46.1.

06
AOAC Official Surplus Method 920.209i
i
Natural Coloring Matters
Identification by Color Reactions
First Action
Surplus 1970
34.017
Properties
As a class, natural coloring matters show much less
tendency to dye animal fiber than do common synthetic
colors. Many crude products contain number of colored
substances, and complete sepn is not practicable. As dil.
solns of most of natural coloring matters are sensitive
to alkalies and some are sensitive to acids, such
reagents must be used with care. Relatively few good
tests are known for common natural colors. (See
967.17i
i (See 37.1.57.)
34.018
Separation
(a) By extraction with ether from neutral
solns.From neut. solns ether exts carotene,
xanthophyll (pigments found in leaves, fats and oils,
egg yolk, carrots, etc.) coloring matter of tomatoes and
paprika, and green chlorophyll. Coloring matter
remains in the ether soln on shaking with 1N NaOH or
1N HC1, no apparent change taking place, altho the
substances may be altered chemically by this treatment.
(b) By extraction with ether from acid solns.From
slightly acid solns ether very readily and completely
exts coloring matter of alkanet, annatto, turmeric, and
red dyewoods, sandalwood, camwood, and barwood. It
exts, in large proportions, flavone coloring matters of
fustic, Persian berries, and quercitron (after hydrolysis),
as well as coloring matter of brazilwood and green
derivatives formed from chlorophyll by alk. treatment.
It exts, in relatively small amt, coloring matters of
logwood, orchil, saffron, and cochineal. Coloring
matters of this group are readily removed from ether by
shaking with alk. solns, but in most cases they rapidly
undergo chemical change.
(c) By extraction with amyl alcohol from acid
solns.From slightly acid solns amyl alcohol exts
major part of coloring matters of logwood, orchil,
saffron, and cochineal. Amyl alcohol exts relatively
small proportions of caramel and anthocyans
constituting red coloring matter of most common fruits.
Identification
34.019
By Color Changes Produced with
Various Reagents
Evap. to dryness ether solns obtained in 34.018 (a)
and (b) (Caution: See Appendix B: Distillation,

Extraction and Evaporation; and Diethyl Ether),

warm residue with little alcohol, and dil. with H2O. Dil.
amyl alcohol soln obtained in 34.018 (c) with pet ether
and ext with H2O. To portions of these somewhat
purified solns of coloring matter apply reagents as
follows:
Hydrochloric acid.Add to soln, first, 1 or 2 drops
HC1; then excess equal to 34 times vol. soln.
Sodium or potassium hydroxide.Make soln slightly
alk. by adding 1drop 10% NaOH or KOH soln. (Red
changing to yellow, especially on warming, may be due
to presence of gallate antioxidants.)
Sodium hyposulfite.Add small Na2S2O4 crystal.
Ferric chloride.Very carefully add small amt of
freshly prepd 0.5% FeCl3 soln, small drop at time.
(Colors are not always obtained when excess is used.)
Alum.To test soln add 1/5 its vol. of 10% K- or
NH4-alum soln.
Uranium acetate.Add 5% UO2(OAc)2.2H2O soln
dropwise. (Caution: See Appendix B: Uranyl Acetate
and Toxic Dusts.)
Sulfuric acid on dry color.Evap. small amt of soln
or of coloring matter in porcelain dish. Cool thoroly and
treat dry residue with 1 or 2 drops cold H2SO4. Colors
are sometimes extremely transitory, and may be
observed only instant acid wets residue.
Table 34:2 shows behavior of certain natural coloring
matters when treated in manner described above.
34.020
By Special Tests
(a) Chlorophyll. Brown phase reaction (Molisch,
Ber. botan. Ges. 14, 16(1896)) may be useful to
characterize chlorophll, when it has not been previously
treated with alkalies. Treat green ether or pet ether soln
of coloring matter with small amt of 10% soln KOH in
MeOH. Color becomes brown, quickly returning to
green.
(b) Annatto (Leach-Winton, Food Inspection and
Analysis, 4th ed,. 1920, pp. 161, 558).Pour on
moistened filter alk. soln of color obtained by shaking
out oil or melted and filtered fat with warm 2% NaOH
soln. If annatto is present, paper absorbs color, so that
when washed with gentle stream of H2O it remains
dyed straw color. Dry paper, add drop of 40% SnCl2
soln, and again dry carefully. If color turns purple,
presence of annatto is confirmed.
(c) Turmeric.Treat aq. or dil. alc. soln of color with
HC1 until shade just begins to appear slightly orange.
Divide mixt. into 2 parts and add some H3BO3 powder
or crystals to one portion. Marked reddening is quickly
apparent, best seen by comparison with portion to
which H3BO3 has not been added. Test may also be
made by dipping piece of filter paper in alc. soln of
coloring matter, drying at 100E, and then moistening
with weak soln of H3BO3 to which few drops HC1 have

been added. On drying again, paper becomes cherryred.

(d) Cochineal.When presence of cochineal is
suspected, acidify mixt. with a it vol. HC1 and shake
with amyl alcohol. Wash amyl alcohol soln of coloring
matter 24 times with equal vols H2O to re-remove
HCl, etc. Dil. amyl alcohol with 12 vols pet ether and
shake with few small portions of H2O to remove color.
Divide combined aq. exts into 2 portions.
To first add, dropwise, 5% UO2(OAc)2.2H2O soln,
shaking thoroly after each addn. In presence of
cochineal, soln turns characteristic emerald-green.
(Girard, Analyse des Matires Alimentaires et
Recherche de leurs Falsifications, 2nd ed., 1904.) U
salts do not turn green in presence of much free acid.
Therefore, add little NaOAc before making this test, or
correspondingly larger amt of UO2(OAc)2 soln must be
added.
To second portion add 1 or 2 drops NH4OH; in
presence of cochineal, soln turns violet. This, however,
is not so characteristic as first test, as many fruit colors
give almost identical reactions. Cochineal is not
decolorized by Na2S2O4 in either acid, neut., or alk. soln
(differs from orchil).
As cochineal lakes often contain Sn, always make
further examination for this metal when H2O-insol.
cochineal compds seems to be present.
(e) Orchil.This coloring matter is either sulfonated
or unsulfonated. Unsulfonated orchil is readily exd by
amyl alcohol from weak acid soln, while extn of
sulfonated color is incomplete even from strongly
acidified soln. Behavior of color towards acids and
alkalies is similar to cochineal, e.g., HC1 produces
yellow shade and alkalies produce bluish shade.
Na2S2O4 reduces orchil, but color is restored by air
oxidn (differing from cochineal). Characteristic
property of orchil is to dye, strip, and redye wool
readily.
(f) Caramel.Number of tests have been developed
for this coloring matter, most of them based upon
insolubility in ether, CHCl3 , or amly alcohol. Probably
most sensitive test is Woodman-Newhall (Mass. Inst.
Tech. Quart. 21, 280(1908)) modification of Amthor
test with slight deviation.
To 1020 mL neut. soln of color in small centrf. tube
add 2 mL 5% ZnCl2 soln and 2 mL 2% KOH soln, stir
well, and centrf. Pour off liq., and add 25 mL boiling
H2O to magma. Mix, centrf., and pour off liq. Repeat
this operation until aq. wash liq. is colorless. Dissolve
ppt with 15 mL 10% HOAc, conc., neutze carefully,
and filter. Divide into 2 poritons. To one add 35 vols
paraldehyde in 50 mL g-s cylinder, and just enough
absolute alcohol to form homogeneous soln (avoid
excess). Caramel is indicated by formation of brownish
ppt on standing. To other portion of caramel soln add

equal vol. freshly prepd reagent consisting of

phenylhydrazine. HC1, 2 parts; NaOAc.3H2O, 3 parts;
H2O, 20 parts. Dark brown ppt is formed in presence of
caramel.
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