SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
AOCS Off icial Method 3b-87
Hexane Residues
latile hydrocarbons remaining in the fats and oils following
rbons are desorbed by heating the sample at 80 C in a closed
on of a calibration factor, hydrocarbons in the headspace are
columns. Results are expressed as hexane in milligrainS per
scope: This
,"*'Aning in
a"t"irir:utio
International
Apparatus
i. ff,. gas chromatograph should have, at a minimum, the
following characteristic s-
(a) Thermostated column oven capable of maintaining
the desired column temperature within t 1 C.
(b) Sanrple inlet system, separately thermostated, that
can be maintained at a minimum temperature of
100 C. If a capillary column is used, the inlet
system must be capable of a 1:i00 split injection.
For seriat analysis a headspace gas chrolnatograph
with automatic sample injection and tempering
bath has been shown to be satisfactory.
(c) Flame ionizatron detector, separately ther-
mostated, that can be maintained at a minimum
temperature of 100 C.
2. Recorder-If the recorder trace is to be used to calcu-
late the composition of the samples analyzed, an elec-
" tronic recorder of high precision is required. The char-
acf.eristics of the recorder should be:
(a) Rate of response below 1.0 seconds (the rate of
response is the time taken for the recording pen to
. introduction of a l00%o signal).
(b) Breadth of paper,25 cm (t0 in.) minimum.
(c) Paper speed, 25-100 cmftr (1040 in.ftrr).
(d) Attenuator switch for range change.
3. Electronic integrator (preferred)-R;pid and accurate
calculation can be performed using an electronic inte-
grator or computer. This must give a linear response
with adequate sensitivity and baseline cor.ection
gonsistent with good chromatographic practice.
Horizontal, nonhorizontal ancl tangential baseline
$hed and silanized diatomaceous earth,
)-180-pm particle size (80- 100 mesh
WAW is suitable). Stationary phase:
Revised 1988
Reapproved f993
Fats and Oils
, expressed in terms of hexane,
d solvents. It is suitable for the
l. This method is equivalent to
and,)'
squalane, consisting of I0%o of the packing. Any
other stationary phase capable of producing the
chromatographic separation required may be
substituted.
(b) Capillary column-The column should be
constructed of glass or fused silica, approximately
30 m long and 0.3 nlm i.d. Stationary phase:
nrethyl polysiloxane (film thickness 0.2 pm).
5. Syringe-l-pl capacity.
6. Syringe- 1 C-pL capacity.
l. Syringe-l000-pl capacity, gas-tight (preferably with
valve).
8. Septum vials-Z}-mL capacity.
9. Septa and aluminum caps suitable for septurn vials,
together with crimping pliers-The septa mttst be resis-
tant to oils and solvents (butyl rubber or red rubber is
recommended).
10. Tongs-suitable for holding septum vials.
11. Heating bath-with clamps for holding septum vials,
thermostatically regulated and capable of maintaining a
temperature of 80 C. For continuous operation, glycerol
is recommended as the heating liquid.
12. Shaking machine.
Reagents
1. Gases-
(a) Carrier-helium (preferred for better resolution)
or nitrogen, minimum purity 99.99 rnol%o, dried
and containing a maximum of 10 mg Orkg.
(b) Flame ionization detector-hydrogen, minimum
purity 99.95 molTo; air or o.2, riry (dew point -59
C maxirnum) and hydrocarbon free (< 2 ppm
hydrocarbons equivalent to CHa).
2. Technical hexane or a light petroleufr-with a compo-
sition similar to that used in industrial extraction, otr
failing these, n-hexane (seg Notes, Caution). For cali-
bration, techrrical extractton hexane is preferred.
3. n-Heptane (internal standard)-analytical reagent
grade. (Cyclohexane is also suitable for use as an inter-
nal standard, provided that the solvent used for the
extraction or calibration has a negligible content of
cyclohexane.) See Notes, Caution.
 SAMPLING AND ANALYSIS OF COMMERCIAL
FATS AND OILS

Hexane Residues in Fats and oirs

Ca3
4' Vegetable oil-solvent free, freshly refined and deodor-
ized' The oil is to be used for calibration Successively immerse the vials up to the
and should be neck
the heating bath (epparatus, 11) at g0
of a similar nature as the sample. It should be free
of
c at intervals
extraction solvent (< 0.0 IVo).If it contains approximately 15 min. This time interval
depends
a greater the duration of the Gc.analysis, which is
amount of solvent, this should be reduced comptt
by laboratory elution of inrernal srandard (n-heptan
deodori zation of the oil. Hfl,samples
lh: ,the Y.c+tl
must be placed in the heating uatrr
,

fh.
intervals such that each iample is tempered
S1m_pling and Sample Storage for exac
60 min.
1' It is essential that loss of solvent from the sample be warm the gas-tight syringe (Apparatus, 7) to
prevented. The laboratory sample should 60
be in a After tempering at 80 c for &actlv 60 minrl
completely sealed container and siored at 4
C. plastic
containers should not be used. Sample analyses
should
be carried out immediately when the sample
container
is opened.

GC Operating Conditions
1- carrier gas flow depends on the carrier
. uri uiu"tl ffi ;1,',fl'#;', ; J : :l3HX'. ; fliil
"T:ffii
gas and the CrxA,
of colu*n being used for the analysis-and shouldtype F-
optfuili zed accordingry. Helium is recommended,
be (An-AB-Ai) xCi
but Where-
nitrogen may be used. some loss of resolution
between
peaks i,lay be experienced with nitrogen. Aru= total peak area of solvent hydrocarbonr
2. The flg* of hydrogen and air or oxygen includirg the area of internal standar
to the flame
ronrzation detector should be optimi ied present in the spiked oil. For identificati
according to
the manufacturer's recornmendations for
the instrument
purposes, a typical chromatogram of
tht
being used. solvent composition shculd b; obtained
3' Injector and detector temperatures should Hydrocarbons that usually make up the tech
be set at
abour lZ0 C. nical hexane are: 2-niethylp.irtur.. 3
4. The column should be maintained at 40 C. methylpentane, methylcyclop.rrtur", ryclo
hexane, etc. Do not incrude peaks due
to thr
Procedure oxidation products, sorne oi which may
i. Determination of present in signiticant amounts.
nearest 0.01 g, Ag= peak area of the sorvent hydrocarbon
(Reagenrs, 4) i ,t: :1; present in the oil to which roiu"rt
has no
vials been added (blank), less the peak area
(Apparatus, 8). S vial with a septum and cap internal standard
of th
(Apparatus,_ 9).
By means of a sydnge (6p*atus, 5 or
6), add technicar hexane (Reagents, 2) Ar= peak area corresponding to the internal
to six stan-
seven vials (the vial with no added solvent
of the dard in the spiked samples
is the blank) Ci= quantity of internal standard added,
according to the following table:
expressed in mglkg (ppm) of oil
I 2 3 4 5 6 7
Vialnumber Note-For an addition of 5 prl- to 5 g of,
rLlle 0 0.5 t 2 4 7 t0 ;iT*? 999, -g
for n-heptane anci t,
S:Icycloh"xine.
780 mg for
=
mgllOO g 0 67 134 268 536 938 t34O Cs= quantity of technical hexane added
to the oil
one vial remains without the addition present in the vial, expressed in mg/kg
of solvent. (ppm)
If n-hexane is used as the calibrating solvent, the of oil
following table applies: Express the results to the third decimal
place.
Calibration factors for the six standards
should be
approximately the sarle. The mean calibration
I Z 3 4 5 6
Vialnumber
should be about 0.45 if n-heptane is
factor
N-l1e 0 0.5 I 2 4 I t07 cyclohexane is used.
used and 0.57 if
mg/100 g 0 66 132 264 528 g24 1320 The factor (F) so evaluated can be used
for deter-
Shake vigorou
mining v <60 _snd ?ppml rr
vent in the shaking
hr. Using a syrin!
l1:,::,r:
nexane r
conraining 0.5 pL of
he mean value, this
deviation hedifficultyofintroducing
septum exactly 5 pL of inte
exactly 0.5 y and, this
prl.,
i. cetermination
to each of the seven vials. must be either
eliminated or repeated.
 ',

SAMPLING AND ANALYSIS OF tOUN,TERCIAL FATS AND OILS

3b- Hexane Residues in Fats and Oils Ca 3b-87

For quantities of hexane between 10 and 20 mgkg Thble 1

''statistical
analysis of results for hexane in oils (1985 collabo-
rative study; duplicate determinations).

ptane Samplea
)ath 23
3xactl
Number of laboratories
quicklY as Possible and close L6 T6 11 11
Number of laboratories after
)60 septum and caq (APParatus,
llnut (Apparatus, 6), add through the s
elimination of outliers 16 169 10
Mean value (in ppm) 2.95 r3.7 t04 1048
intirnal standard (Reagents, 3)'
Repeatability standard
hand for about 1 minute and then immerse the vial up
19 the neck in the heating bath (Apparatus, 11) at 80 C
deviation (Sr) 0.65 r.70 1 .6 7 t.5
for exactly 60 min. Warm t syringe Repeatability coefficient
(Apparatus, 7) to 60 C. After B0 C for of variation (Vo) 22.4 12.2 1.3 6.8
r*uCtty 60 min, use the gas{igh and take frorn Repeatability value
the vial, without removing'it from the bath, 1000 pL (1 r
(S. x2.83) 1.8 4.8 2t 202
mL) of the headspace above the sample. Immediately Reproducibility standard
inject into the gas chromatograph. deviation (SB) 2.6 4.r 28.4 293
3. Number of determinations-Carry out two determina- Reproducibility coeffici ent
rbon tions in rapid succession on each sample. of variation (7o) 89 30 21 28
Reproducibility value
R (Sn x 2,83) 7.4 t2 80 829
Expression of Results
uSample 1, fresh edible peanut
.ained 1. The residual solvent content, W, expressed in mglkg oil + 3.3 ppm technical hexane;
(ppm), is given by the formula: Sample 2, fresh peanut oil after storage + 13 .Z ppmtechnical
hexane; Sample 3, crude rapeseed oil (pressed) + 100 ppm techni-
\rr (AH-Ai)xFxCi
YY- cal hexane; Sample 4, crude rapeseed oil (pressed) + 1000 ppm
,\
technical hexane.
:toth Where-
AH= total peak area of solvent hyCrocarbons,
including the atea of the internal standard. 2. Rerreatability-When the rnean of the duplicate deter-
rrbon Hydrocarbons that usually make up the tech- minations lies within the range of the mean values cited
ras no nical solvents are Z-methylpentane, 3- in Thble 1, the difference between the results of the two
of methylpentane, methylcyclopentane, cyclo- dcterminations, carried out in rapid succession by the
hexane, etc. A typical chromatogram of same operator, using the same apparatus for the analy-
rl st these solvents was shown by the calibration sis of the same test sample, should not be greater than
standards. Do not include peaks due to the the repeatability value (r), which can be obtained from
rdded oxidation products. Some of these products Table 1 by linear interpolation if necessary, for the level
may be present in significant amounts. of hexane in the sample examined.
5go AI = peak area corresponding to internal standard 3. Reproducibility-when the means of the duplicate
in the sample determinations, obtained in two different laboratories
C, of internal srandard added in ms/ks using this standard method for the analysis of the same
=
the oi
tJ;,Xlry laboratory sample, lie within the range of the mean
(ppm) Note-For an addition of 5 pL of values cited in Table 1, the difference between, the
heptane
per5gof mean results obtained by those laboratories should not
be greater than the reproducibility value (R), which can
ruld F =Smlf,?# be obtained from Table 1 by linear interpolarion if
fin necessary, for the level of hexane in the sample exam-
Report as the f ined.
irements for
It should be noted that the reproducibility values
deter-
.Dm).
ffi:rilr; (R) cited in Table 1 apply to the particular case when
the results of single determinations obtained by two
, !"rI- o laboratories are being compared. If when following the
e, this, method described it is desired to compare the final
Preeision
results (which have been derived from the means of
1. The results of one interlaboratory test organized at an duplicate deterrninations) obtained by two laboratories,
international level (for different ,urnp-t".9
statistical results shown in Table
;;;; ,h; the values for (R) should be converted to g5vc proba-
1. bility critical difference values (CrDrr) applicable to
 SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

Hexane Residues in Fats and Oils Ca3

the means of two determinations using the following Administration recommends that exposure not exceed 3
forrnula: ng/M3 for a time-weighted average. Hexane vapor caur
CrDr, = - rzlzy lung irritation and produces neurotoxic effects.

Notes
Caution References
The solvents used in this method-hexane, petroleum ether, 1. Standard Methods
n-heptane and cyclohexane-are all highly flammable.
fo, the Analysis of Oils, Fats
Derivatives, International Union of Pure and Appli
They should not be used near open flame. procedures Chemistry, 7th edn., Blackwell Scientitic Publicatior
requiring the use of these solvents should be canied out in 1987, IUPAC Metho d 2.607 .
a properly operating fume hood. For hexane the TLV is 50 2. Collaborative study results published tn Pure Ap,
ppm in air. The occupational safety and Health Chem. s9 ( tl ):1561 (1981).