Pure & Appl. Chem., Vol. 62, No. 4, pp. 781-793 1990. Printed in Great Britain.

@ 1990 IUPAC

INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY

APPLIED CHEMISTRY DIVISION COMMISSION ON OILS, FATS AND DERIVATIVES*

DETERMINATION OF THE ESTEREMULSIFIERS COMPONENTS CONTENT AFTER HYDROLYSIS AND SILYLATION BY GAS CHROMATOGRAPHY Results of a collaborative study and the standardised method
Prepared for publication by H. BRUSCHWEILER' and A. HAUTFENNE2
'Swiss Federal Laboratories for Materials Testing and Research EMPA, Unterstrasse 11, CH-9001 St. Gallen, Switzerland 'Universite Catholique de Louvain, Unit6 des Proc6des (PRCD) B-1348 Louvain-la-Neuve, Belgium

*Membership of the Commission during the period of the collaborative study (1981-83) was as follows: Chairman: D. Firestone (USA); Vice-chairman: M. Naudet (France); Secretary: A. Hautfenne (Belgium); Titular Members: 0 . Levin (Sweden); J. Pokorng (Czechoslovakia); H. Wessels (FRG); Associate Members: T. Asahara (Japan); J. L. Beare-Rogers (Canada); A. Dieffenbacher (Switzerland); E . Fedeli (Italy); J. Gracian Tous (Spain); E. Kurucz (Hungary); A. T. Moller (Denmark); W. D. Pocklington (UK); W. Prins? (Netherlands); M. Teupel (FRG); A. Visapaa (Finland); G. Zwerenz (Austria); National Representatives: G. Bazan (Argentina); A. Johnson (Australia); B. Jacobsberg (Belgium); R. C. de Araujo Lago (Brazil); D. Chobanov (Bulgaria); A. Tulloch (Canada); G. Holmer (Denmark); A. Gad (Egypt); R. Linko (Finland); J. P. Wolff (France); G. Ostermann (FRG); V. Kapoulas (Greece); N. Bringhi (India); M. McCarthy (Ireland); T. Hashimoto (Japan); P. W. Hendrikse (Netherlands); F. Shorland (New Zealand); W. Zwierzykowski (Poland); R. Stoica (Romania); D. Carr (South Africa); M. Gassiot Matas (Spain); R. Ohlson (Sweden); H. Briischweiler (Switzerland); R. Perin (Turkey); K. Williams4 (UK); A. Waltking (USA); V. Rubajlo (USSR). ?Until 1982 $Deceased 1982
Republication of this report is permitted without the need for formal IUPAC permission on condition that an acknowledgement, wirh full reference together with IUPAC copyright symbol ( 1990 IUPAC), is printed. 0 Publication of a translation into another language is subject to the additional condition of prior approval from the relevant IUPAC National Adhering Organization.

Determination of ester-emulsifiers components content after hydrolysis and silylation by gas chromatography: results of a collaborative study and the standardised method

Abstract A method f o r t h e gas chromatographic d e t e r m i n a t i o n o f t h e compon e n t s o f e s t e r - e m u l s i f i e r s has been e l a b o r a t e d and t e s t e d i n a c o l l a b o r a t i v e study. The method i n v o l v e s s a p o n i f i c a t i o n o f t h e e s t e r - e m u l s i f i e r s i n e t h a n o l i c KOH s o l u t i o n , evaporation o f t h e alcohol, s i l y l a t i o n o f t h e products o f hydrolys i s and d e t e r m i n a t i o n o f t h e components e i t h e r by packed or c a p i l l a r y gas chromatography. The e m u l s i f i e r s analysed i n t h i s s t u d y were mono- and d i g l y c e r i d e s , d i a c e t y l t a r t a r i c a c i d e s t e r o f mono- and d i g l y c e r i d e s and s o r b i t a n monostearate i n o i l (coconut o i l ) . E m u l s i f i e r s p r e s e n t i n o i l were p r e l i m i n a r i l y s e p a r a t e d by column chromatography. The h y d r o l y s i s - s i l y l a t i o n procedure d e s c r i b e d i s a p p l i c a b l e n o t o n l y f o r t h e c h a r a c t e r i z a t i o n and q u a n t i f i c a t i o n o f f u n c t i o n a l components o f e s t e r - e m u l s i f i e r s , b u t a l s o f o r o i l s , f a t s , wax e s t e r s and o t h e r h y d r o l y s a b l e l i p i d s .

INTRODUCTION
E m u l s i f i e r s a r e one o f t h e most i m p o r t a n t c l a s s o f f o o d a d d i t i v e s . Methods and procedures t o analyse e m u l s i f i e r s a r e r e q u i r e d by i n d u s t r y f o r q u a l i t y c o n t r o l purposes and by r e g u l a t o r y o r g a n i s a t i o n s t o t e s t i f l e g a l r e q u i r e m e n t s a r e f u l f i l l e d . A j o i n t FAO/WHO e x p e r t committee on food a d d i t i v e s e l a b o r a t e d s p e c i f i c a t i o n s f o r t h e s e e m u l s i f i e r s (1). E m u l s i f i e r s a r e s u r f a c e - a c t i v e substances which s u s t a i n t h e f o r m a t i o n o f emulsions. Emulsions a r e d i s p e r s e systems o f two l i q u o r s which a r e n o t o r h a r d l y s o l u b l e i n each o t h e r . E m u l s i f i e r s c o n s i s t o f m o l e c u l e s c o n t a i n i n g a p a r t more r e a d i l y s o l u b l e i n a p o l a r s o l v e n t s h a v i n g l i p o p h i l i c p r o p e r t i e s and another p a r t more r e a d i l y s o l u b l e i n p o l a r s o l v e n t s h a v i n g lipophobic properties. Ester-emulsifiers considered i n t h i s r e p o r t a r e p a r t i a l e s t e r s o f e d i b l e f a t t y acids w i t h p o l y a l c o h o l s such as g l y c o l , p o l y g l y c o l , g l y c e r o l , p o l y g l y c e r o l s , s o r b i t a n and c a r b o h y d r a t e s (sucrose). The e m u l s i f i e r s are l i s t e d i n t a b l e 1. They a r e c l a s s i f i e d i n t h e o r d e r o f i n c r e a s i n g number o f h y d r o x y l groups i n t h e p o l a r p a r t o f t h e molecule. E s t e r - e m u l s i f i e r s do n o t c o n s i s t o f one s i n g l e c h e m i c a l compound. They a r e always m i x t u r e s o f substances w i t h d i f f e r e n t degrees o f e s t e r i f i c a t i o n , t y p e s o f f a t t y a c i d s , p o s i t i o n a l isomerism e t c . For example, monoglycerides o f f a t t y a c i d s may c o n t a i n 1-mono-, 2-mono-, 1,Z-di-, 1,3-di- and t r i g l y c e r i d e s o f v a r i o u s f a t t y a c i d s . A l s o f r e e f a t t y a c i d s , g l y c e r o l , d i - and p o l y g l y c e r o l may be p r e s e n t . These m i x t u r e s can b e e s t e r i f i e d f u r t h e r w i t h c i t r i c - , l a c t i c - and s u c c i n i c a c i d . E m u l s i f i e r c o n c e n t r a t e s and e m u l s i f i e r s p r e s e n t i n o i l s and f a t s and i n l i p i d e x t r a c t s o f food may be separated i n t o f r a c t i o n s o f d i f f e r e n t p o l a r i t y , u s i n g chromatographic methods such a s t h i n l a y e r chromatography (TLC) o r l i q u i d chromatography (LC, HPLC). E m u l s i f i e r s such as f a t t y a c i d e s t e r s o f p o l y g l y c e r o l ( 2 , 3 ) , s o r b i t a n (4, 5 ) , sugar ( 6 ) and o t h e r components (1, 7 ) have been h y d r o l y s e d and t h e components o b t a i n e d were determined by gas chromatography as t r i m e t h y l s i l y l d e r i v a t i v e s . The p r o d u c t s o f h y d r o l y s i s o f t h e s e e m u l s i f i e r s a r e shown i n t a b l e 2.

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783

Table 1 CLASSIFICATION OF ESTER-EMULSIFIERS

*
Examples:
Na-,K-,NH4-,Ca-,Mg-salts of stearic-, o l e i c a c i d etc. Sodium s t e a r o y l c i t r a t e Calcium s t e a r o y l - 2 - l a c t y l a t e Sodium s t e a r o y l - 2 - l a c t y l a t e Lactic esters of f a t t y a c i d s Sodium s a l t o f c h o l i c - , d e o x y c h o l i c a c i d

1. C a r b o x y l a t e s 1.1 S a l t s o f f a t t y a c i d s
1 . 2 Esters o f f a t t y a c i d s w i t h hydroxy (mono, p o 1 y ) c a r b o x y l i c a c i d s and its s a l t s 1.3 Further carboxylates 2. S u l f a t e s and S u l f o n a t e s 2.1 Sulfated f a t t v alcohols 12.2 S u l f o n a t e d e s t e r compounds

Sodium l a u r y l s u l f a t e Sodium d i o c t y l s u l f o s u c c i n a t e 1,2-Propylene g l y c o l mono- and d i e s t e r s of f a t t y acids Polyoxyethylene(8)stearate

3 . Glycol e s t e r s o f f a t t y a c i d s ( G l y c o l s ) 3 . 1 Monoesters of f a t t y a c i d s and hydroxy fatty acids w i t h glycols 3.2 Esters o f f a t t y a c i d s w i t h polyoxye t h y l e n e and/or polyoxypropylene g l y cols
4. G l y c e r o l e s t e r s o f f a t t y a c i d s (Glycerides) 4.1 Mono- and d i g l y c e r i d e s o f u n s a t u r a t e d and s a t u r a t e d f a t t y a c i d s 4.2 Monoglycerides e s t e r i f i e d w i t h 4.2.1 A c e t i c a c i d 4.2.2 Citric a c i d 4.2.3 Lactic a c i d 4.2.4 T a r t a r i c a c i d 4.2.5 D i a c e t y l t a r t a r i c a c i d 4.2.6 S u c c i n i c a c i d 4.2.7 Mixed a c i d s 4.3 E s t e r s o f g l y c e r o l and p r o p y l e n e g l y c o l 4.4 E t h o x y l a t e d monoglycerides 4.5 Ester o f p o l y g l y c e r o l 4.6 Polymerised and o x i d i z e d f a t s

Mono- and d i g l y c e r i d e s o f f a t t y a c i d s A c e t y l a t e d monoglycerides ( A c e t o g l y c e r i d e s )

Citric a c i d ester o f monoglyceride

(Citroglycerides) L a c t y l a t e d monoglycerides ( L a c t o g l y c e r i d e s ) T a r t a r y l a t e d monoglycerides D i a c e t y l t a r t a r i c a c i d esters o f monoglycerides S u c c i n y l a t e d monoglycerides Stearyl citric acid ester glycerides Mixed l a c t i c a c i d e s t e r o f p r o p y l e n e g l y c o l and glycerol E t h o x y l a t e d mono- and d i g l y c e r i d e s Polyglycerol esters o f f a t t y a c i d s G l y c e r o l e s t e r s o f f a t t y a c i d s o b t a i n e d from s o j a o i l o x i d i z e d under h e a t Polyglycerol esters o f i n t e r e s t e r i f i e d ricinol e i c acid Pmmonium p h o s p h a t e s Lecithin, hydroxylated l e c i t h i n Sorbitan monostearate Polyoxyethylene ( 2 0) s o r b i t an monostear a t e Polyoxyehtylene(20)sorbitan t r i s t e a r a t e Sucrose e s t e r s o f f a t t y a c i d s ( S u c r o e s t e r s )

4.7 P h o s p h a t e - d e r i v a t i v e s o f mono- and diglycerides 4.8 L e c i t h i n

5. S o r b i t a n ester o f f a t t y a c i d s 5.1 Mono-, d i - and t r i e s t e r s o f f a t t y acids w i t h sorbitan 5 . 2 Mono-, d i - and t r i e s t e r s o f f a t t y acids w i t h sorbitan, ethoxylated
6. Suqar e s t e r s o f f a t t y a c i d s 6.1 Mono, d i - and t r i e s t e r s o f f a t t y a c i d s w i t h mono- and d i s a c c h a r i d e s

names a c c o r d i n g t o IUPAC recommendations, see Appendix

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Table 2
COMPONENTS OF ESTER-EMULSIFIERS AFTER HYDROLYSIS

JAlcohols and Polyalcohols

Acids

I
pal: oleic

Fatty alcohols Glycols Po 1 1 c o l s yg y Glycerols Polyglycerols Sorbitol and i t s anhydrides Carbohydrates mono- and disaccharides

Carboxylic a c i d s acetic-, lauric-, mitic-, stearic-, acid etc.

- Dicarboxylic acids

succinic acid

Hydroxy c a r b o x y l i c a c i d lactic-, tartaric-, c i t r i c acid Inorganic acids phosphoric-, s u l p h u r i c acid

The h y d r o l y s i s - s i l y l a t i o n procedure proposed s i m p l i f i e s t h e c h a r a c t e r i z a t i o n o f complex m i x t u r e s o f e m u l s i f i e r s and may be used f o r t h e q u a n t i f i c a t i o n o f f u n c t i o n a l components i n emulsifiers. The c o l l a b o r a t i v e study presented i n t h i s r e p o r t i s c o n f i n e d t o the gas chromatographic det e r m i n a t i o n s o f t h e products o f h y d r o l y s i s o f samples o f mono- and d i g l y c e r i d e s , d i a c e t y l t a r t a r i c a c i d e s t e r s o f mono- and d i g l y c e r i d e s and s o r b i t a n monostearates i n o i l . The procedure c o u l d be a p p l i e d t o o t h e r e m u l s i f i e r s l i s t e d i n Table 1 and t o o i l s , f a t s , wax e s t e r s (11) and other hydrolysable l i p i d s .

COLLABORATIVE S T U D Y
Samples
Samples and t h e d r a f t method were sent t o p a r t i c i p a t i n g l a b o r a t o r i e s i n A p r i l 1981. The substances were i n t e r n a l standard n-tetradecane and t h e reference substances g l y c e r o l , t a r t a r i c acid, and furthermore a m i x t u r e o f C1o:o-, C12.0-, C 4:Q-, C16:4-, C18:o-, C18:1fatty acids, n-tetradecane and n-tetracosane. The componenks i n t h e m i x t u r e were present i n equal weight r a t i o . The response f a c t o r s o f t h e s i l y l a t e d reference substance versus n-tetradecane had t o be determined. The values were then used f o r t h e c a l c u l a t i o n o f t h e components o f t h e e m u l s i f i e r s samples 1 t o 4 a f t e r h y d r o l y s i s . The e m u l s i f i e r s provided f o r t h e study were: Mono- and d i g l y c e r i d e s (sample 1 ) ; d i a c e t y l t a r t a r i c a c i d e s t e r s o f mono- and d i g l y c e r i d e s (sample 2) ; s o r b i t a n monostearate (sample 3 ) ; and s o r b i t a n monostearate i n coconut o i l (sample 4 ) . For sample 4 the p a r t i c i p a n t s were asked t o perform a p r e l i m i n a r y separation o f e m u l s i f i e r s from t h e o i l u s i n g s i l i c a column chromatography according t o method 2.321 "Determination o f mono-, d i - and t r i g l y c e r i d e s by column chromatography" ( 8 ) .

RESULTS A N D D I S C U S S I O N
Results obtained by s i x l a b o r a t o r i e s are summarized i n Tables 3 chromatograms.

6 . Fig. 1 shows t y p i c a l

Operating conditions
Operating c o n d i t i o n s used by c o l l a b o r a t o r s are shown i n Table 3 . A l l l a b o r a t o r i e s used packed columns. Laboratory 1 and 3 a d d i t i o n a l l y a p p l i e d c a p i l l a r y columns.

Response factors
Response f a c t o r s o f t h e reference substances vs. n-tetradecane were determined. The response f a c t o r f o r g l y c e r o l was h i g h due t o the d e r i v a t i z a t i o n and i n t r o d u c t i o n o f a d d i t i o n a l (Me)3Si-groups. Response f a c t o r s f o r the C g f 0 - t o C14,0f a t t y a c i d s were c l o s e t o one, b u t became lower f o r f a t t y a c i d s w i t h h i g h e r numbers o f C-atoms, i n d i c a t i n g some d i s c r i m i n a t i o n due t o i n s t a b i l i t y o f t h e t r i m e t h y l s i l y l f a t t y a c i d d e r i v a t i v e s a t higher column temperatures.

Determination of copper, iron and nickel

785

The r e l a t i v e s t a b i l i t y o f t r i m e t h y l s i l y l d e r i v a t i v e s o f f a t t y a c i d s i n a gas chromatographic set-up can be t e s t e d by t h e a d d i t i o n o f equal amounts o f hydrocarbons such as hexadecane (C16), eicosane (C o ) , tetracosane (Cz4), octacosane (Cz8) t o t h e f a t t y a c i d s t o be s i l y l a ted. The peaks o f t h e corresponding hydrocarbons should have about t h e same h e i g h t s as t h e Clerland C18: - f a t t y a c i d s ones o f the s i l y l d e r i v a t i v e s . With packed columns, C18.0-, s i l y l d e r i v a t i v e s were n o t separated, b u t could be q u a n t i f i e d applying c a p i l l a r y columns.

Table 3

Operating Conditions Reported by C o l l a b o r a t o r s

Dimension

Phase

remper t u r e s I nj e c - De tor tector

'C) I ni ti a l
_ I

Carrier F i n a l Gas F1ow

Sample Size P1

--

Packed c 1 2 3 4 5 6

umns -

2.4x2.0 2.0x2.2 1.5x2.0 1.8~3 2x 3 3x 3

Glass, Supelcoport AW-DMCS, 80-100 mesh; 3% OV-17 S t a i n l e s s Steel, Chromosorb WHP, 100-200 mesh, 3% D e x i l 300 GC. Glass Chromosorb G AWDMCS, 80-100 mesh, 3?L SE 30. Glass, Chromosorb W, 80-100 mesh, 5% OV-1. Glass, Chromosorb G AWDMCS, 60-80 mesh, s i l i c o n e , 2% OV-1. S t a i n l e s s Steel, Chromosorb W AW-DMCS, 100120 mesh, 3% OV-17. Glass C a p i l l a r y , SE 52.

310
310
310 250 310
280

310 310 310 300 310 280

80 80 3 mir 80 1mir 130

275 275 275 260 275 270

1 Helium 3hl/min Helium 1.6 Zhl/min Nitrogen 0.5 25m l / m i n 3.5 b a r He 1 ium 2-7 5hl/min 3.4 bar Nitroger 3Om l / m i r 0.5 bar N it r o g e r 3hl/mir 1.95 bar Helium 1:lOO lml/min N i troger 0.5 0.5ml/ min 1.03 bar 1:150

100
100

F
3

columns 20~0.32 20~0.32

310

310 310

80

275 300

Glass C a p i l l a r y , 310 Durabond 5, Polymethyl(5% Pheny1)-Siloxan.

I

100

--_

I -

--

--

Statistical evaluation
S t a t i s t i c a l a n a l y s i s o f t h e c o l l a b o r a t i v e study r e s u l t s i s presented i n Tables 4 , 5 and 6. Values o f d u p l i c a t e determinations obtained i n p a r t i c i p a t i n g l a b o r a t o r i e s are reported. Res u l t s were c a l c u l a t e d according t o t h e scheme o u t l i n e d by P o c k l i n g t o n ( 9 ) and IS0 5725 (10) which a l l o w s r e j e c t i o n o f u n s a t i s f a c t o r y r e s u l t s u s i n g t h e Cochran's and Dixon's t e s t (951 confidence l e v e l ) and c a l c u l a t i o n o f standard d e v i a t i o n o f r e p e a t a b i l i t y ( s r ) and reproduc i b i l i t y ( S R ) and t h e i r c o e f f i c i e n t s o f v a r i a t i o n (CV 7;).

Analysis of emulsifier samples

Sample 1, mono- and diglycerides The sum o f t h e mean values o f t h e components g l y c e r o l , C14-, C 6- and C18-fatty a c i d s as determined by a l l c o l l a b o r a t o r s was 100.99% (Table 4 ) . R e p e a t a b i l i t y and r e p r o d u c i b i l i t y values are given i n Table 4.

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Table 4: R e s u l t s f o r Sample 1 Mono- and D i g l y c e r i d e s , (expressed a s a percentage by mass o f sample) Lab. Determination Glycerol
C16:o

ClrtO

Claz0

/yacked folumn

1 2 1 2 1 2 1 2 1
lypi--A;y 3
n Mean

22.19 21.90 18.65b 18.21b 22.32 22.47 21.35 21.85 23.80 22.52 22.25 22.72 column 21.33 21.83 21.44 21.03 7 22.07 0.432 1.96 0.729 3.30

1.73 1.57 2.68 2.71 2.59 2.58 2.65 2.70 2.50 2.09 2.64 2.35 2.70 2.88 3.30 3.14
8 2.55 0.146 5.72 0.462 18.13

23.44 22.63 24.01 25.10 24.49 23.81 24.75 23.85 29.38 26.48 27.55 28.88 23.43 23.32 27.66b 24.121,
8 25.18 1.271 5.05 2.172 8.62

40.76 39.79 56.54 59.44 45.63 42.79 50.15 49.75 96.93b 87.67b 56.30 58.68 56.23 52.26 58.33 50.04

1 2

Sr
CV 76 ( r ) SR CV X ( R )

7 51.19 2.774 5.42 7.032 13.74

b R e s u l t s r e j e c t e d by Dixon t e s t (95% confidence l e v e l )

Sample 2 diacetyl tartaric acid esters of mono- and diglycerides ,

The sum o f t h e mean v a l u e s o f t h e components g l y c e r o l , t a r t a r i c a c i d , C14-, C1 and C 1 8 - f a t t y a c i d s determined by c o l l a b o r a t o r s was 75.02%. The a c e t i c a c i d c o n t e n t was analysed by t h e procedure o f P i c k e t t , u s i n g s a p o n i f i c a t i o n , a c i d i f i c a t i o n w i t h p h o s p h o r i c a c i d , d i s t i l l a t i o n and t i t r a t i o n o f t h e a c i d c o n t e n t . The amount o f a c e t i c a c i d has been found i n t h e c o - o r d i n a t o r ' s l a b o r a t o r y t o b e 19.8 X , g i v i n g a t o t a l o f 94.82 76. The r e c o v e r y i s l o w , due t o l o w v a l u e s f o r t a r t a r i c a c i d o b t a i n e d by some c o l l a b o r a t o r s . T h i s i s a t t r i b u t e d t o s a l t f o r m a t i o n and reduced s o l u b i l i t y o f t h e sodium s a l t o f t a r t a r i c a c i d . C o l l a b o r a t o r 2 suggest e d t o use u l t r a s o u n d f o r d i s p e r s i o n and c o l l a b o r a t o r 5 t o change t h e s o l v e n t , a p p l y i n g a mixed s o l v e n t o f p y r i d i n e and a c e t o n i t r i l e (50:50). I t has been found i n t h e c o - o r d i n a t o r s l a b o r a t o r y t h a t t h e use o f bis(trimethylsily1)trifluoroacetamide (BSTFA) and TMCS, t h e l a t t e r a c t i n g a s a c a t a l y s t , completes s i l y l a t i o n o f s a l t s o f l a c t i c - , t a r t a r i c - and c i t r i c a c i d s . The BSTFA + TMCS-combination has f u r t h e r m o r e t h e advantage t h a t n o p r e c i p i t a t e o f i n s o l u b l e ammonium c h l o r i d e i s formed, a s observed w i t h HMDS + TMCS. T h e r e f o r e i n t h e f i n a l procedure t h e use o f ESTFA + TMCS has been proposed as s i l y l a t i n g agent. The performance o f t h i s combin a t i o n has been e x t e n s i v e l y t e s t e d i n t h e c o - o r d i n a t o r s l a b o r a t o r y . Lower c a r b o x y l i c a c i d s such as formic-, a c e t i c - , b u t y r i c - and p r o p i o n i c a c i d may be a n a l y s e d by gas chromatography u s i n g t h e same procedure. The peaks o f t h e s e a c i d s appear between t h e peaks o f t h e s i l y l a t i n g reagents.

Determination of copper, iron and nickel Table 5: R e s u l t s f o r Sample 2, O i a c e t y l T a r t a r i c A c i d E s t e r s o f Monoand O i g l y c e r i d e s (expressed as a percentage by mass o f sample) Glycerol TartaC14:o ClGt0 C Cletl C18:*

787

Lab.

Oetermination

sum o f C18

ri c
Acid 14.81 13.52 12.55 10.18 13.88 13.88 14.30 14.10 13.15 12.81 10.29 13.91 14.92 13.68 12.47 12.70 8 13.20 1.176 8.91 1.386 10.50 5.76 7.58 13.29 7.92 5.81 5.74 19.85 19.05 13.63 12.01 10.80 16.13 5.89 7.75 3.97 6.37 8 10.10 2.127 21.06 5.182 51.30

Packed column 1 1 2 2 1 2 3 1 2 4 1 2 5 1 2 6 1 2 C a p i l l a r y column 1 1 2 3 1 2 n Mean

0.71 0.70 0.72 0.69 0.65 0.70 0.66b 0.4Eb 0.59 0.80

11.02 13.22 13.92 13.77 14.18 13.41 11.55 12.15 12.68 11.36 11.84 15.74 12.31 11.88 15.88 16.86 8 13.24 1.223 9.24 1.772 13.39

< <
9.59 9.02

< < < < < <

10.86 9.37

32.90 34.57 29.67 28.88 50.16 47.46 35.40 35.80 29.77 28.18 32.77 43.99 18.03 17.80 34.20 38.65

> > > > > > > > > > > >
7.04 7.54

32.90 34.57 39 26 37.90 50.16b 47.46b 35.40 35.80 40.6 3 37.55 32.77a 43.99a 32.24 32.13 34.20 38.65 6 35.94 1.686 4.69 2.943 8.19

7.17 6.79

Sr
CV 76 ( r ) SR CV Z ( R )

0.99b 1.03b 4 0.70 0.077 11.10 0.077 11.10

> >

a R e s u l t s r e j e c t e d by Cochran t e s t (95% c o n f i d e n c e l e v e l ) b R e s u l t s r e j e c t e d by Oixon t e s t (95% c o n f i d e n c e l e v e l )

Sample 3, sorbitan monostearate and sample 4, sorbitan monstearate in coconut oil S o r b i t a n monostearate was a p p l i e d as r e f e r e n c e t o d e t e r m i n e t h e same compound p r e s e n t i n c o c o n u t o i l (sample 4), u s i n g t h e peak a r e a s o f s o r b i t a n monoanhydrides and s o r b i t a n d i a n h y d r i des f o r q u a n t i f i c a t i o n . I n t h e c o l l a b o r a t i v e s t u d y s o r b i t a n monostearate was p r e l i m i n a r i l y s e p a r a t e d from t h e coconut o i l t r i g l y c e r i d e s , by s i l i c a column chromatography, a p p l y i n g procedure 2.321 ( 8 ) . E l u t i o n o f t h e t r i g l y c e r i d e s was performed w i t h benzene and t h e p o l a r e m u l s i f i e r f r a c t i o n was e l u t e d w i t h chloroform/methanol ( 1 : l ) . B o t h p o l a r and a p o l a r f r a c t i o n s were s e p a r a t e l y h y d r o l y s e d and s i l y l a t e d i n o r d e r t o see t h e d i f f e r e n c e s between t h e two f r a c t i o n s . The i n t e r l a b o r a t o r y s t u d y i n d i c a t e d ( t a b l e 6 ) t h a t t h e LC-separation was n o t v e r y r e p r o d u c i b l e . T h i s was o b v i o u s l y due t o d i f f e r e n t c o n d i t i o n s and t h e s i l i c a g e l chosen by c o l l a b o r a t o r s . The p o l a r f r a c t i o n was g e n e r a l l y t o o h i g h . B u t t h i s d i d n o t a f f e c t f i n a l d e t e r m i n a t i o n s o f t h e c o n t e n t s o f e m u l s i f i e r s which were q u a n t i f i e d based on t h e amount o f s o r b i t a n d i a n h y d r i d e s i n t h e p o l a r f r a c t i o n . The amount o f s o r b i t a n monostearate added t o t h e coconut o i l was 4.5%. The average r e s u l t s were 3.90% and 3.97% ( t a b l e 6 ) . Recovery was 87.4%. B e t t e r r e c o v e r y were r e p o r t e d i f t h e q u a n t i t i e s o f p o l a r f r a c t i o n used f o r t h e gas chromatographic d e t e r m i n a t i o n s were increased.

Further applications
The procedure d e s c r i b e d was a p p l i e d i n t h e c o - o r d i n a t o r ' s l a b o r a t o r y t o a n a l y s e o t h e r e m u l s i f i e r s such as l a c t i c a c i d monoglycerides, g l y c e r o l monooleates, sucrose e s t e r s o f f a t t y acids, p o l y g l y c e r o l stearate, s o r b i t a n t r i s t e a r a t e . Furthermore f a t s , o i l s and waxes ( 1 1 ) were analyzed. The one-pot h y d r o l y s i s - s i l y l a t i o n proced u r e d e s c r i b e d a l l o w s t h e d e t e r m i n a t i o n o f components o f v e r y s m a l l f r a c t i o n s ( < 0.1 mg) o f e m u l s i f i e r s , f a t s , o i l s , waxes and o t h e r h y d r o l y s a b l e l i p i d s .

Jaa

COMMISSION ON OILS, FATS AND DERIVATIVES

!L
8l
20

8
12

L
D

12

13

J
3
10

1
10 20

30

3c

Retention Time (min)

F i g . 1 Gas Chromatograms o f S i l y l a t e d F a t t y A c i d s and Hydrolysed and S i l y l a t e d E m u l s i f i e r s . A: M i x t u r e o f clo.o-, C 1 2 : o - , C14:o-, Cl6: o-, C18:o-, C18 , l - f a t t y a c i d s , n - t e t r a d e c a n e , n - t e t r a c o s a n e , s i l y l a t e d B: Sample 1: Mono- and d i g l y c e r i d e s , h y d r o l y s e d and s i l y l a t e d C: Sample 2: D i a c e t y l t a r t a r i c a c i d esters o f mono- and d i g l y c e r i d e s , h y d r o l y s e d and s i l y l a ted D: Sample 3: S o r b i t a n m o n o s t e a r a t e , h y d r o l y s e d and s i l y l a t e d

S i l y l a t i o n : Sample: 1 0 mg; r e a g e n t s : O . l m l p y r i d i n e c o n t a i n i n g 1.0 mg n - t e t r a d e c a n e , BSTFA, O . l m l TMCS; r e a c t i o n : 20 min a t 70 O C .

0.2ml

H y d r o l y s i s : Sample: 1 0 mg i n 2 m l v i a l , 0.25ml 0.5N e t h a n o l i c KOH s o l u t i o n , r e a c t i o n : 3 h a t 8OOC, e v a p o r a t i o n o f e t h a n o l . O p e r a t i o n c o n d i t i o n s : U l t r a #2 ( H e w l e t t - P a c k a r d ) f u s e d - s i l i c a c a p i l l a r y column, 25m x 0.32mm I.D., f i l m t h i c k n e s s : 0.17 wm, c o l . temp.: 80 t o 275OC w i t h 5 OC/min, i n j e c t o r temp.: 3 1 0 O C , d e t e c t o r temp.: 3 1 O o C , flow: 2.0 ml/min H a t 8 O 0 C , d e t . : F I D , s e n s . : 2 6 (HP e 5880A), s a m p l e : 2.0 p l , s p l i t r a t i o 1 : l O O . Peak i d e n t i f i c a t i o n : 1: C . o - f a t t y a c i d , 2: n - t e t r a d e c a n e , 3: Ci8ioE, 6: c16:0-, 7: c18: 8: f a t t y a c i d s , 9: n - t e t r a c o s a n e , a c i d s , 12: glycero!, 13: t a r t a r i c a c i d , 14: s o r b i t a n d i a n h y d r i d e s , drides.

-,

Ei8:l-

4: C1 : o 5: C 2-, 21: 8: s o r b i t a n monoanhydri-

-il

&e;

Determination of copper, iron and nickel Table 6 Results f o r Sample 4 , Sorbitan Monostearate i n Coconut Fat, a f t e r LC-Separation o f Polar F r a c t i o n Sample Test Polar F r a c t i o n portion E m u l s i f i e r c a l c u l a t e d from S o r b i t a n MonoS o r b i t a n Oianhydr i d anhydrid
00 '

789

1
2 3 4 5 6

1 2

1
2 1 2 1 2 1 2 1 2

1000.0 1000.0 999.2 1011.2 982.2 1035.6 985.4 1005.6 1018.2 1012.1

1110.0
987.0 1000.0 1000.0 982.2 984.2

98.5 98.5 132.3 144.5 99.6 284.6 92.6 94.3 93.7 93.3 200.0 220.0 98.5 98.5 99.6 127.5

Packed columns 9.85 3.00 9.85 3.93 13.24 4.96 14.29 3.82 10.14 3.19 3.39 27.48 9.40 4.63 9.38 4.77 9.20 4.60 9.22 4.50 18.01 4.28 22.23 5.03 C a p i l l a r y columns 9.85 3.51 9.85 3.59 10.14 2.23 12.95 2.95

3.83 3.63 4.25 3.59 3.30 3.96 4.12 4.03 4.30 4.20 4.50 4.68 3.59 3.59 3.57 4.43 8 3.97 0.326 8.21 0.408 10.28 4.50 88.2

1
3

1
2

1
2

8 3.90 0.456 11.69 0.845 21.68 4.50 86.66

Added X Recovery ? ;

CONCLUSIONS
O t h e b a s i s o f t h e r e s u l t s t h e Commission decided t o adopt t h e method. The t e x t o f t h e n standardized procedure i s given i n t h e f o l l o w i n g pages.

790

COMMISSION ON OILS, FATS AND DERIVATIVES

6.201 DETERMINATION OF THE ESTER-EMULSIFIERS COMPONENTS CONTENT AFTER HYDROLYSIS AND SILYLATION BY GAS CHROMATOGRAPHY
1. SCOPE
T h i s Standard d e s c r i b e s a method f o r t h e d e t e r m i n a t i o n by gas l i q u i d chromatography o f e s t e r - e m u l s i f i e r s components c o n t e n t a f t e r h y d r o l y s i s ( s a p o n i f i c a t i o n ) and s i l y l a t i o n . By t h i s procedure t h e components determined a r e : Acids, o r t h e i r Na- and K - s a l t s : f a t t y a c i d s , h y d r o x y f a t t y a c i d s , d i c a r b o x y l i c a c i d s t a r t a r i c - , c i t r i c a c i d ) g l y c e r o l p h o s p h o r i c a c i d s and i n o r g a n i c a c i d s (lactic(phosphoric-, s u l p h u r i c a c i d ) and a l c o h o l s and p o l y a l c o h o l s : a l c o h o l s ( f a t t y a l c o h o l s ) , g l y c o l s , p o l y g l y c o l s , g l y c e r o l s , p o l y g l y c e r o l s , s o r b i t o l and i t s anhydrides, c a r b o h y d r a t e s (mono- and d i s a c c h a r i d e s ) .

2. FIELD OF APPLICATION
T h i s Standard i s a p p l i c a b l e t o h y d r o l y s i s p r o d u c t s o f e s t e r e m u l s i f i e r s c o n c e n t r a t e s and t o e m u l s i f i e r s separated from o i l s and f a t s (Note 1). The components o f t h e f o l l o w i n g c l a s s e s o f e m u l s i f i e r s may be determined: - e s t e r s o f f a t t y a c i d s w i t h hydroxy c a r b o x y l i c a c i d s (e.g. sodium s t e a r o y l - 2 - l a c t y l a t e ) - e s t e r s o f f a t t y a l c o h o l s w i t h hydroxy c a r b o x y l i c a c i d s (e.9. sodium s t e a r y l - c i t r a t e ) , - f a t t y a c i d e s t e r s o f mono- and p o l y g l y c o l s (e.9. 1,2-propylene g l y c o l mono- and d i e s t e r s o f f a t t y acids, polyoxyethylene-(8 )-stearate), - g l y c e r o l e s t e r s o f f a t t y a c i d s (mono- and d i g l y c e r i d e s , monoglycerides e s t e r i f i e d w i t h a c e t i c - , c i t r i c - , t a r t a r i c - , and l a c t i c a c i d , e s t e r s o f p o l y g l y c e r o l ) - sorbitan e s t e r s o f f a t t y acids, sugar e s t e r s o f f a t t y a c i d s , - lecithin.

3. DEFINITION
The c o n t e n t s o f a component o f an e m u l s i f i e r i s a q u a n t i t y d e t e r m i n e d by t h e p r e s e n t method and expressed as a percentage by mass r e l a t i v e t o t h e sample.

4 . PRINCIPLE
H y d r o l y s i s ( s a p o n i f i c a t i o n ) o f t h e e m u l s i f i e r s w i t h a l c o h o l i c KOH s o l u t i o n (Note 1). Evaporat i o n o f t h e a l c o h o l . S i l y l a t i o n o f t h e components. I d e n t i f i c a t i o n and d e t e r m i n a t i o n o f t h e t r i m e t h y l s i l y l d e r i v a t i v e s by gas l i q u i d chromatography w i t h packed o r c a p i l l a r y column.

5 . APPARATUS
5.1 5.2 5.3 Screw c a p v i a l s , 2 m l , w i t h c o n i c a l bottom. Screw caps s u i t a b l e f o r v i a l s (5.1) w i t h i n e r t p l a s t i c f a c e d s e p t a (Notes 2 and 3 ) . V o l u m e t r i c f l a s k s , 10 m l 5.4 P i p e t t e s , 0.1, 0.2, 0.5 m l . 5.5 M i c r o s y r i n g e s , 1 5 p l . Gas l i q u i d chromatograph w i t h flame i o n i z a t i o n d e t e c t o r , t e m p e r a t u r e programming and 5.6 integrator. 3 m l o n g and Packed column, m e t a l o r g l a s s , s u i t a b l e f o r chromatograph (5.6), about 2 5.7 2- 4 mm i n s i d e d i a m e t e r f i l l e d w i t h e i t h e r m e t h y l p o l y s i l o x a n e , p h e n y l m e t h y l p o l y s i l o x a n e o r m e t h y l p h e n y l v i n y l p o l y s i l o x a n e (Note 4) on an acid-washed, s i l y l a t e d , f u l l y d e a c t i v a t e d diatomaceous e a r t h (Notes 5 and 6 ) , o r C a p i l l a r y column, g l a s s o r f u s e d - s i l i c a , f u l l y d e a c t i v a t e d , s u i t a b l e f o r chromatograph 5.8 25 m l o n g and 0.2 - 0.4 mm i n s i d e diameter. F i l m t h i c k n e s s 0.1 - 0.2 (5.6), about 15 pm. Coating: m e t h y l p o l y s i l o x a n e , phenylmethylpolysiloxane o r m e t h y l p h e n y l p o l y s i l o x a n e (Notes 4 and 7). H e a t i n a d e v i c e f o r v i a l s (5.1). 5.9 5.10 Needles f o r b l o w i n g a g e n t l e stream o f n i t r o g e n i n t o t h e v i a l s (4.1).

6. REAGENTS
6.1 6.2 6.3 6.4 6.5
Tetradecane, p u r e . P y r i d i n e , pure, k e p t over potassium h y d r o x i d e (Note 8 ) . T r i m e t h y l c h l o r o s i l a n e (TMCS). B i s ( trimethylsily1)trifluoroacetamide (BSTFA) Potassium h y d r o x i d e , 0.5 N e t h a n o l i c s o l u t i o n .

Determination of copper, iron and nickel

791

Tetradecane i n p y r i d i n e , i n t e r n a l s t a n d a r d s o l u t i o n . A c c u r a t e l y weigh about 100 mg o f t e t r a d e c a n e (6.1) i n t o a v o l u m e t r i c f l a s k (4.3). D i l u t e t o volume w i t h p y r i d i n e (6.2). 6.7 Reference s o l u t i o n s w i t h i n t e r n a l standard. A c c u r a t e l y weigh about 100 mg o f r e f e r e n c e s u b s t a n c e ( s ) ( a l c o h o l s , p o l y a l c o h o l s , f a t t y a c i d s , h y d r o x y c a r b o x y l i c a c i d s , o r t h e i r sodium o r potassium s a l t s ) i n t o a v o l u m e t r i c f l a s k s (5.3). Add about 100 mg a c c u r a t e l y weighed t e t r a d e c a n e (6.1). D i l u t e t o volume w i t h p y r i d i n e (6.2). S e v e r a l substances (e.g. f a t t y a c i d s ) may b e p r e s e n t i n a r e f e r e n ce s o l u t i o n . 6.8 C a r r i e r gas: helium, d r y , f r e e f r o m i m p u r i t i e s . 6.9 A u x i l i a r y gases: hydrogen, f r e e from o r g a n i c i m p u r i t i e s , a i r o r oxygen. 6.10 N i t r o g e n . 6.6

7. PROCEDURE 7.1 Hydrolysis (saponification) of sample solutions

A c c u r a t e l y weigh about 10 mg o f t h e homogenized sample i n t o a v i a l (5.1). By means o f a p i p e t t e (5.4) add 0.25 m l o f t h e e t h a n o l i c potassium h y d r o x i d e s o l u t i o n (6.5) (Note 9 ) . Put t h e r e a c t i o n v i a l i n t o a h e a t i n g d e v i c e (4.9) a t a t e m p e r a t u r e o f 70OC. The h y d r o l y s i s i s completed a f t e r about 3 hours. By means o f a n e e d l e (5.10) evaporate c a r e f u l l y a l l t h e e t h a n o l i n a stream o f n i t r o g e n (6.10).

7.2 Silylation of sample solutions

By means o f a p i p e t t e o r s y r i n g e (5.4) add t o t h e d r i e d h y d r o l y s e d p r o d u c t 0.1 m l i n t e r n a l s t a n d a r d i n p y r i d i n e (6.6), 0.2 m l BSTFA (6.4) and 0.1 m l TMCS (6.3) ( N o t e 1 0 ) . H u m i d i t y must s t r i c t l y be excluded. Close t h e v i a l . Shake v i g o r o u s l y . I f necessary d e s t r o y s k i n formed and remove substances from t h e g l a s s w a l l w i t h a s m a l l g l a s s r o d which i s b r o k e n o f f a f t e r use. Heat t h e r e a c t i o n m i x t u r e f o r about 20 m i n u t e s a t 70 O C .

7.3 Chromatography of sample solutions

The s u p e r n a t a n t l i q u o r o f t h e s i l y l d e r i v a t i v e s s h o u l d be i n j e c t e d soon a f t e r t h e d e r i v a t i z a t i o n process. C a r r y o u t two d e t e r m i n a t i o n s , each c o n s i s t i n g o f d u p l i c a t e i n j e c t i o n s o f t h e t e s t s o l u t i o n s and r e c o r d t h e a r e a s o f t h e peaks. The o p e r a t i n g c o n d i t i o n s w i t h gas chromatograph (5.6) be t h e f o l l o w i n g : and columns (5.7 and 5.8) should

- i n j e c t o r t e m p e r a t u r e : 310 O C - d e t e c t o r t e m p e r a t u r e : 310 O C -column t e m p e r a t u r e : i n i t i a l 80 O C program r a t e 5 10 O C / m i n f i n a l 275 O C . - w i t h packed column : f l o w r a t e o f c a r r i e r gas 30 ml/min - w i t h c a p i l l a r y column: f l o w r a t e o f c a r r i e r gas: 1 t o 3 m l / m i n (measured a t 8 0 OC) - i n j e c t i o n by means o f a m i c r o s y r i n g e (5.5) : 1 t o 5 p l (Note 11).

7.4 Identification
I d e n t i f y peaks by comparison o f r e t e n t i o n t i m e o f known substances i n r e f e r e n c e s o l u t i o n (6.7) a f t e r s i l y l a t i o n and chromatography under t h e same c o n d i t i o n s as t h e sample or a p p l y c o u p l e d gas chromatography and mass s p e c t r o m e t r y .

7.5 Silylation and chromatography of reference solution

By means o f a p i p e t t e (5.4) t r a n s f e r 0.1 m l o f t h e r e f e r e n c e s o l u t i o n (6.7) i n t o a v i a l (5.1). With a p i p e t t e (5.4) add 0.2 m l BSTFA (6.4) and 0.1 m l TMCS (6.3) ( N o t e 10). Humidity must s t r i c t l y be excluded. Close t h e v i a l . Shake v i g o r o u s l y . I f necessary remove substances from t h e g l a s s w a l l . Heat t h e r e a c t i o n m i x t u r e f o r about 20 m i n u t e s a t 70 O C . The s u p e r n a t a n t l i q u o r o f t h e s i l y l a t e d r e f e r e n c e s o l u t i o n s h o u l d b e i n j e c t e d soon a f t e r t h e d e r i v a t i z a t i o n process. Operate under t h e same c o n d i t i o n s a s f o r t h e sample s o l u t i o n (7.3). Use d u p l i c a t e i n j e c t i o n s and c o n c e n t r a t i o n ranges o f r e f e r e n c e substances as t o be q u a n t i f i e d i n sample. Check response f a c t o r s p e r i o d i c a l l y .

792

COMMISSION ON OILS, FATS AND DERIVATIVES

8. EXPRESSION OF RESULTS
8.1 Response factor Calculate response f a c t o r o f t h e reference substance vs. i n t e r n a l standard u s i n g t h e reference s o l u t i o n chromatogram. The value o f the response f a c t o r i s g i v e n by t h e formula:

&

where :

: 9 : & : hs :
Q~

: response f a c t o r o f reference substance x mass, i n mg, o f i n t e r n a l standard mass, i n mg, o f reference substance x peak area o f reference substance x peak area o f i n t e r n a l standard

8.2 Calculation of sample component content

Calculate percentage o f mass content o f component x i n the sample by the formula:

: percentage o f mass o f component x i n sample : response f a c t o r o f component x i n sample %is i n mg, o f i n t e r n a l standard i n sample : mass, Els : mass, i n mg, o f sample A', : peak area o f t h e component x i n sample : peak area o f t h e i n t e r n a l standard i n sample

m',

where:

Elis

9. NOTES
1 I f e m u l s i f i e r s a r e present i n o i l s and f a t s , t h e p o l a r e m u l s i f i e r s a r e f i r s t separated . from t h e t r i g l y c e r i d e s by method 2.507 "Determination o f p o l a r compounds i n f r y i n g f a t s " , o r by method 2.321 "Determination o f mono-, d i - and t r i g l y c e r i d e s by column chromatography". The t r i g l y c e r i d e s a r e removed w i t h f i v e 60 m l p o r t i o n s o f benzene and t h e e m u l s i f i e r s are e l u t e d w i t h f i v e 60 m l p o r t i o n s o f dichloromethane/methanol (2/1, V/V) solvents. F i n a l l y phospholipids are e l u t e d w i t h t h r e e 60 m l p o r t i o n s o f methanol. I t i s recommended t h a t t h e t r i g l y c e r i d e s e x t r a c t e d from t h e column a r e a l s o hydrolysed and s i l y l a t e d i n order t o recognize t h e d i f f e r e n c e between t h e p o l a r and apolar f r a c t i o n s . 2. Screw cap v i a l s w i t h magnetic s t i r r e r o r u l t r a s o u n d may be applied.

3. Teflon faced septa are s u i t a b l e .

4. S i l i c o n e s O V - 1 , OV-17, SE-52, SE-54 are s u i t a b l e .

5 . Chromosorbs G, AW-DMCS i s s u i t a b l e .
6. The s t a t i o n a r y phase has t o be f u l l y d e a c t i v a t e d u s i n g an excess o f s i l y l a t i n g agents, otherwise adsorption o f t r i m e t h y l s i l y l f a t t y a c i d d e r i v a t i v e s occurs. The d e a c t i v a t i o n can be t e s t e d with a m i x t u r e o f hydrocarbons such as hexadecane (c16), eicosane ( C 2 0 ) , tetracosane (C24), octacosane (C2, ) and s i l y l a t e d f a t t y a c i d s such as decanoic a c i d (Cl0), tetradecanoic a c i d (C14), octadecanoic a c i d ( C 1 8 ) and docosanoic a c i d (C22). The peak o f t h e corresponding hydrocarbons and TMS-esters o f f a t t y a c i d s should have about t h e same h e i g h t (Donike t e s t ) . I n j e c t i o n o f s i l y l a t i n g agents may l e a d t o the r e q u i r e d d e a c t i v a t i o n o f t h e columns. Higher f a t t y a c i d s and unsaturated f a t t y acids may show g r e a t e r d i s c r i m i n a t i o n . Faster h e a t i n g r a t e s o r h i g h e r i n i t i a l temperatures may reduce these e f f e c t s . F a t t y a c i d methyl e s t e r s are t o be p r e f e r r e d . 7. C a p i l l a r y columns are p r e f e r r e d , as the C I 8 o-, separated and can be q u a n t i f i e d . hexane o r m i x t u r e s o f them may be used. 9. The amount o f e t h a n o l i c KOH s o l u t i o n s p e c i f i e d i s enough t o hydrolyse ( s a p o n i f y ) an a c i d i c d i a c e t y l t a r t a r i c a c i d g l y c e r i d e w i t h a s a p o n i f i c a t i o n value o f 490. For b e t t e r d i s s o l u t i o n o f some e m u l s i f i e r s an a d d i t i o n o f 0.1 m l o f water i s advantageous.

C ,

C ,

* - f a t t y a c i d s are w e l l

8. I n s t e a d o f p y r i d i n e s o l v e n t s such as dimethylformamide, a c e t o n i t r i l e , tetrahydrofurane,

Determination of copper, iron and nickel

793

10.Salts o f h y d r o x y l i c a c i d s such as l a c t i c - , t a r t a r i c - and c i t r i c a c i d s are b e t t e r and more completely s i l y l a t e d w i t h t h e BSFTA + TMCS-combination than w i t h hexamethyldisilazane (HDMS) and TMCS.

1 . For on-column i n j e c t i o n , or d i r e c t i n j e c t i o n , d i l u t e 50 p1 o f r e a c t i o n m i x t u r e (8.2; 1 w i t h 1 m l hexane and, by means o f microsyringe (6.5), i n j e c t supernatant ( 1 p l ) . I n order t o lengthen the l i f e time o f the column, applying on-column i n j e c t i o n s , a pre-column i s u s e f u l .

8.5)

WARNING

- PYRlDlNE

The danger from n o n - p u r i f i e d p y r i d i n e i s g r e a t e r than from t h e pure reagent, t h e associated homologues and i m p u r i t i e s being g e n e r a l l y more t o x i c than p y r i d i n e i t s e l f . Odour and i r r i t a t i o n f u r n i s h c l e a r warning o f a vapour c o n c e n t r a t i o n l i k e l y t o be dangerous.

APPENDIX
I n some cases names commonly used i n e m u l s i f i e r terminology do n o t conform w i t h I U P A C recommendations. The f o l l o w i n g t a b l e l i s t s IUPAC e q u i v a l e n t s f o r some names used i n t h e present document. Behenic a c i d Stearoyl-2-lactylate Capric a c i d Diacetyl t a r t a r i c acid I U P A C Name Docosanoic a c i d

2-0-(2-c-stearoyllactoyl ) l a c t a t e
Decanoic a c i d 2,3-Di-c-acetyltartaric acid M i x t u r e o f e t h o x y l a t e d g l u c i t o l and a n h y d r o g l u c i t o l s (molar r a t i o g l u c i t o l + anhydroglucitols/oxirane I 1/20) M i x t u r e o f e t h o x y l a t e d s t e a r i c a c i d (molar r a t i o s t e a r i c acid/oxirane = 1/8) Propane-1,Z-diol 12-Hydroxy-c&-octadec-9-enoic acid M i x t u r e o f g l u c i t o l , 1,4-, 2,5-, 3,6-anhydroglucitol, and dianhydroglucitols

Polyoxyethylene(20)sorbitan
Polyoxyethylene( 8 ) s t e a r a t e Propyleneglycol Ricinoleic acid Sorb i tan

REFERENCES
1. Food and A g r i c u l t u r e Organization o f t h e U n i t e d Nations, S p e c i f i c a t i o n f o r I d e n t i t y and P u r i t y , Rome 1978. 2. Sahasrabudhe, M.R., J. Am. O i l Chem. SOC. 1967, 44, 376-378 3. Schutze, I., Die Nahrung 1977, 21, 405-415 4. Sahasrabudhe, M.R., J. Am. O i l Chem. SOC. 1969, 46, 8-12 5. Tsuda, T., Nakanishi, H., Kobayashi, S. and Morita, T., J. Assoc. O f f . Anal. Chem. 1984, 67, 1149-1151 6. Tsuda, T. and Nakanishi, H., J. Assoc. O f f . Anal. Chem. 1983, 66, 1050-1052 7. B u x t o r f , U.P., B r k c h w e i l e r , H. and Martin, E., Z . Lebensm. Unters. Forschung 1982, 175, 25-28 8. IUPAC, Standard Methods f o r t h e Analysis o f O i l s , F a t s and D e r i v a t i v e s , 7 t h Ed., C. Paquot, B l a c k w e l l Sci 9 . Pocklington D., Guidelines f o r t h e Conducting o f C o l l a b o r a t i v e Study, IUPAC, Commission on O i l s , Fats and D e r i v a t i v e s , September 1984. 1 O . I S O 5725, P r e c i s i o n o f Test Methods, Determination o f R e p e a t a b i l i t y and R e p r o d u c i b i l i t y by Inter-Laboratory Test, 1981 l l . B r i k c h w e i l e r , H . , Felber, H. and Schwager, F., F a t S c i . Technol, 1989, 91(2), 73-79

Acknowledgements
The Commission wishes t o express i t s thanks t o c o l l a b o r a t o r s i n Denmark, Germany FR, Japan f o r t h e i r p a r t i c i p a t i o n and valuable co-operation.