Analytica Chimica Acta 428 (2001) 83–88

Sucrose effect on reversed-phase liquidchromatography solute retention

Eric Peyrina,∗, Yves Claude Guillaumeb, Catherine Grosseta,Annick Villet a, Anne Ravela, Josette Alarya

a Laboratoire de Chimie Analytique, Faculté de Pharmacie, Domaine de la Merci, 38700 La Tronche, Franceb Laboratoire de Chimie Analytique, Faculté de Médecine et Pharmacie, Place Saint-Jacques, 25030 Besançon cedex, France

Received 28 December 1999; received in revised form 4 September 2000; accepted 20 September 2000

Abstract

The reversed-phase liquid chromatography retention of a series of benzoate ester molecules was investigated over a widerange of sucrose concentrations (c) 0.01–0.8 M using a C18 as the stationary phase and a methanol–water mixture with highorganic fraction (60/40, v/v) as the mobile phase. A theoretical treatment was developed to investigate the effect of sucrosemolecules on the equilibrium between the solutes and the C18 phase and the methanol–water medium, respectively. Thiswas found to be adequate to accurately describe the benzoate ester retention behavior whenc varied. It was expected thatthe addition of sucrose was responsible for two main opposite contributions to solute retention, (i) a net interaction betweenthe solute and the modifier in the mobile phase determining a decrease ink′ values withc up to 0.2 M; and (ii) an increasein the solute affinity for the C18 stationary phase due to the salting-out effect governing a retention increase above 0.2 M.Thermodynamic parameter variations were calculated using van’t Hoff plots and discussed in relation to this retention modelto confirm the respective effects of the modifier on the solute affinity towards the two phases. © 2001 Elsevier Science B.V.All rights reserved.

Keywords:Sucrose; Reversed-phase liquid chromatography; Benzoate ester

1. Introduction

Many papers have investigated the retentionmechanism in reversed-phase liquid chromatog-raphy (RPLC). Adsorption-like (solvophobic) andpartitioning-like models developed successively byHorvath et al. [1] and Dill [2] constitute the two maintheories which have been established to describe themechanistic aspects of solute retention. The distinc-tion between these two models is related to the role

∗ Corresponding author. Tel.:+33-476637145;fax: +33-476518667.E-mail address:eric.peyrin@ujf-grenoble.fr (E. Peyrin).

of the stationary phase. This is minimized in thesolvophobic theory, whereas the partitioning theoryconsiders the bonded phase as an active player in theretention process [3]. However, the variations in re-tention in RPLC when the mobile phase compositionvaries are mainly dominated by the modification in thesolute–mobile phase interaction. For a hydro-organiceluent, it has been demonstrated by Carr et al. [4]that the decreasing retention of weak polar solutes asthe volume fraction of organic modifier increased isdue to the decrease in the strength of the hydropho-bic effect. As well, our group has shown that thestructural organization of a hydro-organic mixture is

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84 E. Peyrin et al. / Analytica Chimica Acta 428 (2001) 83–88

strongly implicated in the solute retention mechanismand is dependent on the nature of interaction existingbetween the organic modifier and the water [5–7].Acetonitrile (ACN) molecules are associated to formhydrophobic aggregates (or clusters) [5,6], whereasmethanol (Me) solution is dominated by hydrogenbonding to obtain mixed clusters of Me/water [7].The effect of increasing ionic strength of the mobilephase on the solute retention has been ascribed tothe increased surface tension and the concomitant in-crease in the energy required for the cavity formationin the eluent [8]. In an effort to extend the investiga-tion of this salting-out effect on the solute retentionin RPLC, the influence of sucrose on the eluentproperties is examined in this work. The aim of thispaper was to examine how sucrose affected the in-teraction between the solute and both the mobile andthe stationary phases and assess the difference withthe salting-out agents which are traditionally used inchromatographic systems. Using a physicochemicalmodel derived from the Wyman linkage relation andpreviously adapted to other chromatographic systems[9–11], the retention factor variation of a benzoateester series on a C18 stationary phase was accuratelydescribed over a wide range of sucrose concentration.A thermodynamic study was then carried out to pro-vide further information on the mechanistic aspectsof solute retention in such a chromatographic system.

2. Theory

The structure of methanol/water (W) mixture de-pends on the existence of hydrogen-bonding betweenthe water molecule and, respectively, the hydrophilic(Eq. (1)) and the hydrophobic ends of the alcoholmolecule (Eq. (2)) [12]

Me + W ↔ MeW (1)

Me + δW ↔ MeWδ (2)

It has been demonstrated that the presence of the MeWassociation is predominant in high methanol fraction[13]. Thus, in the mobile phase used in the study, thebenzoate molecule (B) is expected to be preferentiallysolvated by the Me free species and the MeW clusteras described by the two following equations:

B + Me ↔ BMe (3)

B + MeW ↔ BMeW (4)

In a previous paper [14], the retention behavior ofweak polar solutes was modeled to accurately describethe variation in the equilibrium constantK for the an-alyte transfer from the mobile to the stationary phase,when the volume fraction of methanol varied. Thismodel was related to the following:

K = KBLs

1 + KBMeχMe + KBMeWχMeW(5)

where KBLs, KBMe and KBMeW are the equilibriumconstants for the B molecule transfer from a totalaqueous eluent to the C18 bonded phase, the B solva-tion by the free Me molecule and B solvation by theMeW association, respectively. The constantχ val-ues correspond to the product of the volume fractionand molar density of the organic species Me or MeW[14].

As well, it has been known for many years thatthe addition of sugars such as sucrose or lactose andsalts such as LiCl or NaCl in an aqueous environmentstrengthen the hydrophobic effect between apolarspecies and, then, decrease the apolar solute solubilityin water [15]. Thus, these substances have been namedsalting-out agents. It has been shown by several au-thors that this increase in “hydrophobic interaction”is related to the enhancement of the cavitation energydue to the increased surface tension effect [16–18].The influence of this type of water structure mod-ifier on solute retention has been previously mod-eled for various chromatographic systems [9–11].The model equation derived from the adsorptionisotherm of Gibbs corresponds to the Wyman linkagerelation(

d1G0

d lnc

)T

= RTλ (6)

where1G0 is the free energy change for the solutetransfer from the mobile to the stationary phase,RTthe thermal energy,c the sucrose concentration in themobile phase andλ a constant related to the excessof sucrose molecules at the solute–stationary phaseinterface. Thec dependence on theKBLs constant isgiven by integration of Eq. (6)

KBLs = γ cα (7)

E. Peyrin et al. / Analytica Chimica Acta 428 (2001) 83–88 85

with α and γ constants. When Eq. (7) is integratedinto the Eq. (5), the following is obtained.

K = γ cα

1 + KBMeχMe + KBMeWχMeW(8)

This equation describes the sucrose dependence on theequilibrium constantK for only the salting-out proper-ties of the modifier. In this approach, no allowance ismade for the direct interaction which occurs betweenthe solute and the sucrose molecules. However, it iswell known that the sugars are more apolar moleculesthan either methanol or water molecules [19]. Sucroseis characterized by its strong capabilities to engagevan der Waals (vdW) interactions with weak polar so-lute. The vdW superficial tension (γ ) of sucrose ismuch higher (equal to 41.6 mJ/m2) than the vdW su-perficial tension of water (21.6 mJ/m2) or methanol(18.2 mJ/m2) [20]. Thus, the interaction between thebenzoate ester and sucrose was expected to be largelyfavored in aqueous-methanol media. So, an additionalequilibrium can be written

ρiS+ B ↔ SρiB (9)

wherei varies from 1 ton in relation to the number ofthe different associations between B and S molecules.Thus, the global equilibrium constantK for the solutetransfer can be described by

K = γ cα

η + ∑ni=1εicρi

(10)

with η constant. It is also well known that the retentionfactor for a solute is related to the equilibrium constantK by

k′ = ϕK (11)

where ϕ is the phase ratio. Thus, Eq. (10) can berewritten as follows

k′ = κcα

η + ∑ni=1εicρi

(12)

In this last equation, the retention factor is linked to thesucrose concentrationcand theκ, η, α andε constants,which are dependent on the chemical properties of thesolute in this chromatographic system.

3. Experimental section

3.1. Apparatus

The HPLC system consisted of a HPLC waterspump 501 (Saint Quentin, Yvelines, France), anInterchim Rheodyne injection valve model 7125(Montluçon, France) fitted with a 20ml sample loop, aMerck 2500 diode array detector (Nogent sur Marne,France). A LICHROCART 125 mm× 4 mm RP18column (5mm, particle size) was used with con-trolled temperature in an Interchim oven TM no. 701(Montluçon, France).

3.2. Reagents

All the alkyl (methyl, ethyl, propyl, butyl) benzoateester molecules were synthesized in our laboratory byan esterification reaction [5]. Samples were preparedat a concentration of 10–80 mg/l in methanol. Sodiumnitrate (Merck, Nogent sur Marne, France) was usedas a dead time marker. Water was obtained from an El-gastat option water purification system (Odil, Talant,France) fitted with a reverse osmosis cartridge. Themobile phase consisted of an methanol–water mixture(60/40, v/v). The variation range of the sucrose (Pro-labo, France) was 0.01–0.8 M. The 20ml of each solutewere injected and the retention times were measured.

3.3. Temperature studies

Compound retention factors were determined overthe temperature range 15–45◦C. The chromatographicsystem was allowed to equilibrate at each temperaturefor at least 1 h prior to each experiment. To study thisequilibrium process, the retention time of the propylester was measured every hour for 7 h and again after20, 21 and 23 h. The maximum relative difference ofthe retention time of this compound between thesedifferent measurements was always 0.3%, making thechromatographic system sufficiently equilibrated foruse after 1 h.

4. Results and discussion

4.1. Validation of the model and retention behavior

The retention factor values were determined fora wide range of sucrose concentrations at a column

86 E. Peyrin et al. / Analytica Chimica Acta 428 (2001) 83–88

temperature= 15◦C. All the experiments were re-peated three times. The coefficients of variation ofthe k′ values were<3% in most cases, indicating ahigh reproducibility and good stability for the chro-matographic system. Using a weighted non linearregression (WNLIN) [21,22], the data were fitted toEq. (12). The WNLIN regression method was used tocalculate the optimum parameter values by simultane-ously minimizing theχ2 function with respect to eachof the parameters. After the WNLIN procedure, thecalculated parameters were used to estimate the reten-tion factors for each solute. The correlation betweenthe predicted and experimentalk′ values exhibitedslopes equal to 0.96 withr2 > 0.95. This good corre-lation between the theoretical and experimental valueswas considered to be adequate to verify the model. Allthe benzoate molecules exhibited a similar variationfor the retention factor withc. Fig. 1 represents, boththe experimental and the theoretical curve re-createdusing Eq. (12) for butyl benzoate. It was shown thatthe solute retention was minimal for ac value equal toaround 0.2 M. All the curves were globally related tothe following general equation derived from Eq. (12)

k′ ≈ A√

c

B + D√

c + Ec(13)

where A, B, D and E were coefficients dependentonly on the physico-chemical properties of the solutein the given chromatographic system. Fig. 2 showsthe chromatograms representing the retention of butylbenzoate atc equal to 0.01 and 0.2 M.

For thec values below 0.2 M, the retention varia-tion with c was dominated by the denominator terms,

Fig. 1. Experimental variation (s) in the retention factor forbutyl benzoate in relation to the sucrose concentrationc (M) atT = 15◦C. The theoretical curve (—) was recreated using Eq. (12).

Fig. 2. Chromatograms representing the retention of butyl benzoateat a sucrose concentrationc = 0.01 (A) and 0.2 M (B).

i.e. D√

c and Ec. Whenc increased, the interactionbetween solute and the sucrose molecule governed thedecrease in thek′ value. These results confirmed thatsugar increased the strength of the mobile phase. Thiswas explained by the fact that the sucrose moleculeswere more apolar than the W, Me or the MeW species.The solute was able to engage strong vdW interac-tions with the sugar in such a way that the soluteaffinity for the C18 stationary phase decreased. As

E. Peyrin et al. / Analytica Chimica Acta 428 (2001) 83–88 87

well, it was observed from Eq. (13) that the molecularassociation between S and the benzoate ester impliedthe presence of one (c term) or one-half (

√c term)

molecule of sucrose for one molecule of solute. Atlow concentration, this modifier increased the weakpolar solute affinity for the mobile phase and was incontradiction with the characteristics of the classicalsalting-out agents such as NaCl salt [18]. As well,to the best of our knowledge, no previous exampleof such a paradoxical effect has been observed forsugars or polyols. This could be explained by thefact that, in most cases, the surface tension propertiesof these molecules have been examined over a highconcentration range superior to 0.2 M [23,24].

For c values above 0.2 M, the retention variationwith c was dominated by the numerator terms, i.e.A

√c. The solute retention increase was related to the

salting-out effect of the sugar. The enhancement inthe cavitation energy determined a facilitation in theinteraction between the solute and the C18 stationaryphase. When the sucrose concentration increased, thesugar capability to act on the hydrogen-bonded waterstructure largely counterbalanced the net interactionbetween the solute and the modifier in such a way thatthe solute affinity for the mobile phase decreased.

4.2. Temperature effects on the retention factor andthermodynamic parameter variations

To study the influence of temperature on thek′ val-ues, the same experiments were carried out atT = 20,25, 30, 35, 40 and 45◦C. Thek′ value can be brokendown into enthalpic1H0 and entropic1S0 terms togive the van’t Hoff equation

ln k′ =(

−1H 0

RT

)+

(1S0

R

)+ ln ϕ (14)

If there is no change in the solute interactions in re-lation to temperature, then a plot of lnk′ versus 1/Twould be linear with a slope of−1H0/R and an inter-cept of (1S0/R) + ln ϕ. This provided a convenientway of calculating the thermodynamic parametersfor a chromatographic system if the phase ratio isknown or can be calculated. Usually,1S0 is not pro-vided due to the ambiguity in the calculation of thephase ratio for commercial columns. In this work, aphysical model was used to estimate the phase ratio

Fig. 3. Influence of sucrose concentrationc (M) on 1H0 (kJ/mol)for butyl benzoate.

with a physical constant of the packing material [25].The van’t Hoff plots were all linear for the benzoatemolecules at all sucrose concentrations. The corre-lation coefficients for the linear fits were in excessof 0.98. The typical standard deviations of slope andintercept were, respectively, 0.008 and 0.08. All thebenzoate ester molecules exhibited a similar variationfor the thermodynamic parameters withc. For exam-ple, Figs. 3 and 4 show the variation in1H0 and1S0

with c for the benzoate butyl molecule. The negativevalue of the1H0 and 1S0 terms demonstrated thatthe retention was controlled enthalpically. This wasconsistent with results reported in the literature forvarious chromatographic separations [26,27].

Whenc was infarior to 0.2 M,1H0 value increasedfor all the solutes. It has been previously demonstrated

Fig. 4. Influence of sucrose concentrationc (M) on 1S0 (J/mol/K)for butyl benzoate.

88 E. Peyrin et al. / Analytica Chimica Acta 428 (2001) 83–88

that the vdW interactions are characterized by a de-creasing variation in enthalpic term [28]. Thus, the1H0 value for the solute transfer become more posi-tive due to the vdW interactions engaged with the su-crose in the mobile phase. The entropic term1S0 wasnot significantly affected by the variation of sucroseconcentration forc inferior to 0.2 M. This confirmedthat the solute retention decrease was principally en-thalpically driven for the low concentration value ofthe modifier.

As c increased above 0.2 M, the solute interactionincrease with the C18 bonded phase was governedby the modification of the water structure induced bythe sugar. When the solute was transferred from themobile to the stationary phase, the amount of work re-quired for the exclusion of the sucrose molecule formthe solute–stationary phase interface was reduced byinterpenetration of solvation layers of the two com-ponents. This was characterized by a decrease in theenthalpic and entropic terms of the solute transfer.Based on these results, it can be said that the ther-modynamic trends confirmed the theoretical modeltaking into account the two opposite effects of sucroseon solute retention.

5. Conclusion

This paper described a model that could determinethe contribution of the sucrose modifier to the ben-zoate ester retention on a C18 stationary phase. Usinga simple physico-chemical model, it was possible todescribe the different equilibria which were impliedwhen the compound was transferred from the mobileto the stationary phase. The model was validated by anon-linear regression procedure and confirmed by thethermodynamic variations. It was shown that the su-crose molecule acted on the benzoate ester retentionthrough two reverse contributions, (i) an enhance-ment of the solute affinity for the mobile phase dueto its apolar property (vdW interactions); and (ii) aninteraction decrease with the eluent determined byits donor property for hydrogen-bonding (hydropho-bic effect). This approach showed that the sucrosemolecule was not only a water structure modifier, butalso possessed the capability to interact directly withweak polar solutes.

Acknowledgements

We thank Mireille Thomassin for her technicalassistance.

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