New capillarieswere conditioned by flushing them first with NaOH solution(0.1 mol/L) for 30 min and subsequently with run buffer for10 min. A rinsing step with run buffer for 1 min was usedfor cleaning the capillary between runs. A voltage of 10 kVwas used for separation. When new buffer additives wereintroduced, a short rinsing step with 0.1 mol/L NaOH wasused followed by rinsing with the new buffer. Data acquisi-tion was done with PACE Station software. Origin 6.0 Pro-fessional software from Microcal Software (North Hampton,USA) was used for data analysis.

4 Results and discussion

4.1 Influence of additives

Those parameters (Table 2) were selected which have beenproven in previous studies [27] to make possible the separa-tion of very hydrophobic analytes (log POW = 3–4) with aconventional surfactant. The separation electrolytes studiedare all based on 1.875 mmol/L Na2B4O7 and 60 mmol/LSDS. ACN is added at volume concentrations sA between 0and 25%. At higher volume concentration an unstable elec-tric current and a low reproducibility of retention data wereobserved. Urea is added at concentrations of 0–5 mol/L, atypical range for this additive given by the solubility of thiscompound in aqueous solution. The parameter range ofCaCl2 is between 0 and 7.5 mmol/L. At higher concentration,a turbid solution was observed, presumably due to the lowsolubility of Ca(DS)2.

4.1.1 ACN

ACN is widely used as modifier in MEKC separations due toits ability to reduce retention factors and to widen themigration time window [35, 48–50]. The addition of ACN tothe separation electrolyte induces changes in the dielectricconstant and in the viscosity [49, 50]. The migration timewindow tMC/t0 is widened from 2.74 (sA = 0%, curea = 0mol/L) to 11.91 (sA = 25%, curea = 0 mol/L) (see Fig. S1A inthe Supporting Information). The combination of ACN andurea results in a widening of the migration time windowlarger than that obtainable with ACN as single modifier: tMC/

Table 2. Compositions of separation electrolytes based on 1.875 mmol/L Na2B4O7, 60 mmol/L SDS, pH 9.2

Buffer type I II III IV V VI

ACN (%) 0, 5, 10, 15, 20, 25 0, 5, 10, 15, 20, 25 – 20 – 20Urea (mol/L) – 4 0, 1, 2, 3, 4, 5 0, 1, 2, 3, 4 – 4CaCl2 (mmol/L) – – – – 0, 1.0, 2.5, 5.0, 7.5 0, 0.25, 0.5, 0.75,

1.0, 1.5, 2.0



t0 = 33.36 (sA = 25%, curea = 4 mol/L). The large reduction ofthe electroosmotic mobility meo is the main reason for thisenlargement. meo is decreased linearly from 2.35610-4 cm2/Vs (sA = 0%) to 1.52610-4 cm2/Vs (sA = 25%). The additionof urea has an additive effect on the reduction of meo, but theslope for meo ¼ f sAð Þ is independent of the concentration ofurea. For curea = 4 mol/L m0 decreases from 1.97610-4 cm2/Vs (sA = 0%) to 1.22610-4 cm2/Vs (sA = 25%). It is knownthat the influence of sA on the electrophoretic mobility of themicelles mMC is dependent on the concentration of SDS [35,48, 50]. We observed only a small reduction in mMCj j(from21.4961024 cm2/Vs (sA = 0%, curea = 0 mol/L) to21.3961024 cm2/Vs (sA = 25%, curea = 0 mol/L)), which ismore pronounced if urea is simultaneously added to theseparation electrolyte (from 21.52610-4 cm2/Vs (sA = 0%,curea = 4 mol/L) to 21.19610-4 cm2/Vs (sA = 25%,curea = 4 mol/L)). By the addition of ACN retention factorsare strongly decreased (see Fig. S2 in the Supporting Infor-mation). As expected from Eq. (13) [50], there is a linearrelationship between log k and sA. The slope of the regres-sion line is the solvent strength S and the intercept is thelogarithm of the retention factor kw for a pure aqueousmobile phase.

log k ¼ log kw � SsA (13)

High correlation coefficients were obtained when plottinglog k versus sA (data not shown).

The parameters obtained from plotting the retention datafor the alkyl phenones against the carbon number are givenin Table 3. While the slope b (which is related to the selectiv-ity factor a, Eq. 5) does not follow a clear trend, the intercept ashows a decrease with increasing sA, which is associatedwith the reduction of retention factors. The data of Table 3were used to calculate f ðN�cÞ according to Eq. (6). The resultsfor separation electrolytes of Type II (variation of sA withcurea = 4 mol/L) are presented in Fig. 2A.

These plots illustrate the impact of sA on resolution-de-termining parameters: The widening of the migration timewindow is reflected by the higher value of f N�c;max

� with

increasing sA. The decrease of retention factors is reflectedby the strong shift of N�c;max to higher N�C. The highest reso-lution (at similar selectivity and efficiency) is expected foranalytes with N�C close to N�c;max. In Table 4, the values for�kmax, N�c;max calculated according to Eq. (7) and log POW;max

calculated according to Eq. (11) are listed.According to Giddings [51] the peak capacity corresponds

to the maximum number of components resolvable in onechromatographic run. It is obvious that this magnitude is acentral parameter for method optimization. As in MEKC theefficiency is not independent of k, Kolb et al. [52] suggested tocalculate the overall peak capacity in MEKC from the sum ofseparation numbers (SNs) within a given z range. The SN ofa separation electrolyte is defined as the number of compo-nent peaks that can be placed between the peaks of two con-secutive homologous standards with z and z 1 1 carbon

Figure 2. Plots of f N�c

� (A) for varied sA with curea = 0 mol/L,

cCaCl2 = 0 mmol/L and (B) for varied cCaCl2 with sA = 20% andcurea = 4 mol/L; N�C of the analytes are given as vertical bars,numbering according to Fig. 1; for further experimental detailsrefer to Table 2.

chain atoms, each peak pair separated by a resolution of1.177 [53].

SN ¼ tzþ1R � tz

R

wzþ10:5 þ wz

0:5

� 1 (14)

where tzR and tzþ1

R are the retention times of two adjacentmembers of a homologous series with the carbon numbers zand z 1 1, wz

0:5, and wzþ10:5 = peak widths at half height.

However, a magnitude which is related to the overallpeak capacity as it was defined by Giddings can easily be

determined from the integralZþ 1

� 1

f N�c� �

d N�c using the

function f N�c� �

. In the following discussion this parameterwill be termed separation factor (SF). SF is solely based onthe characteristics of the separation electrolyte. Processes ofband broadening due to injection or instrumental parame-ters are not taken into account. SF is thus independent of theinstrumental setup used. SF was calculated from the datashown in Fig. 2. In Fig. 3 the results are plotted against sA.



Table 3. Obtained regression parameters for plotting log kagainst Nc

Type I Type II

sA (%) b a b a

0 0.273 20.0489 0.320 20.3355 0.315 20.259 0.311 20.48110 0.368 20.482 0.327 20.70715 0.307 20.559 0.293 20.81120 0.257 20.652 0.252 20.93625 0.224 20.742 0.220 21.031

Type III Type IV

curea (mol/L) b a b a

0 0.287 20.150 0.265 20.6481 0.329 20.253 0.242 20.7152 0.314 20.322 0.236 20.8233 0.348 20.453 0.229 20.8754 0.372 20.583 0.236 21.0535 0.384 20.734 - - - -a) - - - -a)

Type V Type VI

cCaCl2 (mmol/L) b a b a

0 0.281 20.076 0.257 20.9390.25 - - - -a) - - - -a) 0.254 20.9380.5 - - - -a) - - - -a) 0.237 20.8320.75 - - - -a) - - - –a) 0.253 20.9111.0 0.273 20.094 0.257 20.8981.5 - - - -a) - - - -a) 0.256 20.8862 - - - -a) - - - -a) 0.318 21.2222.5 0.266 20.094 - - - -a) - - - -a)

5.0 0.263 20.125 - - - -a) - - - -a)

7.5 0.329 20.129 - - - -a) - - - -a)

a) Not determined.For further experimental details refer to Table 2.

These data show that SF is increased from 1.60 (sA = 0%,curea = 0 mol/L) to 4.81 (sA = 25%, curea = 0 mol/L). Urea hasan additional effect. SF is increased from 2.00 (sA = 0%,curea = 4 mol/L) to 6.91 (sA = 25%, curea = 4 mol/L). Thisincrease corresponds to a more than three-fold improvementin the overall peak capacity.

From the data presented in Tables 1 and 3 together withthe experimental data for t0 and tMC (for experimental condi-tions refer to Section 3) and a predefined value for the num-ber of theoretical plates N of 100 000 it is possible to predictthe resolution for adjacent peaks according to Eq. (6). Theresults are shown in Tables S1 in the Supporting Informa-tion for the different separation electrolytes. Table 5 sum-marizes these data, selecting those data providing highestresolution for the most hydrophobic analytes. Without theaddition of urea highest resolution is expected for the mosthydrophobic solutes for sA = 15–20%. If curea = 4 mol/L,

Figure 3. SF dependent on sA determined for curea = 0 mol/L (n)and curea = 4 mol/L (d); for further experimental details refer toTable 2.

highest resolution is expected for sA = 10–15%. The pre-dicted resolution Rs of the critical peak pair under optimumconditions is 1.94.

4.1.2 Urea

Urea is used as an additive in MEKC mainly for the separa-tion of hydrophobic analytes due to its ability to reduceretention factors [54, 55]. Urea changes the water structure inthe separation electrolyte. The dielectric constant [50, 56] andthe viscosity [57, 58] of aqueous solutions of urea areincreased compared to that of pure water. With increasingconcentration of urea, meo was linearly decreased from2.8661024 cm2/Vs (sA = 0%, curea = 0 mol/L) to2.1561024 cm2/Vs (sA = 0%, curea = 5 mol/L) and from1.9261024 cm2/Vs (sA = 20%, curea = 0 mol/L) to1.4261024 cm2/Vs (sA = 20%, curea = 4 mol/L). mMCj j wasdecreased linearly (as also observed by [50, 55]) (from21.6961024 cm2/Vs (sA = 0%, curea = 0 mol/L) to21.5961024 cm2/Vs (sA = 0%, curea = 5 mol/L) and from21.9261024 cm2/Vs (sA = 20%, curea = 0 mol/L) to21.4261024 cm2/Vs (sA = 20%, curea = 4 mol/L).

Employing only urea as modifier the migration timewindow tMC/t0 is widened only to a small extent from 2.44(curea = 0 mol/L) to 3.85 (curea = 5 mol/L) (see Fig. S1B in theSupporting Information), which has also been observed byTerabe et al. [54] and Shi et al. [59]. There is a linear decreasein log k with increasing concentration of urea (see Fig. S3 inthe Supporting Information) as already described by Terabeet al. [54] and others [55]. In the parameter range investigatedthe observed decrease in the retention factor induced by ureais much smaller than that induced by ACN.

The results of the iteration procedure with retention datafor alkyl phenones are given in Table 3. It is interesting tonote that b is increased with increasing curea if sA = 0% while



Table 4. Maxima or poles of f ðN�cÞ

Types I and IIsA (%) 0 5 10 15 20 25

�kmax Type I 1.66 1.85 2.00 2.44 3.34 3.45Type II 2.09 2.41 2.61 3.18 3.91 5.78

N�c;max Type I 0.98 1.67 2.13 3.08 4.58 5.73

Type II 2.05 2.77 3.44 4.49 6.06 8.14logPOW;max Type I 1.72 2.07 2.30 2.77 3.53 4.10

Type II 2.26 2.62 2.95 3.48 4.27 5.31

Types III and IVcurea (mol/L) 0 1 2 3 4 5

�kmax Type III 1.56 1.59 1.69 1.76 1.84 1.96Type IV 2.28 3.00 3.14 3.37 3.34 –

N�c;max Type III 1.20 1.38 1.75 2.01 2.28 2.68

Type IV 3.80 4.93 5.60 6.12 6.69 –logPOW;max Type III 0.65 1.92 2.11 2.24 2.37 2.57

Type IV 3.14 3.70 4.04 4.30 4.59 –

Type VcCaCl2 (mmol/L) 0 1.0 2.5 5.0 7.5

�kmax 1.59 1.68 1.77 1.98 2.08N�c;max 0.99 1.16 1.29 1.60 1.36

logPOW;max 1.72 1.81 1.88 2.03 1.91

Type VIcCaCl2 (mmol/L) 0 0.25 0.5 0.75 1.0 1.5 2.0

�kmax 0.57 – – – – – –kpol – 1.62 0.82 0.66 0.66 0.39 0.26N�c;max 5.87 – – – – – –

N�c;pol – 10.07 6.96 6.22 6.06 4.97 4.66

logPOW;max 4.18 – – – – – –logPOW;pol – 6.28 4.72 4.35 4.27 3.72 3.57

For further experimental details refer to Table 2.

Table 5. Predicted resolution for peak pairs of sassafras oil constituents for different compositions of the separation electrolyte

Predicted resolution

Type IsA = 15%

Type IIsA = 15%

Type IIIcurea = 5 mol/L

Type IVcurea = 1 mol/L

Type VcCaCl2 = 5 mmol/L

Type VIcCaCl2 = 2 mmol/L

1/2 13.12 9.91 12.72 8.50 12.09 10.512/3 20.60 18.07 20.58 14.66 14.97 24.633/4 6.30 6.20 6.04 4.80 3.73 11.334/5 3.75 3.88 3.49 2.98 2.10 8.095/6 5.95 6.46 5.35 4.98 3.20 15.126/7 6.57 7.63 5.60 5.90 3.35 21.777/8 3.13 3.86 2.53 2.98 1.51 14.498/9 2.20 2.81 1.72 2.17 1.04 12.619/10 1.48 1.94 1.13 1.50 0.69 10.1810/11 3.03 3.41 1.90 2.65 1.17 20.45

For peak assignment refer to Fig. 1, for further experimental details refer to Table 2.



there is no clear trend for sA = 20%. As already observed forthe modifier ACN the intercept is strongly decreased withincreasing curea reflecting a significant reduction of retentionfactors. f N�c

� �was calculated from the data presented in Table

3, and the values for tMC and t0. A slight widening of themigration time window (increase in f N�c;max

� ) and a moder-

ate reduction of retention factors reflected by a small shift ofN�c;max to higher N�C induced by the addition of urea can beobserved (data not shown). These effects are much larger forsA = 20%. The calculated maxima �kmax, N�c;max, andlog POW;max are given in Table 4. Independent of sA the max-ima are shifted to higher values. With sA = 0%, SF is onlymarginally improved from 1.35 (curea = 0 mol/L) to 1.52(curea = 5 mol/L), while the improvement is more pronouncedwith sA = 20% (Type IV buffer). Under these conditions SF isimproved from 2.71 (curea = 0 mol/L) to 4.44 (curea = 4 mol/L).

From the data presented in Tables 1 and 3 together withthe experimental data for t0 and tMC (for experimental condi-tions refer to Section 3) and a predefined value for the numberof theoretical plates N of 100 000 it is possible to predict theresolution of adjacent peaks according to Eq. (6) (Table S2 inthe Supporting Information and Table 5). The results predictthat highest resolution for the critical peak pair (Rs = 1.13) willbe obtained with curea = 5 mol/L, if sA = 0%. If sA = 20%,highest resolution is expected for curea = 1 mol/L with a pre-dicted resolution Rs of the critical peak pair of 1.50.

4.1.3 ACN and urea in combination

The data discussed in the last two sections show that syner-gistic effects are present using a combination of the additivesACN and urea. Additive effects were observed for the electro-osmotic mobility. From the linear solvation energy relation-ship (LSER); data determined by Liu et al. [60] the followingalterations in the selectivity are expected to be induced bythese additives: The coefficient s (describing differences inpolar interactions) shows a different dependence on the twoadditives: It is decreased in case of urea, and increased in caseof ACN. The opposite dependence was found for the coeffi-cient b describing the difference in the ability of the mobilephase and the PSP for interactions via hydrogen bonding(phases as hydrogen bond donator, analyte as hydrogen bondacceptor). A strong reduction in the coefficients a (hydrogenbond acceptor properties of the phases) and v (describing cav-ity formation) is observed for both additives. Dodecanophe-none has a retention factor larger than 100 for sA = 25% andcurea = 0 mol/L, while it is 25 for sA = 25% and curea = 4 mol/L. It can be concluded that the separation of very hydrophobicanalytes (log POW.3.5) is only possible with a separationelectrolyte containing simultaneously both modifiers.

4.1.4 CaCl2 as modifier

Divalent metal cations are used in CE for the reduction of theEOF velocity [61]. They interact with the negatively chargedsilanol groups and are adsorbed onto the capillary wall. In

MEKC with the cationic surfactants TTAB and CTAB, diva-lent metal cations (e.g., cCaCl2 = 10 mmol/L) were used for thereduction of the EOF velocity in order to widen the migrationtime window [62]. The use of metal cations in MEKC withanionic micelles, however, has not been reported so far. It islimited to a low concentration of the metal salt due to thehigh Krafft points of the salts of dodecyl sulfate with divalentand trivalent metal counter ions.

The addition of the trivalent metal salts (Al(NO3)3,La(NO3)3, InCl3, or Ce(NO3)3) to a separation electrolyte con-taining 60 mmol/L SDS resulted in the visible precipitationof the dodecyl sulfate salt or in irreproducible measurementswith strong disturbances of the baseline. Another problemwith these trivalent cations was the formation of insolublemetal hydroxides at pH 9.2. Similar effects were observed forthe divalent metal cation Zn21. However, with CaCl2 asmodifier turbid buffer solutions were only observed ifcCa2þ.10 mmol/L.

There is a linear decrease in meo with increasing cCa2þ .With sA = 0% and curea = 0 mol/L, there is a strong reduc-tion of meo from 2.6761024 cm2/Vs (cCaCl2 = 0 mmol/L) to2.0061024 cm2/Vs (cCaCl2 = 7.5 mmol/L) and withsA = 20% and curea = 4 mol/L a more pronounced decreasefrom 1.3661024 cm2/Vs (cCaCl2 = 0 mmol/L) to0.8261024 cm2/Vs (cCaCl2 = 2 mmol/L). mMCj j was slightlyincreased from 21.2761024 cm2/Vs (cCaCl2 = 0 mmol/L) to21.3561024 cm2/Vs (cCaCl2 = 7.5 mmol/L) in separationelectrolyte of Type V. In separation, electrolyte of Type VImMCj j slightly changed from 21.62 to 21.7661024 cm2/Vs

in the range cCaCl2 = 0–2 mmol/L. It should be noted that inseparation electrolytes of Type VI, the condition for therestricted/reverse elution mode is reached. In order todescribe this phenomenon, negative migration times aredefined (for the micelles) analogously to the use of negativemobilities (Fig. 4) [39]. Therefore, negative values were

Figure 4. Dependence of the migration time window tMC/t0 oncCaCl2 for separation electrolytes of Type V (n) and Type VI (s); forfurther experimental details refer to Table 2.



obtained for tMC/t0. For other types of buffer a plot of tMC/t0 isshown in the Supporting Information.

In contrast to the organic modifiers CaCl2 has a negli-gible effect on the retention factors (data not shown). Theparameters obtained from plotting the retention data for thealkyl phenones according to the Martin equation are given inTable 3, a plot of log k against NC is shown in Fig. S4 in theSupporting Information. The parameters a and b can beregarded to be independent of cCaCl2 .

For separation electrolytes of Type VI (with varied cCaCl2)f N�c� �

plotted against N�C is given in Fig. 2B, illustrating theeffect of CaCl2 on the separation of neutral hydrophobicanalytes: The migration time window is widened largely bythe reduction of meo whereas other effects are negligible.With a separation electrolyte containing all three additivesthe restricted elution mode is reached. Values for kmax,N�c;max, and log POW;max corresponding to the maxima orpoles of the functions defined in Section 2 are given inTable 4. As expected, for the normal elution mode the max-ima are only marginally shifted to higher values, while thereis a very interesting dependence observed for the poleswhich (starting from a high value) are shifted to lowervalues with increasing concentration of Ca21. It should benoted that much higher values for f N�c

� �are obtained than

in the normal elution mode (Fig. 2A). Consequently, bring-ing the system into the restricted elution mode has advan-tages for the separation of the hydrophobic solutes investi-gated. The large impact of cCaCl2 on the achievable resolu-tion of adjacent peaks is clearly visible when comparingrecorded electropherograms for sassafras oil constituentswith different compositions of the separation electrolyte(Fig. 5).

From the data presented in Tables 1 and 3 together withthe experimental data for t0 and tMC and a predefined valuefor the number of theoretical plates N of 100 000 it is possibleto predict the resolution of adjacent analytes according to Eq.6 (see Table S3 in the Supporting Information and Table 5).Without the addition of ACN and/or urea there is only asmall improvement in the resolution with increasing cCaCl2due to the widening of the migration window. WithsA = 20% and curea = 4 mol/L, highest resolution is expectedfor cCaCl2 = 2 mmol/L. Under these conditions the predictedoptimum resolution Rs of the critical peak pair is 8.09. How-ever, baseline resolution for all constituents is already expect-ed for cCaCl2 = 0.5 mmol/L.

The SF is a magnitude which can only be calculated for asystem following the normal elution mode with a limitedmigration window, if it is defined as the integralZþ1

�1

f N�c� �

d N�c. In the restricted elution mode (see Fig. 2B)

this parameter can only be calculated for a segment of thef N�c� �

curve. In this case SF is defined as the integral

Zlim N�c;pol

�1

f N�c� �

d N�c . The upper limit of this integral is given

Figure 5. Recorded electropherograms for the separation of sas-safras oil constituents with different compositions of the separa-tion electrolyte: capillary 20/27 cm, 50 mm id, 1.875 mmol/LNa2B4O7, 60 mmol/L SDS, pH 9.2 with (A) 20% v/v ACN, (B) 4 mol/L urea, (C) 4 mol/L urea and 20% v/v ACN, (D) 4 mol/L urea, 20%v/v ACN, 0.75 mmol/L CaCl2, (E) 4 mol/L urea, 20% v/v ACN,2 mmol/L CaCl2; peak assignment (see Fig. 1).

by the tolerable migration time. It cannot exceed N�c;pol. WithsA = 0% and curea = 0 mol/L, SF is moderately increasedfrom 1.43 (cCaCl2 = 0 mmol/L) to 2.26 (cCaCl2 = 5 mmol/L).

4.2 Resolution optimization

The data presented in the previous sections form a databasefor method development. There are several possible criteriafor the selection of optimum conditions. One possible cri-terion is the difference between N�c for the solutes of interest



and the parameter N�c;max or N�c;pol (listed in Table 4). In Fig. 6,N�c;max or N�c;pol is plotted against cCaCl2 for separation elec-trolytes of Types V and VI, the graphs for all six types ofelectrolytes are summarized in Fig. S5 in the SupportingInformation. The values for N�c of the sassafras oil constitu-ents are represented as horizontal lines. Optimum condi-tions can be defined for DN = 0 (DN = N�c 2 N�c;max (orN�c;pol)). According to the data presented in Fig. 6, optimumconditions can be expected for sA = 20%, curea = 4.0 mol/L,cCaCl2 = 2.0 mmol/L. It is also visible that the conditions forsA = 0% and curea = 0 mol/L (separation electrolyte of TypeV) are far from optimum. It should be emphasized that thisprocedure does neither take into consideration the actualvalue of f N�c

� �at N�c;max (compare to Fig. 2) nor changes in

the selectivity parameter b. Therefore, this procedure onlyforms a starting point for an optimization scheme.

As a second optimization criterion f N�c� �

at N�c of thesolute of interest can be defined. The estimation of this pa-rameter is demonstrated in Figs. 2A and B by vertical lines(representing N�c of the sassafras oil constituents (Table 1))intersecting with the f N�c

� �curve. Also with this approach

changes in the selectivity parameter b are neglected. How-ever, for an additive like CaCl2 inducing negligible changesin selectivity the use of this optimization criterion is wellsuited for a fast method optimization.

A third optimization criterion, which can be expected tohave the highest meaningfulness, is the predicted resolu-tion of the critical peak pair (Table 5, Tables S1–S3 in theSupporting Information). According to this criterion theoptimum composition of the separation electrolyte isreached for sA = 20%, curea = 4 mol/L, cCaCl2 = 2 mmol/L. Ifthe selection criterion is defined as the resolution of thecritical peak pair exceeding a predefined threshold value

Figure 6. Maxima n (electrolyte of Type V) or d (electrolyte ofType VI) and poles s (electrolyte of Type VI) of f N

�c

� plotted

against cCaCl2 together with the carbon number equivalents of thesassafras oil constituents (represented as black horizontal lines)(see Table 1), for further experimental details refer to Table 2.

(e.g., 2.00) then the optimum composition of the separationelectrolyte is reached for sA = 20%, curea = 4 mol/L,cCaCl2 = 0.5 mmol/L.

If the predicted resolution Rs of the critical peak pair istaken as optimization criterion, then this criterion has to becalculated with sufficient prediction ability. Therefore, weevaluated the accuracy of the calculation of Rs using N�c asanalyte descriptor and a, b, t0, and tMC as separation electro-lyte coefficients. To this end Rs for adjacent peaks was deter-mined according to standard procedures from recordedmigration times t and peak widths w (Rs = [26(t1 2 t2)]/(w11w2)). In addition, the number of theoretical plates N wascalculated from the recorded migration time t and the peakwidth at half height w0.5 (N = 5.546(tR/w0.5)

2). The predictedresolution Rs, cal for analyte pairs was calculated according toEq. 6 from this experimental value of N, mean values for N�c;1and N�c;2 listed in Table 1, a and b determined for eachseparation electrolyte listed in Table 3 and experimentalvalues for t0 and tMC. In Fig. 7, experimental and predictedvalues are compared for the resolution of peak pairs obtainedwith separation electrolytes of Types I, II, IV, and VI. Theseseparation electrolytes were selected, because they provideadequate resolution for all peak pairs investigated. The linearregression yields Rs,cal = 0.454 1 0.8956Rs,exp with a corre-lation coefficient of R = 0.941. The statistically significantdeviation of the slope of the regression line from 1 (t-test) canbe ascribed to a bias concerning the prediction of high reso-lutions. This bias, however, has no impact on the predictionof optimum conditions for the separation of the analytes ofinterest. Comparing the datasets of each separation electro-lyte via a paired t-test, the results show that on a significancelevel of 0.05, the datasets are not significantly different excepttwo separation electrolytes (Types IV (curea = 2 mol/L) and VI(cCaCl2 = 1 mmol/L)).

Figure 7. Calculated resolution Rs,cal vs. experimentally obtainedresolution Rs,exp for adjacent peaks with separation electrolytesof Types I, II, IV, and VI, for further experimental details refer toTable 2.



The precision of Rs,cal depends on the precision in thedetermination of the parameters a, b, t0, and tMC. Table 6compares values for a, b, mMC, and meo determined in differ-ent capillaries with different batches of the separation elec-trolyte at different days (triplicate measurements) for aseparation electrolyte containing 4 mol/L urea and 20% v/vACN. In Table 7, values for Rs, cal using both datasets of Table6 are compared to each other. From the data obtained at thesecond day much higher resolutions would be expected. Acomparison of the data given in Table 6 suggests that thisdifference in the results might be mainly due to the differ-ence in meo supporting the observation that reducing meo by asuitable modifier (CaCl2) has a strong impact on achievableresolutions. However, substitution of parameters revealedthat it is the accuracy of the parameters a and b which iscrucial for the accuracy of Rs, cal.

Table 6. Comparison of parameters a, b, mMC, meo obtained foridentical composition of the separation electrolyte(1.875 mmol/L Na2B4O7, 60 mmol/L SDS, 20% ACN, and4 mol/L urea), measured at different days with differentbatches of separation electrolyte in different capillaries

b a mMC /1024 cm2/Vs

meo /1024 cm2/Vs

Day 1 0.2356 21.0527 21.289 1.416Day 2 0.2573 20.9391 21.218 1.312

Table 7. Predicted resolution for peak pairs of sassafras oil con-stituents in a separation electrolyte of 1.875 mmol/LNa2B4O7, 60 mmol/L SDS, 20% ACN, and 4 mol/L urea(calculated from data from Table 6) using N�C (Table 1)

Analyte pair Day 1 Day 2

1/2 4.79 6.872/3 9.19 13.023/4 3.17 4.524/5 2.02 2.875/6 3.49 4.916/7 4.32 5.997/8 2.24 3.108/9 1.66 2.299/10 1.17 1.6010/11 2.10 2.85

5 Concluding remarks

Four coefficients, tMC, t0, a, and b, are introduced which aresufficient to fully characterize the separation properties ofseparation electrolytes of different composition in MEKC.The influences of additives to the separation electrolytes canbe characterized using these four coefficients. ACN and ureahave a synergistic influence on the distribution of a hydro-

phobic solute between the micellar and the aqueous phase.This synergistic influence makes it possible to largely reduceretention factors for hydrophobic solutes (here log POW = 3–4) without compromising efficiency. The coefficients alsoshow that the additive CaCl2 can be used to fine-tune the EOFvelocity without having an influence on the retention factorand interestingly with negligible influence on the electro-phoretic mobility of the micelles. With these three modifiers(ACN, urea, and CaCl2) the separation system can easily bebrought into the restricted elution mode, which has advan-tages concerning the achievable resolution for structurallyrelated compounds.

Employing N�c as analyte descriptor and a, b, t0, and tMC

as separation electrolyte coefficients a fast optimization ofthe composition of the separation electrolyte is possible. Tothis end, the function f N�c

� �was defined giving guidelines

for the selection of optimum conditions with respect to theconcentrations of organic modifiers and the EOF modifier inthe normal and in the restricted elution mode.

C. H. thanks for the financial support from the HessianMinistry of Science and Art. Financial support from the FederalCriminal Police Office is acknowledged.

The authors have declared no conflict of interest.

6 References

[1] Roman, G. T., McDaniel, K., Culbertson, C. T., Analyst 2006,131, 194–201.

[2] Li, Q., Chang, C. K., Huie, C. W., Electrophoresis 2005, 26,3349–3359.

[3] Seifar, R. M., Kraak, J. C., Kok, W. T., Anal. Chem. 1997, 69,2772–2778.

[4] Kaneta, T., Yamashita, T., Imasaka, T., Electrophoresis 1994,15, 1276–1279.

[5] Cole, R. O., Sepaniak, M. J., Hinze, W. L., Gorse, J., Oldiges,K., J. Chromatogr. 1991, 557, 113–123.

[6] Gong, S., Liu, F., Li, W., Gao, F. et al., J. Chromatogr. A 2006,1121, 274–279.

[7] Song, L., Zhang, S., Qu, Q., Yu, W., J. Microcol. Sep. 1995, 7,123–126.

[8] Lin, J. M., Nakagawa, M., Uchiyama, K., Hobo, T., Chroma-tographia 1999, 50, 739–744.

[9] Terabe, S., Ishihama, Y., Nishi, H., Fukuyama, T., Otsuka, K.,J. Chromatogr. 1991, 545, 359–368.

[10] Bütehorn, U., Pyell, U., J. Chromatogr. A 1997, 792, 157–163.

[11] Hancu, G., Gaspar, A., Gyeresi, A., Farmacia 2006, 54, 46–55.

[12] Gotti, R., Fiori, J., Hudaib, M., Cavrini, V., Electrophoresis2002, 23, 3084–3092.

[13] Stutz, H., Malissa, H., Jr., Mikrochim. Acta 1998, 129, 271–280.

[14] Spencer, B. J., Purdy, W. C., J. Chromatogr. A 1997, 782, 227–235.



[15] Liu, Y., Gu, J. L., Fu, R. N., J. High Resolut. Chromatogr. 1997,20, 159–164.

[16] Terabe, S., Miyashita, Y., Ishihama, Y., Shibata, O., J. Chro-matogr. 1993, 636, 47–55.

[17] Terabe, S., Miyashita, Y., Shibata, O., Barnhart, E. R. et al., J.Chromatogr. 1990, 516, 23–31.

[18] Norton, D., Shamsi, S. A., Anal. Chim. Acta 2003, 496, 165–176.

[19] Otsuka, K., Wada, M., Ishisaka, A., Terabe, S., Anal. Sci. 2002,18, 101–103.

[20] Palmer, C. P., McNair, H. M., J. Microcol. Sep. 1993, 4, 509–514.

[21] Edwards, S. H., Shamsi, S. A., J. Chromatogr. A 2000, 903,227–236.

[22] Fujimoto, C., Fujise, Y., Kawaguchi, S., J. Chromatogr. A2000, 871, 415–425.

[23] Yang, S., Bumgarner, J. G., Khaledi, M. G., J. High Resolut.Chromatogr. 1995, 18, 443–445.

[24] Lin, C. E., Chen, M. J., J. Chromatogr. A 2001, 923, 241–248.

[25] Esaka, Y., Tanaka, K., Uno, B., Goto, M., Kano, K., Anal.Chem. 1997, 69, 1332–1338.

[26] Jandera, P., Chromatographia 1984, 19, 101–112.

[27] Huhn, C., Pütz, M., Holthausen, I., Pyell, U., Electrophoresis,2008, 29, in press.

[28] Huhn, C., Pütz, M., Dahlenburg, R., Pyell, U., Beiträge zumXIV. GTFCh-Symposium, 14–16 April 2005, Mosbach.

[29] Huhn, C., Pütz, M., Pyell, U., Electrophoresis 2008, 29, inpress.

[30] Bushey, M. M., Jorgenson, J. W., Anal. Chem. 1989, 61, 491–493.

[31] Bushey, M. M., Jorgenson, J. W., J. Microcol. Sep. 1989, 1,125–130.

[32] Terabe, S., Otsuka, K., Ando, T., Anal. Chem. 1985, 57, 834–841.

[33] Pyell, U., J. Chromatogr. A 2004, 1037, 479–490.

[34] Muijselaar, P. G. H., Claessens, H. A., Cramers, C. A., Anal.Chem. 1994, 66, 635–644.

[35] Chen, N., Terabe, S., Nakagawa, T., Electrophoresis 1995, 16,1457–1462.

[36] Ahuja, E. S., Foley, J. P., Analyst 1994, 119, 353–360.

[37] Poothree, K., Leepipatpiboon, N., Petsom, A., Nhujak, T.,Electrophoresis 2007, 28, 767–778.

[38] García, M. A., Marina, M. L., Díez-Masa, J. C., J. Chromatogr.A 1996, 732, 345–359.

[39] Vindevogel, J., Sandra, P., Introduction to Micellar Electro-kinetic Chromatography, Hüthig, Heidelberg 1992.

[40] Foley, J. P., Anal. Chem. 1990, 62, 1302–1308.

[41] Yang, S., Bumgarner, J. G., Kruk, L. F. R., Khaledi, M. G., J.Chromatogr. A 1996, 721, 323–335.

[42] Yang, S., Khaledi, M. G., Anal. Chem. 1995, 67, 499–510.

[43] Herbert, B. J., Dorsay, J. G., Anal. Chem. 1995, 67, 744–749.

[44] Trone, M. D., Leonard, M. S., Khaledi, M. G., Anal. Chem.2000, 72, 1228–1235.

[45] Muijselaar, P. G., J. Chromatogr. A 1997, 780, 117–127.

[46] Ishihama, Y., Oda, Y., Uchikawa, K., Asakawa, N., Anal.Chem. 1995, 67, 1588–1595.

[47] Yang, S., Bumgarner, J. G., Kruk, L. F. R., Khaledi, M. G., J.Chromatogr. A 1996, 721, 323–335.

[48] Bütehorn, U., Pyell, U., J. Chromatogr. A 1997, 772, 27–39.

[49] Lukkari, P., Vuorela, H., Riekkola, M. L., J. Chromatogr. A1993, 655, 317–324.

[50] Liu, Z., Zou, H., Ye, M., Ni, J., Zhang, Y., Electrophoresis1999, 20, 2898–2908.

[51] Giddings, J. C., Anal. Chem., 1967, 39, 1027–1028.

[52] Kolb, S., Welsch, T., Kutter, J. P., J. High Resolut. Chroma-togr. 1998, 21, 435–439.

[53] Muijselaar, P. G., van Straten, M. A., Claessens, H. A., Cra-mers, C. A., J. Chromatogr. A 1997, 766, 187–195.

[54] Terabe, S., Ishihama, Y., Nishi, H., Fukuyama, T., Otsuka, K.,J. Chromatogr. 1991, 545, 359–368.

[55] Pyell, U., Bütehorn, U., Chromatographia 1995, 40, 175–184.

[56] Schick, M. J., J. Phys. Chem. 1964, 68, 3585–3592.

[57] Dworschak, A., Einsatzmöglichkeiten der Mizellaren Elek-trokinetischen Chromatographie, Ph. D. Thesis, Universityof Marburg, Germany, 2000.

[58] Hood, G. R., Physics 1933, 4, 211–214.

[59] Shi, W., Zhang, J., Wang, L., Zou, H. F., Zhang, Y. K., Chin. J.Chem. 1997, 15, 144–153.

[60] Liu, Z., Zou, H., Ye, M., Ni, J., Zhang, Y., J. Chromatogr. A1999, 863, 69–79.

[61] Brechtel, R., Hohmann, W., Rüdiger, H., Wätzig, H., J. Chro-matogr. A 1995, 716, 97–105.

[62] Dworschak, A., Pyell, U., J. Chromatogr. A 1999, 848, 387–400.


Separation of very hydrophobic analytes by micellar electrokinetic chromatography. III....

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