A GUIDE TOCAROTENOIDANALYSIS INFOODS

Delia B. Rodriguez-Amaya, Ph.D.

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A GUIDE TO CAROTENOID ANALYSIS IN FOODS

Delia B. Rodriguez-Amaya, Ph.D.Departamento de Cincia de AlimentosFaculdade de Engenharia de AlimentosUniversidade Estadual de CampinasC.P. 6121, 13083-970 Campinas, SP., Brasil

Open-column Method 41

OPEN-COLUMN METHOD

Analytic methods for carotenoids are classified bychromatographic technique into open-column (OCC)and high-performance liquid chromatography (HPLC)methods. The OCC method herein described wasput together in 1976 (Rodriguez-Amaya et al. 1976a)from the best procedure for each step available atthe time. It has been used in several laboratories inBrazil, evaluated in terms of repeatability for provita-min A carotenoids in different food samples, alongwith the assessment of the extraction and saponifi-cation steps (Rodriguez-Amaya et al. 1988), and com-pared with HPLC methods in terms of provitamin Acarotenoids in different foods (Carvalho et al. 1992)and of major carotenoids in cassava leaves (Adewusiand Bradbury 1993). Additionally, results obtained withthis method compare well with those obtained bysome HPLC methods, as shown for -carotene andlycopene in tomatoes (Table 3), -carotene and -carotene in carrot (Rodriguez-Amaya 1989), andmajor carotenoids of mango, guava, and papaya(Wilberg and Rodriguez-Amaya 1995). In fact, thereseems to be more coherence between the results fromthis method and those of some HPLC methods thanamong results of HPLC methods.

The method as described below may appear te-dious because it has the capacity to identify and quan-tify the range of carotenoids in the sample, includingminor carotenoids. If the objective is to determineonly major provitamin A and nonprovitamin A caro-tenoids, the analysis is much simpler because onlyone to five carotenoids need to be separated, identi-fied, and quantified.

This method is also efficient for isolating caro-tenoids that are not available synthetically or com-mercially to serve as standards for HPLC.

Precautions

Review the precautionary measures discussed inGeneral Procedure and Sources of Errors in Caro-tenoid Analysis and put them into practice.

Reagents and Apparatus

Acetone, reagent-grade or distilled, left in the re-frigerator for about 1 hour.

Mechanical blender with stainless steel or glasscup, or mortar and pestle.

Vacuum filtration deviceBuchner funnel or sin-tered glass funnel (porosity 3), suction flask, andwater aspirator.

Separatory funnel with Teflon stopcock. Potassium hydroxide solution10% in methanol.

Dissolve 10 g reagent-grade potassium hydroxidein 100 mL methanol, mix, and cool.

Chromatographic glass tube2.5 cm i.d. 30 cm,tapering at the bottom.

AdsorbentMix magnesium oxide andHyflosupercel (diatomaceous earth) (1:1 or 1:2 w/w) by shaking until well mixed. This can be donemanually by shaking the adsorbent in a containerbig enough to allow thorough mixing. If needed,activate the adsorbent already mixed by heating inan oven at 110 C for 4 hours. After cooling in adesiccator, store in tightly closed container. Whenalumina (activity grade III) is used, mix neutral alu-mina (activity grade I) with 6% water thoroughly(until no lumps can be observed). This is best doneusing a rotary evaporator flask without applyingvacuum. Store in tightly closed container.

Eluting solventPetroleum ether, reagent gradeor distilled (b.p. 3060 C), with increasing per-centage of diethyl ether (peroxide-free), acetone,and water (v/v).

Anhydrous sodium sulfate. Rotary evaporator. Ultraviolet-visible recording spectrophotometer. Volumetric flasks, beakers, Erlenmeyer flasks, and

other common laboratory glassware. Silica gel thin-layer chromatography (TLC) plates

and TLC development tank. TLC developing solvent3% methanol in benzene.

(Because of the high toxicity of benzene, this de-

42 A Guide to Carotenoid Analysis in Foods

veloping solvent has been replaced in our labora-tory with 5% methanol in toluene, the chromato-graphic behavior being equivalent.)

Reagents and solvents for chemical testspyri-dine, acetic anhydride, hydrochloric acid, sodiumborohydride, 95% ethanol, and iodine.

Extraction

Weigh a portion of the homogeneous, representativesample. The weight depends on the carotenoid con-tent of the sample, varying from 2 g of dark greenleafy vegetable to 100 g of low-carotenoid fruit orvegetable. Blend in a mechanical blender for 3060seconds with enough cold acetone to cover and celiteor Hyflosupercel. Alternatively, grind sample with amortar and pestle, with enough cold acetone to coverand celite or Hyflosupercel. Filter with suction througha Buchner funnel or sintered glass funnel. Wash theblender or mortar, funnel, and residue with smallamounts of acetone, receiving the washings in thesuction flask with the extract. Return the residue tothe blender or mortar, add fresh acetone, and macer-ate again. Filter and wash as before. Repeat the ex-traction and filtration until the residue is devoid ofany color and washings are colorless (usually 3 timesis enough).

Partitioning to Petroleum Ether

Put about 100 mL of petroleum ether in a separatoryfunnel and add a small portion of the acetone ex-tract. Add distilled water slowly, letting it flow alongthe walls of the funnel. To avoid formation of anemulsion, do not shake. (Once formed, an emulsioncan be broken by adding acetone or saturated saltsolution and swirling the funnel to mix. When anemulsion is difficult to break, it is better to start theanalysis over rather than proceed with an analysisthat can give an erroneous result.) Let the two phasesseparate and discard the lower aqueous-acetonephase. Add another portion of the acetone extractand repeat the operation until all of the extract hasbeen transferred to petroleum ether, then wash 4-5times with water to remove residual acetone. Collectthe petroleum ether phase and dry with sodium sul-fate (add sodium sulfate until some crystals becomeloose). If saponification will be carried out, skip thisdrying step. If at any time during this process thelower phase appears colored, collect it and add it backin portions to the upper phase.

The partitioning to petroleum ether is more diffi-cult with the extract obtained from the first extrac-tion, which contains water, lipids, and other compo-nents of the sample. You may want to separate this

first extract so that you can partition it more care-fully. The carotenoids of the succeeding extracts areeasily transferred to petroleum ether.

If your sample has appreciable amount of xan-thophylls, add some diethyl ether to petroleum etherto facilitate the transfer of these xanthophylls fromacetone to petroleum ether.

Saponification

Do this step only when absolutely necessary (seeGeneral Procedure and Sources of Errors in Caro-tenoid Analysis). To the carotenoid solution in pe-troleum ether, add butylated hydroxytoluene (0.1%in petroleum ether) and an equal volume of 10% po-tassium hydroxide in methanol. Flush with nitrogenbefore stoppering the flask. Let stand in the dark atroom temperature overnight (up to about 16 hours).Wash with distilled water to remove the potassiumhydroxide. Do this as in the partition procedure byadding portions to a separatory funnel, each additionfollowed by washing with water (adding water, al-lowing the phases to separate, and discarding thelower aqueous phase). When all of the carotenoidhas been added to the funnel, wash an additional 4-5times with water to get rid of the potassium hydrox-ide. Collect the carotenoid phase and dry with so-dium sulfate.

Concentration

Concentrate carotenoid solution so that it can be in-troduced into the chromatographic column in thesmallest volume possible. Decant solution to a round-bottom flask and rinse the receiving flask and sodiumsulfate with small amounts of petroleum ether, com-bining washings with the carotenoid solution. If diffi-cult-to-wash xanthophylls are present, rinse also witha small amount of ethyl ether. Connect the flask tothe rotary evaporator and concentrate the solution toabout 1020 mL, the temperature not exceeding 40C.

Chromatographic Separation

Preparing the ColumnMount a chromatographic glass tube on a suctionflask. Place a small glass wool plug at the bottom ofthe chromatographic tube. Add adsorbent loosely upto a height of 20 cm and apply a moderate vacuumfrom a water aspirator (the vacuum should be con-tinuously applied from this point on). Use a flat in-strument (such as an inverted cork mounted on a rodor a tampering rod, the diameter just a little bit smaller

Open-column Method 43

than that of the glass tube so that it fits snugly intothe tube) to press down the adsorbent and flatten thesurface (the packed column should about 12 cm high).Top the column with a 1-cm layer of anhydrous so-dium sulfate to ensure that no residual water getsinto the adsorbent. Test the column with petroleumether. Pass about one bed volume of petroleum etherthrough the column (the adsorbent surface must besmooth and the solvent flow even) and adjust thevacuum so that the solvent flow is about 23 dropsper second. Once petroleum ether is added to thecolumn, keep the top of column covered with solventuntil the chromatography is completed.

Developing the ColumnPour the carotenoid solution into the column and letthe sample layer go down almost to the surface ofthe sodium sulfate layer before adding the rinse (pe-troleum ether) from the round-bottom flask. The ob-jective is to keep the carotenoids in as small a vol-ume as possible to diminish band broadening and toprevent the separation from initiating before the en-tire carotenoid sample has reached the adsorbent top.

Once the petroleum ether rinse almost reachesthe surface of the sodium sulfate layer, develop themagnesium oxideHyflosupercel (1:2) column suc-cessively with 50 mL each of 1%, 2%, and 5% ethylether and then 2%, 5%, 8%, 10%, 15%, and 20%acetone in petroleum ether. Depending on the caro-tenoid composition, the amount of acetone can there-after be increased by 10% and then 20% up to pureacetone. Tightly adsorbed carotenoids can be elutedby 5% and then 10% water in acetone. Hexane maybe used instead of petroleum ether.

For the activated magnesium oxideHyflosupercel (1:1) column, start development with1% acetone and continue increasing the acetone per-centage as described above.

For succeeding analyses modify the above de-velopment pattern according to the carotenoid com-position of the sample to obtain the best separationwithin the shortest possible time. Some of the solventmixtures may be deleted or the volume reduced,whereas others may have to be increased. With ex-perience it may not be necessary to develop the col-umn with the entire series of solvents, even when acommodity is being analyzed for the first time. In thiscase, add only a small amount of the eluting solvent.If no separation is observed, go to the next solvent.If, by bypassing a solvent, two bands appear to fusetogether, go back to the previous solvent. Examplesof separation patterns are shown in Figure 20.

Monitor separation of the carotenoids visually and

collect each separated fraction as it leaves the col-umn. Change suction flasks rapidly but gently so thatbreaking the vacuum will not perturb the adsorbentcolumn. Alternatively, when all of the carotenoidshave been separated, let the column dry, remove theadsorbent from the glass tube by tapping the invertedcolumn gently, cut the remaining bands, and extractthem with acetone or acetone with 510% water forthe more polar carotenoids. Dilute or concentratefractions eluted with petroleum ether or petroleumether containing small amount of diethyl ether to asuitable volume for spectrophotometric reading.

Because acetone affects the absorption of caro-tenoids in petroleum ether, remove acetone from frac-tions eluted with petroleum ether containing acetoneor transfer carotenoids eluted with acetone or ac-etone with water to petroleum ether. To do this, putthe fraction in the separatory funnel (there is no dan-ger of emulsion formation at this point) all at once,add petroleum ether if necessary, and then add dis-tilled water. After phase separation, discard the lowerphase. Wash with water an additional 34 times, col-lect the petroleum ether phase, dry with sodium sul-fate, transfer to a suitable volumetric flask, and ad-just the volume with petroleum ether.

Record the spectrum of all carotenoids in a 1-cmcuvette from 330 nm (lower for phytoene and phyto-fluene) to 550 nm.

For some food samples, better separation isachieved with an alumina column or unresolved frac-tions from the magnesium oxideHyflosupercel col-umn are rechromatographed on another magnesiumoxideHyflosupercel column or on an alumina col-umn. Pack the alumina column by filling the chro-matographic tube (a smaller tube than that used forthe first separation is usually used forrechromatography) with the adsorbent (usually neu-tral alumina, activity III) and tapping gently to ac-commodate the adsorbent better in the tube. Appli-cation of a vacuum is not required, but the column isalso developed with petroleum ether or hexane con-taining an increasing amount of diethyl ether and thenacetone.

Thin-layer Chromatography

After recording the spectra, concentrate all fractionsand apply on a silica gel thin layer. Develop the thin-layer gel with 5% methanol in toluene. All caroteneswill run with the solvent front. Xanthophylls will bedistributed in the chromatogram according to the typeand number of substituents present. For example,monohydroxy carotenoids will be situated in the middle,trihydroxy carotenoids will remain in the origin, and