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`OATING and CHEMICAL

LABORATORY

CCL REPORT NO. 139

APPLICATION OF THE INTERNAL STANDARDIZATION METHOD FOR THE

GAS CHROMATOGRAPHIC DETERMINATION OF DRYING OIL FATTY ACIDS

BY

GEORGE G. ESPOSITO

AMCMS CODE NO. 5 026.11.8/4 20 5 A_ r - ,,

DA PROJECT I-H-O-24401-A-110-05 FiL!

20 MARCH 1963 /LrL•-,, _

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TABLE OF CONTENTS

Page No.

TITLE PAGE ............ ............................................

ABSTRACT ...........................................................

INTRODUCTION ....................................................... . 1

DETAILS OF TEST .................................................... 1 - 2

RESULTS ............................................................ 2 - 3

DISCUSSION ......................................................... 3

REFERENCES ......................................................... 3

APPENDIX ........................................................... 4

Tables I - V ..................................................... 5 - 7

DISTRIBUTION LIST .................................................. 8 - 9

UNCLASSIFIED

Report No CCL # 139

Copy Number

APPLICATION OF THE INTERNAL STANDARDIZATION METHOD FOR THE

GAS CHROMATOGRAPHIC DETERMINATION OF DRYING OIL FATTY ACIDS

By

George G. Esposito

20 March 1963

AMCMS CODE NO. 5026.11.84205

Dept. of the Army Project No.I-H-0-24L4o0-A-l 10-05

Coating and Chemical LaboratoryAberdeen Proving Ground

Maryland

UNCLASSIFIED

AUTHOR: REVIEWED BY: YGE G e ITOE M. ROSENFELD, Chief

Chemical Cleaning & Chemical Cleaning &Corrosion Branch Corrosion Branch

APPROVED BY:

C. F. PICKETT, Technical DirectorCoating and Chemical Laboratory

ii

ABSTRACT

This investigation was conducted to determine a suitable method for obtain-ing quantitative data from the chromatographic separation of methyl estersprepared from oils used in organic coatings. The internal standard method iscompared to the total area technique and found to be necessary for accurateanalysis.

The sample is saponified and the fatty acids isolated. A known weight ofmargaric acid, the internal standard, is added to a known amount of samplefollowed by methyl ester formation and separation of the esters on a polyestercolumn by gas chromatography. The unreacted fatty acids are calculatedindividually and materials, such as dimers, trimers and adducts are determinedby difference.

iii .

I. INTRODUCTION

Gas chromatography has been used as an analytical tool to determine thefatty acid composition of oils and fats by separation of methyl esters onvarious polyester columns. Most of the data found in the literature uses thenormalization of peak areas (total area) as the method of quantitative analysis.In this method the total area under a chromatogram is assumed to represent allthe material in the sample. Since some of the fatty acids present in dryingoils are of the polyunsaturated type, it was logical to assume that somereaction would occur during the preparation of coating resins which wouldrender them unsuitable for chromatographic analysis by the total area method.Treatment of oils; such as heat bodying and reaction with unsaturated monomers,is commonly practiced to produce high molecular weight compounds of fatty acids

which are conducive to forming rapid drying coating materials. Drying oils areused in the preparation of alkyd resins and during the process are subjectedto considerable heat, causing reaction of some polyunsaturated acids. In orderto account for the reacted unsaturated fatty acids, an investigation wasinitiated to determine the applicability of the internal standard method tothis type of material.

A modified total area method (7) was reported for the determination ofpolymerized fatty acids by a technique requiring precise sampling of knownmixtures of pure methyl esters and comparing the total area obtained to thatof an equal volume of sample, the difference being attributed to polymerizedfatty acids. This method requires daily calibration and is based on exactsampling, which introduces error into the method and provides an approximationof polymerized fatty acids.

The use of an internal standard has been reported for the determinationof fatty acids in meat fat (3) and the fatty acid content of milk (2). Idenand Kahler (5) used methyl margarate, as an internal standard, for thedetermination of fatty acids in tall oil and found some to contain as low as85% fatty acids. The use of an internal standard for the determination offatty acids in drying oils could not be found in the literature and is thesubject of this investigation. The amounts of reacted and unreactad fattyacids found in alkyd resins, heat bodied oils and cyclopentadiene treated oilsby the internal standard method are shown in this report.

The method employed in this study is essentially the one used by Iden andKahler (5) for the determination of tall oil fatty acids with changes made forthe isolation of fatty acids and preparation of methyl esters. The resin wassaponified using the ASTM unsaponifiable method D555-54 and the unsaponifiablewas removed before isolating the fatty acids. A weighed amount of margaricacid was added to a known weight of fatty acids and methyl esters wereprepared using boron triflouride etherate in methanol and the methyl esterswere separated on a polyester column. An inert gas was used at all stages toprotect the fatty acids from oxidation by the atmosphere.

II. DETAILS OF TEST

Chromatographic Unit

The equipment used to obtain the chromatograms was a Model 500 LinearProgrammed Temperature Gas Chromatograph (F&M Scientific Co.) equipped with a

Brown Electronik recorder (Minneapolis-Honeywell).

Operating Conditions

Detector cell temperature, °C. 300Detector cell current, ma. 160Injection port temperature, *C. 330Helium flow at exit, cc./minute 85Column temperature, 0C. 190

An 8-ft. length of !-inch copper tubing was packed with 10% diethy-lene glycol succinate on acid washed chromosorb W. The column was conditionedat 2000G. until bleeding was at a minimum.

Isolation of Fatty Acids and Esterification

The sample is treated using ASTM Method D 555-54 for determination ofunsaponifiable matter in drying oils. After the unsaponifiable has beenseparated, the water layers are combined and made acid with 18 N H2 SO4 . Theliberated fatty acids are extracted using 75, 50, and 50 ml. portions of chloro-form and the combined chloroform extracts washed with 50 ml. aliquots of wateruntil acid free. Evaporate chloroform in 600 water bath with a slow current ofinert gas flowing over surface of sample. Bring to constant weight in 60'vacuum oven.

The method of Metcalf and Schmitz (6) using boron trifluoride-methanol reagent is used to prepare the esters because it is rapid, simpleand less hazardous than other procedures. This method is simplified stillfurther by substituting boron triflouride ethyl ether (purified) for borontriflouride gas.

Ill. RESULTS

The detector response is not the same for different methyl esters andcalibration is necessary when a thermal conductivity detector is used. Methylpalmitate is assigned the correction factor of 1.0 and all the other methylesters are related to it. The correction factors obtained experimentally byrunning known weights of pure methyl esters were in good agreement with thecalculated values obtained using the equations described by Horrocks, Cornwelland Brown (4).

Refined coconut oil fatty acids were adulterated with commercial dimeracids and examined before and after adulteration by gas chromatography usingan internal standard. This was done to establish the feasibility of using aninternal standard when examining methyl esters containing materials ofextremely low vapour pressures. Dimer acids are C3 6 acids and are the typethat would be encountered in fatty acids extracted from heat bodied oils andalkyd resins. The results are shown in Table I and the necessity for usingan internal standard is illustrated by the comparison with total area results.

Table II shows a comparison of the methods for the determination of fattyacids from a soybean alkyd. Two different alkyds prepared from linseed oil wereanalyzed and the total area and internal standardization figures given in TableIll. Duplicate results were obtained by repeating the entire procedure and not

2

just the gas chromatography step. The total area results are in error and theamount of polymerized fatty acids formed is not apparent.

Heat bodied and cyclopentadiene treated oils were tested and the lowerfatty acids values obtained with an internal standard are shown in Tables IVand V and indicates that a high percentage of polymerized material is present.A modified total area method (7), which provides approximate results, was usedto confirm that the internal standard results were in proper al:ignment. The95% confidence interval statistical method (1) was applied to the results fromthe modified total area technique and gave a 40% to 46% range for heat bodiedfatty acids and 35% to 43% range for the cyclopentadiene treated fatty acids.The broad range is indicative of poor reproducibility from three observations.

IV. DISCUSSION

Varying amounts of polymerized fatty acids will form when drying oils areincorporated into coating resins. The total area gas chromatographic procedureis inadequate when polymerized fatty acids are present and it is necessary toanalyze these materials by gas chromatography using an internal standard.Margaric acid, a C1 7 carboxylic acid, is not natural occurring and forms amethyl ester possessing a retention time different than the natural occurringacids, thus making it suitable as an internal standard..

V. REFERENCES

I. Dean, R. B. and Dixon, W. J.r., Anal. Chem., 23,636 (1951).

2. Gehrke, C. W., Goerlitz, D. F., Richardson, C. 0. and Johnson, H. D.,J. Diary Sci., 43, 439 (1960).

3. Hornstein, I., Alford, J. A., Elliott, L. E. and Crowe, P. F., Anal.Chem., 32, 540 (1960).

4. Horrocks, L. A., Cornwell, D. G. and Brown, J. B., J. Lipid Research,2, 92 (1961).

5. Iden, R. B. and Kahler, E. J., JAOCS, 39, 171 (1961).

6. Metcalfe, L. D. and Schmitz, A. A., Anal. Chem., 33, 363 (1961).

7- Zielinski, W. L., Moseley, W. V. and Bricker'I R. C., Offic. DigestFederation Paint & Varnish Production Clubs, 33, 622 (1961).

3

APPENDIX

Tab I es

'4

TAB LE I

GAS CHROMATOGRAPHIC ANALYSIS OF A MIXTURE OF COCONUT OIL FATTY ACIDSAND DRIMER ACIDS USING THE INTERNAL STANDARD METHOD

Methyl Ester Found %

Caprylate 6.8Caprate 4.9Laurate 45.4Myristate 15.7Palmitate 7.6Stearate 4.1Oleate 4.8Linoleate 0.9

Total 90.2*

*Mixture contained 90.1% coconut fatty acids and 9.8% dimer acids.

TABLE II

ANALYSIS OF SOYBEAN OIL FATTY ACIDS FROM ALKYD RESIN COMPARISONOF INTERNAL STANDARD AND TOTAL AREA METHODS

Internal Standard Total AreaMethyl Ester % %

Palmitate 15.0 16.8Stearate 3.7 4.2Oleate 19.3 21.7Linoleate 45.7 51.3Linolenate 4.4 4.9Miscellaneous 0.9 1.1

Total .89.0 100.0

5

TABLE III

ANALYSIS OF LINSEED OIL FATTY ACIDS FROM ALKYD RESINSCOMPARISON OF INTERNAL STANDARD AND TOTAL AREA METHODS

Alkyd A Alkyd BMethyl Ester Method 1* Method 2** Method 1 Method 2

Palmitate 5.7; 5.6. 7.4; 7-3 6.1; 5.9 6.8; 6.6Stearate 3.6; 3.5 4.7; 4.4 4.1; 3.8 4.6; 4.2Oleate 19.5; 19.3 25.5; 25.2 21.0; 20.5 23.0; 23.0Linoleate 13.0; 13.2 17.3; 17.1 15.4; 15.8 17.3; 17.5Linolenate 33.2; 34.4 44.3; 45.0 42.5; 43.5 47.7; 48.7Miscellaneous 0.7; 0.7 0.8; 0.9

Total 75.7 76.7 100.0 100.0 89.1 89.5 100.0 100.0

", Internal standard.

"Total area.

TABLE IV

ANALYSIS OF HEAT BODIED LINSEED OILCOMPARISON OF INTERNAL STANDARD AND TOTAL AREA METHODS

Mod if iedInternal Standard Total Area Total Area*

Methyl Ester % % %

Palmitate 5.5 11.5 ---Stearate 4.2 8.5Oleate 18.5 39.5Linoleate 9-7 20.1Linolenate 9"8 20.4 ---

Total 47.7 100.0 43.0

"* Method (7) produces approximate results.

6

TABLE V

ANALYSIS OF CYCLOPENTADIENE TREATED OILCOMPARISON OF INTERNAL STANDARD AND TOTAL AREA METHODS

ModifiedInternal Standard Total Area Total Area*

Methyl Ester % % %

Palmitate 5.3 14.7 --

Stearate 3.7 10.2 --

Oleate 15.2 41.0 --

Linoleate 6.5 18.5 --

Linolenate 5.6 I5. --

Total 36.3 100.0 39.0

* Method (7) produces approximate results.

7

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9

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