Analytical Methodologies for the Determination of ...biodiesel.org/reports/19960227_gen-221.pdf · ANALYTICAL METHODOLOGIES FOR THE DETERMINATION OF BIODIESEL ... GC Injection Technique

FINAL REPORT

PROJECT TITLE:

ANALYTICAL METHODOLOGIES FOR THE DETERMINATION OF BIODIESELESTER PURITY - DETERMINATION OF TOTAL METHYL ESTERS

NBB CONTRACT #:520320-l

SUBMITTED BY:

RICHARD W. HEIDEN, PH.D.R. W. HEIDEN ASSOCIATES

P.O.BOX 5001LANCASTER, PA 17606-5001

717-299-6860FAX: 717-299-6868

FEBRUARY 27, 1996

TABLE OF CONTESTS

.4 . Disclaimer

I . Esecut ive SummaryConclusionsRecommendationsSummary

PAGE

1 - 5

I I . Introduction 5

III. Literature Review 6-13

IV. Statistical Requirements 14-19

V . Method Laboratory Evaluation 20-36

Precision and Accuracy Studies 21-33GC

Injection TechniqueHot On-Column vs Cold On-ColumnInternal Standards

HPLCPrecision

GC Interferences

Potential Application to Diesel BlendsGC -- 34-35HPLC 36

Potential ApplicatGCHPLC

ion to Biodiesel Made f rom Ethanol36-3736

VI . Research Needs 38-40

VII. Draft GC Yethod for the Determination of Total MethylEsters of FAME Biodiesel 41-45

VIII. References 46-47

A. DISCLAIMER: The use of tradenames, brandnames ormanufacturers' names herein in no way constitutes arecommendation of the products or wares mentioned.

R. W. HEIDEN ASSOCIATES/!?ATIONAL BIODIESEL BOARD 27 FEB 96

I. EXECUTIVE SUMMARY

CONCLUSIONS

1) The accuracy and repeatability of commonly used gaschromatographic (GC/FID) determinations of methyl ester contentof biodiesel is limited by a host of variables which need to becontrolled to ensure reliable results.

Accuracy is limited by the complexity of the calibration process,the purity of substances used as standards, the changes instandards caused by handling and storage, the inability toidentify and quantify all esters in the sample, the complexity ofthe samples themselves, for example as caused by mixtures ofalkyl esters formed by compounded starting materials, and variouspotential interferences causing peak overlaps or misidentifi-cation.

2) Only methods capable of the highest precision and accuracy aresuited for this determination.

The precision and accuracy requirements for total methyl esteranalysis are high, determined by the need to distinguish 95-98xspecified purity from 100% purity reliably.

3) A modified GC standard method is offered for immediateimplementation as a short term stop gap. This is fordetermination of total methyl esters in biodiesel made from puremethanol.

.4 modified version of an AOCS standard method with cool on-columninjection, a moderately polar megabore capillary column,autoinjection, and computer data analysis with gaschromatography/ flame ionization (GC/FID) detection is presentedas the method of immediate choice to address the short term needfor high precision and accurate determinations of total methylesters by gas chromatography.

4) High performance liquid chromatography (HPLC) with masssensitive detectors, such as evaporative light scattering (ELSD)affords a highly precise estimation of the ester content with thepotential for greatly reduced calibration needs, and directapplication to diesel blends and ethyl esters. However, thistechnology is relatively new and not yet established forbiodiesel work.

5) Research is needed to improve the soundness of data from thelaboratory, and reduce the effort needed to generate such data.

R. W. HEIDEN ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

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RECOMMENDATIONS

1 . The a t tached procedure f o r de terminat i on o f t o ta l methy les ters by gas chromatography (GC) shou ld be implementedimmedia te ly . Because o f i d i osyncras ies assoc ia ted wi th the GCapproach th i s shou ld be v i ewed as a shor t t e rm s top gap , wh i l eresearch cont inues t o f ind more re l iab le and eas i e r t o implementmethods.

2 . Research i s needed immedia te ly t o improve the qua l i ty o f t o ta lmethy l e s te r de terminat i ons , and to f ind ways t o r educe thee f f o r t r e q u i r e d t o o b t a i n t h e d e s i r e d r e s u l t .

H i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) in con junct ion wi thvar i ous mass de tec to rs , s u c h a s t h e d e n s i t y d e t e c t o r , o f f e r s t h ep o t e n t i a l t o s a t i s f y t h i s n e e d . T h e s e d e t e c t o r s h a v e p o t e n t i a lf o r r e l i a b l e a n d r e p r o d u c i b l e d e t e r m i n a t i o n s a n d a r e d u c t i o n i ne f f o r t a s s o c i a t e d w i t h c a l i b r a t i o n a n d i n t e r p r e t a t i o n , a n d ,t h e r e f o r e , s h o u l d b e i n v e s t i g a t e d t h o r o u g h l y .

See a l so Sec t i on VI I on research recommendat i ons .

SUMMARY

The work presented here enta i l s a rev iew o f l i t e ra ture concern ingt h e a n a l y t i c a l c h a r a c t e r i z a t i o n o f m e t h y l e s t e r s o f f a t s a n d o i l so f b i o l o g i c a l o r i g i n , f o l l o w e d b y a l a b o r a t o r y s t u d y o f s e l e c t e dmethods. The purpose i s t o l ay the f oundat i on f o r as t a n d a r d i z e d p r o c e d u r e f o r r o u t i n e l y d e t e r m i n i n g t h e t o t a l e s t e rp u r i t y , a n d c o n s i s t e n t w i t h t h i s p u r p o s e t o f i n d s u i t a b l ea n a l y t i c a l m e t h o d s .

A gas chromatograph i c procedure i s o f f e red here f o r immedia teimplementat i on , whi ch meets shor t t e rm labora tory needs . Thismethod represents a subs tant ia l improvement over ear l i e r c ommonlyused methods. The gas chromatograph i c approach i s l imi ted ,however , b y v a r i o u s i d i o s y n c r a s i e s , a n d t h e s e l i m i t a t i o n s y i e l d aca l l f o r immedia te research on promis ing t e chno log ies , such asHPLC wi th mass sens i t i ve de tec tors , and ways t o r educe laboratorye f f o r t t o p r o d u c e r e l i a b l e r e s u l t s .

The underlying theme of the work is perhaps best summarized byt h e q u e s t i o n : “What is the most economical way to obtain theaccuracy and repeatab i l i ty demanded by b i od iese ls p e c i f i c a t i o n s ? “ . T h e q u e s t i o n o f w h e t h e r e s t a b l i s h e d a n a l y t i c a lmethodo log ies and method s tandards are read i ly adaptab le t o th i stheme is addressed here . P r o s p e c t i v e a n a l y t i c a l m e t h o d s a r ef i r s t s c r e e n e d d u r i n g t h e l i t e r a t u r e r e v i e w u s i n g a s e r i e s o fp r e s e l e c t e d c r i t e r i a . A m o n g t h o s e c r i t e r i a a r e s t a t i s t i c a lmethods which help def ine the demands on the analytical method


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f o r r e p e a t a b i l i t y . Sound cho i ces f rom the s c reen ing are fur there v a l u a t e d u s i n g l a b o r a t o r y t e s t s t o d e t e r m i n e t h e r e p e a t a b i l i t y( p r e c i s i o n ) a n d i d e n t i f y f a c t o r s w h i c h i n f l u e n c e a c c u r a c y .

The l i t e ra ture on methy l e s te r de terminat i ons i s r ev i ewed wi th afocus on cliromatographic d e t e r m i n a t i o n s o f w e i g h t p e r c e n t .Chromatograph i c methods were inves t iga ted because o f l imi ta t i onsi n t h e a p p l i c a t i o n o f s p e c t r o s c o p i c a n d c h e m i c a l t e s t s a n d t h ew i d e s p r e a d use o f chromatographi c ins t rumentat i on in theb i o d i e s e l i n d u s t r y . Fat ty ac id methy l e s ter (FAME) ana lys i s i sthe f o cus o f much research over severa l decades , but thea p p l i c a t i o n o f t h e r e s u l t s o f t h i s r e s e a r c h t o t h e a n a l y s i s o fb i o d i e s e l i s r e l a t i v e l y r e c e n t . P i o n e e r e d In the U.S. , most o ft h e s p e c i f i c a p p l i c a t i o n d e v e l o p m e n t o f c h r o m a t o g r a p h i c t e c h n i q u eto b i od iese l i s now bes t documented in the European l i t e ra ture .T h i s i s c l e a r l y t h e r e s u l t o f a n o n - g o i n g p u s h t o c o m m e r c i a l i z eb i o d i e s e l t h e r e . T h e r e c e n t o b j e c t i v e o f e s t a b l i s h i n g s u i t a b l em e t h o d s f o r e n f o r c i n g s t r i c t s p e c i f i c a t i o n s f o r t h e w e i g h tp e r c e n t t o t a l m e t h y l e s t e r c o n t e n t o f b i o d i e s e l f u e l s d i f f e r sapprec iab ly f r om that o f mos t ear l i e r work , where the e f f o r t s a reconcentra ted on de termin ing the numerous spec i f i c impur i t i e sthought t o pose po tent ia l o r r ea l p rob lems .

A ser i e s o f c r i t e r ia a re presented whi ch were used t o makep r e l i m i n a r y e v a l u a t i o n s o f p r o s p e c t i v e m e t h o d s f o r t o t a l m e t h y le s t e r d e t e r m i n a t i o n s . Four main c r i t e r ia were used t o choosemethods f o r exper imenta l eva luat i on : 1 ) p o t e n t i a l f o r t h e h i g h e s ta t t a i n a b l e p r e c i s i o n a n d a c c u r a c y ; 2 ) c omparab le cos ts o fi n s t r u m e n t a t i o n ; 3 ) f l e x i b i l i t y o f i n s t r u m e n t a t i o n , i . e .p o t e n t i a l a p p l i c a t i o n t o o t h e r b i o d i e s e l a p p l i c a t i o n s , a n d o t h e rl a b o r a t o r y n e e d s ; 4 ) e s t a b l i s h e d t e c h n o l o g i e s . O t h e r f a c t o r s ,s u c h a s a n a l y s i s t i m e , o p e r a t i n g c o s t s , e a s e o f m e t h o d e x e c u t i o n ,c a l i b r a t i o n a n d s t a n d a r d i z a t i o n d e m a n d s , t o x i c i t y a n d h e a l t hhazards o f hand led chemica l s , a n d t h e a v a i l a b i l i t y o fins t rumentat i on were a l so cons idered . O n e g o o d p o s s i b i l i t y f o rBPLC d e t e c t i o n , t h e d e n s i t y d e t e c t o r , was i gnored because o f theu n a v a i l a b i l i t y o f i n s t r u m e n t a t i o n a t t h e t i m e o f t h e s t u d y .

T h e r e s u l t s o f t h e c r i t e r i a e v a l u a t i o n i n d i c a t e t h a t g a schromatograph i c methods w i th f l ame i on i za t i on de tec t i on (GC/FID)o f f e r the mos t immed ia te promise , b e c a u s e o f t h e p o t e n t i a l f o rh i g h p r e c i s i o n a n d a c c u r a c y , and the widespread use andpopular i ty o f GC ins t rumentat i on . HPLC with evaporative l ights c a t t e r i n g d e t e c t i o n , a more recent t e chno log i ca l advance , a l s ooffers the potential for highly repeatable determinations oft o t a l m e t h y l e s t e r s , e x t e n s i o n t o b i o d i e s e l i m p u r i t i e s , a n d is

R. W. HEIDEN ASSOCIATES/KATIONAL BIODIESEL BOARD 27 FEB 96

P.4GE 3

used as a comparison to the GC approach in the experimentale v a l u a t i o n . The requ i rements f o r ins t rumentat i on are presentedand the costs for purchase of e ither HPLC or suitably equipped GCins t rumentat i on are comparab le .

V a r i a b i l i t y i n t h e m e a s u r e m e n t p r o c e s s d i c t a t e s a s c i e n t i f i cunders tand ing o f the demands on ana ly t i ca l per fo rmance t or e l i a b l y d e t e r m i n e t h e p u r i t y o f b i o d i e s e l , a n d a s t a t i s t i c a lbasis is developed and presented to make appropriate methodd i s t i n c t i o n s . The s ta t i s t i ca l r equ i rements f o r methods capab leo f d i s t ingu ish ing be tween 95 or 98 and 100 % pur i ty arep r e s e n t e d , and these requ i rements d i c ta te the need f o r methodswi th the h ighes t prec i s i on and accuracy . S t a t i s t i c a l m e a s u r e s o fv a r i a b i l i t y a r e u s e d i n t h e e x p e r i m e n t a l e v a l u a t i o n o f t h emethods.

Two GC approaches and one HPLC approach were evaluatede x p e r i m e n t a l l y i n d e t a i l u s i n g t h e s t a t i s t i c a l e s t i m a t e s o fv a r i a b i l i t y ( r e p e a t a b i l i t y ) , t e s t s o f p o s s i b l e i n t e r f e r e n c e s , a n dt h e p r o s p e c t s f o r a p p l i c a t i o n s t o b l e n d s o f b i o d i e s e l w i t hpe t ro l eum, a n d t o e t h y l e s t e r s . In each case , the methods weremod i f i ed f r om pub l i shed methods and opt imized f o r b i od iese ld e t e r m i n a t i o n s o f t o t a l m e t h y l e s t e r s . The GC modi f i ca t i ons o fan AOCS s tandard f o cus on in j e c t i on cond i t i ons opt imized f o rb i o d i e s e l d e t e r m i n a t i o n s , a n d t w o v a r i a t i o n s o f t h e b e s t p o s s i b l ei n j e c t i o n c o n d i t i o n s w e r e e v a l u a t e d .

The resu l t s show that the coo l on co lumn in jec t i on GC techn iquep r o v i d e s t h e b e s t r e p e a t a b i l i t y . T h e c o o l , on-column methodg ives a prec i s i on o f p lus o r minus 0 .34 % r e l a t i v e , c o m p a r e d t o aprecision of plus or minus 0.73% for the HPLC, and plus or minus2.87% for hot on column GC. Analysis t ime for the HPLC approachis l e ss than 8 minutes , whereas the total t ime for a GC run ofthe methy l e s te rs i s 40 minutes , thereby making possible morer e p l i c a t i o n s ( w h i c h c a n b e u s e d t o i m p r o v e t h e s t a t i s t i c s ) f o renhanced s ta t i s t i ca l r e l iab i l i ty w i th the HPLC method . The HPLCmethod i s a l so r ead i l y adapted t o e thy l o r mixed es te rs andes ters b l ended w i th pe t ro l eum d iese l . The eva luat i on o fp o t e n t i a l i n t e r f e r e n c e s s u c h a s f r e e f a t t y a c i d s , a n d p o s s i b l ea p p l i c a t i o n t o e t h y l e s t e r s o r b i o d i e s e l b l e n d s s h o w s t h a t t h eHPLC appears t o o f f e r cons iderab le advantage and f l ex ib i l i ty .

V a r i o u s f a c t o r s w h i c h l i m i t a c c u r a c y a r e d i s c u s s e d . Among thosef a c t o r s t h e a v a i l a b i l i t y o f s u i t a b l y c e r t i f i e d r e f e r e n c em a t e r i a l s , and errors in the preparat i on and hand l ing o fs tandards l oom as impor tant cons idera t i ons . For the GC method,each ind iv idua l methy l e s ter must be quant i ta ted wi th a s tandardand th i s poses a ser i ous cha l l enge t o the ana lys t as thec o m p l e x i t y o f t h e e s t e r i n c r e a s e s , f o r e x a m p l e , t h e r e s u l t o fm i x t u r e s o f a l c o h o l s i n t h e s t a r t i n g m a t e r i a l s .


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The HPLC method with mass selective detection offers potentialadvantages in this regard, also, with the possibility for minimalsubstance calibration. But the application of this technology tomethyl ester purity determinations is less well established, andrequires further validation and research.

Continued research is necessary to achieve reliable, costeffective approaches to the chromatographic determination of thetotal weight percent of the methyl ester content, and theresidual glyceride impurities. Research needs (Section VII) arepresented to amplify and further improve the methods andencourage the development of newer technology which promisesimproved method performance, reliability, and a reduction in theeffort required to obtain precise and accurate results.

A draft procedure utilizing the cool on-column gas chromatographytechnology investigated in this study is attached.

II. INTRODUCTION

The transesterification of triglyceryl esters with simplealcohols affords an elegant pathway to materials potentiallysuited for diesel fuels. The starting materials for theconversion are, however, often of complex origin and lead topotential diesel products with varying extents of esterconversion, and fuel properties.

This biodiesel is comprised of numerous esters plus impurities,such as byproducts, residual reactants, free acids, and variousglycerides. Such a concoction poses a challenge to analysts whoneed to address on one hand impurities at the 0.1% level whichimpact engine performance, and on the other, purity at the95%-100% level. Both are necessary for complete fuelcharacterization.

The commercialization of biodiesel fuels dictates the need forenforceable specifications. The purity of the fatty acid methylesters used in these fuels must be well defined to ensureconsistent product quality and engine performance. Clearly,reliable analytical methodologies for determining biodieselquality are needed to form a solid basis for enforcement ofstrict specifications of 95 % or better purity.

The following work entails a review of existing literatureconcerning the analytical characterization of methyl esters ofoils and fats of biological origin, followed by a laboratorystudy of selected methods. A procedure which addresses shortterm needs is offered here for immediate implementation.Research needs are presented to amplify and further improve themethod and encourage the development of newer technology whichpromises improved method performance and reliability.

R. W. HEIDER: ASSOCIATES/KATIOWAL BIODIESEL BOARD 27 FEB 96

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III. LITERATURE REVIEW

METHYL ESTER PURITY DETERMINATIONS

The need for methyl ester purity evaluations is a two headeddragon. On one head the tot a 1 methyl ester content by weightpercent is needed to predict important fuel properties, and toclearly establish the identi y and purity of samples which areunknown. On the other head, the level of many importantimpurities such as glycerol, mono and diglycerides at levels ofaround 0.1% (a factor of I/ 1 000 or less of the main estercomponents) or lower appears necessary for optimum engineperformance (I). The two differing needs pose a seriouschallenge to the analyst, and while a complete scan of theimpurities by one practical analytical protocol remains a lofty,yet unattained goal, separate analysis schemes for optimizedprecision and accuracy appear necessary.

The following summarizes the work on indirect and direct methodsfor determining methyl ester purity. The indirect methods aredefined as those methods which attempt quantification ofimpurities, and the direct methods are those that determine thetotal methyl ester content by weight.

Chromatographic methods are the focus of much of this review,since these methods are widely used in the biodiesel industry,and gas chromatography is already specified (3,3) or proposed forASTM.

INDIRECT METHODS FOR DETERMINING PURITY

The overall objective of indirect methods, then, is to determineall the impurities, sum them to arrive at a total impurityfigure, and subtract that amount from 100 to arrive at percentpurity. All of the impurities must be determined to arrive at asatisfactory determination.

Chemical, spectroscopic, and chromatographic methods are allapplied to the determination of specific impurities, such asfree fatty acids, soaps, water, glycerol, sterols and otherunsaponifiable matter, mono and diglycerides, and residualreactants-the triglycerides and alcohol, in biodiesel. A recentmonograph (4) summarizes much of the analytical methodologycurrently used for biodiesel in Europe, and should be consultedas a background primer on biodiesel analysis. A recent symposiumaddressed the need for standardized approaches to biodiesel

R. 6. HEIDEN ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

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analysis, and updated the status of several of the EuropeanCommunity methods (1,2). These methods or variations of them areused throughout the biodiesel industry, and chromatographicmethods which allow for direct estimates of weight percent arethe focus of industry.

Chemical and spectroscopic techniques are generally acceptablefor biodiesel when other suitable alternatives for determiningweight percent are inappropriate. These methods are based onfunctional group population estimates, however, and additionalanalytical information from complementary techniques is usuallynecessary to arrive at a weight percent figure. Some routinespectroscopic techniques such as mid range infrared and NMR areinsufficiently sensitive or specific for determinations of manyspecific impurities of biodiesel at the 0.1 % range in a methylester matrix. Others, such as various forms of direct massspectrometry are usually too complex and expensive for routineapplication in a QC environment. On the other hand, the cost ofchromatographic instrumentation is well within the grasp of mostQC labs and can provide direct weight percent data.

Gas Chromatography

Most of the current focus of the biodiesel community is on gaschromatographic methods, possibly due to the historicaldevelopment of gas chromatographic technologies, such as thecapillary column, the availability and flexibility ofinstrumentation, and the lure of the relatively low cost of flameionization detectors (FID). Usually the object of gaschromatography is to separate the various molecular entities of asample, and this is often accomplished readily with capillary GC.

Derivatization, a separate sample treatment which makessubstances essentially non volatile more volatile, makes possiblethe determination of many of the impurities by gaschromatography. Glycerol, sterols, free fatty acids, residualalcohols, mono and diglycerides are all readily derivatized withthe silylating agent BSTFA (5), and this forms the basis ofcurrent GC methods for impurity determinations.

Freedman et a1.(6) published the classical basis for determiningBSTFA derivatized mono and diglycerides and residual


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t r i g l y c e r i d e s a s a m e a n s f o r r a p i d m o n i t o r i n g o f b i o d i e s e lt r a n s e s t e r i f i c a t i o n p r o c e s s m i x t u r e s b y G C . Severa lm o d i f i c a t i o n s o f t h i s m e t h o d f o r b i o d i e s e l a n a l y s i s h a v ea p p e a r e d , d i f f e r i n g i n c o l u m n s t a t i o n a r y p h a s e , l e n g t h a n dd iameter , i n j e c t i o n p a r a m e t e r s , i n t e r n a l a n d e x t e r n a ls t a n d a r d i z a t i o n p r o c e d u r e s ( 7 - 1 1 ) .

An AOCS s tandard ,Cd lib-91 (II), d e s c r i b e s t h e d e t e r m i n a t i o n o fmono and d ig lycer ides , based on BSTFA der ivat i za t i on . Bandiol iet a l . (8) i n v e s t i g a t e d t h e u s e o f c o o l o n c o l u m n i n j e c t i o nt e c h n i q u e s , and th i s i s incorpora ted in to the method presentedb y P l a n k (10) f o r b i o d i e s e l . P lank makes a c r i t i ca l assessmento f methods used f o r de termin ing the acy lg lycero l (mono , d i , andt r i g l y c e r i d e s ) c o n t e n t o f b i o d i e s e l f u e l s a t t h e 0 . 1 % l e v e l b ygas chromatography. BSTFA der ivat i za t i on methods are a l sor e p o r t e d f o r d e t e r m i n a t i o n s o f g l y c e r o l ( 1 2 ) a n d r e s i d u a lmethanol (13) in biodiesel FAME.

High Performance Liquid Chromatography (HPLC)

The HPLC approach to ana lys i s o f l i p ids has b l ossomed in the las tt en years . Advances in HPLC detect ion have resulted in renewedi n t e r e s t i n t h i s t e c h n i q u e f o r d e t e r m i n a t i o n o f l i p i d i m p u r i t i e ssuch as the mono, d i a n d t r i g l y c e r i d e s ( 1 4 ) . S p e c i f i ca p p l i c a t i o n s t o b i o d i e s e l , h o w e v e r , a r e f e w .

R a t h e r t h a n s e p a r a t e t h e l i p i d i m p u r i t i e s i n t o i n d i v i d u a lcompounds and determine each entity , the emphasis of much work isd i r e c t e d a t s e p a r a t i o n o f l i p i d s i n t o c l a s s e s o f c o m p o u n d s , s u c ha s p r o p o s e d b y C h r i s t i e ( 1 5 ) u s i n g e v a p o r a t i v e l i g h t s c a t t e r i n gd e t e c t i o n . This HPLC approach di f fers from that of GC whereeach compound i s separated and de termined ind iv idua l ly . Thees ters as a g roup o f subs tances are separated f r om the var i ousg lycer ide compounds . T h e l u r e i s t h e p o t e n t i a l s i m p l i f i c a t i o n o fthe ana ly t i ca l ca l ibra t i on process t o but a f ew compounds , w i tht h e p o s s i b i l i t y o f u n i v e r s a l c a l i b r a t i o n .

Examples o f app l i ca t i ons o f HPLC to b i od iese l a re pub l i shed .B r u n s ( 1 6 ) s t u d i e d t h e a p p l i c a t i o n o f e a r l y e v a p o r a t i v e l i g h ts c a t t e r d e t e c t i o n (ELSDJ t e c h n o l o g y t o t h e d e t e r m i n a t i o n o f m o n o ,d i a n d t r i g l y c e r i d e s i n m e t h y l e s t e r s . The ELSD offers highs e n s i t i v i t y f o r l o w v a p o r p r e s s u r e s u b s t a n c e s , s u c h a s t h o s ef o u n d i n b i o d i e s e l . Trathn igg and Mi t t l ebach used dens i tyd e t e c t i o n ( 1 7 ) t o m o n i t o r t r a n s e s t e r i f i c a t i o n r e a c t i o n s f o rb i o d i e s e l p r o d u c t i o n .


PAGE 8

Thin Layer Chromatographv (TLC/FID)

Thin layer chromatography (TLC) with flame ionization detectionis used for classical class type separations in lipid anal-ysis (18), and can form the basis of quantitative estimates.Manual sample application to the sample plates is exacting forthe best quantitative work, and a robot may be needed to do thison a routine basis. Limits of detectability are reported in therange of tenths of a percent, which is considerably higher thanthat required for routine monitoring of glyceride impurities atthe 0.1% level in the European Community nations.

TOTAL METHYL ESTER DETERMINATIONS

Introduction

The analysis of methyl esters of fatty acids is the subject ofextensive research over several decades. The focus of thatresearch is on detailed investigations of the composition oftriglycerides, and other lipid (fat) components in livingsystems, food and industrial oils. The objective of theanalytical research is mainly to define the type of fatty acidgroups attached to the glycerol backbone of fats and biogenicallyderived oils.

Availability of Standard Procedures

Many standard analytical procedures are published for thecharacterization of fats and oils by the AOCS (American OilChemists Society) AOAC (Association of Official AnalyticalChemists) and ASTM, and these are applied to numerous differentanalytical challenges with varying degrees of success. Gaschromatography forms the basis of much of the current analyticalmethodology for biodiesel, and methods used for determining the


PAGE 9

total methyl ester content are based on these standards,particularly those published by the AOCS.

Weight Percent and Functional Group Methodologies

A whole spectrum of organic analytical techniques is applied tofatty acid methyl ester characterization. While many differentmethodologies are available, these can be roughly categorizedinto two groups. In the first, the total methyl esters aredetermined, and weight percent is derived directly from weightstandards, as is done in various chromatographic techniques. I nthe second, the weight percent is indirectly derived from anestimation of the population of functional groups (functionalgroups are the specific chemical building blocks of a moleculewhich determine molecular properties) on a substance's molecularframework.

The application of functional group methodologies is complicatedby the fact that biodiesel represents numerous substancessomewhat similar rather than a distinct single substance.Without additional detailed analytical information aboutimpurities and chain length distribution, the accuracy of theweight percent estimates in the functional group tests isgenerally limited. While acceptable in many other instances thehigh degree of precision and accuracy required for biodieseldeterminations limits the utility of functional group methods.Chromatographic separation which defines the specific chemicalcomposition can greatly enhance the value of spectroscopictechniques.

Mid range infrared (IR) and nuclear magnetic resonance (NMR)areexamples of widely used spectrometric techniques which focus inon the functional groups of a substance. Various chemical testssuch as saponification number or acid number are aimed atfunctional group populations, also.

Chromatography

Chromatographic methods, such as gas chromatography (GC), highperformance liquid chromatography (HPLC) and thin layerchromatography (TLC) separate the many individual compounds offamilies of substances such as those found in biodiesel. Theseseparation tools are applied widely to the characterization offatty acid methyl esters, oils and fats, and are the most usefuin obtaining quantitative determinations of weight percentdirectly.


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O n l y r e l a t i v e l y r e c e n t l y i s t h e r e a c o n c e r t e d e f f o r t t o r e f i n ea n a l y t i c a l m e t h o d s f o r t h e p u r p o s e o f d e f i n i n g t h e p u r i t y o f t h em e t h y l e s t e r s o f f a t t y a c i d s u s e d i n d i e s e l f u e l s u b s t i t u t e s( b i o d i e s e l ) . The s tar t ing po in t o f many o f these methods i s thegas chromatograph i c separat i on work used t o de f ine the fa t tya c i d s a t t a c h e d t o t r i g l y c e r i d e s , o r o f f r e e f a t t y a c i d s , o r t h emakeup o f var i ous mono and d ig lycer ide subs tances , by convers i ono f t h e f a t t y a c i d r e s i d u e s t o f a t t y a c i d m e t h y l e s t e r s .

M e t h o d s w h i c h s p e c i f i c a l l y a d d r e s s t h e a n a l y t i c a l r i g o r s r e q u i r e dt o m e e t t h e t i g h t p r o p o s e d s p e c i f i c a t i o n s f o r t h e t o t a l p u r i t y o fb i od iese l fue l s by gas chromatography , the most w ide ly usedt e c h n o l o g y t o d a t e , a r e u n a v a i l a b l e . N e w e r t e c h n o l o g i e s o f f e rt h e p o s s i b i l i t y f o r r e d u c e d t e d i u m t o a c h i e v e a p p r o p r i a t es t a n d a r d i z a t i o n a n d c a l i b r a t i o n . G iven the complex i ty o f thesea n a l y s e s , t h a t p o s s i b i l i t y , i f r e a l i z e d s a t i s f a c t o r i l y , w o u l do f f e r a c o n s i d e r a b l e a d v a n t a g e .

Gas Chromatographic (GC) Methods

The app l i ca t i on o f gas chromatography to the ana lys i s o f methy le s t e r s i s e x t e n s i v e . A l r e a d y m e n t i o n e d i s t h e e a r l y a p p l i c a t i o no f t h i s t e c h n i q u e t o t h e c h a r a c t e r i z a t i o n o f o i l s a n d f a t s .

S a m p l e s o f o i l o r f a t a r e t y p i c a l l y c o m p l e t e l y s a p o n i f i e d( conver ted t o a f a t ty ac id soap ) and then conver ted t o the methy les ter by a der ivat i za t i on reac t i on wi th a subs tance such as boront r i f l u o r i d e i n m e t h a n o l . The methy l e s ters are then ana lyzedu s i n g a c a p i l l a r y c o l u m n a n d f l a m e i o n i z a t i o n d e t e c t i o n . T h i s i sa s t a n d a r d a p p r o a c h t o d e r i v i n g t h e f a t t y a c i d d i s t r i b u t i o n a n dis the basis for methods published by ASTM, AOAC and AOCS.T y p i c a l l y , th i s approach i s used to ampl i fy the meaning o f s implet e s t s w h i c h d e t e r m i n e t h e t o t a l f a t c o n t e n t b y e x t r a c t i o n s .These GC methods f o rm the bas i s o f current e f f o r t s t o de terminet h e t o t a l m e t h y l e s t e r c o n t e n t o f b i o d i e s e l ( 1 9 - 2 2 ) .

Severa l approaches t o the gas chromatograph i c de terminat i on o ft h e t o t a l m e t h y l e s t e r s a r e r e p o r t e d . T h e s e d i f f e r p r i m a r i l y o nt h e b a s i s o f r e s o l u t i o n , a c c u r a c y a n d p r e c i s i o n , t h e t y p e s o fco lumns and de tec tors ava i lab le .

COLUMNS AND INJECTOR TYPES

Both non polar and polar columns are used depending on thep a r t i c u l a r a d v a n t a g e s r e q u i r e d f o r a s p e c i f i c d e t e r m i n a t i o n . A


PAGE 11

challenge with non polar types such as the 5% phenyl/dimethylsilicone phases is the separation of the main types of Cl8 fattyacid methyl esters in many vegetable oils, i.e. the stearate,oleate, linoleate, and linolenate. Such non-polar columns, whichare widely used for determinations of ester impurities by BSTFAderivatization, make quantification difficult, and areinappropriate where the highest precision and accuracy arerequired.

Short columns for low resolution requirements are evaluated. Alow resolution GC/FID method was developed by Freedman et al. (6)for the purpose of monitoring transesterifications of soybeanoils for the conversion to biodiesel. The column has a non polarstationary phase with split injection.

The AOCS standard methods Ce 1-62 (20), and ASTM method D 1983-90(19) are methods for determining fatty acid composition by GLC ofmethyl esters with packed column technology. Ackman (23) hascommented on the advantages of capillary columns compared topacked columns. Ce le-91 (22) and Ce lc-89 (21) determine thefatty acid composition by capillary GC techniques. Bothcapillary methods employ split injection. Method Ce le-91utilizes a polar cross bonded polyethylene glycol stationaryphase with split or heated on-column injection. The Ce lc-89standard method for determining the cis/trans unsaturation ratiosand unsaturation positional isomers of fatty acids (as theirmethyl esters) utilizes a highly polar cyanopropyl column, with asplit injection technique.

Non polar, so-called "boiling point" columns are also used bysome investigators for methyl ester separations, a possibleadvantage when chain branching of the hydrocarbon backbonerequires investigation. These columns have difficulty inseparating the important unsaturated methyl esters such as oleic,linoleic. and linolenic.

QUANTITATION

Slob-er and Lanza (24) have published a detailed study on factorswhich affect the accuracy and precision associated with thequantitative analysis of food fatty acids by capillary gas

P\. W. HEIDEN ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

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chromatography of methyl esters using the split injectiontechnique. Their study includes the results of calibration workwith correction factors. The data indicate that the correctionfactors vary in a complex way on concentration of analyte and thechemical structure of the analyte. The correction factors mustbe calculated for each analysis system, as a significant portionof the correction is the result of analyte decomposition andirreversible adsorption to the active sites in the sample flowpath.

HPLC

Several methods are reported for determining the total methylester content of biodiesel. A precision and accuracy of plus orminus 1% are reported f17,25). Trathnigg and Mittlebach (17)investigated separating the biodiesel into its esters andimpurities using a combination of a cyanopropyl bonded phasecolumn and a styrene divinyibenzene column with a densitydetector and chloroform mobile phase.

ANALYTICAL PRACTICES IN THE US

In a study hampered by confidentiality concerns, the currentpractices of industrial firms manufacturing biodiesel in the U.S.were surveyed. The results of this study indicate that themethods currently in use for impurities are based on BSTFAderivatization, and AOCS standards for iodine value, acid number,and glycerol, and AOCS standards Ce le-91 for methyl esterdistribution, and total methyl ester determinations.

R. W. HEIDEK ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

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IV. STATISTICAL REQUIREMENTS

ACCURACY AND PRECISION

Summary

The accuracy and prec i s i on requ i rements f o r the var i ous methodsare examined here. Th is i s no t a comprehens ive rev iew , butrather an attempt to determine approximate boundary parameters ,so that methods are more eas i l y eva luated . Becausechromatograph i c methods are the sub jec t o f the eva luat i ons in thec o m i n g s e c t i o n s t h e y a r e t h e f o c u s o f t h i s d i s c u s s i o n .

Whi l e the accuracy o f a de terminat i on i s r e la ted t o var i ouss a m p l e r e l a t e d c o n s i d e r a t i o n s a n d i s d i f f i c u l t t o p r e d i c t , au s e f u l q u e s t i o n t o p o s e f o r t h e t o t a l m e t h y l e s t e r a n a l y s i s i s“what 1s the maximum allowable precision(standard dev ia t i on )which makes poss ib l e a v iab le d i s t inc t i on be tween 100% pure t o ta lmethy l e s ter and the min imum a l l owab le pur i ty ( e . g . a proposeds p e c i f i c a t i o n o f 9 8 % p u r i t y ) ? “ . Th is in format ion a l l ows f o r anin formed dec i s i on o f the min imum requ i rements f o r prec i s i on andaccuracy f o r the methodo logy , and a l so the sample ana lys i sr e p l i c a t i o n r e q u i r e m e n t s .

The es t imated s tandard dev ia t i on assoc ia ted wi th a par t i cu larm e t h o d o l o g y i s a s t a t i s t i c a l s t a t e m e n t o f t h e p e r f o r m a n c e o f amethod and i t s prec i s i on . S i g n i f i c a n c e t e s t i n g u t i l i z i n g ts t a t i s t i c s a n d c o n f i d e n c e l i m i t s w a s u s e d h e r e t o e s t i m a t e t h emaximum s tandard dev ia t i ons a l l owed to d i s t ingu ish the d i f f e rencebetween 100% pur i ty and o ther l eve l s o f pur i ty a t d i f f e rentp r o b a b i l i t i e s (g/IO, 95/100, and 99/100) a n d r e p l i c a t i o n s . Theab i l i ty t o d i s t ingu ish be tween pure 100% methy l e s ter and twod i f f e r e n t l e v e l s o f p u r i t y , 9 8 a n d 95%, w a s c a l c u l a t e d .

Method Precis ion Requirements

Tables 1 and 2 l ist the est imated maximum al lowable method errorsin t e rms o f the s tandard dev ia t i on . The number of determinationsfo r each o f bo th pure , 100 % s tandard , and sample required toa c h i e v e a p a r t i c u l a r s t a n d a r d d e v i a t i o n i s n , w h i l e t h e p i s aprobabalistic c o n f i d e n c e l e v e l . I f t h e a c t u a l s t a n d a r d d e v i a t i o nf o r a s i n g l e d e t e r m i n a t i o n i s g r e a t e r t h a n o r e q u a l t o t h a tappear ing in the tab le then the d i f f e rence be tween the t rue meansof sample and standard might be zero at a particular confidencei n t e r v a l .

2. W. HEIDEN ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

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TABLE 1

MAXIMUM ALLOWABLE STANDARD DEVIATION FOR DISTINGUISHINGDIFFERENCES BETWEEN 98% AND 100% PURITY

NUMBER OF REPLICATES EACH FOR SAMPLE AND STANDARD

N=

CONFIDENCE LEVELPROBABILITY

P

9/10 (90%)

19/20 (95%)

99/100 (99%)

2 3 4

STANDARD DEVIATIONPLUS OR MINUS PERCENT

0.68 1.15 1.46 1.70

0.46 0.88 1.16 1.37

0.20 0.53 0.76 0.94

R. W. HEIDE?: ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

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TABLE 2

MAXIMUM ALLOWABLE STANDARD DEVIATION FOR DISTINGUISHINGDIFFERENCES BETWEEN 95% AND 100% PURITY

NUMBER OF REPLICATES EACH FOR SAMPLE AND STANDARD

N =

CONFIDENCE LEVELPROBABILITY

P

9/10 (90%)

19/20 (95%)

99/100 (99%)

2

1.71

1.16

0.50

3 4

MAXIMUM ALLOWABLESTANDARD DEVIATION

PLUS OR MINUS PERCENT

2.87 3.64

2.21 2.84

1.33 1.91

5

4.25

3.43

2.36


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__ _.. ----_. . . . ---

A quick review of the tables reveals that the requirements fordistinguishing between 95 and 100 percent are considerably lessdemanding than those for distinguishing between 98 and 100%.For example, a precision of less than plus or minus 1.16% isneeded for a 19/20 confidence (95%) level to distinguish between95 and 100% by analysis of duplicated samples and standards. Incontrast, a precision of less than plus or minus 0.46% isnecessary to distinguish 98 and 100 percent with duplicateanalyses of samples and standards.

Stated in another way a method with a precision of plus or minus1.15 % would require quadruplicate determinations of bothstandards and samples to achieve 95% confidence of a differencebetween 98 and lOO%, and just duplicate samples for the 95%confidence of a true difference between 95% and 100%. This ishypothetical, in that it assumes that in the analysis of actualsamples there are no biases (these are some), and that theprecision of the determination of the samples and standards isidentical (which should be true,but there are no guarantees).That is, the real situation is worse taking into account biases,and sample related measurement variability.

ACCURACY

Factors affecting the accuracy of methods for the determinationof total methyl esters include the following:

1) The precision of the method.

2) The ability to quantitate overlapping peaks.

3) Interferences from unknown substances

4) Accurate assignment of peaks including minor peaks, andpeaks less than 1%.

5) The inertness of the sample flowpath duringchromatographic separation.

6) The accuracy of certifications of standards.

Because of the small differences between 100% and 95% puresubstances the need to control these factors is acute.

I) Precision- in absence of analytical biases which alter theanalytical result, precision determines accuracy. Highprecision, as discussed above, is key for determining the successof a method, and becomes critical on a practical level, when thereplication needed to have a certain degree of confidence isconsidered. Put in another way, the rate of sample throughnnt,eventually limits the degree of replication possible for J. givenmethod.

R. W. HEIDEN ASSOCIATES/NATIONAL BIODIFSEL BOARD 27 FEB 96

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2) The ability to quantitate overlapping peaks is especiallyimportant if the response factors of the two overlapping esterpeaks differ. The variation of response factors as a complicatedfunction of chemical structure and concentration is discussed ina previous section. To distinguish between 98% pure and 100%pure samples dictates special care for the main and minorcomponents. Resolution, or the ability to separate the maincomponents chromatographically is critical.

Eliminated from further consideration here are methods which failto resolve the main Cl8 fatty acid methyl esters, such as gaschromatographic methods based on polydimethylsilicone stationaryphases used in BSTFA derivatization methods.

Accurate quantification of overlapping peaks is becoming more ofa concern to fatty methyl ester analysts and to achieve the bestpossible quantification of often complex chromatograms computerswith data processing techniques are required. Possibly, moreadvanced techniques of detection, such as N/MS are necessary toanalyze biodiesel made from mixtures of alcohols.

3) Interferences defined here are substances which are not fattyacid esters. Peak overlap with methyl esters is a potentialconcern, as is spurious high or low level peaks, which in thelatter case might lead to several peaks at levels of less than 1%-easy to ignore, or to mistakenly consider as methyl esters.

In GC, any peak which is unidentified is a potential interferencebecause the whole chromatogram is scanned. Some possibleinterferences include fatty acids and some hydrocarbons.

4) The identity of peaks is normally accomplished througha comparison of retention times of the components of standards tothose of the unknown sample. Unidentified esters go uncounted,and the presence of minor amounts of alcohols in addition tomethyl could cause low estimates of ester content.

As an example. two or three small unidentified peaks just lessthan 1% could determine whether a fuel passes or failsspecifications for 98% purity. Peaks down to 0.1% need to beidentified and quantified to meet such a specification.

5) The inertness of the sample flowpath is particularly importantin chromatography because microscopic amounts of

R. W. HEIDE?; ASSOCIATES/KhTIONAL BIODIESEL BOARD 27 FEB 96

P.L\GE 18

substances are introduced to the analysis system, and to ensurethat every component passes through to the detector is extremelyimportant.

Problems associated with adsorption, destruction of substancesare particularly acute with gas chromatography. The inlet of thegas chromatograph is susceptible to such problems because of thehigh temperatures of injection, and residues left behind fromprevious samples. Unsaturated methyl esters are particularlysusceptible because they are readily oxidized at highertemperatures. The analysis system must be inert.

6) The availability of certified reference standards ofsufficient quality is a key requirement for accurate results.Vendors often provide certificates of analysis. Once suchstandards are in hand adequate procedures to maintain the purityof the standards is necessary.

Many warnings are issued regarding the susceptibility of themethyl esters to light, heat and oxygen. The degree to whichthis impacts ester analysis is unclear. Some compounds, such asthe highly unsaturated compounds, are relatively unstable andreactive towards oxygen. However, relating this information toroutine practice in the laboratory to avoid losses of even 1 or2%, which is likely to be acceptable in many other applications,is difficult.

As yet, NIST has no certified reference materials for methylester determinations. Nixtures of esters are available from NIHand AOCS through private standards companies. Private companiesare the biggest source of standard materials. The companiessometimes provide a certificate of analysis to accompany the soldmaterial. There are, however, no independent checks on thequality of these materials, and provisions are lacking instandard methods to check the purity in the laboratory, wherechanges in the purity by esposure to air, heat or light arelikely.

One such company was contacted to determine the level ofvariation in purity of pure substances and mixtures used forstandards. The pure materials are sold as >99 % purity, and arechecked by gas chromatography and thin layer chromatography priorto customer delivery. These tests may be insufficient to definepurity adequately.

R. W. HEIDES ASSOCIATES/XATIONAL BIODIESEL BOARD 27 FEB 96

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“_.~. ._- _ ._-.- _-.. . -

METHODS EVALUATIONS

INTRODUCTION

To routinely distinguish with confidence pure 100% methyl estersfrom 95-98X purity requires the highest attainable accuracy andprecision. Based on this need, two modified methodologiescapable of the highest precision and accuracy were chosen todetermine which is best suited for routine determinations ofbiodiesel purity. These methodologies were based on gaschromatography (GC) and high performance liquid chromatography(HPLC), and were optimized for biodiesel needs. Two variationsof injection conditions of the GC approach were tested. Theresults show that GC with direct, cool, on column injectionprovides the best precision.

CRITERIA FOR METHOD SELECTION

The following criteria were considered in selection of methods:

*Potential for the highest attainable precision and accuracy*Technology status-state of technology development*Analysis time/sample throughput*Flexibility of required instrumentation/potential adaptability

to other biodiesel analyses*Operating costs

Maintenance of equipmentChemicals/gasesDegree of trainingMan time

"Method performance-precision/accuracy*Capital costs of instrumentation*Instrumentation availability*Toxicity and health hazards of handled chemicals"-'Ease of method execution/degree of tedium*Calibration and standardization demands

RATIONALE

Four main criteria were used to choose methods for experimentalevaluation:

1) Potential for the highest attainable precision and accuracy asdescribed in the statistical requirements previously discussed.


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2) The comparable costs for a new GC/FID and isocratic HPLC/ELSDare similar and within the range of 35-$40,000 sourced from highprofile vendors. These instruments are equipped in a way whichmakes them suitable for the high precision work required here.These costs only reflect those associated with the instruments,and do not reflect any facilities modifications necessary toaccommodate the instruments. More sophisticated and expensivedetection instruments such as FTIR or MS which presumably offermore specificity were not considered because of substantialincreases in costs and manpower skills.

3) The flexibility of instrumentation, i.e. potentialapplications to other biodiesel analyses, and other laboratoryneeds.

4) Established methodologies- gas chromatography/flame ionization(GC/FID) is already used widely for biodiesel analysis, isalready specified as the technique of choice for impurityanalysis, and is the heart of proposed ASTM specifications forbiodiesel analysis.

The potential for the highest attainable precision and accuracyand the widespread use and popularity of GC/FID instrumentationin biodiesel analytical laboratories are the most heavilyweighted criteria above. The GC/FID approach is, therefore, thefocus of this evaluation. HPLC with recent ELSD detectiontechnology offers the potential for highly precise determinationsof methyl esters, extension to biodiesel impurities, and is usedas a comparison to the GC approach.

GC METHODS

The highest precision and accuracy attainable by GC is affordedby direct on-column injection techniques (27). In particular,cool on-column technologies avoid common GC pitfalls associatedwith hot injection, such as needle discrimination, degradation ofeasily oxidized or thermally degraded components, and samplecomponent discrimination, all of which impact accuracy andprecision negatively. The sample is placed directly into the


PAGE 21

column, rather than onto insert liners with inertness which isquestionable. Questions regarding the accuracy of splitting, acommon GC injection technique for methyl esters, are avoided withthe on-column approach. The potential for sample discriminationeffects is greatly reduced by the on-column approach, and thatgives this technique the best chance for high laboratoryrepeatability and interlaboratory reproducibility.

Cool, direct on-column and heated-on column injection techniqueswere evaluated by repetitive analyses of a methyl ester mixturesimilar to that derived from soy transesterification to determineprecision. Although cool, on-column injection is intuitivelypreferable, some advantage may be realized for certain olderinstruments with packed column inlets which are readily convertedto direct, heated, on-column injection. Because the simplest,least possible interactive sample flow path in the injection zonewas sought, split injection was not included in the evaluation.Yodifications of .4OCS method Ce-le-91, a capillary GC method fordetermining the fatty acids in edible oils and fats (by analysisof their methyl esters), were used for the evaluation. since nosuitable alternative procedure could be found in the publicdomain for determining methyl esters which would meet theprecision and accuracy requirements of biodiesel analysis.

The modifications of this method are as follows:

1) The injector is direct on-column (this is an option of AOCSmethod Ce-le-91, but only the split injection method is providedtherein).

2) The column is a 30 M 0.53mm megabore with 1.0 urn film (we aresacrificing some resolution to : a) gain capacity for higherworking concentrations, which allows for better quantification ofminor and trace peaks, and higher peak areas for major peaks); b)to simplify the sample flow path, and allow for direct needleinsertion into the column. This avoids usage of retention gapswhich can complicate the injection process.

3) Helium is the carrier gas (caution: Helium is an asphyxiant).Helium is an option of Ce-le-91.

&) The column temperature program is modified to accommodate 2)and 3) above.

R. W. HEIDES ASSOCIATES/YATIONAL BIODIESEL BOARD 27 FEB 96

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HPLC METHOD

A precision of plus or minus 1% is reported for analyses ofbiodiesel by HPLC ( 17,25), and, therefore, HPLC is potentiallysuited for routine biodiesel analyses of total methyl esters.The HPLC approach offers better control of injection conditions,where many of the problems of GC are encountered. Loopinjectors, for example, allow for manual, highly controlled, andrepeatable injection volumes, and thermal or oxidativedegradation of the sample during injection is far less of aconcern than in GC.

Among methods considered were those employing refractive index,density, flame ionization and evaporative light scatterdetection. Refractive index detection of the separatedcomponents is highly dependent on the response factors of theindividual methyl esters (17), and FID detection, at best, offerslimitations in response factors, as in GC/FID.

Density detectors used in a previous study (17) were not marketedcommercially for HPLC in the U.S. during the time of this study,and therefore, were removed from consideration. The toxicity ofmobile phase solvent components was also considered andhalogenated solvents, such as chloroform, were deemed lesspreferable than hydrocarbon solvents in this regard, and methodsusing such solvents were given low priority.

A modification of the methods by W. W. Christie (15) andBruns(l6) were used to evaluate the use of evaporative lightscattering detection (ELSD). In these methods, classes ofcompounds, such as mono, di and triglycerides, similar to thosefound as impurities in biodiesel, are separated by normal phasechromatography with hydrocarbon solvents. The families ofcompound classes emerge as single or but a few peaks whencompared to the many peaks which emerge from a high resolution GCcolumn.

This approach offers promise in extensions to biodieselapplications, and the potential advantage of reduced calibration,since the highly sensitive detector responds directly to changesin mass, a universal indicator. Initial studies of biodiesel

R. W. HEIDEY ASSOCIATES/SATIOYAL BIODIESEL BOARD 27 FEB 96

PAGE 23

showed that the methyl esters emerge from the column as one peak,rather than five or more, which is clearly a positive sign for asignificant advantage over high resolution, and individual peakidentification and quantitation.

The modifications are as follows:

1) The method is applied to methyl esters of fatty acids, asfirst reported by Bruns (16).

2) The solvent mobile phase is isocratic, i.e. is constant,rather than a more complicated gradient form. Conditions for theseparation of hydrocarbons, methyl esters and triglycerides weredeveloped using hydrocarbon solvents such as hexane or pentane.

3) The detector is recent technology.

COMPARISON OF INSTRUMENTATION REQUIREMENTS FOR GC AND HPLC

The cost of instrumentation requirements for an isocratic HPLCwith ELSD to do these methods are comparable to those for theGC/FID with accessories, and in the range of $ 35-40,000.Table 3 lists various requirements for each instrument.

A difference in requirements between the instruments is in thesophistication of the injection equipment and the computerizeddata analysis sophistication. To achieve adequate precision, amanual injection loop is possible for HPLC, and the autoinjectoris optional (though desirable). For GC, however, theautoinjector is necessary for optimum precision. Because of thesimplified chromatogram of the HPLC compared to the highresolution GC, only a good recording integrator is absolutelynecessary. In GC, the complicated tracings of the chromatogramdictate the highest flexibility for data analysis.

EXPERIMENTAL

Standard Solutions

Pure standards, standard mixes and samples of the methyl esterswere prepared for analysis by weighing approximately 100.0 mg ofthe pure standards in a 10 ml volumetric flask, and dilution withhexane. Approximately 1 mg/ml dilutions were prepared forroutine injections by I:10 dilution.

Evaluation of methyl undecanoate internal standards was performedon stock solutions prepared as above with an additional 40.0 mgof internal standard material.


PAGE 24

INJECTION

DETECTION

DATA HANDLING

OVEN

PUMP

MISCELLANEOUS

TABLE 3INSTRUMENTATION REQUIREMENT DESCRIPTION

GC

Autoinjector

High Precision Syringe

Cool, direct on-column

Flame ionization

Computer w/chromatog-raphy software/ data

handling

Standard, high precisionoven, T maximum at least280 degrees C

None

carrier gas purifiersto less than 2 ppm oxygenand moisture.

HPLC

Manual valve

Loop

Evaporative lightscatter

Recording integrator

None

High Precision,pulseless HPLCcapable of 3.0ml/Min.

detector gas filters


PAGE 25

Instrumentation and Operating Conditions

Gas Chromatograph

Analyses were performed with a Varian 3400 equipped with flameionization, a packed injector modified for direct capillaryon-column injection, and a computer data station. The column wasa moderately polar, 0.53mm megabore column with 1.0 urn film, asdescribed in the attached draft procedure. The injectortemperature was either 250 degrees C for the experiments withheated, on-columm, or 50 degrees for experiments with cool,on-column injection. The design of the cool on column variationdescribed here is inadequate in a high throughput lab, because ofthe excessive time required to achieve the set injectortemperatures between runs. A more conventional cool, on-columndesign is necessary for routine work. Detector temperature was250 degrees C. Helium was used as carrier gas at a flow rate of5.0 ml per minute. Detector gases were set according tomanufacturer recommendations.

Samples (2 ul) were injected manually into the column using asyringe with a 4 inch needle to place the sample at an adequatedistance away from the cool inlet to prevent recondensation ofsample in the inlet. Heated injector samples were introducedwith a collar fitted with the 4 inch syringe to allow forinjection into the heated area of the injection port with thesame syringe. A smooth, rapid depression of the plunger was usedfor all injections. The column was started at 40 degrees andthen heated at 15 degrees/minute to 120 degrees, 4 degrees perminute to 250 degrees, then held for 2 minutes. Total run timewas about 40 minutes.

Liquid Chromatograph

Analysis was performed using a Waters 510 pump with a Rheodyne7125 loop injector and a 5 ul injection loop. The detector was aSedex 55 evaporative light scattering unit, and the output wassent to a computer data station. The detector was modified toallow for room temperature cooling and routine operatingtemperatures just above 22 degrees. The nitrogen flow rate wasadjusted with a pneumatic pressure gauge set at 2.1. The columnwas a 5um cyanopropyl bonded phase 25 cm x 4 mm ID. Flow rate ofhexane or pentane was 0.8 ml./minute. Total run time was 6-8minutes.

R. W. HEIDEW ASSOCIATES/NATIOKAL BIODIESEL BOARD 27 FEB 96

PAGE 26

GC RESULTS

Example GC Chromatograms

Figure 1 shows the separation of methyl esters typical of thosefound in a transesterified soy sample (Mixture G). The mainpeaks are those of the three main Cl8 methyl esters of stearic.oleic, and linoleic acids. Also present are the Cl6 methylesters of palmitic and palmitoleic acids and a small amount ofCl4 methyl ester of myristic acid. Figure 2 shows the separationof a standard material containing higher C22 and C24 methylesters. as well.

INJECTION TECHSIQUE

Comuarison of Hot and Cold On-Column GC Injection Techniques

The precision of determining the total methyl ester content ofthe mixture depicted in Figure 1 was calculated from the standarddeviations associated with measurements of individual esters.This figure of merit is the square root of the sum of thev-ariances for each ester. Table 4 lists the different compoundsin the samples and their respective standard deviation for bothhot and cold on-column injection. Ester 16-O was used as aninternal standard to correct for variations in injection volume,and consequently was not listed.

The results show that the precision of the cool injection is morefavorable to repeatable determinations than the hot variation.This result was not a surprise because of the many known vagariesassociated with hot injection, as already discussed. Theseresults clearly suggest that cool on column techniques afford anadvantage over hot injection.

Hot injection is, consequently. not recommended fordeterminations of total methyl esters in biodiesel.

R. W. HEIDEN ASSOCIATES/NATIOSAL BIODIESEL BOARD 27 FEB 96

P.\GE 27

ia C I$-On-

AliF, c ‘ct.’ -ME ClS-0 1

-1% C-&o'_%2ech-l _ME C17-0 -

FIGURE 1GC CHROMATOGRAM TRACE OF MIXTURE G METHYL ESTERS

‘0 '10 '20 '30 '40! mVolts- _ _

is2.235

F=-- i8-32i i9.i93; 20.569

i!-c - 23.400 22.325

L- 27*10i r_ --

/

7 LO.l/L - 28.515i_ 29,847

b36.314

31.524\ 32.493


PAGE 28

-

ME C16-O*-u&c (L-i -

ME C22-O*-hEC 22-f$yL .c-q 7 -2 --LL

FIGlJRE 2GC CHROMATOGRAM TRACE OF STANDARD MIXTURE

I 10 '5 '10 '15 '20 '25) mVolts

18.496

- 22.97423.571

m 27.26227.714r-

2 8 . 6 9 0I

i=-- - 30.02c

‘F==- 31.289 -gx

71 7lQ_ _ _ I_"77 CCQ-LIIIU

1+=- 35.072r-_ 3&?

7c C?,z

\

‘$L38.636-r

39.119

R. ii. HTI3ES .~SSOCIATES/4ATIOSAL BIODIESEL BS.%RD 27 FEB 96

PAGE 29

Internal Standards

A review of possible internal standards was undertaken. The mostwidely used internal standards for methyl ester analysis are oddcarbon methyl esters. Apparently, such esters are sometimespresent at measurable levels in manufactured methyl esters forbiodiesel, and, therefore, only one ester, methyl undecanoate, ispotentially a suitable internal standard (26). The precision ofthe cold on column technique was compared using the Cl1 methylester and a Cl6 ester inherent to the sample. The results ofthat test indicate that the precision of the method is adverselyaffected 'by the Cl1 ester, and, therefore, the use of an internalstandard is, at this time, not recommended.

HPLC RESULTS

Example of HPLC Chromatograms

The HPLC/ELSD trace of a mixture of methyl esters is shown inFigure 3. A key feature of these chromatograms is the singlepeak containing the fatty acid methyl esters. This peak isclearly resolved from any hydrocarbon or glyceride contaminantsin the sample. Hydrocarbons intentionally added in the form ofmineral oil emerge from the column well ahead of the ester peak,and the neutral glyceride series begins to emerge well after themethyl ester peak. Continuous runs of 7 to 8 minutes wereadequate to fuily quantitate the methyl ester peak.

Evaluation of Precision

The precision of the method was estimated by manual replicateinjections. The sample injected was the same as that used forevaluation of GC precision consisting of methyl esters similar tothose of a preparation from soy oil. The estimated relativestandard deviation with seven replicates (N=7) is plus or minus0.73%. This shows that the method is capable of good precision,and with an analysis time of just 7 minutes, capable of the rapidreplication required for improving precision of the mean.

GC INTERFERENCES

Possible interferences in the determination of the total methylester content were evaluated. Among those potentialinterferences are free fatty acids, commonly found as impurities,

R. W. HEIDER ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

PAGE 31

FIGURE 3HPLC/ELSD CHROMATOGRAM OF METHYL ESTER MIXTURE G

‘0.00 '0.25 0.50 '0.75 ‘1.00Volts

i-_--.___-

-4.6i3

/------------ -/

D1,. w. HEIDEK ASSOCIATES/NATION~%L BIODIESEL BOARD 27 FEB 96

PAGE 32

FIGURE 4GC CHROMATOGRAM OF HYDROCARBONS SIMULATING DIESEL

‘0 '10 '20 '30 '40 '50 '60 '701 mVolts

-

-

---

-

__

-

-

---


PAGE 33

and hydrocarbons, such as those associated with diesel fuel.This evaluation is not all inclusive, and any substance whichelutes during the emergence of the fatty acid methyl esters is apotential confounding factor, if unidentified, or unresolved fromthe methyl ester peaks.

Figure 4 shows a GC chromatogram trace of a mixture of CIO-C25hydrocarbons similar to those found in diesel fuel. A regularpattern of peaks appears over a considerable range of the methylester series. These peaks are broader than those of the esters,and occur near or directly on top of several ester peaks. Thebreadth of compounds found in diesel is actually much larger thanthose represented in the chromatogram, and clusters of peaks soclose in characteristics are usually found in diesel. Clearly,diesel or similar hydrocarbon contaminants would pose aquantification interference.

Free fatty acids are frequent impurities in biodiesel, and can bechromatographed. Figure 5 shows a GC chromatogram trace of thethree free fatty acids, myristic, stearic and oleic. Free fattyacids are much less volatile than the corresponding methylesters, but nonetheless can emerge in a region of methylesters. The oleic and stearic acids emerge from the columnafter the C24 methyl esters, but the myristic (C14) acid emergesin a region near to the C20-2 ester. By mathematical inference(which is legitimate because of the predictability of fatty acidelution) the Cl6 acid cluster emerges near the C22-2 methylester, and the Cl2 acids, near the C18-2 ester. The acid peakshave much less than perfect shapes with considerable tailing onthe downside, and, therefore, can pose a mathematical challengeto automatically correct peak overlaps accurately.

POTEEJTIAL APPLICATION TO DIESEL FUEL BLENDS

GC/FID

The potential application of the GC method to diesel fuel blendsis complicated by the complexity of hydrocarbons found in diesel,many of which could elute in the range of methyl esters as isshown in Figure 4. Biodiesel blends of 20-30 % would probablyreauire reliable sample pretreatment measures to separate the_ _esters from the hydrocarbon components.

R. W. HEIDEX ASSOC IATES/NAT IONAL BIODIESEL BOARD 27 FEB 96

P.AGE 34

--

-

-

a

_

-

-

-

‘!+YPdmc 1

-_-

S_rC -OLlac. -

--

FIGURE 5GC CHROMATOGRAY OF FREE FATTY ACIDS

MYRISTIC, STEARIC, OLEIC

I ‘0 '10 '20 '30 '40 '50mVolts

_c,

1 1 8 , 4 0 9

; 22.906

/! 36.332

I+ 40.121/ 40.5123f 42.371

j 44.868

Ft. w. HEIDE?; ASSOCIATES/KATIONAL BIODIESEL BOARD 27 FEB 96

P.4GE 35

HPLC/ELSD

The methyl esters are well separated from hydrocarbons which arelikely in diesel fuels. As an example, a trace of mineral oiladded to methyl ester mixture G emerges almost a minute beforethe methyl ester peak.

POTENTIAL APPLICATION TO ETHYL ESTERS

GC/FID

When ethanol is the starting material (or one of the startingmaterials) ethyl esters are formed with properties which differsubstantially from those of the methyl esters. To determine thedegree of success the method proposed here applies to ethylesters the retention characteristics of two ethyl esters, thestearate and oleate were analyzed and compared to the results ofmethyl esters. The results show that the method would requiremodification to address ethyl esters.

Figure 6 shows a GC chromatogram of the ethyl stearate andoleate. The peaks at retention times, 35.75 and 36.62,respectively show that these esters are clearly separated fromone another. These times are very close to those of the CZZ-1and C22-2 methyl esters. Also, compared to the retention timesof the methyl esters (27.3 and 27.7 minutes) these areconsiderably longer, and, in fact, close to the end of thestandard run. In a normal run of 40 minutes the likelihood ofloss of C22 and higher ethyl ester data is high. Clearly the GCmethod will require modification to accommodate the ethyl esters.

BPLC/ELSD

The analysis of ethyl stearate/oleate mixtures leads to a singlepeak in the area of the methyl ester mixture peaks, and possiblya slight shift to lower retention times.

R. W. HEIDEX ASSOCIATES/NATIOKAL BIODIESEL BOARD 27 FEB 96

PAGE 36

----

--

___

-

-

--

-

R. W. HE

PAGE 37

FIGURE 6GC CBROYATOGRAM OF ETHYL STEARATE AKD ETHl-L OLEATE

0.0 '2.5 '5.0 '7.5 '10.0 '12.5 '15.0m.v_n_l,ts- +.3!45 I.- .I.111.0,s 7.703 ? cc?-___-

:DEN ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

VI . RESEARCH NEEDS

1 1 PROBLEM:

GC of methyl esters is designed to separate and quantiindividual compounds of methyl esters. The result ischromatogram which can contain ten or more substances.

tate thea

To obtainaccurate quantitative data each compound must be clearlyseparated from one another and also individually calibrated.

When the starting alcohol is a mixture rather than a pure form ofmethanol or ethanol a mixture of esters is formed duringtransesterification. Multiply the number of different types ofesters in the original starting material by the number ofdifferent alcohols and now you get the total number of esterpeaks in the chromatogram. If 10 esters make up the first sampleof biodiesel made from pure methanol, then 20 make up the totalwith a mixture of methanol and ethanol. The greater complexityof the alcohol starting materials compounds the laboratory effortto quantify, and magnifies the likelihood of errors incalibration. Also, non esters are more likely to beinterferences, with so many potentially quantifiable peaks.

NEED:

Research is needed to find ways to simplify the calibration in GCwork while retaining the precision and accuracy and flexibilityneeded to do this work on a practical basis. The evaluation ofmethods for determining the total methyl esters using techniqueswhich allow for a simplified approach to calibration, and greaterselectivity for biodiesel esters. The new methods should striveto equal or exceed the total precision and accuracy available inexisting methods, and, if requiring different instrumentationshow promise for determining glyceride impurities in biodiesel.

R. W. HEIDEK ASSOCIATES/XATIOKAL BIODIESEL BOARD 27 FEB 96

P4GE 38

2) PROBLE?I:

R e f e r e n c e s t a n d a r d s f o r t h e c a l i b r a t i o n o f t o t a l m e t h y l e s t e rd e t e r m i n a t i o n s i n t r o d u c e b i a s e s w h i c h a r e e a s i l y o v e r l o o k e d . Forexample, pure chemica l s a re o f t en s ta ted co be be t te r than 99%pure , but the exac t pur i ty i s no t a lways s ta ted , and thec e r t i f i c a t i o n p r o c e s s C3ii dif fer from company to company.S t a n d a r d snbstances a l so have a she l f l i f e , a s do the prepareds o l u t i o n s , and the she l f l i f e o f the mixtures i s unknown.

NEED

a) A rap id method f o r check ing the pur i ty o f r e f e rence s tandardsw o u i d g r e a t l y l e s s e n t h e u n c e r t a i n t y a s s o c i a t e d w i t h u s e o f“pure ” chemica l s tandards . D e v e l o p e s p e c i f i c , p r a c t i c a ll a b o r a t o r y p r o c e d u r e s f o r i d e n t i f y i n g t h e p u r i t y o f a n dm a i n t a i n i n g t h e i n t e g r i t y o f s t a n d a r d s u s e d i n t h e l a b o r a t o r y .

b) An eva luat i on o f the cond i t i ons f o r s to rage and use o fs tandard so lu t i ons and pure methy l e s te r s tandards i s needed ,with a recommended pract ice . E v a l u a t e t h e s t a b i l i t y o find iv idua l compounds , a n d i d e n t i f y p r o s p e c t i v e a v e n u e s f o rmainta in ing the s tandards a t t h e i r c e r t i f i e d i n i t i a l p u r i t i e s .The she l f l i f e o f var i ous s tandards needs t o be es tab l i shed andthe appropr ia te procedures f o r mainta in ing and recogn iz ings t a n d a r d s a n d s t a n d a r d s o l u t i o n s a t t h e i r c e r t i f i e d v a l u e s n e e dt o b e i d e n t i f i e d .

c) T h e p r a c t i c e s r e q u i r e d t o e s t a b l i s h t h e e x a c t p u r i t y o freference standards need to be evaluated and compared to what isprac t i ced current ly by organ izat i ons manufac tur ing and vendor ings tandards .

3 ) PROBLEY:

Interna l s tandards are used in chromatographi c ana lys i s t o ad jus tf o r v a r i a b l e i n j e c t i o n v o l u m e s , wandering compound retentiont imes , a n d q u a n t i t a t i o n . In GC espec ia l ly , t h e i n t e r n a ls tandard can p lay a ma jor ro l e , a n d a s u i t a b l e i n t e r n a l s t a n d a r do f f e r s t h e p o t e n t i a l f o r e n h a n c e d c e r t a i n t y a s s o c i a t e d w i t h p e a ki d e n t i f i c a t i o n , and manual in jec t i on , thereby reduc ing ins t rument

R . W. BEIDEX ASSOCI.4TES/XBB 27 FEB 96

P.4GE 39

costs considerably. The application of odd number carbon fattyacid methyl esters is limited by the prevalence of naturallyoccurring low level odd number acid methyl esters, which candistort the quantification process. Internal standards can alsopose a problem if their chromatographic properties differssubstantially from those of the compounds of interest. There arecurrently no suitable internal standards.

YEED:

The concept of internal standardization as applied to the highprecision and accuracv requirements of biodiesel needs study toevaluate its applicability to total methyl ester determinations.Evaluate and recommend candidate internal standards, and testexperimentally in gas chromatographic determinations of totalmethyl esters.

4) PROBLEM:

Extension of GC methods to petroleum/biodiesel blends iscomplicated by interferences in ester determinations by petroleumhydrocarbons.

NEED:

Investigate ways to estend GC methodology to biodiesel blends,test and recommend the "best" possible adaptation.

51 PROBLEM:

The development of laboratory analytical technology for analyzingcomponents in biodiesel has undergone considerable advances inthe last ten years, particularly in HPLC detection technology.The implementation of gas chromatography capillary techniques tothis application is well over ten years old, and still posesnumerous challenges in quantitation.

NEED:

Investigate technologies associated with BPLC detection and itspotential application to biodiesel glyceride determinations andtotal methyl ester determinations. Test and recommend the "best"possible technology.

R. 6. HEIDEK ASSOCIATES/NBS 27 FEB 96

PAGE 40

DRAFT

WEIGHT PERCEKT TOTAL METHYL ESTER CONTENT BY GC- 'l/29/96

IKTRODUCTION/SCOPE

This method is applicable to the determination of total methylester content of pure biodiesel fuels comprised of the methylesters of fatty acids (FAME) having 8-24 carbon atoms. Thisincludes methyl esters derived from animal fats, vegetable andmarine oils. As such, the method permits quantitative separationof methyl esters containing saturated and unsaturated residues offatty acids. This method fails to measure the ester content ofpolymerized substances or esterified osidation products of fattyacids.

This method does not purport to address all of the safetyproblems, if any, associated with its use. It is theresponsibility of the user of this method to establishappropriate safety and health practices and determine theapplicability of regulatory limitations prior to use.

ADDITIOKAL APPLICATIOYS

This method may also be used to determine the unsaturationcontent of FAME by an estimation of the iodine number throughmethod AOCS Cd lc-85. The density (specific gravity), vaporpressure as a function of temperature, boiling pointdistribution, are among the possible properties which may becalculated for pure materials having the composition determinedby this method.

The biodiesel fuels are first pretreated by passage through amicrocolumn dissolved in hexane, then analyzed by gaschromatography with flame ionization detection, using cool,on-column injection techniques. Autoinjection of externalstandards is used for calibration. The solutions are analyzedusing a moderately polar megabore (0.53mm) capillary column.

Each individual ester is determined by comparison to standardsolutions of pure ester compounds. Response factors areestablished for each compound and used to convert raw data intoweight percent figures. The weight sum of the individual estersis the total methyl ester content.

REFERENCE

This method is a modification of AOCS standard method Ce-le-91(22), to accommodate the requirements for determination of total


PAGE 41

methyl esters in biodiesel. Specifically, the followingmodifications are introduced for optimized precision andaccuracy:

1) Only cool on column injectors with stable inlet temperatureson injection at less than 50 degrees C are specified (this is anoption in the .4OCS method).

2) A megabore (0.53 mm) column with 1.0 urn film thickness isspecified. Here, some resolution capability is sacrificed with again in sample capacity, decreased analysis times, and simplicityin sample and gas flow path. The temperature program and carrierflows are also modified to accommodate the megabore column.

3) Samples are pretreated to remove polar substances insolublein hexane.

b) An autoinjector system is required to attain the necessaryprecision.

5) ,4 computer/data system is necessary.

6) Helium is specified as carrier gas for safety reasons.

EQUIPXENT AND APPARATUS

1) A gas chromatograph capable of multiple temperatureprogramming with direct, cool, on-column capillary injection anda flame ionization detector (FID). .4n autosampler is necessary.

2) Chromatography software/computer interface/with integratorcapability, data automation capability preferred.

3) Polyethylene glycol bonded phase capillary column, 30 M x0.53mm ID, 1.00 urn film thickness or equivalent (betterresolution, but lower sample capacity with thinner films),capable of baseline resolution of C18-0, C18-1, and C18-2 methylesters @ IO:50 weight percent, and a resolution equal to orbetter than for the separation of C22:0, and C22:1.

4) A high precision 10 ul autoinjector syringe.

5) Analytical micro balance capable of 5 decimal (O.Olmg)readings.

6) Cap sealed borosilicate volumetric flasks, IO and 100 ml.

7) Carrier gas purification system to remove oxygen and moisturetraces to below 2 ppm.

R. W. HEIDES ASSOCIATES/NATIONAL BIODIESEL BOARD 27 FEB 96

PAGE 42

CHEMICALS AND REAGENTS

Solvents:

All solvents used in this method are HPLC or GC high purity, lowresidue grade.

Standards

High purity >99% substances for standards, certified by thevendor with plus or minus tolerances. Mixtures of esters fromthe vendor should be certified for composition and weight contentof methyl esters within acceptable tolerances. To minimizehandling of the standards, the exact weight of esters in a givenampule, and the total weight should be specified.

Gases

1) Carrier and makeup gas- helium 99.995% minimum with moistureand oxygen removal capability to less than 2 ppm.

2) Hydrogen and air for FID, zero grade, or hydrogen, 99.9%minimum free of organic impurities and oxygen.

SAMPLE PREPARATION/CLEANUP

Approximately 100.00 mg of transesterified product is transferreddirectly to the inside of an RP CN disposable micro column(Waters CN Seppak was found to work). Wet the upper half of themicrocolumn with pentane. Allow to stand 2 minutes. Then pass10 ml of hexane through the column at a rate of about 10ml/minute following the manufacturers recommendations, and into a100 ml volumetric flask. Dilute to volume with hexane.

REPLICATION

At minimum samples and standards should be prepared in duplicateand each duplicate analyzed in duplicate.

GC COLUMN CONDITIONS

Carrier: He, 5.0 ml/minInjector T: 50 cDetector T: 250 CColumn Program:

40 C /O min 15 C/min to 120/O min 4 C/min to250C/hold 2 min.


PAGE 43

INJECTIONS

A 2 ul aliquot is injected rapidneedle into the septum.

lY, w ith fdll depress

STANDARDS AND CALIBRATIOX

ion of the

Standards are prepared from either certified standard mistureswith a stated composition within IO percent relative of eachcomponent in the unknown sample, or by manual preparation ofmixtures from pure, certified standard materials. The sample isfirst run with results compared to a prechosen standard mixturethought to resemble the sample. If the area of each componentgreater than 1% (of the total methyl ester area) is within %I0 ofthe calibrated standard component, then the results calculatedfrom the standard stand. If not, then each component outside ofthe 10 % range must be calibrated separately. Response factorsfcr components less than 1.0% down to Cl2 are arbitrarilyassigned a value of 1.00, compared to a Cl8 methyl ester standardand additional calibration of such components is unnecessary.

The pure standard material in milligrams should be weighed to thenearest 0.01 milligram, and quantitatively transferred to a 100ml volumetric flask for analysis. The weight of commerciallyavailable standard mixtures should be certified for weightcontent, in addition to composition. For these mixtures completedirect transfer of the content with careful washings of solventare recommended to avoid unnecessary handling and subsequentlosses of slightly volatile materials.

CALCULATIONS

The mass of all individual components is summed to obtain thetotal methyl ester content. This is then divided bl- the totalsample weight and multiplied by 100 to obtain a percentage total.

Obtain the area for each peak and espress it as percent of thetotal fatty acid methyl ester.

The individual components are calculated from the data in thefollowing way.

Area !S4MPLE COYPONEKTi/Area (STD)=Hass(SAYPLE COMPOSEXT)/massiSTAZD.4RD~

R. W. BEIDES .4SSOCIATES/N+.TIONAL BIODIESEL BOARD 27 FEB 96

PAGE 41

orMass(SAMPLE CO!lPONENT)=Area (SAMPLE CO?IPONENT) X mass (STANDARD)

Area (STANDARD)

Total methyl ester percent (TEP)=

TEE' = 100 X Sum of masses/ Total sample weight

ACCDRACY AND PRECISION

The precision of the method is evaluated by a series of replicateinjections. The precision is calculated as the square root ofthe sum of the variances associated with each component. A totalprecision of plus or minus 0.5% relative is attainable for totalmethyl ester determinations using this method.

The accuracy of the method is unknown and depends on at leastthree factors. The first is the accuracy of certified referencestandards, and the biases introduced by laboratory mixturepreparation and handling. The second is errors in integration,e.g. those introduced by unresolved peaks, shoulders, etc. Thethird is interferences.

Standards certified on the basis of purity and weight areavailable from severai commercial sources. Certificationtolerances for the standards are unspecified at the moment, butconsideration of the total bias introduced by these tolerancesmust be given, and clearly should be at a minimum withinacceptable limits with consideration of the limits of total estercontent of biodiesel specifications.

QUALITY COKTROL/QUALITT ASSURATCE

At minimum, samples and associated standards should be analyzedin duplicate. A quality program should be established whichreflects an on-going monitoring cf the precision of the methodand subtle changes in the analysis system, and which assists inidentifying the degree of replication required to validatebiodiesel specifications.

R. W. BEIDES ASSOCIATES/XATIONAL BIODIESEL BOARD 27 FEE 96

PAGE 45

VIII. REFERENCES

1) International Conference on Standardization and Analysis ofBiodiesel, Session 2, "Interaction Between Engine and Fuel",Vienna, November 6-7, 1995-to be published.

2) International Conference on Standardization and Analysis ofBiodiesel, Session 1, .4nalytical Methodology, Vienna, November6-7, 1995-to be published.

3) Steven Howell, NBB communication, January 3, 1996.

4) J. Bailer, P. Hodl, K de Hueber, M. Mittlebach, C. Plank, H.Schindlbauer, "Handbook of Analytical Methods for Fatty AcidMethyl Esters Used as Diesel Fuel Substitutes", Fichte,University of Technology, Vienna, 1994, 71 pp.

5) C. F. Poole, "Advances in Silylation of Organic Compounds forGC", in "Handbook of Derivatives for Chromatography", Heyden,London, 1979, pp, 152-200.

6) B. Freedman, W. F. Kwolek and E. H. Pryde, JAOCS, 63(10):1370-75 (1986).

7) c. Mariani, P. Bandioli, S. Venturini, E. Fedeli, Riv. Ital.Sostanze Grasse, 68: 549-551 (1991).

8) P. Bandioli, "Analysis of Different Glycerides in Biodiesel:the Italian Experience", Poster Session delivered at theInternational Conference on Standardizaion and Analysis ofBiodiesel, Vienna, November 6-7, 1995.

9) c. Plank, "Quantitative Determination of Mono-, Di, andTriglycerides in Fatty Acid Methyl Esters by Capillary GasChromatography", in "Handbook of Analytical Methods for FattyAcid Methyl Esters Used as Diesel Fuel Substitutes", Fichte,University of Technology, Vienna, 1994, pp. 29-38.

10) C. Plank, "Critical Assessment of the Gas ChromatographicDetermination of Acylglycerols in Biodiesel at the 0.1% Level",Poster Session delivered at the International Conference onStandardization and Analysis of Biodiesel, Vienna, November 6-7,1995.

11) AOCS Official Standard Cd lib-91, AOCS, Champaign, IL.

12) M. Mittlebach, "Gas Chromatographic Determination of FreeGlycerol Involving Derivatization", in "Handbook of AnalyticalMethods for Fatty Acid Methyl Esters Used as Diesel FuelSubstitutes", Fichte, University of Technology, Vienna, 1994, pp.22-26.

R. W. HEIDEN ASSOCIATES/NATIONAL BIODIESEL BOARD '7 FEB 96

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13) M. Mittlebach, personal communcation, November 7, 1995.

14) J. Liu, T. Lee, E. Bobik, Jr., M. Guzman-Harty and C.Hastilow, JAOCS, 70(4): 343-347 (1993).

15) W. W. Christie, J. Lipid Research, 26:507-512 (1985).

16) A. Bruns. Fat Science and Technology, 90:289-293 (1988).

17) B. Trathnigg and M. Mittlebach, J. Liq.Chrom.,lS(l): 95-105(1990).

18) B. Freedman, E. H. Pryde and W. F. Kwolek? JAOCS, 61(7):1215-1220 (1984).

19) Standard ASTM D 1983-90. ASTM, Philadelphia, PA.

20) AOCS Official Standard Ce l-62, AOCS, Champaign, IL.

21) 4OCS Official Standard Ce lc-89, AOCS, Champaign, IL.

22) AOCS Official Standard Ce le-91, AOCS, Champaign, IL.

23) R. G. Ackman, JAOCS, 66(3): 293-301 (1989.

24) H. T. Slover and E. Lanza, JAOCS, 56(12): 933-943 (1979).

25) K. Komers, J. Machek, F. Skopal, "Determination of Degree ofConversion of Rape Oil to Biodiesel", Poster Session delivered atthe International Conference on Standardizaion and Analysis ofBiodiesel, November 6-7, 1995.

26) Paul Gaines, personal communication, Calgene Chemical,Skokie, Illinois, (1995).

27) M. S. Klee, "GC Inlets-An Introduction", Hewlett PackardCorporation, Wilimington, DE, 1990, p. 4.

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