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TECHNICAL COMMUNICATIONS

Procedure Modification of AOAC Official Method 962.17,


Volatile Oil in Spices
ERTL: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 4, 1997
FRAN ERTL
Botanicals International, 2550 El Presidio St, Long Beach, CA 90810-1193

he modification of AOAC Official Method 962.17,


Volatile Oil in Spices, was requested by the Executive
Committee of the Technical Group (ECTG) of the
American Spice Trade Association (ASTA). Numerous reported problems with the current ASTA and AOAC method are
associated with the reproducibility and repeatability.
After an extensive search through ASTA member companies, we concluded that because the current method is not defined clearly it leads to many variables left to the analysts discretion. Each variable should be properly identified and fixed
to remove the cause of variability in final results.
To identify sources of variation, the ruggedness test was performed according to AOAC guidelines. Five spices were selected on the basis of market volume, volatile oil content, and
oil density. Ruggedness was tested in 2 consecutive phases.
The following spices were analyzed: (1) black pepper, one
of the spices with the largest volume traded in the United
States; (2) coriander, which has the lowest volatile oil content
among spices; (3) allspice, whose volatile oil is heavier than
water; (4) cloves, whose volatile oil is heavier than water and
also has the highest oil content; (5) oregano, one of the herbs
with the largest volume traded in the United States.
The variable parameters in the first phase are presented in
Table 1.
A volatile oil yield of >5% on each variable was considered
significant. For the first phase, use of 20 mesh particle size and
a cold grinder such as Brinkmann yielded significantly more
volatile oil (>5%), in agreement with unpublished data from the
spice industry.
The only issue of concern was poor repeatability. This was
traced back to the cleaning procedure for the traps. A new cleaning procedure versus ASTA-recommended procedure was assigned as one of the factors for the second phase. We also included the 2 most predominant contributors to the volatile oil
yield, particle size, and grinder type in the second phase to confirm our original finding. Temperature was also included in the
second phase to double-check the marginal result observed in

Submitted for publication April 26, 1996.


The recommendation was approved by the Methods Committee on
Additives, Beverages and Food Process Related Analytes, and was adopted
by the Official Methods Board of the Association. See Official Methods
Board Actions (1996) J. AOAC Int. 79, 111A, and Official Methods
Board Actions (1996) The Referee, October issue.

the first phase. The variable parameters in the second phase are
presented in Table 2.
Results of the second phase confirmed the initial findings:
The type of grinder is the most important factor that affects the
yield of volatile oils. A 20 mesh product has marginally better
yield than a 40 mesh product and significantly higher yield than
a 60 mesh product. Problems with repeatability were corrected
by the new cleaning procedure. No significant difference was
found between crank and clevenger traps, 5 versus 8 or 12 h
distillation periods, and between low and high temperature settings.
962.17, Volatile Oil in Spices
First Action 1962
Final Action 1965
Revised First Action 1996
(Applicable to determination of steam-volatile oil in spices.)
Caution: See Appendix B, safety notes on safe handling of
alkaliespotassium hydroxideand on safe handling of acidschromic and sulfuric acids. Chromium(VI) is extremely
toxic and a suspected carcinogen. Do not discharge into sewers
or waterways. See material safety data sheets or equivalent for
all reagents. Dispose of waste solvents in an appropriate manner compatible with applicable environmental rules and regulations.

A. Principle
Ground spice sample is boiled in H2O or NaCl solution. Released volatile oil (vapor form) is condensed and collected in
trap and then measured through trap calibration.

B. Apparatus
(a) Volatile oil trap.5 mL, without stopcock, clevenger
receivers crankcase oil, ASTM 5.0 0.1 mL subdivisions (Figure 962.17). Available from Lurex (clevenger trap, Part No.
720-1075, and crankcase trap, Part No. LX 4448-1002), Rt 40
& Oak Rd, Buena, NJ 08310-0688, USA. Once a year, calibrate
trap for xylene retention by running 3 blanks. Use average volume.
(b) West condenser.400 mm long with drip tip and
Ts 24/40 ground joints.

(c) Centrifugal grinding mill.Set with 0.75 mm mill


screen. Cold grinder can be used. (Note: To preserve volatile oil
constituents, mill must not generate heat during sample grinding.)
(d) Erlenmeyer flask.2 L; equipped with Ts 24/40 ground
joint.
(e) Stirring hot plate.Suitable for 2 L flask with variable
voltage transformer.
(f) Stirring bars.Teflon-coated, egg-shaped.
(g) Pipet filler.
(h) Cylinder.2 L, graduated.
(i) Tubing.Tygon tubing Norton, 79 mm id, or equivalent.
(j) Refrigerated circulating bath.Capable of maintaining
20C.
(k) Insulating tape.
(l) Blender.
(m) Stainless steel funnels.
(l) Volumetric pipets.5 mL.

C. Reagents
(a) Xylene.Reagent grade.
(b) Sodium chloride (NaCl) solution.10%.
(c) Antifoam B.Silicone emulsion containing 1% hydrogenated tallow glycerides (Dow Corning or equivalent).
(d) Chromic acidsulfuric acid solution.For cleaning
volatile oil trap. Mixture of 97.8% sulfuric acid, 0.5% chromium trioxide, and 1.7% H2O. Store at room temperature.
Chromic acidsulfuric acid solution is stable up to 2 years.
(e) Potassium hydroxide (KOH) solution.0.05%. For
rinsing volatile oil trap.
(f) Dishwashing liquid.

D. Determination
Blend all samples from a given lot in mixer and weigh 200 g
representative test sample. Ginger and turmeric samples must
be cracked before grinding. Set grinder with 0.75 mm mill
screen. Grind test sample. More than 99% sample must pass
through 0.85 mm sieve during the first run. Regrind oversize
until sample passes sieve.
Mix ground sample thoroughly and store in refrigerator (2
6C) in tightly sealed container until analysis.
Clean volatile oil trap by soaking overnight in chromic
acidsulfuric acid solution, C(d). Rinse with tap H2O followed
by rinsing with KOH solution, C(e). Rinse again with tap H2O.
Check cleanliness of trap by adding small volume of distilled
H2O. Water should flow down the wall of trap smoothly. Otherwise, repeat cleaning procedure. Immediately before use,
clean trap again with dishwashing liquid and test for cleanliness
as above.
Before removing aliquot for analysis, mix sample in container by rotating, to avoid stratification. Allow sample to
equilibrate to ambient temperature.
Weigh test sample as specified in Table 962.17. Quantitatively transfer sample to 2 L Erlenmeyer flask. Add ca 1200 mL
H2O or NaCl solution, C(b). (See Table 962.17 for distillation
solvent.) Add 23 drops of antifoam. Insert stirring bar.

Before placing volatile oil trap on flask, ensure that trap is


clean. Place trap on flask by using small amount of nonvolatile
stopcock grease on ground joints. Fill collection arms with H2O
or NaCl solution as specified in Table 962.17. (Note: For allspice, anise, and cloves add trap additive to graduated portion;
see Table 962.17.) Add 2 mL xylene with 5 mL pipet and verify
added volume by using volatile oil trap calibration [see B(a)].
Repeat analysis if part of xylene passes back into flask during
distillation.)
Connect trap to condenser with small amount of nonvolatile
stopcock grease and connect condenser to refrigerated circulating bath set at 20C. Insulate neck of trap and flask with insulating tape.
Heat flask on stirring hot plate with constant stirring. Adjust
trap and condenser so that drops flow down the wall of trap and
do not drop directly on surface of liquid. Gently swirl flask
several times during initial heating until sample is at rolling boil
to eliminate scorching of sample on walls. Maintain reflux rate
of 515 drops/10 s. Distill 8 h or until 2 consecutive readings
taken at 1 h intervals show no change of oil volume in trap. If
separation of oil is not satisfactory, add 12 drops of dishwashing liquid to trap through condenser or 23 mL saturated NaCl
solution.
Cool to 20C either by allowing trap to stand at ambient
temperature or by immersing in H2O bath. If trap is read at
temperature other than 20C, include it in report. Read volume
of oil collected to the nearest 0.01 mL.

E. Calculations
Calculate concentration of volatile oil in test sample as follows:
Volatile oil, % (v/w) =

Vo
100
Ws

where Vo = volume of oil collected in trap, mL, and Ws = weight


of test sample, g.
When 2 mL xylene is used as trap additive, calculate concentration of volatile oil in test sample as follows:
Volatile oil, % (v/w) =

Vo Vx
100
Ws

where Vx = volume of residual xylene, mL.


Ref.: JAOAC 17, 70, 371(1934); 18, 611(1935); 21,
435(1938); 22, 598(1939); 25, 700(1942); 33, 575(1950); 36,
752(1953); 37, 390(1954); 38, 548(1955); 42, 312(1959); 45,
212(1962); J. AOAC Int. 80, 901906(1997)

F. Specific Gravity of Volatile Oil


Determine specific gravity at 25/25 as in Methods 948.22
(see 40.1.05) and 950.48 (see 40.1.06), using 1 mL Sprengel
tube.

G. Refractive Index of Volatile Oil


See 950.50 (see 40.1.09) and 950.51 (see 40.1.10).

H. Eugenol in Volatile OilFinal Action 1965


Measure 2 mL volatile oil (transfer pipet) into Babcock milk
bottle, 989.04A(a) (see 33.2.27). Add 20 mL 3% KOH solution, shake mixture 5 min, heat 10 min in boiling H2O bath,
remove, and cool to room temperature. When liquids separate
completely, add enough KOH solution to bring residual oil
within graduated portion of neck and note volume. Calculate %
by volume from difference between volume sample used and
residual oil.
CAS-97-53-0 (eugenol)

I. Volatile Oil and Resin in GingerFinal Action 1965


Caution: See Appendix B, safety notes on monitoring equipment, distillation, flammable solvents, and diethyl ether.
Place 50 g ground ginger in Soxhlet extractor and extract
completely with ether (ca 4 h). Transfer extract to 300 mL flask
and evaporate ether on steam bath until solvent is no longer
detected. Add 50 mL H2O to residue and determine yield of
volatile oil (using trap for oils lighter than H2O), specific gravity, and refractive index, as in DG.
Transfer residue in flask to separator and extract resin with
ether. Transfer to tared beaker, evaporate ether on steam bath,
and dry to constant weight in vacuum desiccator.
Ref.: JAOAC 19, 98, 411(1936).

Explanation of Ruggedness Results


Refer to the Statistical Manual of the AOAC (1975), Youden
& Steiner, page 35. Ruggedness study results are presented in
Tables 3 and 4.
Example 1: In case of coriander analysis in phase I, the effect of altering the particle size is calculated by adding the %
volatile oil collected when the s, t, u, and v combinations are
applied versus w, x, y, z combinations:
s+t+u+v
4
0.536 + 0.572 + 0.429 + 0.464
=
4
= 0.50

Particle size, option I =

w+x+y+z
4
0.500 + 0.536 + 1.000 + 0.929
=
4
= 0.74

Particle size, option II =

Example 2: Oregano phase II, the effect of altering the type


of trap:
s+v+x+y
4
3.250 + 2.875 + 3.375 + 2.875
=
4
= 3.094

Trap type, option I =

t+u+w+z
4
3.375 + 2.875 + 3.000 + 2.875
=
4
= 3.031

Trap type, option II =

The percent volatile oil collected in phases I and II determined by using the same type of calculations as above are presented in Table 5.
There has been an error in phase I concerning distillation
temperature. High temperature was applied to s, u, v, w and z
instead of just s, v, w, z. Therefore, the calculation for this factor
has been modified as follows:
Temperature, option I =

s+u+v+w+z
5

Temperature, option II =

t+x+y
3

To compensate for this mistake, the effect of temperature


was evaluated again in phase II. The results clearly indicate that
temperature setting is not a major contributor to volatile oil recovery.

Table 962.17. Weights, distillation solvents, and trap


additives for various spices
Sample weight,
g

Distillation
solvent

Allspice

40

10% NaCl

Xylene, 2.0 mL

Anise
Basil
Bay leaf
Caraway
Cardamon
Celery seed

50
50
60
50
50
50

10% NaCl
H2O
H2O
H2O
H2O
H2O

Xylene, 2.0 mL

Cloves
Coriander
Cumin
Dill seed
Fennel
Fenugreek

10
70
50
50
50
50

10% NaCl
H2O
H2O
H2O
H2O
H2O

Xylene, 2.0 mL

Ginger
Marjoram
Nutmeg
Mace
Oregano
Black pepper
White pepper
Rosemary
Sage
Savory
Tarragon
Thyme
Turmeric

50
40
30
20
40
50
40
50
50
40
40
50
50

10% NaCl
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O

Spice

Trap additive

Table 1. Variable parameters in first phase of study


Variable

Option I

Option II

Particle size
Repeatability measurement within laboratory
Trap type
Grinder type
Distillation time

60 mesh grind; 99% through U.S. 60 mesh


Analyst 1
Clevenger
Brinkmann, cold grinder
5h

20 mesh grind; 99% through U.S. 20 mesh


Analyst 2
Crank
Regular grinder
8h

Temperature setting for distillation


Flask size

High temperature, distillation rate, 2 drops/s


1000 mL

Low temperature, distillation rate, <1 drop/s


2000 mL

Table 2. Variable parameters in second phase of study


Variable
Cleaning procedure
Grinder type
Temperature setting for distillation
Particle size
a
Repeatability
Distillation time
Trap type
a

Option I

Option II

New cleaning method


Brinkmann, cold grinder

Current ASTA method


Regular grinder

High temperature, 2 drops/s

Low temperature, <1 drop/s

20 mesh, 99% through U.S. 20 mesh


Analyst 1
5h
Crank

40 mesh, 99% through U.S. 40 mesh


Analyst 2
12 h
Clevenger

The contribution of the trap cleaning procedure has been removed.

Table 3. Ruggedness study results for phase I


Volatile oil, %
Variable
Particle size
60 mesh grind
a
20 mesh grind
% difference = 8.2 (avg)
Repeatability
Analyst 1
a
Analyst 2
% difference = 9.7 (avg)
Trap type
Clevenger
a
Crank
% difference = 1.2 (avg)
Grinder type
a
Cold grinder
Regular grinder
% difference = 11.7 (avg)
Distillation time
5h
a
8h
% difference = 2.8 (avg)
Temperature
High
a
Low
% difference = 4.3 (avg)
Flask size
a
1000 mL
2000 mL
% difference = 2.6 (avg)
a

Black pepper

Coriander

Allspice

Cloves

Oregano

1.885
2.012

0.50
0.741

3.90
4.032

18.95
19.275

3.375
3.288

(6.3)

(32.5)

(3.3)

(1.7)

(2.6)

1.848
2.05
(9.9)

0.536
0.706
(24.1)

3.928
4.005
(1.9)

19.025
19.2
(0.9)

3.125
3.538
(11.7)

2.0
1.898

0.616
0.625

3.962
3.97

19.225
19.0

3.35
3.312

(5.1)

(1.4)

(0.2)

(1.2)

(1.1)

2.035
1.862
(8.5)

0.759
0.482
(36.5)

3.99
3.942
(1.2)

19.15
19.075
(0.4)

3.538
3.125
(11.7)

1.912
1.985
(3.7)

0.608
0.634
(4.1)

3.90
4.032
(3.3)

18.95
19.275
(1.7)

3.312
3.35
(1.1)

1.94
1.963
(1.2)

0.572
0.702
(18.5)

3.958
3.975
(0.4)

19.025
19.20
(0.9)

3.325
3.342
(0.5)

1.962
1.935
(1.4)

0.634
0.607
(4.3)

3.99
3.942
(1.2)

19.15
19.075
(0.4)

3.35
3.312
(5.6)

Factor on which the percentage difference is based.

Table 4. Ruggedness study results for phase II


Volatile oil, %
Variable

Black pepper

Oregano

Coriander

Allspice

Cloves

1.612
1.762

3.094
3.031

0.75
0.759

3.796
3.816

18.594
18.609

% difference = 1.7 (avg)


Grinder type
a
Cold grinder
Regular grinder

(8.5)

(2.0)

(1.2)

(0.5)

(0.1)

1.825
1.55

3.25
2.875

0.866
0.643

3.792
3.821

18.984
18.219

% difference = 11.1 (avg)


Temperature
a
Low
High

(15.1)

(11.5)

(25.8)

(0.8)

(4.0)

1.688
1.688

3.0
3.125

0.75
0.759

3.796
3.816

18.672
18.531

% difference = 1.0 (avg)


Particle size
a
20 mesh
40 mesh
% difference = 4.8 (avg)
Repeatability
Analyst 1
a
Analyst 2

(0)

(4.0)

(1.2)

(0.5)

(0.8)

1.762
1.612
(8.5)

3.094
3.031
(2.0)

0.804
0.705
(12.3)

3.816
3.796
(0.5)

18.688
18.516
(0.9)

1.75
1.625

3.094
0.031

0.776
0.732

3.696
3.916

18.688
18.516

% difference = 2.0 (avg)


Distillation time
5h
a
12 h

(7.1)

(2.0)

(5.7)

(5.6)

(0.9)

1.688
1.688

3.00
3.125

0.768
0.741

3.733
3.879

18.766
18.438

% difference = 0.5 (avg)


Trap type
a
Crank
Clevenger

(0)

(4.0)

(3.5)

(3.8)

(1.7)

1.65
1.725

3.094
3.031

0.777
0.732

3.792
3.821

18.594
18.609

% difference = 0.5 (avg)

(4.3)

(2.0)

(5.8)

(0.8)

(0.1)

Cleaning procedure
a
New method
ASTA

Factor on which the percentage difference is based.

Table 5. Percent volatile oil collected in phases I and II


Spice

1.900
0.500
4.000
19.300
2.875

1.750
0.536
3.940
19.000
2.875

2.300
1.000
4.190
19.800
3.775

2.100
0.929
4.000
19.000
3.625

1.800
0.786
3.833
19.062
3.000

1.850
0.857
3.750
18.750
3.375

1.600
0.679
4.000
18.125
2.875

1.800
0.714
3.683
18.500
2.875

Phase I
Black pepper
Coriander
Allspice
Cloves
Oregano

1.900
0.536
3.830
18.800
3.375

1.840
0.572
3.940
19.000
3.375

1.900
0.429
3.830
19.000
3.375

1.900
0.464
4.000
19.000
3.375
Phase II

Black pepper
Coriander
Allspice
Cloves
Oregano

1.850
0.964
3.583
19.375
3.250

1.800
0.857
4.000
18.750
3.375

1.500
0.571
3.767
18.125
2.875

1.300
0.607
3.833
18.125
2.875

Figure 962.17. Apparatus for volatile oil in spices.

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