S AMPLING AND ANALY S IS OF C OMME R C IAL F ATS AND OILS

•• A OC S Official Method C a 18-79

R eapproved 2009

Analysis for C hromium, C opper, Iron, Nickel,

and Manganese in Triglyceride Oils by
Atomic Absorption S pectrophotometry Using
a Graphite F urnace
DE F INITION
V egetable and animal oils dissolved in methyl isobutyl ketone (MIBK ) or MIBK + HNO3 are analyzed for
metals by direct injection into a graphite tube furnace. Calibration is based on MIBK or MIBK + HNO3
solutions of organometallic reference compounds.
S C OPE
This method is intended for the analysis of crude and refined vegetable oils and animal fats that contain
trace metals that may be present in the form of oil-soluble adducts. The detection limit of this method
is approximately 0.1 mg/kg (0.1 ppm). This method has a lower detection limit than A OCS Method Ca
15-75, which has a detection limit of approximately 2 mg/kg (2 ppm). Therefore, when lower detection
limits may be required, as in the case of contractual trade methods, A OCS Method Ca 18-79 should be
used.

A PPA R A TUS
1. A tomic absorption spectrophotometer— with Zeeman or Smith-Hieftje background correction and graphite tube furnace.
2. V acuum desiccator— containing a suitablefresh desiccant (see A OCS Specification H 9-87).
3. Weighing bottles— 10— 15 mL capacity.
4. Hot plate— with rheostat temperature control.
5. Oven.
6. Beakers— 25 and 100 mL .
7. Pipets— graduated, to deliver 5, 10, and 20 mL .
8. Eppendorf pipets— 5, 10, 20, 30, 40, and 50 pL , with disposable tips.
9. Balance— analytical, 200 g capacity with ±0.0001 g sensitivity.

R E A GE NTS
1. Organometallic standards— National Institute of Testing (NIST); Conoco (Ponca City, OK ), USA , or equivalent.
(a) tris(l-phenyl-l,3-butanediono)chromium III.
(b) bis(l-phenyl-l,3-butanediono)copper II.
(c) tris(l-phenyl-l,3-butanediono)iron III.
(d) Nickel cyclohexanebutyrate.
(e) Manganous cyclohexanebutyrate.
2. 4-methyl-2-pentanone (methyl isobutyl ketone, MIBK )— reagent grade (see Notes, Caution).
3. X ylene— reagent grade (see Notes, Caution).
4. 2-Ethylhexanoic acid— practical grade.
5. 2-Ethylhexylamine— practical grade.
6. Nitric acid— Spectral or Ultrex grade (see Notes, Caution).

PR E PA R A TION OF S TA NDA R DS
A ll standards should be prepared in a fume hood and operators should wear gloves.
1. Chromium stock standard, 100 pg/mL — Dry about 2 g tris(l-phenyl-1,3-butanediono)chromium III 1 hr at 110°C. Weigh
103.1 mg into a 25 mL beaker, add 5 mL xylene and 5 mL 2-ethylhexanoic acid, and gently warm on hot plate without refluxing,
swirling occasionally until the salt dissolves. Cool to ambient temperature. Transfer the solution to a 100 mL volumetric flask,
rinsing the beaker with five 10 mL portions of MIBK , then dilute to exactly 100 mL , mix, and store at 20 ± 2°C. This standard
stock solution should be stable for at least 6 months.
2. Copper stock standard, 100 pg/mL — Dry about 0.13 g bis(l-phenyl-l,3-butanediono)copper II 30 min at 110°C. Weigh
60.6 mg into a 25 mL beaker, add 4 mL xylene and 4 mL ethylhexylamine, and gently warm on hot plate without refluxing,
swirling occasionally until the salt dissolves. Cool to ambient temperature. Transfer the solution to a 100 mL volumetric

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Copyright © 2009 by the American Oil C hemises' Society. A ll rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
S AMPLING AND ANALY S IS OF C OMME R C IAL F ATS AND OILS
C a 18-79 • Analysis for C hromium, C opper, Iron, Nickel, and Manganese in Triglyceride Oils by AAS

flask, rinsing the beaker with five 10 mL portions of MIBK , then dilute to exactly 100 mL , mix, and store at 20 ± 2°C. This
standard stock solution should be stable for at least 6 months.
3. Iron stock standard, 100 pg/mL — Dry about 0.2 g tris(l-phenyl- 1,3-butanediono)iron III for 1 hr at 110°C. Weigh 97.1
mg into a 25 mL beaker, add 5 mL xylene and 5 mL 2-ethylhexanoic acid, and gently warm on hot plate without refluxing,
swirling occasionally until the salt dissolves. Cool to ambient temperature. Transfer the solution to a 100 mL volumetric
flask, rinsing the beaker with five 10 mL portions of MIBK , then dilute to exactly 100 mL , mix, and store at 20 ± 2°C. This
standard stock solution should be stable for at least 6 months.
4. Nickel stock standard, 100 pg/mL — Desiccate about 0.15 g nickel cyclohexanebutyrate for 48 hr over suitable fresh desic­
cant. Rapidly weigh 67.6 mg into a 25 mL beaker, add 5 mL xylene and 5 mL 2-ethylhexanoic acid, and gently warm on hot
plate without refluxing, swirling occasionally until the salt dissolves. Cool to ambient temperature. Transfer the solution to
a 100 mL volumetric flask, rinsing the beaker with five 10 mL portions of MIBK , then dilute to exactly 100 mL , mix, and
store at 20 ± 2°C. This standard stock solution should be stable for at least 6 months.
5. Manganese stock standard, 100 pg/mL — Desiccate about 0.15 g manganese cyclohexanebutyrate for 48 hr over suitable
fresh desiccant. Rapidly weigh 71.4 mg into a 25 mL beaker, add 5 mL xylene and 5 mL 2-ethylhexanoic acid, and gently
warm on hot plate without refluxing, swirling occasionally until the salt dissolves. Cool to ambient temperature. Transfer the
solution to a 100 mL volumetric flask, rinsing the beaker with five 10 mL portions of MIBK , then dilute to exactly 100 mL ,
mix, and store at 20 ± 2°C. This standard stock solution should be stable for at least 6 months.

WOR K ING S TA NDA R DS

1. Fe— Pipet 10 pL of 100 ppm stock standard into a 25 mL volumetric flask. A dd 15 mL MIBK , 12 drops concentrated
HNO3, swirl, dilute with MIBK to a final volume of exactly 25 mL , and mix completely.
2. Cu— Pipet 10 pL of 100 ppm stock standard into a 25 mL volumetric flask, dilute with MIBK to a final volume of exactly
25 mL , and mix completely.
3. Ni— Pipet 10 pL of 100 ppm stock standard into a 25 mL volumetric flask. A dd 15 mL MIBK , 600 pL concentrated
HNO3, swirl, dilute with MIBK to a final volume of exactly 25 mL , and mix completely.
4. Mn— Pipet 10 pL of 100 ppm stock standard into a 25 mL volumetric flask, dilute with MIBK to a final volume of exactly
25 mL , and mix completely.
5. Cr— Pipet 10 pL of 100 ppm stock standard into a 25 mL volumetric flask, dilute with MIBK to a final volume of exactly
25 mL , and mix completely.

PR OC E DUR E
1. A ccurately weigh 1.0 g ± 0.1 mg of oil into a 10 mL volumetric flask, dilute to volume with appropriate solvent system [see
(a) through (e) immediately following] and mix (see Notes, 1).
(a) Fe-MIBK + 250 pL coned HNO3/10 mL MIBK
(b) Ni-MIBK + 250 pL coned HNO3/10 mL MIBK
(c) Cu-MIBK
(d) Mn-MIBK
(e) Cr-MIBK
2. Set up instrument for operation with the following graphite furnace conditions:

Dry 30 sec 100°C

Char 20 sec 800°C
A tomize 20 sec 2300°C ‘2
A rgon purge gas 5— 6 L /min

^2300°C is the upper limit for P-E HGA -2000 with grooved
graphite tubes. If either HGA -2100 or HGA -2200 is
available, atomization at 2500°C is preferred.
3. Following the manufacturer’s instructions, set up the instrument, and adjust to the recommended wavelength (Table 1).
4. Record absorbance readings for the standard curve of interest by preparing at least a five-point curve. By varying injection
volume, a single working standard solution can be used to establish the entire curve. Inject 10, 20, 30, 40, and 50 pL volumes
of working standard solution in which the absorbance of at least three determinations at each injection volume agree within ±
0.005 absorbance units. Plot the curve for the metal of interest using the average absorbance as the Y axis and concentration
in pg as the X axis.
5. Sample analysis—
(a) Inject 20 pL of test solution as a first estimate. If an unacceptable absorbance value is obtained (either a low absorbance
value or a high absorbance value that exceeds the working linear range of the standard curve), the sample injection vol­
ume must be adjusted accordingly (see Notes, 2). With P-E grooved graphite tubes, individual injection volumes must
not exceed 50 pL .

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Copyright © 2009 by theAOC S
 S AMPLING AND ANALY S IS OF C OMME R C IAL F ATS AND OILS
C a 18-79 • Analysis for C hromium, C opper, Iron, Nickel, and Manganese in Triglyceride Oils byAAS

(b) For samples with unusually low metal content (see Notes, 3), multiple sample injections are permissible, if, after each
injection, the sample is allowed to proceed through the dry and char cycle, and is then interrupted for each additional
injection until the final aliquot is injected, in which case the dry/char/atomize cycle is not interrupted, but allowed to
continue through atomization (see Notes, 4).
(c) The absorbance value is then compared to the standard curve and the pg value for the sample is obtained from the
X axis.
6. Repeat Procedure, 3-5 for each analysis using the appropriate source lamp (as noted in Table 1) and conditions.

C A L C UL A TIONS
. (1000)(C)(SV )(F)*
1. Metal, ppm = ---------------------------------
(IV )(SM)
Where—
C = metal in mg from calibration graph
SV = final volume of sample flask
IV = volume, mL of test solution injected into furnace
SM = test sample mass in grams
F = dilution factor*, where F = volume of dilution flask/aliquot taken
*Use dilution factor only when dilution is made because initial sample preparation is too concentrated.

PR E C IS ION
1. Several laboratories participated in the collaborative study of the graphite furnace technique used to determine copper, iron,
nickel, chromium, and manganese at levels below 1 pg/g and mostly at 0.2 pg/g and under. Results showed a relative stan­
dard deviation (RSD) of 15 and 30% at levels of 0.2 pg/g and 0.1 pg/g, respectively. A t the 0.05 pg/g level, RSD values of
50% and above were frequently observed.

NOTE S
Caution
Methyl isobutyl ketone (MIBK ) is flammable and a dangerous fire risk. Explosive limits in air are 1.4— 7.5%. A void inhalation
and ingestion. It is absorbed by the skin. The TL V is 50 ppm in air.
X ylene is flammable and a dangerous fire risk. The TL V is 100 ppm in air.
Nitric acid— Use effective acid-resistant fume-removal device whenever heating acids or performing reactions that liberate
acid fumes. When diluting acids, always add acid to water unless otherwise directed in a method. K eep acids off skin and protect
eyes when working with acids. If acids come in contact with skin or eyes, wash immediately with large amounts of water. Do not
store oxidizing acids (perchloric, nitric, sulfuric) near organic materials. Mixing organic materials with these acids, particularly
perchloric, could result in an explosion.

NUMB E R E D NOTE S
1. A chieving a 10% wt/vol MIBK solution for C 16 and C 18 fatty acid triglycerides is readily accomplished down to an iodine
value (IV ) of 65— 70. Below this IV , solubility decreases to approximately 2% wt/vol for triglycerides with an IV of 0-10.
L auric stearins, however, are soluble in MIBK at 7-8% wt/vol.
2. The following is a guide to the range of linear response to the metals of interest:
Fe 2.4 ng
Cu 8.0 ng
Ni 11.0 ng
Mn 2.0 ng
Cr 2.0 ng
3. For a sample with either an unusually low metal content or a troublesome sample matrix, the “standard method of addition”
technique is recommended.
4. It is recommended that the purge gas not be interrupted during the atomization cycle. Past experience indicates that during the
final stage of triglyceride decomposition when moving from the char to the atomization mode, a last surge of smoke is liberated
from the sample. The background corrector is capable of compensating the magnitude of the smoke scatter effect. However,
when operating in a purge gas interrupt mode during atomization, a nonuniform and poorly reproducible smoke pattern results
in an apparent scatter of sample response. Operating in “purge gas on” mode during atomization may cause some loss in sensi­
tivity, but with “purge gas on” it transforms or mixes the smoke into a uniform gray field that is reproducibly corrected.

R E F E R E NC E S
1. Olejko, J .T .,/. Am. Oil Chem. Soc. 53:480 (1976).

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Copyright © 2009 by theAOC S
S AMPLING AND ANALY S IS OF C OMME R C IAL F ATS AND OILS
C a 18-79 • Analysis for C hromium, C opper, Iron, Nickel, and Manganese in Triglyceride Oils by AAS

Table 1
R ecommended wavelengths.
Element Wavelength, nm

Fe 248.3
Cu 324.8
Ni 232.0
Mn 279.5
Cr 357.9

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Copyright © 2009 by theAOC S