CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

Anna Rocco1

Zeineb Aturki1

Giovanni D’Orazio1

Salvatore Fanali1

Veronika Solínová2

Jan Hlavácek2

Václav Kasicka2

1Institute of ChemicalMethodologies,National Council of Research,Monterotondo Scalo,Rome, Italy

2Institute of OrganicChemistry and Biochemistry,Czech Academy of Sciences,Prague, Czech Republic

Received July 21, 2006Revised November 9, 2006Accepted November 9, 2006

Research Article

CEC separation of insect oostatic peptidesusing a strong-cation-exchange stationaryphase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with astrong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of75 mm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, andtemperature on peptides’ resolution was evaluated. Baseline separation of the studied IOPswas achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were per-formed in the short side mode where the stationary phase was packed for 7 cm only. Theselection of the experimental parameters strongly influenced the retention time, resolution,and retention factor. An acidic pH was selected in order to positively charge the analyzedpeptides, the pI’s of which are about 3 in water buffer solutions. A good selectivity andresolution was achieved at pH ,2.8; at higher pH the three parameters decreased due toreduced or even zero charge of peptides. The increase in the ionic strength of the bufferpresent in the mobile phase caused a decrease in retention factor for all the studied com-pounds due to the decreased interaction between analytes and stationary phase. Raising theACN concentration in the mobile phase in the range 40–80% v/v caused an increase in bothretention factor, retention time, and resolution due to the hydrophilic interactions of IOPswith free silanols and sulfonic groups of the stationary phase.

Keywords:

CEC / Insect oostatic peptides / Peptides / SCX-stationary phaseDOI 10.1002/elps.200600452

Electrophoresis 2007, 28, 1689–1695 1689

1 Introduction

CEC is a modern separation technique, developed by Jor-genson and Lukacs [1] and by Tsuda et al. [2] in the early1980s following the work of Pretorius et al. [3] that is widelyapplied to the separation of mainly uncharged compounds.CEC is a hybrid method that combines the high selectivity ofHPLC and the high efficiency of CE. Recent results focusingon the improvement of column technology expanded theselectivity of this miniaturized technique, that at present isable to perform rapid separations of many classes of com-pounds also including charged species such as chiral and

nonchiral drugs, metabolites, food additives, pesticides, pro-tein digest, peptides, etc. [4–10].

In CEC, both the mobile phase and the analytes aretransported towards the detector by a relatively strong EOFgenerated by the application of a relatively high electric field.Furthermore, in the case of charged/chargeable compounds,also electrophoretic mobility is responsible for this transport.The selectivity is controlled by appropriate interactions be-tween analytes and stationary phase during the run [11–13].Different approaches have been used in order to retain thestationary phase into the capillary, e.g., the stationary phase(i) can be bonded to the capillary wall, (ii) packed, or (iii)polymerized into a capillary tube [14–23].

The flat (piston-like) profile of EOF, unlike the parabolicprofile of pressure-induced hydrodynamic flow, and its inde-pendence of the particle size reduce band broadening, i.e.,increase separation efficiency and make possible theemployment of smaller particles and longer columns.

In any case, most of the literature reports the use of RPmaterials, especially C18-modified silica particles with di-ameters of 3–5 mm. Usually such stationary phases are pow-erful tools for the separation of uncharged compounds,while in the case of basic analytes their applicability cannot

Correspondence: Dr. Salvatore Fanali, Institute of ChemicalMethodologies, National Council of Research, Area della Ricercadi Roma I, Via Salaria Km 29300, I-00016 Monterotondo Scalo,Rome, Italy.E-mail: [email protected]: 139-0690672269

Abbreviations: IOP, insect oostatic peptide; SCX, strong cationexchange; SCX-SP, SCX-stationary phase; TMOF, trypsin modu-lating oostatic factor

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

https://www.researchgate.net/publication/235993331_Capillary_Electrophoresis_in_Chiral_Analysis?el=1_x_8&enrichId=rgreq-1b8b2242-88a0-4f9b-8832-cb2bea2c898c&enrichSource=Y292ZXJQYWdlOzYzNTI5MTU7QVM6MTM5NDU4Mzg1MDI3MDcyQDE0MTAyNjA4Nzg0NjM=

1690 A. Rocco et al. Electrophoresis 2007, 28, 1689–1695

be satisfactory due to some drawbacks, e.g., severe tailingbecause of the interactions between analytes and free silanolspresent on the silica surface. Such problems have been suc-cessfully resolved in HPLC by either adding amino deriva-tives in the mobile phase and/or eliminating silanol groups(end-capped phases). Unfortunately, the second approachcannot be used in CEC because end-capped silica sorbentwould exhibit very low EOF which is not suitable for CEC asit needs a relatively high EOF as driving force for analytesseparations [24].

In order to have a stationary phase with specific char-acteristics for CEC applications, strong-cation-exchange(SCX) phases have been introduced. These stationary phases,where sulfonic acid groups are linked to the silica surface viahydrophilic polymers [25], are able to generate a sufficientlyhigh EOF over a wide pH range and are selective regardlessof analytes because of electrostatic interactions between thesolutes and their surface.

In this work, a silica-based SCX-stationary phase (SCX-SP) was selected for the analysis of some insect oostatic pep-tides (IOPs) and their synthetic analogs by CEC. The sulfonicacid groups of SCX-SP, dissociated even at low pH (pKa ,2),allow to operate with a high EOF in acidic conditions (pH ,3),necessary to have positively charged peptides [26, 27].

IOPs are antigonadotropic insect hormones occurring indifferent species, such as the fleshfly Neobellieria bullata ormosquito Aedes aegypti. These peptides, alternatively alsocalled trypsin-modulating oostatic factors (TMOFs), affectthe reproduction processes of insects and are considered as apotential new type of biologically degradable insecticides.TMOF in A. aegypti (Aed-TMOF), a native decapeptide withthe amino acid sequence H–Tyr–Asp–Pro–Ala–Pro–Pro–Pro–Pro–Pro–Pro–OH, was first described by Borovsky et al.[28] as an inhibitor of the biosynthesis of trypsin-likeenzymes in midgut. It prevents degradation of swallowedblood and thus indirectly stops oocyte growth [29]. Borovskyet al. also suggested the probable structure of the decapep-tide as left-handed helix [29]. Lately, analogs with shortenedC-terminal sequences, cyclic structures, and analogs withproline substituted by dehydroproline or tyrosine substitutedby homotyrosine were synthesized [30–32], and character-ized by NMR and circular dichroism spectra [32, 33]. Theirbiological activities and mechanism of action were tested inthe fleshfly N. bullata with resulting 20–80% reductioneffects on reproduction depending on their structure [34, 35],whereas no effect on oocyte maturation was found in thetests on mice [36]. IOPs and their analogs were also analyzedand separated by CZE in acidic (pH 2.25–2.4) as well asalkaline (pH 8.1) BGEs [37].

In this paper, the optimization of the CEC separation ofthe nine synthetic peptides previously achieved by CZE wasinvestigated by studying the interactions between IOPs andSCX phase; the influence of the pH value, the ionic strengthof buffer, the organic solvent content, the applied voltage andthe temperature on retention time, retention factor and res-olution were evaluated.

2 Materials and methods

2.1 Chemicals

All chemicals used in this study were of analytical reagentgrade with no further purification. Phosphoric acid andammonia solution (30%) were from Carlo Erba (Milan, Italy);sodium hydroxide was from Fluka Chemie (Buchs, Switzer-land). ACN and methanol (MeOH) were obtained from BDH(Poole, UK). Ultrapure water was used throughout the studyand was obtained with a Milli-Q system (Millipore/Waters,Milford, MA, USA).

2.2 Peptide synthesis

IOPs were prepared by solid-phase synthesis on a poly-styrene/divinylbenzene polymer resin with 2-chloro-tritylchloride linker and using 9-fluorenyl-methoxycarbonyl(FMOC)/t-butyl chemistry as described elsewhere [30, 31];for their amino acid sequences, see Table 1. Amino acidswere coupled in dimethylformamide (DMF) by means ofdicyclohexylcarbodiimide (DCCI)/1-hydroxybenzotriazole(HOBt). The synthesized peptides were purified by pre-parative HPLC and characterized by amino acid analysis,MS, analytical HPLC, and CZE [30, 31, 37].

Table 1. IOPs and their amino acid sequences

Peptide number Peptide sequencea)

1 YD2 YDP3 YDPA4 YDPAP5 YDPAP2

6 YDPAP3

7 YDPAP4

8 YDPAP5

9 YDPAP6

a) Sequences are presented in the single letter code. A, alanine;D, aspartic acid; P, proline;Y, tyrosine.

2.3 Instrumentation

An FS 100b ultrasonic bath (Decon, Hove, UK) wasemployed to sonicate the solutions. A Micro pH 2001 pHMeter (Crison, Barcelona, Spain) was used to measure pH.An Agilent Technologies 3DCE system (Agilent Technolo-gies, Waldbronn, Germany) equipped with an UV-visiblespectrophotometric DAD and an air thermostating systemwas used to carry out CEC experiments. UV-absorptiondetection was set at 205 nm. The vial carousel was at a tem-perature of 207C. The inlet and outlet capillary ends werepressurized with 10 bar during CEC analysis. The 3DCEChemStation software (Rev.A.09.01, Agilent Technologies)


Electrophoresis 2007, 28, 1689–1695 CE and CEC 1691

was employed to collect and process data. All samples wereinjected by pressure. The fused-silica capillaries (75 mm id,375 mm od) were purchased from Composite Metal Services(Hallow, UK). Capillaries were packed by using a Series 10HPLC pump (Perkin-Elmer, Palo Alto, CA, USA).

2.4 Samples

Standard stock solutions of peptides (0.1 mg/mL) were pre-pared in water and stored at 2217C. Working standard solu-tions were prepared weekly by adding the appropriate vol-ume of each of the stock solutions directly in a vial anddiluting with water in order to obtain the desired final con-centrations for each compound (5 mg/mL).

The stock buffer solution of 100 mM sodium phosphatewas prepared by diluting the appropriate volume of phos-phoric acid in water and adjusting to the desired pH withconcentrated sodium hydroxide solution. Mobile phasesused for electrochromatographic experiments were dailyprepared by mixing appropriate volumes of stock buffersolutions, water, and organic solvents in order to obtain a10 mM aqueous buffer concentration.

2.5 Column preparation

Fused-silica capillaries were packed in our laboratory withSCX 3 mm particles (X-tec, Bromborough, UK) (Spherisorbporous silica (Waters, Elstree, UK), surface area with 8 nmpores, pore volume of 0.5 cm3/g, functionalized with propylsulfonic acid, with a carbon loading of 4% w/w).

The SCX-SP was suspended in acetone and the slurrypacked into the capillary (30 cm was the packed length). Thecolumn was then flushed with water for 30 min and the inletand outlet frits prepared with a heated wire at about 7007C for5 s. The excess of stationary phase was eliminated by flushingthe capillary with water. The outlet frit was outside coatedwith epoxy resin. The detection window was prepared byremoving the polyimide stratum with a razor (about 1.5 cm).

2.6 Computational methods

The retention factor kCEC was calculated as kCEC = (tr2tEOF)/tEOF, where tr is the retention time of the peptide and tEOF isthe elution time of toluene.

3 Results and discussion

3.1 Selectivity

Based on our experience in CEC, preliminary experimentswere carried out analyzing the studied IOPs in a capillarycolumn (100 mm id633.0 cm total length) packed with C18

5 mm silica particles (23.0 cm). Different mobile phasesbased on mixtures of Tris-formate buffer at pH range 2.5–4.0and ACN (40–80%, v/v) were tested in order to achieve the

baseline separation of the nine IOPs. Besides the stationaryphase exhibited some selectivity towards the studied com-pounds, no satisfactory results were achieved (results notshown). Therefore, a SCX-SP was considered for furtherexperiments. It has been reported that with these chromato-graphic phases, due to the presence of charged sites on thesurface, strong electrostatic interactions with oppositelycharged sample compounds can take place. Therefore, theretention mechanism is mainly based on cation exchange,however, it is noteworthy that electrophoretic mobility ofsolutes can also play an important role in the separationprocess.

As previously reported, in aqueous solution at pH 2.7–3.0the effective charge of IOPs is close to zero, i.e., these peptidesare neutral at this pH [37]. Hence, in order to positivelycharge these compounds, analyses were performed in amobile phase at pH 2.3 with the following composition:100 mM sodium phosphate buffer/water/ACN (10:20:70,v/v/v) and applying a separation voltage of 20 kV. A first cap-illary column was packed with the SCX-SP for 23.0 cm (Leff/Ltot 24.5:33.0 cm, id 75 mm). Under these conditions, theelectrophoretic migration direction of peptides was the sameas the direction of the EOF and because these compoundseluted after the EOF marker, strong electrostatic interactionsbetween the peptides and SCX took place. In any case, nodifferent elution order was observed as compared to CZEseparation [37]. We achieved a complete separation of allcompounds in about 60 min, with noticeable low signal of themost retained compounds. Considering the long analysistime necessary to elute the studied IOPs, experiments weredone applying a low pressure at the inlet of the packed capil-lary in order to speed up and improve the separation. How-ever, due to the relatively low pressure applied because of thelimitation of the instrument (maximum pressure 12 bar), noremarkable improvement was observed. Therefore, consider-ing that analytes were resolved with high resolution factors,the short side mode was applied preparing a capillary packedfor 7.0 cm (Leff/Ltot 8.4:33.0 cm, id 75 mm) and running theexperiments. Figure 1 shows the CEC separation of the ninestudied peptides using the short side mode. As can beobserved, baseline separation of all peptides was achieved in arelatively short time (less than 25 min), with a high efficiency,from 39 000 to 330 000 plates/m, and almost no peak tailing.Repeatability of retention time of unretained compound(EOF marker, toluene) and retention times of IOPs was stud-ied and good values of RSD were obtained (1.5–4.5%,days = 3, n = 5) while the intraday repeatability of retentionfactor was in the range 1.3–1.9% (n = 7).

3.2 Effect of pH, ionic strength, and organic modifier

of the mobile phase

In order to optimize the CEC separation of the IOPs theeffect of the buffer pH, the buffer ionic strength and theorganic solvent content in mobile phase on resolution andretention times of analyzed peptides was studied.



Figure 1. Separation of IOPs by SCX-CEC. Experimental condi-tions: mobile phase, 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v); capillary column, 75 mm id6375 mmod packed with 3 mm SCX stationary phase (packed length,7.0 cm, effective length, 8.4 cm, total length 33 cm); applied volt-age, 20 kV, temperature, 207C, pressure, 10 bar applied to bothelectrode vials, injection by pressure 10 bar60.2 min, UV-absorption detection at 205 nm. tEOF, elution time of EOF marker;for peak identification see Table 1.

Taking into account the previously published data onCZE separation of IOPs [37], a relatively low pH was select-ed in order to positively charge these analytes; chargestatus of peptides is crucial for their electrostatic interac-tions with chromatographic sorbent and, therefore, evensmall pH changes can strongly influence the retention ofpeptides. Over the pH range 2.1–3.0, the retention factors,kCEC, of the IOPs weakly increased from pH 2.1 untilpH 2.8, while at pH 3, they showed a little reduction (seeFigs. 2a and b). This can be explained considering that (i)the ionization of sulfonic groups is partially suppressed atlow pH (2.1) and (ii) at pH 3 the effective charge of thepeptides becomes equal to zero with a reduction of reten-tion time and retention factor, but the separation of com-pounds is still possible due to hydrophilic interactions. AtpH above 3 (pH 4, results not shown), a considerablereduction of retention time was observed and a drop in res-olution and efficiency had occurred as a consequence ofrepulsion between sulfonic groups and peptides that startedto be negatively charged. Figure 3 shows plots of EOF ve-locity versus mobile phase pH, in the range of 2.1–4.0, using100 mM sodium phosphate buffer/water/ACN (10:20:70v/v/v) and an applied voltage of 20 kV. The EOF velocityincreased relatively steeply between pH values 2.1 and 2.3and then raised less steeply and linearly until pH 4.0. Thistrend can be explained considering that in the pH range2.1–2.3 some residual sulfonic acid groups will dissociatewhile the further increment is mainly due to the ionizationof the silanol groups.

Figure 2. Influence of the buffer pH of the mobile phase on valuesof the retention factor of IOPs, kCEC (a and b). For other conditionssee text and Fig. 1.

Figure 3. Effect of mobile phase pH (range 2.1–4.0) on EOF using100 mM sodium phosphate buffer/water/ACN (10:20:70 v/v/v)and an applied voltage of 20 kV. For other conditions see text.

The increased ionic strength of the buffer in the mobilephase is usually responsible for decreased zeta potential,double-layer compression, and reduced EOF. Using theexperimental conditions reported in Fig. 1 and changing thebuffer concentration in the range 5–20 mM, we did notobserve a remarkable change of EOF (data not show). How-ever, we observed a general decrease in retention time of allstudied compounds causing a pronounced decrease in thekCEC, as the electrostatic interactions between the positivelycharged peptides and negatively charged surface of the sor-bent are reduced. This effect was remarkable mainly for themost retained compounds where, for example, the value ofkCEC changed from 21.14 to 9.94 (see Figs. 4a and b).

The effect of ACN concentration in the mobile phase wasalso investigated by increasing the volume percentage from40 to 80 whilst keeping constant the buffer concentration of10 mM and pH 2.3. Concentrations of ACN lower than 40%were not studied because a loss of peptide resolution espe-cially for the less retained compounds was recorded.



Figure 4. Effect of buffer concentration in the mobile phase onthe retention factor of IOPs, kCEC (a and b). For other conditionssee text and Fig. 1.

As can be observed in Figs. 5a and b, increasing the ACNconcentration caused an increase in both retention factor,kCEC, and resolution. This trend is characteristic in the pres-ence of hydrophilic interactions which can take place be-tween analytes and polar groups present in the system (sila-

Figure 5. kCEC values of IOPs versus percentage of ACN (a and b).For other conditions see text and Fig. 1.

nol groups of the silica backbone and sulfonic acid groups inour case) via hydrogen bond [38, 39]; a higher percentage ofACN enhances these interactions. As a consequence, themost polar solute is the most retained.

The influence of ACN on EOF velocity was studied: thisorganic modifier affects viscosity, zeta potential, and therelative permittivity (dielectric constant) of the mobile phase.In most of the works, the EOF increases with the increasingcontent of ACN while keeping the ionic strength and pHconstant. In this study, only going from 60 to 70% of organicmodifier, a remarkable increase in EOF velocity was observed(data not shown).

Figures 6. Influence of applied voltage on retention factor (kCEC) ofthe IOPs (a and b) separated by SCX-CEC. For experimental con-ditions see text and Fig. 1.

Figure 7. Influence of applied voltage on retention time (tr) ofIOPs separated by SCX-CEC. For experimental conditions, seetext and Fig. 5.



Figure 8. CEC separation of IOPs at 30 kV using 100 mM sodiumphosphate buffer/water/ACN (15:15:70 v/v/v) with SCX stationaryphase. For other experimental conditions, see text and Fig. 1.

3.3 Effect of applied voltage and temperature

The applied voltage was changed in the range of 15–30 kV inorder to study its effect on retention time and separationfactor. As can be observed in Figs. 6a and b, a generalincrease in k was achieved by raising the applied voltage witha maximum between 20 and 25 kV. The increase in voltagealso had a strong influence on retention time of all studiedpeptides (see Fig. 7). At 30 kV the baseline separation ofanalytes was achieved in less than 10 min, by using the sameconditions of Fig. 1 with the exception of buffer concentra-tion (15 mM) (see Fig. 8). The effect was more remarkablefor the most retained peptides, e.g., those with six and fiveproline residues in the structure. The relationship betweenEOF and applied voltage was linear in the range of 15–30 kVwith a correlation coefficient of r2 = 0.9924.

The influence of capillary column temperature on theseparation of peptides was also explored performing CECseparations changing temperature in the range of 10–307C,with increments of 57C. As expected, the retention times ofpeptides were shorter at higher temperature and, as a con-sequence, we also noted a reduction in resolution. The peakefficiency, instead, was not significantly affected by this pa-rameter (results not shown).

4 Concluding remarks

A capillary electrochromatographic method was developedfor the separation of nine IOPs. The use of RP CEC did notallow to achieve baseline separation of these peptides underisocratic conditions because of their very similar amino acidsequences and rather high and close hydrophilicity. Experi-ments carried out by employing a capillary packed with anSCX stationary phase were very effective in achieving thebaseline resolution of the studied peptides. Therefore, the

results of this study demonstrate that the SCX stationaryphase can be very helpful for the separation of related syn-thetic peptides by CEC and complementary to other sta-tionary phases (e.g., C18 and C8), especially in the case ofcharged analytes. Under isocratic elution conditions, theseparation of the studied compounds was achieved in a rela-tively short time (less than 10 min using the highest appliedvoltage, 30 kV).

The influence of the pH value and the ionic strength ofthe buffer, the organic modifier (ACN) content in the mobilephase, and the applied voltage and the capillary temperatureon IOPs separation were also examined in order to under-stand better the interaction between these peptides and theSCX stationary phase. Both cation exchange and hydrophilicinteractions resulted to be essential for the separation of an-alyzed compounds.

We are very grateful to Dr. P. Myers, X-tec, Bromborough,UK for the gift of the SCX stationary phase. This work was sup-ported by CNR-AVCR project in the years 2004–2006.

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CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

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