Introduction

N-Acyl amino acid surfactants, which are anionic aminoacid-type surfactants, are of immense importance bothfrom the industrial and the domestic viewpoints becauseof their biodegradability and low toxicity [1]. Micelleformation is an important property of surfactants andhas been the subject of intense research [2, 3, 4, 5, 6, 7,8]. The aggregation number of a micelle is of the sameorder as that for general anionic surfactants (ca.40�100) and increases with increasing concentrations ofsurfactant and/or salt [4, 5, 8]. Furthermore N-acylamino acid surfactants are useful for chiral discrimina-tion. There are two types of chiral discrimination; one inwhich the D-L interaction is more favorable, and a sec-ond where the D-D or L-L interaction is more favorable.The latter is especially important from the standpoint ofimproving optical purity. The chiral discrimination ofN-acyl amino acid surfactants has been reported in the

solid state [9, 10], Langmuir monolayer [11, 12, 13, 14,15], and even for micelles [2, 16]. This effect in the ex-panded state of a Langmuir monolayer and micelle,however, is much smaller than in the solid and con-densed state of a Langmuir monolayer. However, theability of N-acyl amino acid surfactants to act as chiralselectors have been confirmed for both micelles [17, 18,19] and copper complexes [20, 21]. Therefore, thesecompounds have enormous potential as column-packingmaterial for chiral chromatography.

In this study, the effects of the size of the hydrophobicresidue of N-hexadecanoyl amino acid surfactants on theKrafft temperature (KT) and the enthalpy of solution areinvestigated by both solubility measurements and dif-ferential scanning calorimetry (DSC). It is known thatthe KT is an important property of a surfactant becausemicelle particles appear in aqueous solution only abovethe KT. This has obvious applications in industrialprocesses where the KT is critical. Thus, it is also

ORIGINAL CONTRIBUTIONColloid Polym Sci (2003) 281: 363–369DOI 10.1007/s00396-002-0784-y

Akio Ohta

Noriaki Ozawa

Satoru Nakashima

Tsuyoshi Asakawa

Shigeyoshi Miyagishi

Krafft temperature and enthalpy of solutionof N-acyl amino acid surfactants and theirracemic modifications: effect of theamino acid residue

Received: 7 June 2002Accepted: 8 August 2002Published online: 22 October 2002� Springer-Verlag 2002

A. Ohta (&) Æ N. Ozawa Æ S. NakashimaT. Asakawa Æ S. MiyagishiDepartment of Chemistry and ChemicalEngineering, Faculty of Engineering,Kanazawa University, 2-40-20 Kodatsuno,Kanazawa, Ishikawa 920-8667, JapanE-mail: akio-o@t.kanazawa-u.ac.jp

Abstract The Krafft temperaturesand the enthalpies of solution of sixkinds of N-hexadecanoyl amino acidsurfactant (Gly, Ala, Val, Leu, Ile,and Phe) were obtained from bothsolubility measurements and differ-ential scanning calorimetry. It wasshown that the Krafft temperatureof N-hexadecanoyl amino acid surf-actant increased with decreasing sizeof the amino acid residue except forthe case of phenylalanine. On theother hand, the enthalpy of solutionwas endothermic and increased withdecreasing size of the amino acidresidue except for the cases of gly-cine and phenylalanine. It was found

from these results that the D-L inter-action was superior to the L-L inter-action in solid state of N-hexa-decanoyl amino acid surfactantsalt for both the alanine and phe-nylalanine systems. It was suggestedby ab initio calculations that thedifference of the magnitude of thepeptide-peptide hydrogen bondingwas the dominant factor for thechiral effect.

Keywords N-Acyl amino acidsurfactant Æ Krafft temperature ÆEnthalpy of solution Æ Amino acidresidue Æ Chiral effect

important to investigate the effect of the amino acidresidue on the KT of N-acyl amino acid surfactants. Inaddition, we also studied the chiral effects of N-hexa-decanoyl amino acid surfactant on the KT and theenthalpy of solution. These are discussed from theviewpoint of homochiral (L-L or D-D) and the heterochiral(D-L) interactions by use of theoretical calculations.

Experimental

Materials

Glycine (Gly), L-alanine (Ala), L-valine (Val), L-leucine (Leu),L-isoleucine (Ile), L-phenylalanine (Phe) and their racemic mixtures(DL-form) were purchased from Peptide Institute and NacalaiTesque, and used without further purification. N-Hexadecanoylamino acids (C16-amino acid; see Fig. 1) were synthesized by thereaction of an amino acid with hexadecanoyl chloride as describedpreviously [2] and were re-crystallized from mixtures of either di-ethyl ether-ethanol or acetone-methanol. Their purities werechecked by HPLC and DSC and by observing no minimum on thesurface tension vs. concentration curves at 298.15 K. The meltingpoints were measured by DSC and are listed in Table 1. They weredissolved in 1 mM excess aqueous solution of sodium hydroxide.Auramine (guaranteed reagent, Kanto) was used as a fluorescenceprobe for determination of critical micelle concentration (cmc). Theconcentration of the probe molecule was 1·10–5 M.

Solubility

The solubility of N-hexadecanoyl amino acid was measured byHPLC (Japan Optics, BIP-1). After dissolving the N-hexadecanoylamino acids in an aqueous solution of 0.4 M NaCl, the solutionswere kept at 2 �C to deposit the solid Na N-hexadecanoyl aminoacid salts. The addition of NaCl was necessary in order to raise theKT above 0 �C for almost all surfactants. The concentration of allthe solutions prepared was ca. 0.1 mM. After the samples reachedequilibrium in a thermostat at a given temperature, the supernatantsolution was pipetted by syringe equipped with a disc filter (poresize 0.4 lm). A reversed phase chromatography column (TosohTSKgel ODS-120A) and an UV-VIS monitor (Tosoh model UV-8000) were used as a packed column and a detector, respectively.The eluate was a mixed solution of methanol (80%) and 30 mMaqueous solution of phosphoric acid (20%).

DSC

Differential scanning calorimetry (DSC) experiments were made byusing a DSC7 (Perkin-Elmer) thermal analyzer. A sample solution

was prepared in the same way as for the solubility measurements,and was sealed in a large volume capsule (Perkin-Elmer) using anO-ring-sealed 60 ll stainless steel container. The experiments weredone on the solutions in the concentration range 5 to 20 mM usingabout 50 mg of the samples. After the samples were kept at 0 �Cfor 5 h, they were heated at a rate of 0.5 K/min. At least four runswere performed for each system.

Critical micelle concentration

Fluorescence measurements were carried out on a Hitachi fluo-rescence spectrophotometer F-2000 for determination of cmc val-ues of N-acyl amino acid surfactants. Excitation and emissionwavelengths were 410 and 470 nm, respectively. The ratio of thefluorescence intensity in an aqueous solution containing no surf-actant (I0) against that for a surfactant solution (I) was used as anindication of microviscosity and was measured as a function ofconcentration of surfactant and temperature.

Results

Solubility

Figure 2 shows the logarithm of the solubility of N-hexadecanoyl amino acid sodium salts obtained byHPLC as a function of temperature. For C16-DL-Ile, itssolubility could not be determined because its KT wasbelow 0 �C. From Fig. 2 it can be seen that the solu-bility rises abruptly at a particular temperature in allsystems. The temperatures correspond to the KT andthese values are summarized in Table 2. The order ofKT was C16-Ile<-Leu<-Val<-Phe<-Ala, which in-creased with decreasing size of residue except for C16-Phe. For the C16-Ala and C16-Phe systems, the KT ofthe DL-form was higher than that of the L-form. Con-versely, for the C16-Val, C16-Leu, and C16-Ile systems,the KT of the DL-form was lower than that of theL-form. Furthermore, the solubility of C16-Phe below theKT was smaller by one order of magnitude comparedwith the other systems, and its temperature dependencewas much larger. This means that the enthalpy of solu-tion of C16-Phe is substantial as described in the nextsection.Fig. 1 Chemical structure of N-hexadecanoyl amino acid

Table 1 Melting point of various surfactants

Surfactant Melting point(K)

C16-Gly 393.2C16-L-Ala 374.3C16-DL-Ala 388.8C16-L-Val 366.9C16-DL-Val 375.1C16-L-Leu 367.1C16-DL-Leu 355.1C16-L-Ile 367.1C16-DL-Ile 351.2C16-L-Phe 364.6C16-DL-Phe 371.6

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DSC

The thermograms of a 10 mM aqueous solution of NaN-hexadecanoyl amino acid surfactants in the presenceof the solid deposit are shown in Fig. 3. It can be seenthat an endothermic peak accompanies the dissolutionof the surfactant around the KT. In this measurement,KT could be defined as the temperature correspondingto the onset of the peak and these values are shown inTable 2. The KT values determined by this methodgave a similar result to those obtained from the sol-ubility measurements; C16-Leu<-Val<-Phe<-L-Ala<-DL-Ala�-Gly. The enthalpy of solution wascalculated from the peak area and is shown in Table 2.Except for C16-Gly and C16-Phe, enthalpy decreasedwith increasing size of the amino acid residue. Whenthe KT of the DL-form is higher than that of theL-form, the enthalpy of the DL-form is correspondinglygreater than that of the L-form. The enthalpy ofsolution of C16-Phe is surprisingly large compared tothe others.

Critical micelle concentration

Figure 4 shows a dependence of fluorescence intensityratio (I/I0) on concentration for several amino acidsurfactants. The cmc was determined as the concentra-tion at which the fluorescence intensity increasessharply. The cmc determined by this method is in goodagreement with those obtained using other techniques,such as the surface tension method [22]. The results areshown in Fig. 5. With the exception of C16-Ala, theorder of cmc reflects the magnitude of hydrophobicity ofthe amino acid residue (i.e. C16-Phe<-Ile<-Leu<-Val). It was confirmed that the concentration at the KTwas equivalent to the cmc. Contrary to the melting pointand KT, the difference in cmc between the L- and DL-form of amino acid surfactants was not obvious. Weinterpret this result as meaning the optical isomer wasnot developed in a micelle where surfactants were rela-tively loosely assembled compared with the solid state.

Discussion

Krafft temperature

We have shown that the KT of N-hexadecanoyl aminoacid surfactants increased with decreasing the size of thehydrophobic residue of amino acid except for the caseof phenylalanine. This suggests that the increase in sizeof the hydrophobic residue makes the molecular packingof surfactant loosen, thereby causing the KT of surfac-tant to decrease. A comparison of the KT for C16-Leuand C16-Ile indicates that the molecular packing ofsurfactant becomes looser when the branch point of theresidue is situated nearer to the main chain. Since boththe solubility and the cmc of C16-Phe was the smallest,we conclude that this was the most hydrophobic

Fig. 2 Temperature depen-dence of solubility ofNaC16-amino acid salt in aqueous1 mM NaOH and 0.4 MNaCl. Solid circle L-form,open circle DL-form

Table 2 Krafft temperatures and enthalpies of solution of varioussurfactants

Surfactant KT bysolubility

KT by DSC Dhsol

K K kJ mol–1

C16-Gly – 327.2 36.7±2.0C16-L-Ala 311.2 313.9 48.8±2.1C16-DL-Ala 324.1 327.4 61.8±3.3C16-L-Val 294.6 302.6 41.0±1.5C16-DL-Val 291.4 295.7 38.1±2.1C16-L-Leu 290.8 291.7 33.6±1.5C16-DL-Leu 285.5 286.2 28.5±2.3C16-L-Ile 285.8 – –C16-L-Phe 302.1 305.3 104.0±5.2C16-DL-Phe 307.1 309.5 165.0±3.0

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Fig. 3 Thermograms of the10 mM of the aqueous solu-tions of Na C16-amino acidsalt. Solid line L-form, dottedline DL-form

Fig. 4 Dependence of afluorescence intensity ratio(I/I0) on concentration forseveral amino acid surfac-tants in aqueous 1 mMNaOH and 0.4 M NaCl.(1) C16-Val at 308.15 K, (2)C16-Leu at 308.15 K,(3) C16-Ile at 308.15 K, (4)C16-Ala at 318.15 K

Fig. 5 Critical micelleconcentration vs temperaturecurves. (1) C16-Val, (2)C16-Leu, (3) C16-Ile, (4)C16-Phe*, (5) C16-Ala.(The lines are guides for theeyes.) * by surface tensionmeasurement [25]

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surfactant in this study. Furthermore, the aromatic ringof phenylalanine is large and bulky compared to theother amino acid residues. The KT of C16-Phe, how-ever, is relatively high and deviates from the seriesmentioned above. Rather than causing steric hindrance,access between the benzyl groups leads to a favorableinteraction in the solid state, possibly stabilized by p-por CH-p interactions between the aromatic rings [23].

The chiral effect of the amino acid can be divided intotwo types. Firstly, where the KT of the DL-form is higherthan that of the L-form (C16-Ala and -Phe), and sec-ondly where the opposite is the case (C16-Val, -Leu, and-Ile). In the former case, the DL-form of the surfactant isobviously a racemic compound, while it seems to be aracemic mixture in the latter case. The KT of C16-DL-Ala is almost identical to that of C16-Gly. It is reason-able to suppose that C16-Gly, which has the smallestresidue, can be oriented in the solid state most efficiently.This result suggests that a formation of the molecularpair between the L-form and the D-form of C16-Ala isvery effective at enhancing the molecular packing in thesolid state, as is the case for C16-Gly. This is a questionto be considered later. A large residue may prevent themolecular packing. Thus, the DL-forms of C16-Val,-Leu, and -Ile have a lower KT than the L-forms. Fromthe viewpoint of residue size, phenylalanine, whichcontains an aromatic ring, can be considered an excep-tional case. As mentioned above, the interaction be-

tween the benzyl groups is favorable and is enhanced bya combination of the L- and D-forms. Note that the KTof C16-L-Val is higher than that of C16-DL-Val, contraryto the melting point. This may suggest that the nature ofthe counter ion affects the chiral discrimination.

Enthalpy of solution

The enthalpy of solution Dhsol obtained in this studycorresponds to the difference between the enthalpy ofsurfactant in the micellar state h(micelle) and that in thesolid state h(solid).

Dhsol ¼ hðmicelleÞ � hðsolidÞ ð1Þ

As we have mentioned in the previous section, the in-crease in size of the hydrophobic residue causes loosermolecular packing in the solid state and weaker hydra-tion of surfactant in the micellar state. The effect raisesthe values of h(solid) and h(micelle). Therefore, the dif-ference of Dhsol among C16-Ala, C16-Val, and C16-Leuis principally attributable to h(solid), while that betweenC16-Ala and C16-Gly is caused by the variance ofh(micelle). The Dhsol value of C16-Phe was greatest of allin this study. This is because C16-Phe has both thelargest h(micelle), which is due to the contact ofthe hydrophobic benzyl group with water molecules inthe micellar state, and the smallest h(solid), which is due

Fig. 6 a Chemical structure ofdimer of sodium N-acetyl alan-inate. b Top view of optimizeddimer geometries. c Side view ofoptimized dimer geometries

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to the interaction between the benzyl groups in the solidstate.

The chiral effect of the amino acid on the enthalpy ofsolution followed two trends. For C16-Ala and C16-Phe, the Dhsol of the DL-form was higher than that of theL-form, whereas the converse was true for C16-Val andC16-Leu. This classification is equivalent to that for theKT. This fact supports the above conclusion that C16-DL-Ala and C16-DL-Phe form racemic compounds whileC16-DL-Val and C16-DL-Leu are just mixtures. Since theracemic effect was very small in a micelle [2], the enth-alpy of formation of racemic compound Dhrac could beevaluated by subtracting the Dhsol value of the DL-formfrom that of the L-form, under the condition that thetemperature-dependence of enthalpy might be ignored.

Dhrac ¼ hDLðsolidÞ � 12 hLðsolidÞ þ hDðsolidÞ½ �

¼ hDLðsolidÞ � hLðsolidÞ ¼ Dhsol;L � Dhsol;DL

ð2Þ

The values obtained for Dhrac of C16-Ala and C16-Phewere –13 kJ mol–1 and –61 kJ mol–1, respectively. Thesevalues are much larger than the interaction energiesbetween either methyl or benzyl groups and correspondto the energy of hydrogen bonding. The combination ofthe L- and D-forms of C16-Ala or C16-Phe enhances thecontribution of hydrogen bonding between adjacentpeptide groups of the surfactant. This is especially sig-nificant for C16-Phe where the value of Dhrac is verylarge. In addition to the increased hydrogen bondingbetween the peptide groups and hydrophobic interac-tions between the benzyl groups, contact between theacyl moiety is also enhanced for the C16-DL-Phe system.

Calculated structure of Na N-acetyl alaninate dimer

We have applied ab initio calculation to clarify the dif-ference between the L- and DL-form of Na C16-Ala. Thedimer of sodium N-acetyl alaninate (see Fig. 6a) wasemployed as a model compound for simplification ofcalculation in this study. The dimerization energy andthe optimized geometry were calculated by using therestricted Hartree-Fock (RHF) procedure with the 6–31+G* basis set. All calculations were performed usingGaussian 98 [24].

Obtained geometries of L-L and D-L dimers are shownin Fig. 6b. Each dimer is built by coordination of twocarboxylates to two sodium ions, simultaneously. Thetop views of the two structures seem to be similar to each

other. Indeed the calculated dimerization energies were –93.856 and –93.869 kJ mol–1 for the L-L and D-L dimers,respectively. However, the difference is clear from theside view of the structures in Fig. 6c. In the D-L dimer,each alanine residue is situated in the trans positionthrough the bonding between carboxylates, while in thegauche position for the L-L dimer. Although this explainsthe small difference in the dimerization energies betweenthe L-L and D-L dimers obtained by ab initio calculations,it does not explain the difference in Dhrac obtained byDSC measurements. Of course in the experimental sys-tem, the interaction between not only the dimers but alsothe neighboring molecules are investigated. In this case,one of the most significant interactions is the hydrogenbonding between adjacent peptide groups. When thiswas taken into consideration, it was found that the di-mers could interact with the two adjacent molecules atthe two hydrogen bond lines (Fig. 6b and Fig. 6c). Itshould be noted that one line is parallel to the other inthe D-L dimer, whereas the two lines intersect each otherin the L-L dimer (Fig. 6c). This suggests that the DL-formof Na N-acyl alaninate can create a sheet structure sta-bilized by hydrogen bonds between the peptide groupsmore effectively than the L-form. Therefore, Na N-acylDL-alaninate becomes a racemic compound in the solidstate. This could explain why Na N-acyl DL-valinate andDL-leucinate are less stable than their L-form counter-parts, since the larger hydrophobic residues may hinderthe stacking in the sheet-like structure. Conversely, inthe case of Na N-acyl DL-phenylalaninate, contact be-tween the benzyl groups in the sheet structure is an ad-vantageous factor and results in the racemic compound.

Conclusions

The Krafft temperature of N-hexadecanoyl amino acidsurfactant increased with decreasing size of the aminoacid residue, except for the case of phenylalanine. Theenthalpy of solution was increased with decreasing sizeof the amino acid residue, except for the cases of glycineand phenylalanine. Experimental results showed that theD-L interaction was superior to the L-L interaction in thesolid state of N-hexadecanoyl amino acid surfactant saltfor both the alanine and phenylalanine systems, whilethe L-L interaction was advantageous for both the valineand leucine systems. It was suggested that both thepeptide-peptide hydrogen bonding and the steric hin-drance of the amino acid residue govern the chiral effect.

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