美国油脂化学协会 AOCS Official Method Ce 1f-96 (R2001) 通过毛细管气相色谱法测定氢化及精炼油脂中的顺式和反式脂肪酸

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SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

AOCS Official Method Ce 1f-96

Reapproved 1997 • Revised 2001

Determination of cis- and trans- Fatty Acids

in Hydrogenated and Refined Oils and Fats

by Capillary GLC

DEFINITION

This method consists of the gas–liquid chromatography (GLC) conditions optimized to identify and

quantify the trans fatty acid isomers in vegetable oils and fats (References, 1). The fatty acid methyl

esters (FAME) of the sample are separated on a capillary gas chromatography column having a high-

ly polar stationary phase, according to their chain length (CL), degree of (un)saturation, and geome-

try and position of the double bonds [DB(s)].

SCOPE

This method is specially designed to evaluate, by a single capillary GLC procedure, the level of trans

isomers as formed during (high-temperature) refining or during hydrogenation of vegetable oils or

fats (see Notes, 1 and 2). The method may also be used to report all other fatty acids, for example to

obtain saturated fatty acid (SAFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty

acid (PUFA) levels from the same sample and same analysis.

APPARATUS

1. Gas ch ro m at ograph—equipped with a cap i l l a ry injection

system (pre fe rably split mode, operated at a split ratio of

ap p rox i m at e l y 1:100) and flame ionization detector

(FID), cap able of meeting the fo l l owing re q u i re m e n t s :

injection port temperat u r e, 250°C; detector temperat u r e,

250°C; oven temperat u re conditions as given in Table 1.

Typical results with these described conditions are show n

in example ch ro m at o grams (Fi g u res 1–5).

2. Column—highly polar stationary phase, such as one of

the following:

(a) CP™-Sil 88, 100 or 50 m ×0.25 mm i.d., 0.20 µm

film (Chrompack, Middelburg, The Netherlands).

(b) SP-2650, 100 m ×.025 mm i.d., 0.20 µm fi l m

(Supelco Inc., Bellefonte, PA, USA).

(Supelco Inc.).

(d) BPX-70, 120 m or 50 m ×0.22 mm i.d., 0.25 µm

film (SGE Inc., Austin, TX, USA).

3. Recording instrument.

4. Electronic integrator or chromatography software.

REAGENTS

Unless otherwise stat e d , use only re agents as specified in ISO

6353 (parts 2 and 3) (Refe r ences, 2) if listed there; if not, then

use re agents of re c og n i zed analytical grade and water of at

least grade 3 as defined in ISO 3696 (Refe rences, 3).

1. Carrier gas—helium, nitrogen, or hydrogen, GC quali-

ty, dried, and oxygen removed by suitable filters.

2. Internal standard (for calculating fatty acid data as mg

per g oil)—tridecanoin, 5.0 mg/mL in chloroform. This

solution is stable up to 1 week if stored in refrigerator

in well sealed amber bottle. (See Notes, 3).

PROCEDURE

1. Sample preparation—

(a) P rep a re the methyl esters from the tri g ly c e ri d e s

f rom the oils or fats to be analy ze d , using the

b o ron tri fl u o ride method as descri b e d, for ex a m-

ple, in AOCS Official Method Ce 2-66 or IUPAC

2.301 (References, 5 and 6).

( b ) B e fo re test portions are taken from samples, the

samples should be mixed thoro u g h l y. Solid samples

should be melted to ensure proper mixing.

2 . C h ro m at ograp hy —

( a ) Set up the gas ch ro m at o graph with the temperat u re

and column as described in Table 1. Measure the

ave rage carrier gas linear velocity as indicated in

Table 1, with a split ratio of ap p rox i m at e ly 1:100.

( b ) Inject 0.5 to 1 µL of the methyl esters (concentra-

tion approximately 7 mg/mL) from the test sample

into the gas ch ro m at ograph. Compare the re s u l t

with the example chromatograms (Figures 1–5). If

the sep a r ation obtained is not identical to the

Table 1

Proposed optimal GLC conditions for identification and quantification of trans isomers in refined and hydrogenated veg-

etable oil samples (see References, 3).

Stationary phase SP-2340 SP-2560 CP™-Sil 88 BPX-70

Temperature conditions Isotherm 192°C Isotherm 170°C Isotherm 175°C Isotherm 198°C

Column head pressure (kPa) 125 125 130 155

Linear velocity of carrier gas (He) 15 cm/sec 16 cm/sec 19 cm/sec 17 cm/sec

Page 2 of 6

SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

Ce 1f-96 • Determination of cis- and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats

example ch ro m at o grams, small ch a n ges in ove n

t e m p e rat u re may be re q u i re d . If so, decrease or

i n c rease the oven temperat u re with subsequent

s t eps of 1°C until a good sep a ration is obtained.

These small corrections might be re q u i r ed to

correct for batch differences between columns and

i n s t rument temperat u re control and ge n e ra l ly fa l l

within a ra n ge of only a few degrees (plus or

minus) at maximum from the indicated value. The

20:1cpeak will elute earlier relative to 18:3ccc if

the oven temperature is increased (see Notes, 4).

3. Performance check—

(a) If the GLC system is set up properly, the separa-

tion obtained should allow identifi c ation of the

small amount of the nat u ra l ly present 18:1 11c i s

isomer next to the 18:1 9cis peak in (high-temper-

at u re) re fined oils such as soybean oil. The two

1 8 : 1ci s o m e r s should be cl e a rly sep a r ated (see

Figures 1–5).

(b) The 20:1 nat u ral isomer should be positioned

exactly between the last eluting 18:3 trans isomer

(t rans, cis, cis) and the 18:3c c c (linolenic acid)

peak in (high-temperature) refined oils.

sis, in (high-temperature) refined oils a small peak

for the 18:1 t ra n s i s o m e r, two ap p r ox i m at e l y

e q u a l ly sized 18:2 t ra n s i s o m e r s, and 4 (some-

times 5) 18:3 t ra n s i s o m e rs should be obtained

(see Figures 1–5).

(d) For partially hydrogenated oils and fats, the sepa-

ration of the 18:1 13t ra n s and the 18:1 9c i s

i s o m e rs should be visible on the ch ro m at ogra m .

This is required for an accurate peak split between

cis and trans.

4. Peak identification—

( a ) For (high-temperat u re) re fined oils and fats, the

t ra n s i s o m e rs are limited in number because only

ge o m e t rical isomers, with the DB(s) on the same

n at u ral position, are fo rm e d. For C1 8 fatty acids

these specific isomers are 18:1 9t; 18:2 9c1 2 t

and 9t1 2 c; and 18:3 t c t , c c t , c t c , t c c 9, 12, 15-

i s o m e rs (in some samples the 18:2 9t1 2tand 18:3t t c

i s o m e r s are found as well in ve r y small amounts).

(b) For part i a l ly hy d roge n ated oils and fats the t ra n s

DB-containing isomers are identified using the

e q u ivalent chain length (ECL) concept (Refe r-

ences, 7; see Table 2). For accurate peak identifi-

cation with this system, the ECL values have to be

determined after suitable calibration with available

cis and trans fatty isomer standards.

SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

Ce 1f-96 • Determination of cis- and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats

Page 3 of 6

Figure 4. Chromatogram of methyl esters from a physicall y

refined rapeseed oil sample using 50 m ×0.25 mm ×0 . 2 0

µm CP™-Sil 88 column (Chr o m p a ck). The t ra n s fatty acid

isomers are indicated in the chromatogram.

Figure 5. Chromatogram of methyl esters from a high-tem -

perature-refined rapeseed oil sample, using 50 m ×0.22 mm

×0.25 µm BPX-70 (SGE). The t ra n s fatty acid isomers are

indicated in the chromatogram.

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标签： #管 #测定 #相色谱法测定

摘要：

本文详细介绍美国油脂化学协会（AOCS）官方方法Ce 1f-96 (R2001)，该标准规范通过毛细管气相色谱法精确测定氢化及精炼油脂中的顺式和反式脂肪酸含量。该方法适用于氢化植物油、精炼油脂及含此类油脂的食品基质，能够高效分离并定量C16至C22范围内的顺反异构体，尤其对于部分氢化油脂中常见的反式十八碳一烯酸（C18:1 trans）具有高灵敏度与重现性。通过采用固定相极性色谱柱（如SP-2560或CP-Sil 88）配合程序升温程序，可在单次分析中同时获得脂肪酸组成及顺反比例数据，避免红外光

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美国油脂化学协会 AOCS Official Method Ce 1f-96 (R2001) 通过毛细管气相色谱法测定氢化及精炼油脂中的顺式和反式脂肪酸

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