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46.1.03 other.) Following color tests may be applied to sepd

AOAC Official Surplus Method 930.38 dyes to confirm identity:

Color Additives

(Synthetic Organic) in Foods

First Action

(Amaranth, ponceau 3R, ponceau SX, erythrosine,orange I*, light green SF yellowish, fast green FCF,

guinea green B, brilliant blue FCF, indigotine, naphthol

yellow S**, sunset yellow FCF, tartrazine yellow

AB**, yellow OB**, orange SS*, and oil red XO*.)

34.007 Preparation of Solution—

Official First Action

(a) For foods containg oil-soluble dyes.—Shake oil

or melted fat with equal vol. alcohol, 90% by vol. wash

alc. ext with several portions of pet ether to free

coloring matter from fats, and then evap. alc. ext to

dryness in casserole. Treat residue with 40 ml pet ether,

and shake pet ether soln with two or three 5 ml portions

2-4% NaOH soln (to remove annatto, turmeric, etc., if

present). Pet ether soln will contain yellow OB, yellow

AB, orange SS, and oil red XO.

(b) For foods containing no oil-soluble dyes or from

which these dyes have been removed.—Obtain aq. soln

as free as possible from suspended matter, alcohol,

acids, alkalies, and salts. Dye soln should preferably be

0.01-0.05%. Soln obtained in examination of colored

food products rarely requires further diln. but with com.

food colors care must be taken that concn is not too

great.

34.008 Separation—OfficialFinal Action

(a) Yellow AB and yellow OB.— Ext pet ether soln of

these dyes, 34.007 (a), 3 times with ½ its vol. 13 N

H SO . Shake each acid ext successively with 22 4portions (equal vols) of pet ether, using same 2 portions

of pet ether for each acid portion. Ext each of 2 latter

pet ether portions with 20 ml 13 N H SO , same acid2 4portion successively for both pet ether portions. Finally

ext second of these pet ether portions with another 20

ml portion 13 N H SO . (Original pet ether portion 42 4times, and third times.) Combine acid exts, dil. with

H O, re-ext with pet ether, and evap solv. Yellow AB2will be found in practically pure state.

Combine pet ether solns (original and subsequent

solns left after acid washings), wash with small potions

of H O to remove excess acid, and evap. soln Yellow2OB will remain as residue (This method is not

absolutely quant., but it is accurate enough to sep.

either dye with comparatively little contamination from

(1) Shake 5 ml neut. pet ether soln of dye in test tube

with 5 ml mixt. of 1 part 40% HCHO soln and 4 parts

Ac O. Both coloring matters are extd by Ac O, yellow2 2AB giving red soln in few sec, and yellow OB, under

same conditions, giving orange soln.(2) To 1 ml alc. soln of dye (0.005-0.01%), add 0.1

ml HCHO, 0.1 ml H SO , and finally 8.0 ml H O.2 4 2Yellow AB yields red soln, unaltered by addn of excess

NH OH and somewhat intensified by further addn of 4excess HOAc. Yellow OB gives yellow or orange soln.

(3) To 1 ml alc. soln of dye (0.005-0.01%), add 0.1

ml Cu-pyridine soln (5 g CuSO .5H O and 10 ml4 2pyridine dild to 100 ml with H O) and 8.0 ml H O.2 2Yellow AB yields pink soln, becoming purple on addn

of excess NH OH. Yellow OB gives colorless or bluish4soln.

(b) Amaranth, ponceau 3R, ponceau SX, etythrosine,

orange I, light green SF yellowish, fast green FCF,

guinea green B, brilliant blue FCF, indigotine,

naphthol yellow S, sunset yellow FCF, and

tartrazine.—To soln obtained in 34.007(b) add 1 part

HOAc to every 7 parts soln. Ext with three 50 ml

portions amyl alcohol. Drain lower layer and reserve

for futher treatment. Wash amyl alcohol ext in rotation

with 25 ml portions 5% NaCl soln until washings are

colorless or nearly so. Add washings to original aq.

colorless or nearly so. Add washings to original aq.

soln. Dil. amyl alcohol ext with equal vol. pet ether and

wash with 25 ml portions H O until all color is extd.2Coloring matters obtained are orange I and guinea

green B. For their sepn see (1) below.

Treat amyl alcohol-pet ether soln with 10 ml portions0.1 N NaOH or with 10 ml portions NH OH (1+9), to4remove erythrosine. Acidify original soln and washings

(from which 3 named dyes were removed) with HC1 (1

vol. acid to 40 vols soln) and ext in 50 ml vols with

three 50 ml portions amyl alcohol. Resrve lower aq.

layer for further treatment. Wash amyl alcohol ext with

25 ml portions 0.25 N HC1 until washings are colorless

or nearly so. Combine washings with aw. soln above.

Ext amyl alcohol with several 25 ml portions H O until2all color is extd. Coloring matters obtained are ponceau

3R, ponceau SX, and naphthol yellow S. For their sepn

see (2).

Treat original soln and washings (from which 6named dyes were removed in 50 ml vol. with three 50

ml portions glycerol dichlorohydrin. Reserve upper sq.

layer for further treatment. Wash dichlorohydrin ext in

rotation with several 20 ml portions 25% NaCl soln.

Combine washings with aq. soln above. Dil.

dichlorohydrin ext with 2 vols Ccl and ext with several425 ml portions H O until all color is extd. Coloring2matters obtained are light green SF yellowish, fast


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green FCF, and Brilliant blue FCF. For their sepn see To prove presence of light green SF yellowish in

(3). presence of brilliant blue FCF proceed as follows: Dil.

Further acidify origianl soln and washings (from solv. with equal vol. pet ether and remove color with

which the 9 named dyes were removed) with HC1 ( 1 small portions H O. Treat 20 ml soln with 4 ml 10%

vol. acid to 40 vols soln) and ext in 50 ml vols with NaOH soln and boil 5 min. Brilliant blue FCF isthree 50 ml portions amyl alcohol. (If color intensity changed to red; light green SF yellowish is changed to

soln was not too strong, all coloring matter should have yellow. Acidify with 10 ml HOAc, which changes

been extd by solv.) Discard lower colorless or nearly brilliant blue FCF to violet and light green SF yellowish

colorless layer and wash out dyes from amyl alcohol to green. Treat with ca 3 g Zn dust and heat until soln

ext in rotation with several 25 ml portions H O until all is decolorized. Filter, make slightly alk. with NH OH2color is extd. Coloring matters obtained are indigotine, and then acid with HOAc, and bring to boil. In presence

amaranth, tartrazine, and sunset yellow FCF. For their of light green SF yellowish, deep green soln is formed

sepn see (4). while brilliant blue FCF remains colorless.

(1) Orange I and guinea green B.—Ext combined (4) Indigotine, amaranth, tartrazine, and sunset

colors with two 20 ml portions glycerol dichlorohydrin. yellow FCF .—To sep. indigotine, heat to boiling small

Discard colorless upper aq. layer, dil. solv. with 2 vols portion of soln, which should be neut. or faintly acid,

CC1 , and ext orange I in rotation with several 10 ml and add few crystals of Na S O until all dyes are4

portions H O, and guinea green B with several 10 ml reduced. On adding few drops HOAc and shaking with2portions 25% alcohol. air, indigotine is quickly restored, while amaranth,

(2) Ponceau 3R, ponceau SX, and napthol yellow tartrazine, and sunset yellow FCF are destroyed.

S.— Acidify combined colors with HC1 (1 part acid to If pos. test for indigotine is obtained, add several

10 parts soln) and ext naphthol yellow S with two 20 ml decigrams urea to remainder of mixed dye soln, heat

portions washed EtOAc or amyl acetate. (Ponceau 3R and while mixt. is boiling add 1 or 2 drops 10% NaNO

and ponceau SX are not extd appreciably and remain in soln. Indigotine is converted to pale yellow isatine

aq. layer.) Wash solv. with 5 ml portions 1 N HC1 to sulfonate, while amaranth, tartrazine, and sunset yellow

remove traces of ponceaus. Remove naphthol yellow S FCF are little affected. Acidify resultant mixt. with

from combined EtOAc or amyl acetate exts with 5 ml H SO (1+4), using 1 part dil. acid to 10 parts soln. Ext

portions NH OH (1+9). Ext remaining ponceau soln in 25 ml portions with three 50 ml portions n-butyl4with 20 ml portions amyl alcohol and wash out excess alcohol. Drain lower layer and pass successively thru

of acid twice with few ml portions H O. Dil. amyl all separators. Reserve aq. layer if colored; if not2alcohol with equal vol. pet ether and remove color with colored, discard.

small vols H O. Prep. following soln: 13.5 ml H SO , 100 g anhyd.2Treat 10 ml of this soln with 1 ml HC1, 2 ml satd Br- Na SO . amd enough H O to make 1 L. Ext the butyl

H O (Caution: See Appendix B, Laboratory Safety -2Bromine & Chlorine), and lastly 3 ml satd hydrazine

sulfate soln; immediately pour into test tube contg 10

ml 2 N Na CO and 2 drops 1% alc. -Naphthol. (Light2 3orange soln indicates ponceau 3R; deep brownish-red

soln indicates ponceau SX.) Add 5 ml ether to soln, mix

well, and drain lower aq. layer which, if colored,

contains ponceau SX. To ether ext add equal vol. HC1;

formation of purplish soln confirms presence of

ponceau 3R.

(3) Ligh green SF yellowish, fast green FCF, and

brillian blue FCF.—Treat combined colors with equalvol. 2 N Na CO and ext in 25 ml vols with two 50 ml2 3portions n-butyl alcohol. Drain lower aq. layer contg

fast green FCF and wash out last traces from solv. with

25 ml portions 2 N Na CO . Reserve washings and add2 3to aq. soln for confirmatory tests. Light seen SF

yellowish is colorless in the solv. while brilliant blue

FCF imparts bluish-green color.

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alcohol successively with 25 ml portions of this soln

until washings are colorless. Reserve them for amaranth

and tartrazine. Dil. the butyl alcohol with equal vol. pet

ether and remove sunset yellow FCF with H O.2Confirm spectrophtric.

Acidify reserved soln with HC1 (1 vol. acid to 20 of

soln) and ext with two 30 ml portions amyl alcohol.

(This exts both amaranth and tartrazine while isatine

compd, being less readily extd, remains in lower layer

and is discarded.) Remove coloring matter with several

10 ml portions H O. To portion of soln add 5 drops2NH Oh and few Na S O crystals. (This treatment4 2 2 4completely destroys amaranth, leaving tartrazine

practically unaltered.)

Add excess of HC1 and speedily ext dye with small

vol. amyl alcohol, from which soln tartrazine can be

removed with 0.25 N HC1. Treat another 10 ml portion

of neut. dye soln in test tube with 2 ml 20% NH Cl soln4and 1 ml 25% KCN soln (Caution: See Appendix B.,

Laboratory Safety, Cynides), and heat in boiling H O2

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bath 5 min. Cool rapidly, acidify with 2 ml HC1, and

ext with 10 mlamyl alcohol (Caution). Drain lower

layer and discard. Remove tartrazine with 5 ml portions

0.25 N HC1; amaranth is converted to lower sulfonated

to lower sulfonated dye and is not removed at this acidconcn. Dil solv. with equal vol. pet ether and ext dye

with small vols H O (amaranth is modified to brownish-2red dye).

Reference: USDA Bull. 1390, Supplement 1(1930)

© AOAC INTERNATIONAL

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