ISSN 0036�0244, Russian Journal of Physical Chemistry A, 2014, Vol. 88, No. 6, pp. 1076–1077. © Pleiades Publishing, Ltd., 2014.Original Russian Text © V.I. Deineka, M.S. Lapshova, L.A. Deineka, 2014, published in Zhurnal Fizicheskoi Khimii, 2014, Vol. 88, No. 6, pp. 1079–1081.

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Adding cyclodextrins (CDs) to a mobile phaseunder the conditions of reversed phase high perfor�mance liquid chromatography (RP HPLC) is widelyused to separate enantiomers and change the selectiv�ity of the separation of structural isomers or varioustypes of compounds [1]. This effect is associated withthe formation of inclusion complexes between CDsand substrates in the mobile phase. The complexationconstants can be calculated from the quantitativechange in the retention of substrates [2]. If compoundA forms a complex with CD in a molar ratio of 1 : 1(ACD):

A + CD ↔ ACDwith the apparent complexation constant KACD, and if

(a) the modification of the stationary phase can beignored and

(b) the ACD complex is not absorbed on thereversed phase because of the hydrophilicity of theexternal surface of CD, the following correlation istrue:

, (1)

where k0 and ki are the retention factors of sorbate iwith and without the addition of CD with the concen�tration [CD] in the mobile phase, respectively.

However, CDs are capable of first forming inclu�sion complexes with alkanes [3], so the modificationof grafted octadecyl (as well as octyl) radicals by CDscannot be excluded a priori. We may assume that suchcomplexes can modify the stationary reversed phase,thereby increasing the retention of polar compoundsas if the activity of residual silanol groups wereincreased [4]. Second, the inability of the inclusioncomplexes of compounds in CD to absorb on reversed

ACD CD0 1 [ ]i

kK

k= + ⋅

phases is also not evident in general, since a number ofexperimental data indicate that small molecules(to which alkyl groups immobilized on a sorbent canbe assigned as well) can strengthen inclusion com�plexes. Calculations using the MM+ method (Hyper�Chem 8.0) thus showed that the cavity of CD canaccomodate two alkane molecules: if one is an inocu�lated radical, the other can be a molecule from themobile phase [4].

A great many works are known for flavonoids, inwhich their inclusion complexes with various CDswere the subjects of research; however, there are veryfew similar works on anthocyanins (which constituteone of the most important classes of flavonoids). It isknown that upon the addition of a CD, a rise in pHleads to discoloration of solutions of colored anthocy�anins, which exist predominantly in a charged flavil�ium form in a strongly acidic medium (pH < 1) [5]. Itis therefore believed that inclusion complexes areformed not by anthocyanins in flavilium form, but byanthocyanins in colorless form [6].

Our calculations using the MM+ method (in theHyperChem 8.0 environment) showed that inclusioncomplexes of cyanidin�3�glucoside and pelargonidin�3�glucoside can be formed (Fig. 1); however, the freerotation of the glycosidic radical relative to the antho�cyanidinic framework should displace anthocyaninsfrom the cavity of a CD. The removal of the glycosidicradical (a transition from anthocyanins to anthocyani�dins) should promote complexation, as is confirmedby the literature data, e.g., by the ability of pelargoni�din to form inclusion complexes in the flavilium formdirectly [7].

If our assumption about the interfering influence ofglycosidic substituents is true, then complexationshould be promoted not only by their removal, but also

Using Reversed Phase High Performance Liquid Chromatographyto Study the Complexation of Anthocyanins with β�Cyclodextrin

V. I. Deineka, M. S. Lapshova, and L. A. DeinekaBelgorod State National Research University, Belgorod, 308015 Russia

e�mail: deineka@bsu.edu.ruReceived August 27, 2013

Abstract—It is shown by means of reversed phase high performance liquid chromatography (RP HPLC) withmobile phases containing additions of β�cyclodextrin that 5�glucosides of cyanidin and pelargonidin formstronger inclusion complexes than 3�glucosides; this is explained by the steric interference of the glucosideradical.

Keywords: reversed phase high performance liquid chromatography, anthocyanins, complexation withβ�cyclodextrin, complexation constants.

DOI: 10.1134/S0036024414060089

SHORTCOMMUNICATIONS

RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 88 No. 6 2014

USING REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 1077

by their movement from position 3 to the position 5.This assumption can hardly be verified by means ofspectrophotometry because equilibrium in the solu�tion is established over several hours and 5�glicosides(in contrast to 3�glicosides) of anthocyanidins are notstable. It is especially convenient here to use the RPHPLC technique with the addition of CD in themobile phase.

In this work, we used the additions of β�CD in themobile phase during the chromatographic run of apartially hydrolyzed extract of rose flowers, whichcontained 3,5�diglucosides, 3� and 5�glucosides ofcyanidin (the main component) and pelargonidin(Fig. 2).

According to our data, the retention of 3,5�diglu�cosides and 3�glucosides even increases slightly whenthe concentration of β�CD in the mobile phase grows.This confirms the appropriateness of our assumptionabout a certain modification to the stationary phase byCD. However, the drop in the retention in the case of5�glucosides is more than evident, which agrees withthe removal of steric hindrances, thus promoting thecomplexation with CD. Finally, our calculations usingEq. (2) allows us to assess the apparent complexationconstants with no correction for the rise in the reten�tion of polar compounds due to the CD’s modificationof the stationary phase: 10.7 and 38.0 for cyanidin�5�glucoside and pelargonidin�5�glucoside, respectively.Note that in this case, the rise in the complexationconstant is evidence of the introduction of anthocyaninin the cavities of CDs with the B ring (Fig. 1), and is inqualitative agreement with the change in the constantsof phenolic acid complexation with β�CDs and thecomplication of the structure of the phenolic ring [8].

REFERENCES

1. S. R. Gratz, B. M. Gamble, and A. M. Stalcup, Ency�clopedia of Separation Science, Ed. by I. D. Wilson(Elsevier Science, London, 2000), p. 3079.

2. F. G. Sanchez, A. N. Diaz, J. Lovillo, and L. S. Feria,Anal. Chim. Acta 328, 73 (1996).

3. M. S. Lapshova, E. V. Zakharenko, V. I. Deineka,L. A. Deineka, R. N. Saenko, and M. Yu. Tret’yakov,Nauch. Vedom. BelGU, Ser. Est. Nauki 15 (20), 129(2012).

4. V. I. Deineka, Russ. J. Phys. Chem. A 81, 396 (2007).

5. T. Yamada, T. Komiya, and M. Akaki, Agric. Biol.Chem. 44, 1411 (1980).

6. O. Dangles and R. Brouillard, J. Chem. Soc., PerkinTrans. 2, 247 (1992).

7. Y. Matsui, A. Sunouchi, and T. Yamamoto, J. Incl. Phe�nom. Mol. Recogn. Chem. 32, 57 (1998).

8. I. P. Anisimovich, V. I. Deineka, L. A. Deineka, andV. F. Selemenev, Sorbts. Khromatogr. Protsessy 10, 401(2010).

Translated by E. Boltukhina

A

B

12

3

5

Fig. 1. Cyanidin�3�glicoside in a cavity of β�CD. Thestructure of the complex in vacuum is optimized using theMM+ method in the HyperChem 8.0 software package.

0

20 4 6 8 10 tR, min

200

400

V, mV

A

B

C

D

1

2

3 4

5 6

Fig. 2. Retention of anthocyanins in mobile phases with β�CD. Column: Diasphere�110 5C18, 100 × 4.0 mm; mobilephase: CH3CN 10 vol % and H3PO4 0.5 vol %; 1 mL/min.Detector: 515 nm. Concentration of β�cyclodextrin: (A) 0,(B) 2.16 × 10–3, (C) 6.48 × 10–3, and (D) 8.63 × 10–3.Anthocyanins: (1) cyanidin�3,5�diglucoside, (2) pelar�gonidin�3,5�diglucoside, (3) cyanidin�3�glucoside, (4)cyanidin�5�glucoside, (5) pelargonidin�3�glucoside, and(6) pelargonidin�5�glucoside.