International Journal of Applied Science and Engineering2005. 3, 2: 81-88

Int. J. Appl. Sci. Eng., 200 5. 3, 2 81

Chlorophyll Removal From Edible Oils

Levente L. Diosady*

Department of Chemical Engineering and Applied ChemistryUniversity of Toronto

200 College St., Toronto, Ontario, Canada M5S 3E5

Abstract: The chlorophyll content of canola oil, especially when extracted from frost-damagedseed is high, and results in dull, dark brown oil unless very large quantities of bleaching clay areused. We found that treatment with phosphoric acid, under vacuum in the absence of moistureprecipitates most of the chlorophyll. At 140°C 2400 mg/L phosphoric acid precipitated 98 % ofthe chlorophyll in canola oil after 15 minutes of gentle stirring under vacuum of ~ 30 mmHg(4kPa). Chlorophyll levels were reduced from 23.8 mg/L to less than 0.5 mg/L. This was furtherreduced to > 0.05 mg/L with caustic refining and bleaching. A similar reduction in chlorophylllevel was obtained with 5000 mg/L sulfuric acid at room temperature. Mass spectrometric analy-sis indicates that the chlorophyll is irreversibly converted to oil insoluble pheophorbides by thetreatment. A mechanism is proposed. It was shown that the precipitated pigment may be left inthe crude oil, as it will be removed during caustic refining. Thus the process requires only theaddition in front of the caustic refining step of a heat exchanger and a vacuum mixing vesselwith 15 min. residence time. The process was demonstrated on pilot and full scales. It has beenpatented and commercially implemented.

Keywords: Chlorophyll removal; edible oil processing; canola oil; pheophorbides.

* Corresponding author: e-mail: diosady@chem-eng.toronto.edu Accepted for Publication: September 23, 2005

© 2005 Chaoyang University of Technology, ISSN 1727-2394

1. Background

Chlorophyll is a critical compound in thedevelopment and metabolism of plants, and isalways present to some extent in oilseeds.During oilseed processing chlorophyll isreadily extracted by hexane into the crude oil.If it is present in sufficiently large concentra-tion, chlorophyll imparts a greenish color tothe crude oil. Chlorophyll is thermally de-composed to the pigment pheophytin, whichresults in a dull, dark-colored oil. This pig-ment contributes to an off-flavor and may alsopromote the oxidation of the oil, thus reduc-ing its storage stability.

While canola seeds always contain signifi-cant quantities of chlorophyll, if the seeds aredamaged by early frost the chlorophyll con-tent of the oil can increase dramatically. Sincethe growing season for canola is barely ade-quate in some of the more northern growingregions, a large percentage of the crop is al-ways at risk of frost damage.

The problem of chlorophyll in canola oil isindustry-wide in Canada. While soya oil con-tains typically less than 1 ppm chlorophyll,typical Canadian canola contains 13-30 ppm.The problem is much less severe in Europe,

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82 Int. J. Appl. Sci. Eng., 2005. 3, 2

where winter rape is grown, which readilymatures in that climate. The presence of highlevels of chlorophyll in crude canola oil cre-ates very serious processing problems for therefiner. Although chlorophyll and pheophytinmay be removed by increasing the amount ofbleaching clay used, this is expensive and notalways completely effective, since the clayalso adsorbs 1/3 to 3/4 of its weight in oil.Thus it is very important for the Canadiancanola processing industry to develop suitabletechnology for dealing with the presence ofchlorophyll in the seed, either by altering theextraction process to minimize chlorophyllextraction with the oil, or by the removal ofchlorophyll from crude oil during the refiningoperations.

The scientific literature and our own re-search experience indicated that a number ofunconventional approaches may successfullyremove chlorophyll from oil. We previouslyinvestigated four completely unrelated ap-proaches: membrane filtration, ion exchange,supercritical fluid extraction and solvent ex-traction [1].

There were indications in the literature thatchlorophyll and its derivatives may be presentin the oil not in true solution, but as a suspen-sion of very small particles, probably colloi-dal in size, which would permit the removalof chlorophyll particles from the oil using mi-crofiltration [2]. Our work indicated, thatchlorophyll is present in true solution, andwas not removed to any significant extent byeven ultrafiltration.

While it is possible to exchange the Mgatom of chlorophyll with other cations underslightly acidic conditions in an ion-exchangereaction [3] leaving a negative charge on therest of the molecule, reversible absorption ofthe chlorophyll on an ion exchange resinproved to be possible, but impractical.

Extraction of the chlorophyll with super-critical carbon dioxide was promising, buttechnologically too difficult at present.

Ideally the problem would be eliminated bynot extracting chlorophyll into the crude oil at

all. However, a major change in the extractionprocess would be difficult to implement, andtherefore we decided to investigate a series ofchemical treatments for chlorophyll extractionthat can be readily integrated into the conven-tional refining process.

2. Experimental techniques

2.1. Materials

Crude canola oil was obtained from Procterand Gamble Canada Inc., Hamilton ON.

All chemical reagents used were obtainedfrom J.T. Baker Chemicals Co, Phillisburg NJ.The reagents were analytical grade. Foodgrade phosphoric acid (85%) was used.

Natural and acid activated bleaching clays(Vega Plus and Filtrol 105) were obtainedfrom Pembina Mountain Clay Co., Winnipeg,Manitoba.

Chlorophyll-a and chlorophyll-b standardswere bought from Aldrich Chemical Co. Mil-waukee, Wisconsin.

2.2. Analytical

The concentrations of chlorophyll and otherchlorophyllic pigments were determined spec-trophotometricly, using AOCS standardmethod Cc 13d-55 [4].

Free fatty acids were determined by titra-tion in ethanol, according to the AOCS stan-dard method Ca 5a-40 [4].

Fatty acid distribution was determined bygas chromatographic determination of fattyacid methyl esters, using the AOCS standardmethods Ce 2-66 and Ce 1-62 [4].

2.3. Laboratory procedures

To test the effect of chemical addition onchlorophyll content, 300 g of degummedcrude canola oil was weighed into a 500 mLErlenmeyer, and deaerated using water vac-uum at ~ 30 mm Hg ( ~4 kPa) for 15 min. Theselected amount of reagent was added to the

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Int. J. Appl. Sci. Eng., 2005. 3, 2 83

deaerated oil, which was then heated undervacuum to the desired reaction temperature,with gentle mixing by a magnetic stirrer. Thesystem was held at the reaction temperaturefor 10 to 60 min. then cooled to somewhatabove ambient temperature under vacuum.

The oil was centrifuged at 5000 rpm for 10min. in an IAC centrifuge with 4x500 mLfixed angle rotor, to remove the precipitatedpigments. The residual chlorophyll content ofthe supernatant was determined. In some ex-periments the oil was refined and bleachedwithout first removing the precipitate fromthe chemical treatment.

In selected tests the oil was caustic refinedand bleached. Some 300g oil was weighedinto a 500 mL flask, and a 20% excess of 3NNaOH, based on the free fatty acid content ofthe oil, was added with gentle mixing. Thesystem was heated to 65°C for 15 min. andallowed to settle for 1 h. The oil was decanted,and filtered through a coarse filter paper. Itwas then washed twice with 5% (v/v) hot wa-ter to remove traces of soap.

Some 250 g of the refined oil was weighedinto a 500 mL flask, and with gentle stirring apre-determined amount of clay (0 to 3% w/w)was added. The slurry was rapidly heated un-der vacuum to 105°C, and held for 15 min. Itwas then allowed to cool to ~ 40°C, and fil-tered through a Whatman No. 1 filter paper.

3. Results and discussion

Initial tests and the literature indicated thatphosphoric acid added prior to caustic refin-ing can remove some of the chlorophyll fromdegummed oil.

The effects of temperature and concen-trated phosphoric acid (85%) addition levelswere investigated. We found that at tempera-tures up to 100°C the chlorophyll removalwas very slow. The treatment produced a finecolloidal precipitate that was not immediatelyfilterable. After settling for several days(!) thesuspension aggregated, and could be filtered,resulting in 50-85% chlorophyll removal. At

temperatures above 100°C treatments of10-50 min. precipitated chlorophyll, whichwas immediately filterable.

We systematically explored the effects oftemperature, phosphoric acid addition leveland residence time on the residual chlorophylllevel. The results are represented in Table 1.Both increased temperature and addition levellowered the chlorophyll content. At additionlevels at 2000 mg/L phosphoric acid removedmore than 90% of the chlorophyll in all ex-periments in this series. At the highest tem-perature tested, 160°C, more than 96% of thechlorophyll was precipitated at addition levelsabove 1200 mg/L, while lower levels resultedin only small decreases in chlorophyll (Table1).

At temperatures below 120°C the separa-tion of the precipitated chlorophyll was diffi-cult. This indicates that moisture present inthe system interferes with the process. Athigher temperatures the precipitation is com-plete in 15 min., and further contact does notresult in any further decrease in residualchlorophyll level. Thus a treatment of 15 min.at 120°C with 2400 mg/L phosphoric acidwas selected as a practical compromise,which results in 95+ % chlorophyll removal

The treated oil had a slightly increased freefatty acid level, as expected. Some of themeasured acidity was due directly to the titra-tion of the phosphoric acid, while some 0.2%was due to acid hydrolysis of the oil.

The treatment imparted a reddish hue to theoil, resulting in an increased in the Lovibondreading from 6.0R to 8.5R. This was at leastpartially due to the removal of the greenchlorophyll, which masked some of the redtint in the crude. When crude and phosphoricacid treated oils were refined and bleached,the same final FFA and Lovibond readingswere obtained with both. The untreated re-fined oil contained 1.7 mg/L chlorophyll,compared to <0.05 mg/L for the treated oil.

Although soybeans are typically muchlower in chlorophyll than canola seeds, testswith soybean oil also gave excellent results.

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84 Int. J. Appl. Sci. Eng., 2005. 3, 2

Starting with chlorophyll levels of approxi-mately 1 mg/L, the treatment resulted inchlorophyll contents of <0.05 mg/L.

The mechanism of the chlorophyll precipi-tation was not immediately obvious. Phos-phoric acid is a strong mineral acid, but it isalso a good chelating agent, and therefore weinvestigated the effect of a number of otheracids, phosphate salts and chelating agents. Atlow temperature these treatments had nomeasurable effect. At 140°C only minor re-ductions in chlorophyll were observed withmost of the reagents tested, only phosphoricacid had a major effect. The results are pre-sented in Table 2.

Table 1. Effect of temperature and phosphoricacid addition level on chlorophyll removalfrom canola oil containing 26.8 mg/kgchlorophyll

H3PO4 added2400

ppm

2000

ppm

1200

ppm

1000

ppm

800

ppm

Temperature Chlorophyll left in oil, mg/kg

100°C 0.4 1.9 6.7 8.7 20.3

120°C 0.6 2.0 4.9 0.3 19.4

140°C 0.3 1.0 2.5 8.6 17.0

160°C 0.2 0.5 0.8 4.5 16.9

As other acids did not remove appreciablequantities of chlorophyll, the H+ is clearly notthe actual reagent. Curiously, phosphate saltswere also ineffective in the presence of anorganic acid. Typical chelating agents werealso ineffective.

Sulfuric acid is a strong oxidizing agent,and darkens the oil. In high temperaturetreatments with concentrated sulfuric acid thismade the chlorophyll measurement impossi-ble. Niewiadomski [5] indicated that sulfuricacid oxidizes and precipitates gums, proteins

and pigments, including chlorophyll, from theoil, and therefore a range of conditions forsulfuric acid treatment were investigated. Atroom temperature 2000 mg/L H2SO4 additionresulted in significant reduction in chlorophyll.Addition levels above 4000 mg/L H2SO4 re-duced the chlorophyll content by 98%. Thetreatment did not increase the Lovibond redvalue in the treated oil, and the refined andbleached oil had a lower red value than theuntreated oil.

Mass spectrometric analysis [6] of the oilsamples and of chlorophyll standards dis-solved in pure octane shoved that the phos-phoric acid treatment converted the chloro-phyll to oil-insoluble pheophorbides (Figure1).

We believe that reaction proceeds in theabsence of water only, by protonation of thecarbonyl group, followed by de-alkylation.This releases the non-polar aliphatic sidechain, leaving the porphyrin structure with acarboxylic acid side-chain, which is oil in-soluble.

The proposed mechanism predicts that asthe side-chain is completely removed, the pre-cipitation of the chlorophyll is irreversible:the removal of the phosphoric acid by neu-tralization, or the addition of moisture wouldnot re-solubilize the chlorophyll, or the pre-cipitated pheophorbide into the oil. This leadto integrating the acid treatment into the re-fining process. Phosphoric acid was added tothe crude oil at 140°C under vacuum withgentle mixing. After 15 min., the oil wascooled and caustic refined, without first re-moving the precipitated chlorophyll break-down products. The precipitate was removedwith the soapstock, and the oil was bleached.

The phosphoric acid treatment readily fitsinto the normal refining practice, as it uses achemical that is already added to canola oilduring or after degumming. The processwould require the addition of a heated vac-uum mixing vessel and a centrifuge. Sulfuricacid treatment, while it is simpler in someways, requires more acid, and requires the

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Int. J. Appl. Sci. Eng., 2005. 3, 2 85

introduction of another chemical into theprocess, which has some potential labelingand safety implications. The process blockdiagram is presented in Figure 2.

Comparison of the chlorophyll levels afterrefining and after bleaching indicated no sig-nificant differences in chlorophyll contentbetween samples that had the precipitate re-moved prior to refining, and those that elimi-nated that separation step. After bleachingboth of treated oils contained <0.05 mg/Lchlorophyll.

The reaction mechanism indicates that thechlorophyll may be removed at any stage inthe refining process, where the chlorophyll isstill intact, and the process allows the oil to beheated to a high temperature, in the absenceof water. Thus the phosphoric acid treatmentmay be introduced prior to either acid de-gumming or bleaching for physically refiningplants, since both of these process stages arefollowed by a filtration or centrifugal separa-tion step. We tested this hypothesis on a largepilot-plant scale. The results are presented inTable 3.

Table 2. Effect of chemical treatments on chlorophyll removal

Reagent Residual chlorophyllppm

Chlorophyll reduction%

Initial chlorophyll content 23.8 0Citric acid (aq) 22.09 7.2Potassium dihydrogenphosphate (aq) 22.48 5.5

Hydrochloric acid 19.76 17.0Sulfuric acid* n/d n/dMaleic anhydride (aq) 21.59 9.3Sodium EDTA 22.43 5.8Acetic acid 22.32 6.3Sodium sulfate + aceticacid 22.07 7.3

EDTA + acetic acid 22.32 6.3Potassium dihydrogenphosphate + acetic acid 22.41 5.9

Malic Acid 22.5 4.3Monoglyceride citrate 22.5 4.3Phosphoric acid 0.30 98.7Conditions:

Temperature 140°CAddition level 2000 ppmContact time 10 minutesAll figures are averages of three replicate treatments with a relative standarddeviation of less than 10%.

* Not measurable, due to the dark color of the oil.

Levente L. Diosady

86 Int. J. Appl. Sci. Eng., 2005. 3, 2

N

N N

N

R

Mg

OH3CO2CO

O

Chlorophyll a, R = CH3Chlorophyll b, R = CHO

C O CH 2 CH

CH

C

3

R CH R1

H

C O CH 2 CH

CH

C

3

R CH R1

H

C OH CH 2 CH

CH

C

3

R CH R1

O

O

O

+H

Protonate carbonyl group

De-alkylate

R = porphyrin structure of chlorophyll-a

R1 = C15H31 - aliphatic side-chain

Figure 1. Mechanism of chlorophyll removal

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Int. J. Appl. Sci. Eng., 2005. 3, 2 87

Figure 2. Flow diagram for pre-refining chlorophyllremoval

Table 3. Effect of phosphoric acid treatment, withand without the removal of the prcipitatedpheophorbides prior to caustic refining

With treatmentWith Without

Withouttreatment

separationChlorophyll content

Material

ppm ppm ppmOriginal oil 23.2 23.2 23.2Phosphoricacidtreated n/a 2.7 n/aCausticrefined 18.2 0.8 0.5Bleached 1.7 <0.05 <0.05

The elimination of the precipitate separa-tion step makes the process readily adaptableto typical refineries. The addition of a heatexchanger that heats the oil to 140°C and a

vacuum mixing vessel with 15 min. residencetime are the only modifications required. Theblock flow diagram is presented in Figure 3.Pilot scale tests have confirmed the laboratoryresults, and after a full-scale test in Procterand Gamble Inc.'s Hamilton plant, the processwas patented [7], and successfully imple-mented in a full scale caustic refining plant.

Figure 3. Flow diagram for integrated chlorophyllremoval

Acknowledgements

The author would like to thank Joseph Al-berti, Ahmed Hussein and Chris Beharry fortheir technical assistance, and ProfessorD.G.B. Boocock for his theoretical insight.The work was financially supported byNSERC, the URIF program of the Govern-ment of Ontario, and Procter and Gamble Inc.

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88 Int. J. Appl. Sci. Eng., 2005. 3, 2

References

[ 1] Diosady, L. L. 1998. Removal of Chlo-rophyll from Canola Oil. In: "Proceed-ings of World Conference and Exhibitionon Oilseed Processing and Edible OilRefining: Advances in Oils and Fats". S.S. Koseoglu, K. C. Rhee, and R. F. Wil-son, Editors, American Oil Chemists’ Society (AOCS) Publications, Cham-paign IL, 2: 59-63.

[ 2] Brimberg, U. I. 1982. Kinetics ofbleaching of vegetable oils. Journal ofthe American Oil Chemists’ Society, 59:2-74.

[ 3] Kephart, J. C. 1955. Chlorophyll deriva-tives and their uses. Economic Botany, 9:3-38.

[ 4] Anonymous. 1980. American Oil Chem-ists' Society Official and TentativeMethods of Analysis, 3rd ed. WashingtonD.C.

[ 5] Niewiadomski, H. I., J. Bratowska and E.Mossakowska. 1965. Content of chlo-rophyll and carotenes in rapeseed oil.Journal of the American Oil Chemists’ Society, 42: 731.

[ 6] Hussein, A. A. 1992. "Removal of chlo-rophyll from canola oil". M.A.Sc. Thesis,University of Toronto, Department ofChemical Engineering and AppliedChemistry.

[ 7] Diosady, L. L., L. J. Rubin, A. A. Hus-sein and C. Beharry. 1995. Chlorophyllremoval from edible oils. US Patent5,315,021.