High-Performance Liquid Chromatographic Analysis of Phenolic

Chapter 5

High-Performance Liquid Chromatographic Analysis of Phenolic Compounds in Foods

Downloaded by UCSF LIB CKM RSCS MGMT on November 25, 2014 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0506.ch005

Amrik L. Khurana Whatman Specialty Products, Inc., 341 Kaplan Drive, Fairfield, N J 07004

HPLC analysis of various food phenolic materials is discussed. Several mechanisms such as reverse phase, normal phase and anion-exchange to separate phenolic compounds on C-8, C-18, weak cation exchange and amino phases are elaborated. HPLC-resolution of the natural phenolic components such as anthocyanins,flavones,carotenoids, beet pigments, curcumins, mangiferin and gingerolsis reviewed. Isolation and analysis of phenolic components produced from degradation of natural products during food processing is also discussed.

The phenolic components are widely distributed in nature, have been identified and quantified in water (1-5). The natural phenolic components from various food sources are distributed as colorants likeflavones,anthocyanins, beet pigments, curcumins from Curcuma Longa L., gingerols from ginger root and mangiferin from mango fruit (6-12). Gas chromatographic (13-16) and HPLC (6-12) chromatographic techniques have been generally applied to analyze various phenolic components. Most GC methods require derivatization of phenolic compounds (13-15) In the case of HPLC, resolution of food phenolics has been reported on C-18 reverse phases (6-9). The present paper describes a summarized review of HPLC of various naturally occurring food phenolic components along with other possible mechanisms of their separation beside the reported resolution on C-18 reverse phased. Several mechanisms such as reverse phase, normal phase and anion-exchange to resolve phenolic compounds on C-8, C-18, weak cation-exchange and amino phases will be discussed. These HPLC bonded phased were obtained from Whatman Specialty Products Inc., Fairfield, N.J. and can also be prepared by reacting silane materials such as octydimethyl chlorosilane, octadecyldimethyl chlorosilane, carboxypropyldimethyl methoxysilane and aminopropyl dimethyl chlorosilane with silica gel. Various HPLC columns were packed by slurring materials in methanol and applying 5000 psi pressure. Figure 1 represents reverse phase resolutions of simple phenolic compounds such as phenol and 1-naphthol on C-8 and C-18 columns by methanol-water system as mobile phase. Weak-cation exchanger (WCX) with carboxylic acid functionality is generally used to resolve various amino components by cation-exchange mechanism with a counter-ionic salt incorporated in the mobile phase. The same bonded phase can also be used to perform reverse phase or normal phase separation

0097-6156/92/0506-0077$06.00/0 © 1992 American Chemical Society In Phenolic Compounds in Food and Their Effects on Health I; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

In Phenolic Compounds in Food and Their Effects on Health I; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

1.65

3.60

I I 1.55 1.90

•Imites

max

Figure 1. Resolution of phenol and 1-naphthol on C-18 and C-8 columns (11cm χ 4.6mm, I.D.) Mobile phase: water: methanol (40:60, v/v) at 0.9ml/min; À : 254nm; 1. phenol and 2. 1naphthol. A= C-18 and B= C-8 columns

1

max

7.49 8.45

•Inutes

Figure 2. Reverse phase resolution of phenol and 1-naphthol on WCX column (11cm χ 4.6mm, I.D.) Mobile phase: water: ethanol (80:20, v/v) at 0.2ml/min; À : 254nm; 1. phenol and 2. 1naphthol

JUL


5. KHURANA

HPLC Analysis of Phenolic Compounds in Foods

79


when used in nonionic form. Figure 2 shows reverse phase separation of phenol and 1-naphthol on WCX column by using water-methanol mixture as mobile phase. The normal phase separation on the same column is shown in Fig. 3. In this case, hexane-ethanol mixture is used as a mobile phase. Figure 4 shows anion-exchange resolution of the components on amino column. In this case, ammonium phosphate has been used as a counter ion in the mobile phase. Flavonoids A group of naturally occurring substances derived from flavone (phenyl-y-pyrone) are called flavonoids. The citrus fruit exclusively contains the methoxylatedflavones(17) which can be detected in leaves as well as in all fruit parts:flavedo,albedo, membranes, juice and seeds. RP C-18 and normal phase silica gel columns have been used to analyzeflavonesfrom various foods (8). Cellulose triacetate phase has been used to achieve enantiomeric resolution of racemic flavonones such as naringenin, hesperetin, eriodicytiol, homoeriodicytiol, pinocembrin, isosakuranetin, pinostorbin and sakuranetic (18) with methanol as mobile phase. The mechanism of chiral recognition on cellulose triacetate has not been understood in detail. Presence of two hydroxyl groups in positions 5 and 7 (Fig. 5) gave the best results. The 6-methoxylated and 6-hydroxy flavanone were not resolved. Substitution in theringA was considered to be important for chiral recognition, although these substitutions were not in the vicinity of chiral center. It may be due to absorption of a molecule in such a way that discrimination between the two optical antipodes was easier. It is noteworthy that resolution of enantiomeric hydroxyflavonesand methoxylatedflavonesmay also be achieved on commercially available γ-cyclodextrin chiral phase which separates the racemic isomers with three benzene or cyclohexaneringsin the system. Resolution of polymethoxylated and acetylatedflavoneshas been reported (8,19) on RP-18 and silica gel phases using aceonitrile-water (40:60, v/v) and ethanol (100%) as mobile phases respectively. The polymethoxylated flavones are found in orange juice while acetlyatedflavonesoccur in soy flakes. Anthocyanins Red, blue and purple colors exhibited by flowers, fruits and other plant tissues are due to anthocyanins. The major anthocyanins are derived from substitution on theflavyliumcation (Fig. 6). There has been increased interest in the use of water soluble plant extracts as food colorants due to official delisting of artificial food colors. RP-18 columns have been used to analyze these compounds (20-21) Resolution of the anthocyanin molecules can also be accomplished by normal phase or cation-exchange chromatography. HPLC resolution of anthocyanin glucosides and galactosides form cowberry and chokeberry fruits on C-18 columns (20-21) requires the use of formic acid in the mobile phase to deactivate residual silanols on the silica surface. The analysis of these anthocyanin glucosides and galactosides under acid conditions may lead to hydrolysis which can interfere with their results. P-Cyanins and Ameranthin The phenolic coloring matters such as β-cyanins and amaranthin occur in red beetroot (22-25) and in the leaf and stems of the plant amaranth. The structures of pigments are given in Figure 7. These differ from amaranthin with respect on the substituent on position 5. Betanine in solution is known to degrade to botanic acid (BA) and cyclo-dopa-5-O-glucoside (CDG). The regeneration process involves a SchifFs base condensation of the nucleophilic amine of CDG with aldehyde of Β A (26). Reverse phase HPLC on C-18 columns has been used to analyze βcyanins, the degradation products and amaranthin from red beetroot and amaranth (9,27) Modifiers


In Phenolic Compounds in Food and Their Effects on Health I; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992. minutes

Figure 3. Normal phase separation of phenol and 1-naphthol on WCX column (11cm χ 4.6mm, I.D.) Mobile phase: hexane: eihanoi: water (300:38:0.75, v/v) at 0.2ml/min; 1. 1naphthol and 2. phenol

13.60

I J

18.55

max

minutes

Figure 4. Anion-exchange resolution of phenol and 1-naphthol on Amino column (11cm χ 4.6mm, I.D.) Mobile phase: 0.1M ammonium phosphate: ethanol (80:20, v/v) at lml/min. A : 254nm. 1. phenol and 2. 1-naphthol

1.63 2.00

2



KHURANA


R

1

Figure 5.

Figure 6.

Structure of Flavonoids

Structure of Anthocyanins (Flavylium cation)

BETANIOIN. R=H Η

H(

Ν

UETANINE. R=GLUCOSE

coo10

11' ^12

AMARANTHINE

J13 1

4

Γ ι Ί

19

HOO Η

Ϊ16 Η

1

R=2 0-(D-GLUCOSYLURONIC ACID)-D-6LUCOSYL

8

COOH 20

Figure 7. Structure of β-Cyanins and Amaranthine


82

PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I


such as picric acid and tetrahydroammonium phosphate were used in the mobile phase. These pigments have carboxylic acid groups in their skeleton and it will be quite possible to achieve resolution of such phenolic compounds by using an anion-exchanger with a computer ionic agent in the mobile phase. fnrnimins anH Related Compounds Thephenoliccoloringpigments,curcumin( 1,7-bis-4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5dione) and its isomers demethoxy curcumin (l-(4-hydroxy-3-methoxy-phenyl)-7-(4-hydroxyphenyl)1,6-heptadiene-3,5-dione)andbisdemethoxycurcumin(l ,7-bis-(4-hydroxyphenyl)-l ,6-heptadiene-3,5 dione) are found in the rhizomes of the plant Curcumin Longa L. (Zingiberaceae). These curcuminoids have found their use as coloring agents for food and drugs and are regarded as drug/drug model and antiheptotoxic agents (12). The photo-oxidation of curcuminoids produce vanillin, 4-hydroxybenzaldehyde, ferulic aldehyde, p-hydroxybenzoic acid, vanillic acid and ferulic acid (10). Normal phase HPLC on amino columns have been used to analyze curcumin and its isomers (11,12) The photo-oxidation phenolic compounds of curcumin ant its isomers have also been analyzed by normal phase HPLC on Whatman PartiSphere-5 WCX - column (11). The reverse phase analysis of curcumins and their isomers has been reported on C-18 HPLC Column (28). A normal phase silica gel column has been used in an attempt to resolve curcuminoids. It has been reported that 1,3-diketone groups of the curcumin system interact with the active sites on silica surface. It has been further observed that the possibility of the hydroxy groups on the same skeleton interacting with the surface of the stationary phase to provide a normal phase separation cannot be ruled out (11). The amino columns can also be used to resolve the curcuminoids by an anion exchange mechanism which will require a counter ion like ammonium formate in the mobile phase. Mangiferin Mangiferin (Fig. 8) is a well known member of xanthones and occurs as a glucoside in mangoes. It can be easily distinguished fromflavonesand quinones by spectral data. Mangiferin is known for its cardiotonic, spasmolic, diuretic, choliretic and antiphlogistic actions (29). HPLC resolution of mangiferin from two terpinoid coumarins, colladin and colladonin which are found in C. Triquetra along with mangiferin, has been reported on RP-C18 column. Sulfuric acid (0.2%) has been used in the mobile phase to suppress the ionization of residual silanols (29). Other commercially available amino cyano (PAC form Whatman, Inc.) or cyano columns which are used for resolution of sugars may also be used to resolve mangiferin form other materials. It is also possible to resolve the mangiferin by an anion exchange mechanism on PAC column. Gingerols Gingerols are the phenolicflavorcomponents derived from ginger root. The gingerols undergo retroaldol degradation on injection into gas chromatography (7). C6, C8, CIO aldehydes and gingerone are formed as a result of retro-aldol reaction. These components have been analyzed on an RP-C18 column (6). A Whatman - WCX (weak-cation exchange) column has also been used to analyze the same components by a reverse phase mechanism. A gradient starting with 100% water to 100% methanol was used. The unionized carboxylic acid group of the weak - cation exchanger exhibits a reverse phase mechanism under neutral conditions (10). Capsaicins Capsaicins such as nordihydrovapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and



5.

KHURANA


83

homodihydry ocapsaicin are heterocyclic phenolicflavorcomponents. Reverse phase HPLC on a C-18 column has been used to analyze these components (30). Water :acetonitrile (54.7:45.3, vJ.v) has been used as a mobile phase. The electochemical detection of capsaicin can provide a higher degree of sensitivity and specificity as these are very electroactive due to the easily oxidized phenolic functional group. It is easy to identify these phenolic compounds on the basis of hydrophobicities and retention times (31-33) An improvement in resolution can be achieved by decreasing the acetonitrile with methanol as modifier. In short, the phenolic components in food can be resolved on various HPLC phases like C-8 and C-18 by reverse phase mechanismJ. weak cation-exchanger both by reverse and normal phase mechanisms and dialkyl amiopropyl phase by anion-exchange mechanism. Various optical isomeric phenols may be separated on types of commercially available chiro-phases like cellulose triacetate and cyclodextrins.

Figure 8. Structure of Mangiferine

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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I

22. Stark, D.; Reznik, H. Z. Pfanzenphysiol. 1979, 94, 183. 23. Stark, D.; Engel, U.; Rezmik, H. Z. Phanzenphysiol. 1979, 101, 215. 24. Vincent, K.T.; Scholt, R.G. J. Agric. Food Chem. 1978, 26, 812. 25. Von Elbe, J.H.; Huang, A.S.; Attoe, E.L.; Nank, W.K. J. Agric. Food Chem. 1986, 34, 512. 26. Schwartz, S.J.; Von Elbe, J.H. Z. Levensm Unters Forsch. 1983, 176, 448. 27. Schwartz, S.J.; Von Elbe, J.H. J. Agric. Food Chem. 1980, 28, 580. 28. Amakawa, K.E.; Hirata, K.; Ogiwara, T.; Ohnishi, K. Jpn. Anal. 1986, 33, 586. 29. Simova, M.; Tomov, E.; Pangarova, T.; Palvlova, N. J. Chromatogr. 1986, 351, 379-382. 30. Chiang, G.H. J. Food Sci. 1986, 51, 499. 31. Hoffman, P.G.; Lego, M.C.; Galetto, W.G. J. Agric. Food Chem. 1983, 31, 1326. 32. Rouseff, R. In Liquid chromatographic analysis of food and beverages. Charalambous, G. Ed Academic Press, Orlando, Fl. 1979, 1, 16. RECEIVED July 16,

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High-Performance Liquid Chromatographic Analysis of Phenolic

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