34.1.
18
AOAC Official Method 948.14
Succinic Acid
in Eggs
Ether Extraction Method
First Action 1948
Final Action
A. Apparatus
(a) Continuous extractor.—See Figure 937.05 (see 33.2.08).
(b) Chromatographic tube.—Approximately 17 od ´ 250 mm,
plugged at constricted end with either cotton or glass wool.
B. Reagents
(a) Solvent.—tert-Butyl alcohol–CHCl3 (1 + 4). Store over gran-
ular anhydrous Na2SO4.
(b) Glycerol indicator solution.—Dissolve 75 mg mono ammo-
nium salt of 3-(4-anilino-1-naphthylazo)-2,7-naphthalenedisulfonic
acid in 50 mL glycerol, warming on steam bath.
(c) Phenol red indicator.—Rub 100 mg phenolsulfonphthalein
in mortar with 5.7 mL 0.05M NaOH until dissolved; then dilute to
100 mL with H2O.
C. Preparation of Solution
(a) Liquid or frozen eggs.—Weigh 200 g test portion into 1 L
Erlenmeyer, add 500 mL H2O, and mix well, avoiding violent shak-
ing. Add 75 mL 0.5M H2SO4 and mix well. Add 125 mL 20%
phosphotungstic acid solution (w/v), dilute to 1 kg with H2O, and
shake 1 min. Divide between two 24 cm rapid folded filter papers.
Transfer 250 mL filtrate to 400 mL beaker, evaporate to ca 50 mL,
add another 250 mL to same beaker, and evaporate to 10 mL. If ma-
terial starts to bump when volume becomes low, transfer to steam
bath.
If <200 g test portion is available, take 100 g test portion and half
quantities of reagents, and dilute to 500 g with H2O. Filter precipitate
on Bchner with suction, collecting as much filtrate as possible. Use
total weighed filtrate for evaporation.
(b) Dried eggs.—Weigh 50 g test portion into 400 mL beaker,
and with heavy stirring rod make into smooth paste with H2O. Trans-
fer to 1 L Erlenmeyer and add enough H2O to make total weight of
700 g. Add 50 mL 0.5M H2SO4 and mix well. Add 75 mL 20%
phosphotungstic acid solution (w/v) and proceed as in (a), beginning
“. . . dilute to 1 kg with H2O, . . .”.
D. Extraction
Place 15 g (NH 4) 2SO 4 in dry extractor. Transfer evaporated
material, C(a) or (b), to inner tube of extractor by washing
through small funnel with enough H2O to make total volume of
40 mL, add 0.5 mL H2SO4 (1 + 1), and mix by raising and lower-
ing inner tube. Rinse beaker with 50 mL ether and pour rinsings
into inner tube of extractor. Connect efficient condenser to ex-
tractor and proceed with extraction as in 937.05D (see 33.2.08),
placing 150 mL ether in extraction flask and extracting 3 h or as
long as necessary for complete extraction.
To determine time necessary for complete extraction, transfer ca
20 mg succinic acid, accurately weighed, to extractor containing
20 g (NH4)2SO4, add enough H2O to give total volume of 40 mL, and
proceed with extraction as above. After 3 h add 10 mL H2O to extrac-
tion flask, evaporate ether on steam bath, and titrate. If recovery is
<95%, extract another 20 mg succinic acid for longer period and ti-
trate. Continue until 95% recovery is obtained, and use this period of
extraction for determination.
To flask containing ether extract add 5 mL H2O and evaporate ether
on steam bath. Using graduated 5 mL pipet, neutralize contents of flask
with saturated Ba(OH)2 solution, using phenolphthalein. Adjust vol-
ume to 20 mL with H2O, add 90 mL alcohol, heat almost to boiling on
steam bath, and cool. Add ca 0.5 g filter-aid and filter with suction
through suitable filter, such as Caldwell crucible covered with thin layer
of glass fiber filter overlaid with small amount of filter-aid added from
suspension in H2O. Rinse flask with 3 portions of alcohol (9 + 2), trans-
ferring each rinsing to crucible and sucking dry before adding another
portion. Reserve filtrate for determination of lactic acid, 937.05E (see
33.2.08), beginning line 6, “To expel alcohol, evaporate . . .”, using en-
tire filtrate and modifying calculations.
Transfer contents of crucible to 100 mL beaker with 15–20 mL
H2O, acidify to Congo red paper with 1–2 drops H2SO4 (1 + 1),
warm on steam bath, and refilter with suction, rinsing beaker with
three 10 mL portions H2O, transferring each rinsing to crucible,
and sucking dry before adding another. Evaporate filtrate to ca
5 mL, neutralize with 1M NaOH, transfer with H2O to 50 mL
beaker, and evaporate to dryness on steam bath.
E. Preparation of Partition Column
Place 5 g H2SiO3, (reagent grade “100-mesh” silicic acid powder,
suitable for chromatography [Mallinckrodt Speciality Chemical Co.,
No. 2847 or equivalent]) in glazed porcelain evaporating dish and add
0.5 mL freshly prepared glycerol indicator solution. Then add maxi-
mum amount of glycerol (1 + 1) that gel will hold without becoming
sticky (usually 1–3 mL) and 1 drop (ca 0.05 mL) ca 1M NH4OH. Grind
into uniform powder with pestle, make into slurry with ca 30 mL of sol-
vent, and transfer to chromatographic tube, which is clamped vertically.
Apply 5–10 lb (34.5–68.9 kPa) air pressure to top of tube until solvent
just disappears into top of gel; release pressure, add 1 mL CHCl3 con-
taining ca 5 mg CH3COOH, and again apply pressure until solvent just
disappears into gel. Release pressure, add 5 mL solvent, and once more
apply pressure just long enough for solvent to disappear into gel. Pres-
sure should never be left on with no liquid above gel; gel would then dry
and crack, becoming useless.
F. Determination
To dry residue of sodium succinate, D, add 2 mL solvent and
3 drops H2SO4 (1 + 1), and stir with glass rod until all particles are
moistened (material should be acid to Congo red paper). Add anhy-
drous Na2SO4 in 0.5 g portions until material is dry (not gummy).
Stir, and decant onto prepared partition column, pouring it slowly
down side of tube in order to keep surface of gel level. Apply pres-
sure until solvent just disappears into gel. Again wash beaker with
1 mL solvent, pour onto column, and with stirring rod transfer resi-
due in beaker to column. Wash beaker with another 1 mL solvent,
transfer to column, wash inside of tube with 1 mL solvent, and ap-
ply pressure until solvent just disappears into gel. A light placed
adjacent to column, but not so close as to heat it, increases visibility
of bands. Fill tube with solvent and apply pressure. Let CH3COOH
band pass out of tube. When front of succinic acid band reaches
constricted portion of tube, start collecting eluate in 50 mL gradu-
ate. Continue collecting until band has passed entirely from col-
umn or until lower edge of any following band reaches 2–5 mm
above narrowest portion of constriction of tube and until enough
eluate collects to ensure removal of succinic acid from column.
To ensure complete removal of succinic acid from column when
there is no following band, determine total amount of eluate to be
collected by preparing solution of known amount of sodium
succinate, transferring free acid to column, eluting, etc., as above,
© 2000 AOAC INTERNATIONAL
and titrating 25 mL and successive 10 mL fractions of eluate until
last fraction requires <0.2 mL 0.01M alkali to neutralize. Total
amount of eluate required is amount to collect in determination.
Add 10 mL H2O to flask and titrate with 0.005M Ba(OH)2, using
phenol red indicator. As end point approaches, stopper flask and
shake vigorously to extract acid completely from solvent phase.
Correct titration for blank determination on equal volume of eluate
from blank column. 1 mL 0.005M Ba(OH)2 = 0.59 mg succinic
acid. If crystallographic identification of barium succinate
[JAOAC 32, 787(1949)] is not desired, 0.01M NaOH may be used
for titration.
References: JAOAC 31, 134(1948); 32, 787(1949).
CAS-110-15-6 (succinic acid)
Revised: March 1996

© 2000 AOAC INTERNATIONAL