JSS, Análisis de Carotenoides Columna C30

7/24/2019 JSS, Anlisis de Carotenoides Columna C30

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Bhm 955

Volker Bhm

Institute of Nutrition, Friedrich-Schiller-University Jena, 07743Jena, Germany

Use of column temperature to optimize carotenoidisomer separation by C30 high performance liquidchromatography

Five xanthophylls and six carotenes were eluted from a YMC C30column with a binarymobile phase gradient consisting of methyl tert-butyl ether and methanol. The effect

of column temperature on the separation was tested between 158C and 258C. A mix-

ture of prominent lycopene isomers was additionally analyzed to optimize their sepa-

ration in relation to the main xanthophylls and carotenes. At column temperatures

below 208C (15Z)-b-carotene co-eluted with echinenone, a frequently used internal

standard. For most carotenoids an optimal separation was achieved at 23 l 18C

within one hour. In contrast, some (Z)-isomers of lycopene showed a better resolution

at higher column temperatures. While these isomers were separated at 308C and at

358C, another (Z)-isomer co-eluted with (E)-lycopene at 358C. Thus, column tempe-

rature is a noteworthy parameter in high performance liquid chromatography of caro-

tenoids.

Key Words:Column temperature; Carotenoids; b-Carotene isomers; Lycopene iso-mers; C30 column;

Ms received: May 21, 2001; revised: September 7, 2001;accepted: September 10, 2001

1 Introduction

Reversed phase materials and lime are the stationaryphases hitherto most widely used for the separation ofcarotenoids. Carotenoid geometrical isomers were mainlyinvestigated with b-carotene. More than 30 years ago,Sweeney et al. [1] analysed isomers ofb-carotene to eval-uate their provitamin A activity, using a column with cal-cium hydroxide. They separated three b-carotene iso-mers, without exact structural knowledge about the two(Z)-isomers. In the last decade, calcium hydroxide wasoften used to separate geometrical isomers of carotenes[25].

Polymeric ODS columns (e.g. VYDAC TP columns) werefrequently recommended for the separation of geometri-cal isomers of carotenoids. Schierle et al. [6] separatedthe four b-carotene isomers (elution order: (E)-, (9Z)-,(13Z)-, (15Z)-b-carotene) in 25 minutes, using

0.6 mL min 1 methanol/tetrahydrofuran (99: 1, v/v) at308C on a VYDAC 218TP54 column. Nyambaka et al. [7]found the same elution order ((E)-, (9Z)-, (13Z)-b-caro-tene) when analysing vegetables on a VYDAC 201TP col-umn. Saleh et al. [8] showed this elution order to hold withacetonitrile/methanol/dichloromethane (80: 18: 2, v/v/v)

as mobile phase on a VYDAC 218TP54 column. Usingthe same mobile phase on a VYDAC 201TP51 column,the elution order changed to (E)-, (13Z), (9Z)-b-carotene.This elution order is comparable to that obtained in inves-tigations in food [9], using methanol/dichloromethane(94:6, v/v) ona 201TP column.

A new approach to the separation of geometrical isomersof carotenoids is the use of C30columns. Using a gradientmethod at 26.68C on a C30column, Sharpless et al. [10]eluted the fourb-carotene isomers in the following order:(15Z)-, (13Z)-, (E)- and (9Z)-b-carotene. On eluting a C18column with a gradient, (13Z)- and (15Z)-b-carotene co-eluted with a retention time higher than that of (E)- and(9Z)-b-carotene. In two other studies [11, 12], a C30col-umn was eluted isocratically with 1.0 mL min 1 of metha-nol/methyl tert-butyl ether (89:11, v/v) at ambient tem-perature. The elution order of the b-carotene isomers(15Z)- a (13Z)- a (E)- a (9Z)-) within one hour was compar-

able to that in the above-mentioned separation achievedby Sharpless et al. [10]. Lycopene, however, was noteluted within 2 hours [11].

Often, ambient temperature (controlled or uncontrolled) isused for HPLC separation of carotenoids. In contrast,Sander et al. [13] separated two b-carotene isomers usingsubambient temperatures (17 to +308C). Lane et al. [14]separated four b-carotene isomers (elution order: (E)-,(9Z)-, (13Z)-, (15Z)-) on a Suplex pKb-100 column at 08Cusing 0.8 mL min 1 methanol/tert-butyl ether/water(80:20:5, v/v/v). Our own investigations using a poly-

J. Sep. Sci. 2001, 24, 955959

Correspondence: Dr. Volker Bhm, Institute of Nutrition,Friedrich-Schiller-University Jena, Dornburger Str. 2529,07743 Jena, Germany.E-mail:[email protected]:+49 (0)3641 949632

OriginalPaper

i WILEY-VCH Verlag GmbH, D-69451 Weinheim 2001 1615-9306/2001/12120955$17.50+.50/0


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meric C18 column (VYDAC 201TP54) within a temperaturerange of +308C to 78C resulted in dramatic changes inthe elution order of four b-carotene isomers ((E)- and(9Z)-, (13Z)- and (15Z)-b-carotene) with decreasing tem-perature [15]. Polymeric C30 columns showed a better

resolution of carotenoid geometrical isomers compared topolymeric C18columns [16]. However, for this extendedlength alkyl stationary phase it has been only scarcelyinvestigated how column temperature affects the separa-tion. Bell et al. [17] compared the temperature depen-dence of carotenoid retention on three different lengths ofalkyl stationary phases. Besides C18- and C30-materialthey investigated a C34 column, the latter two columnswere prepared in their laboratory. They isocratically elutedthe C30and C34column each with 95% methanol and 5%methyltert-butyl ether using temperatures between 4.68Cand 49.48C. Increasing retention was observed forcarotenoids on long-chain stationary phases above 358C

with increasing temperature, which was unexpected. Thisstudy did not include lycopene. This carotenoid, presentmainly in tomatoes and tomato products, is often eluted inisocratic methods after all other carotenoids, increasingthe analysis duration approximately two to three timescompared to the shown chromatograms [17]. Anotherstudy separated different isomerized carotenoids isocrati-cally at ambient temperature using a binary mobile phaseof methyl tert-butyl ether (MTBE) in methanol. The con-centration of MTBE in the mobile phase was adjusted foreach carotenoid to give optimal resolution [18]. Thus, aseparation of complex mixtures of carotenoids, such asoccur in food and serum, is not possible without co-elutionof some isomers.

The aim of the presented study was to investigate howcolumn temperature can be used to optimize gradientreversed phase liquid chromatographic separation of 11carotenoids, including three (Z)-isomers ofb-carotene, ona YMC C30column. In addition, a mixture of lycopene iso-mers was analysed using different temperatures in orderto improve analyses in food and serum.

2 Materials and methods

2.1 Chemicals

Standard solutions in cyclohexane/toluene (8:2,v/v) con-taining 210 mg/L of canthaxanthin, a-carotene, (E)-b-carotene, (9Z)-b-carotene, (13Z)-b-carotene, (15Z)-b-car-otene, b-crytoxanthin, echinenone, lutein, lycopene,zeaxanthin (all gifts from Hoffmann-La Roche, Basel,Switzerland). To prepare the working solutions, thesestandard solutions were diluted daily 1:50 or 1:100 withthe mobile phase. 20lL of these working solutions wereinjected for HPLC analysis. Cylcohexane and toluenewere of analytical grade, while methanol and methyl tert-

butyl ether, both used as mobile phase, were HPLC qual-ity.

2.2 Iodine isomerization

Lycopene standard solution in cyclohexane/toluene (8:2,v/v) was used for isomerization. Iodine crystals wereadded to the standard at ca. 1% of the carotenoid weightand photoisomerization performed according to themethod of Zechmeister [19]. The resulting mixture of iso-mers was analysed chromatographically as describedbelow.

2.3 Equipment

HPLC pump model L-6200 (Merck, Darmstadt, Germany),sampler model AS-2000, diode array detector modelL-4500 (Merck), column oven model CTO-10AC (Shi-madzu, Duisburg, Germany).

2.4 Column

25064.6 mm, YMC C30 (YMC, Schermbeck, Germany),particle diameter 5lm, pore diameter 200 , column tem-perature (see results and discussion), preceded by a1064.0 mm ProntoSil 120-5-C18 H guard column(Bischoff, Leonberg, Germany), particle diameter 5lm,pore diameter 120 .

2.5 Mobile phaseAs mobile phase methyl tert-butyl ether (solvent A) andmethanol (solvent B) were used at a flow rate of 1.3 mLmin 1. The gradient procedure was as follows: 1) Initialconditions 10% solvent A and 90% solvent B, 2) a 35-minlinear gradient to 45% solvent A, 3) a 10-min linear gradi-ent to 60% solvent A, 4) 60 % solvent A and 40% solventB for 15 min, 5) a 5-min linear gradient to 10% solvent A.This gradient procedure was used for column tempera-tures between 15 8C and 198C. For the temperatures 20258C the steps 4) and 5) were slightly modified as follows:4) 60% solvent A and 40 % solvent B for 11 min, 5) a4-min linear gradient to 10% solvent A.

3 Results and discussion

Five xanthophylls and six carotenes were analysed on aC30high performance liquid chromatography column withvariation of the column temperature between ambientconditions (258C) and 158C. Within this temperaturerange the xanthophylls lutein, zeaxanthin, canthaxanthin,b-cryptoxanthin, and echinenone (often used as internalstandard) eluted after 1020 min. In contrast, the caro-

956 Bhm J. Sep. Sci. 2001, 24, 955959


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tenes b-carotene (four isomers), a-carotene, and lyco-pene showed higher retention times with lycopene elutingafter approximately one hour. Figure 1 shows the k9values of the carotenoids analysed for all column tem-peratures investigated. All xanthophylls were well sepa-rated within the whole temperature range. The resolutionof the carotenes was also good. Only echinenone, theinternal standard in many studies, and (15Z)-b-caroteneco-eluted at column temperatures below 208C. The fourb-carotene isomers showed the same elution order (15Z)-a

(13Z)-a

(E)-a

(9Z)-) as recently seen on a polymeric C18column [15]. However, a nearly baseline separation ofthese four isomers on the C18 column needed approxi-mately one hour and a column temperature below 08C.Bell et al. [17] showed a surprising effect of column tem-perature on the capacity factors of (13Z)-and(15Z)-b-car-otene as well as that of three (Z)-isomers of a-carotene.As expected, the capacity factors decreased with increas-ing temperature. At temperatures above approximately358C the k9 values actually increased, indicating anincrease of interactions of the solutes with the stationaryphase.

Another observation from Figure 1 is the large differenceink9value between lycopene and all other carotenoids ofapproximately 10. This distance in elution could bedecreased by increasing the relative portion of methyl tert-butyl ether in the mobile phase. However, it can also beused for lycopene (Z)-isomers, often eluting before the

J. Sep. Sci. 2001, 24, 955959 Use of column temperature to optimize carotenoid isomer separation 957

Figure 1.Capacity factors of 11 carotenoids at column tem-peratures between 158C and 258C (further chromatographicconditions see text).

Figure 2. Chromatographic separation of (E)-lycopene andfour lycopene (Z)-isomers (2550 ng per isomer) using col-umn temperatures between 228C and 258C as well as 308Cand 358C (further chromatographic conditions see text).1,2,3, 5= lycopene isomers of yet unknown structure, 4= (E)-lycopene.


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(E)-isomer. To test the effect of column temperature onthe elution order of lycopene isomers, a photoisomerizedlycopene was analysed. Five lycopene isomers werecompared with regard to their retention behaviour. Theseisomers were recently analysed for their antioxidant activ-ity and consisted of (E)-lycopene and four (Z)-isomers ofyet unknown structure [20].

The structural characterization of the (Z)-isomers is partof a separate investigation currently in process. Accordingto their mass spectra (results not shown), they all possessthe same mass as (E)-lycopene. This fact and their UV/Vis spectra lead to the assumption that they are (Z)-iso-

mers rather than oxidation products of lycopene. Between158C and 258C the five lycopene isomers eluted within40 65 min, filling a large part of the carotenoid-free spacein the above discussed chromatograms. Three (Z)-lyco-pene isomers eluted as a group before the (E)-isomer,one (Z)-isomer shortly behind it. The three first (Z)-iso-mers resulted in only two peaks up to a column tempera-ture of 238C. Increasing the temperature improved theseparation of the first two isomers. However, good resolu-tion of all five isomers was not achieved within the tem-perature range up to 258C. Thus, the two higher tempera-tures 308C and 358C were additionally tested. Figure 2

shows the chromatographic separation of these five lyco-pene isomers for the temperatures 22258C and 308C aswell as 358C. While the three (Z)-isomers eluting before(E)-lycopene were well separated at 308C and at 358Cthefourth (Z)-isomer co-eluted with (E)-lycopene at 358C.

Looking at the chromatograms of the photoisomerizedlycopene, two further main isomer peaks elute between20 and 30 min within the temperature range 15258C(data not shown). The first one showed a retention timeslightly lower than a-carotene. Co-elution of a-carotenewith this yet unknown lycopene isomer was observed forcolumn temperatures below 178C. The second mentioned

lycopene isomer peak eluted after the (9Z)-b-carotene,well separated from it. Both peaks drew closer withincreasing temperature.Figure 3shows a chromatogramof the mixture of 11 carotenoids at 238C, which seems tobe the optimal temperature. This chromatogram addition-ally includes the two mentioned lycopene isomers (allmarked with X), which are formed in the (E)-lycopenestandard solution during storage.

Thechromatogram of a human plasma extract in Figure 4shows the applicability of the optimised HPLC conditionsfor routine analysis. In this chromatogram five yet

958 Bhm J. Sep. Sci. 2001, 24, 955959

Figure 3.HPLC chromatogram of 11 carotenoids (1443 ng) at a column tem-perature of 23.08C (further chromatographic conditions see text). 1= lutein,2= zeaxanthin, 3= canthaxanthin, 4=b-cryptoxanthin, 5= echinenone,6= (15Z)-b-carotene, 7= (13Z)-b-carotene, 8=a-carotene, 9= (E)-b-carotene,10= (9Z)-b-carotene,11= (E)-lycopene,X = lycopene isomers of yet unknownstructure.

Figure 4.HPLC chromatogram of human plasma extract (for sample preparationsee [21]) at a column temperature of 24.08C (further chromatographic conditionssee text). For peak indentification see Figure 3.


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unknown lycopene (Z)-isomers (all marked with X) areseparated from the (E)-isomer.

4 Concluding remarks

Column temperature is an important tool for optimizationof high performance liquid chromatograpy of carotenoids.For routine analysis a column temperature of 23 l 18Cseems to be the best compromise. Gradient elution at thistemperature permits separation of many prominent caro-tenoids, including a wealth of (Z)-isomers, within onehour.

Acknowledgements

Hoffmann-La Roche (Basel, Switzerland) is gratefully ac-knowledged for supplying the carotenoid reference mate-rials. In addition, the author thanks I. Schmuck for hertechnical assistance.

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J. Sep. Sci. 2001, 24, 955959 Use of column temperature to optimize carotenoid isomer separation 959

JSS, Análisis de Carotenoides Columna C30

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