The separation and determination of fatty acids by isotopic dilution and radiogas—liquid chromatography

T,,h,nra. Vol. 28. pp. 405 to 407. 1981 Printed in Great Britain. All rights reserved

0039.9140.x1 Oflo405.0350?.00 0 Copyright 0 19x1 Pergamon Press Ltd

THE SEPARATION AND DETERMINATION OF FATTY ACIDS BY ISOTOPIC DILUTION AND RADIOGAS-LIQUID CHROMATOGRAPHY D. A. BEARDSLEY School of Health and Applied Sciences (Chemistry Section), Leeds Polytechnic, Leeds, England (Received

3

November 1980. Accepted

23 December

1980)

Summary-A number of static phases have been evaluated for the GLC separation of fatty acids. Of those investigated, only AT 1200 was capable of resolving the isomeric forms of the acids. A radiogasliquid chromatographic method incorporating isotopic dilution analysis has been developed for the determination of n-butyric acid. The proposed method has been applied to the determination of the acid in hydrolysed butter fat and milk chocolate extracts.

Gas-liquid chromatography has been applied to food analysis for about 30 years. The work up to 1974 has been reviewed by Dickes and Nicholas’ and the more recent applications are the subject of a Talanta Review.’ Radiogas-liquid chromatography has been reviewed by James3 Karmen,4 and Scott.’ In principle it can be used to identify and determine radioactive components in any mixture which can be separated by gas-liquid chromatography. Volatile fatty acids can be determined by the Reichert, Polenske and Kirschner process$ this method, however, requires a relatively large sample weight and is timeconsuming. The hydroxamic acid index method’ has the advantage of using a smaller sample weight, but is empirical. The use of GLC for the determination of fatty acids is well documented. Its application to the determination of propionic acid in food has been reviewed by Walker and Greens and Phillips and Sanders9 have described a method for the determination of butyrate esters in fats, by hydrolysis to butyric acid and use of valeric acid as an internal standard. This paper describes a radiogas-liquid chromatographic procedure involving radioactive isotopic dilution analysis for the determination of fatty acids. The sample solution in suitable form is mixed with a i4C-labelled fatty acid of known original specific activity, and subjected to GLC separation. The resulting specific activity of the fatty acid is determined by splitting the effluent gas from the column into two streams, one of which is fed to a flame-ionization detector (FID) and the other, after passage over heated copper(H) oxide, to a gas-flow proportional counter. The weight of fatty acid in the sample is calculated by using the equation for direct isotopic dilution analysis:

y=y,

where y = weight of fatty acid in the sample aliquot, y, = weight of “C-labelled fatty acid added, S, = specific activity of the labelled fatty acid before isotopic dilution and S2 = specific activity of the labelled fatty acid after isotopic dilution. The method has the advantage that quantitative separation of the desired acid is not essential. It is only necessary to determine the specific activity of a pure sample of the fatty acid with adequate precision. A constant splitting-ratio and a linear mass response of the FID over the mass range are, however, essential.

g-1 (

2

> 405

EXPERIMENTAL Apparatus

The radio-GLC system consists of a Pye series 104. chromatograph with Row-controller units and twin injection heaters. The column is coupled to a flow-splitter assembly giving a nominal 1:I split. One effluent stream is fed to an FID detector, the signal from which is integrated

electronically with a Vidar 6300 digital integrator and recorded on a two-channel recorder. The other effluent stream passes through a quartz tube packed with copper(H) oxide and heated to 700” and then through magnesium perchlorate for removal of water. The carbon dioxide thus obtained is passed into a Panax radiogas detector system incorporating a gas-flow proportional counter (EHT 148OV). with cosmic-guard (EHT SSOV), and anticoincidence gate circuit for measurement of the “C01. The signal is fed to the recorder and integrated with a scalar coupled through the ratemeter. The carrier-gas is an argon+arbon dioxide mixture (95:5), at a flow-rate of 40 ml/min. The proportional-counter efficiency, given by efficiency =

1OOXCXF

AxV

where C = number of (ml/mink A = activity volume (ml) is claimed system is fully described

%

counts, F = carrier-gas flow-rate applied (dpm) and V = counter to be 94.5% for carbon-14. The by Simpson.”

406

SHORT COMMUNICATIONS

Reagents

Propionic, n-butyric, isobutyric, n-ualeric, isooaleric and hexanoic acid solution (500 pg/ml). Prepared by dissolving

sealed tube followed by subsequent injection of the liquid phase onto a column of 10% AT 1200 (free from phosphoric acid) indicated that the peak was associated with a reaction of AT 1200 with phosphoric acid. Details of the nature of the static phase could not be elicited from the manufacturers so this aspect was not pursued. For further work a column of 10% AT 1200 alone on Chromosorb W (AW-DCMS) was used. It was further found that after a prolonged exposure to samples containing phosphoric acid degradation of the column occurred and the extra peak reappeared in the chromatograms.

I g (accurately weighed) of the acid in water and diluting to 100 ml with distilled water, and further dilution of 5 ml to 100 ml with distilled water.

Development of the method for determination qf n-butyric acid

“C-labelled

n-butyric acid solution. An ampoule

of sodium butyrate (250 @Ci, 59 mCi/mmole, Radiochemical Centre, Amersham) was opened, 1 ml of 1% phosphoric acid was pipetted into it and the contents were washed out into a 5-ml standard flask with distilled water, mixed with 1 ml of 2500-&ml non-active n-butyric acid solution and diluted to volume with distilled water. The concentration was determined by reverse isotopic dilution analysis.

Selection 0s the column Various columns were tested for isothermal separation of the six fatty acids. A 5-ft column of 5% “free fatty acid phase” on Chromosorb G (AW-DCMS) operated at 145” at a flow-rate of 40 ml/min after overnight conditioning at 180” gave severe tailing of the peaks and very poor resolution of the acids. The results for 10% Carbowax ZOM-TPA on Chromosorb W (AW-DCMS) (column A, 5 ft, flow-rate 40 ml/min, temperature I lo”, conditioned overnight at 200” with flow), Chromosorb 101 (SO-100 mesh) (columns B and C, flowrate 4Oml/min, conditioned overnight at 250” with flow), and 10% AT 1200 + 1% H,PO, on Chromosorb W(AW) (SO-100 mesh) (column D, 5 ft, flow-rate 40ml/min, temperature 1lo”, conditioned overnight at 175” with flow) are given in Table 1. Column A did not fully resolve the acid pairs propionic/ isobutyric and n-butyric/isovaleric. Lowering the temperature to 90” permitted resolution of the latter pair. The 5-ft column B gave incomplete resolution when the two isoacids were present. Increasing the length to 9 ft (column C), and the temperature to 180” gave complete resolution but relatively long retention times. At higher temperatures resolution of the acids was not complete. It has been claimed that the use of a 5% “free fatty acid phase” coating on Chromosorb 101 improves the resolution of the lower fatty acids, but we observed no improvement in resolution with either a 5-ft or a 9-ft column. All the Chromosorb 101 columns suffered gradual degradation with repetitive injection of aqueous solutions of the acids and this was enhanced in the presence of phosphoric acid, which was used to liberate the acids from the saponified samples. The columns darkened near the injection port and erratic baselines were obtained. Column D, with the packing marketed by Alltech Associates Inc.,. proved to be the ideal phase for the resolution of the acids (Table 1). but the chromatographs showed an additional peak at a retention time of about 75 min. The area of this peak was directly related to the volume of aqueous phase injected. It was found that if the column was prepared without phosphoric acid present in the packing, the extra peak did not appear. Prolonged exposure of AT 1200 to water and 1% phosphoric acid at 140” in a Table 1. Retention times (min) for fatty acids Column Acid Propionic Isobutyric n-Butyric Isovaleric n-Valerie Hexanoic

A

(5 ft,B140”)

(9 ft,C1800)

D

2.8 3.0 4.0 4.8 6.8 10.4

2.9 3.2 4.4 4.9 6.4 9.7

9.0 11.0 14.0 17.0 22.0 36.0

3.3 5.4 7.4 9.5 13.7 18.3

For the determination the essential requirement is precise measurement of y,, S,, and S1. S, and S2 are independent of the mass isolated. so the reproducibility of the sample injection volume is not important. It is essential, however, that the splitting ratio for the carrier-gas should remain constant during the analysis, No variation was observed over an 8-hr period of continuous operation. Variation from day to day might occur because of slight differences in gas pressure when setting up the GLC equipment. It is concluded that no significant error should result for a batch of analyses done within a period of continuous operation of the equipment. The accuracy and precision of determination of the specific activity were tested by repeated injection of “C-labelled n-butyric acid solution (Table 2). The linearity of the FID was established by injection of aqueous n-butyric acid samples over the concentration range I@500 pg/ml. A straight-line relationship was found (correlation coefficient = 0.99). As a test of the procedure the method was applied to the determination of n-butyric acid in the products from hydrolysis of a milk chocolate extract and pure butter fat. The results were then compared with those obtained with valeric acid as internal standard (Phillips and Sanders method’). Analytical procedure

An accurately weighed 0.1-g portion of the rendered fat sample was introduced into a 50-ml conical flask and 3 ml of 0.5M ethanolic potassium hydroxide were added. The flask was covered with a watch-glass and heated on a steam-bath for 15min, then the watch-glass was removed and the ethanol evaporated. The flask was allowed to cool. and 3 ml of distilled water were added. When a clear solution had been obtained, 3 ml of 0.5% phosphoric acid were added to precipitate the insoluble fatty acids. The

Table 2. Reproducibility of the determination of the specific activity of “C-labelled n-butyric acid Number of counts Proportional Electronic counter integrator I5486 16024 16850 15770 15951 15789 15950 16680 16900 15480 15112 14401

39557 41121 41421 40208 40160 41821 41970 42288 43490 38660 38174 35912

Specific activity (S) 0.40, 0.39, 0.40, 0.39, 0.39, 0.39, 0.380 0.39, 0.389 0.40, 0.39, 0.40,

n = 12; S = 0.395; std. devn. = 0.007; C of V = 1.8%.

SHORT

407

COMMUNICATIONS

Table 3. Analyses of samples Isotopic dilution method

Internal standard method Sample weight 9 Butter fat Milk chocolate extract

R1

n,

CV,, 9:

Rz

n2

CV2, 7: C.+.5:

S,

n,

CVI, 7;;

SZ

n2 cv*, 7: C& 7;

0.1418 0.93,,* 13 0.87& I9

3.1 2.8

0.900* 10 0.84& I9

2.2 3.1

3.64 3.6,

0.414t

7

2.3

0.213

7

2.1

3.63

0.1511 0.24,:

2.3

0.84a*

1.8

0.96

0.44st

IO

1.6

0.35,

10

1.9

0.92

8

8

* Electronic integration. t Standard 545 pg/ml. $ Triangulation.

solution was then filtered through a moistened 7-cm Whatman No. I filter paper into a lO-ml standard flask. The precipitate was washed with water and the solution made up to volume with distilled water. For the internal standard method, the standard solution was made by mixing 5 ml of 500~pg/mI n-butyric acid solution with 1 ml of ZSOO-pg/mlvaleric acid solution; 5 ml of sample extract were mixed with I ml of 25OO+g/ml valeric acid solution. For the isotopic dilution method, 1 ml of standard “C-labelled n-butyric acid solution was mixed with I ml of sample extract. All the pipetting was done with a Finnpipette (precision 1%) and l-pl samples were injected into the GLC column. Calculation of results In the internal standard method the n-butyric acid content (“/, C,) was calculated from %C1=!z?!!& ?. R, = peak-area ratio for the sample; R2 = peak-area ratio for the standard; W = sample weight

where

(8). For the isotopic dilution method the equation already given was used.

indicate that the method compares very favourably with the internal standard method. In both methods a reproducible injection volume is not essential. Problems associated with variation in response factors do not arise. The proposed method can be applied to any fatty acid provided it is sufficiently volatile and,can be obtained in a suitable radio-labelled form, The investigation showed that only one static phase, AT 1200, was capable of good resolution of all six acids, though FFAP and Chromosorb 101 were suitable for the unbranched acids. A method has been described,” for determination of acid derivatives by use of both 3H and 14C labelling to quantify the degree of derivative formation. It is hoped to develop a substoichiometric method for such samples to avoid determination of the mass involved. Acknowledgement-Many thanks are due to D. Little for invaluable technical assistance given during this research work. REFERENCE8

RESULTS AND DISCUSSION

The results of the analyses are given in Table 3. The method was tested for concentrations lower by a factor of 10, with a X%&ml n-butyric acid solution and the 14C standard diluted by a factor of ten. The mean, based on 10 results, was found to be 50.5 pg/ml with a standard deviation of 0.5 pg/ml. The reproducibilities of the specific activity measurements and of the linearity of response of the FID over the concentration range ICr5OO~g/ml meet the criteria for precise determination of the n-butyric acid by the proposed method. The analytical results together with the statistical analysis given in Table 3

I. G. J. Dickes and P. V. Nicholas, Gas Chromatography in Food Analysis, Butterworths, London, 1976. 7 G. J. Dickes. Taianta. 1979. 26, 1965. 3. A. T. James, ‘New Biochemical Separations, A. T. James and L. J. Morris (eds.), Van Nostrand, London, 1964. 4. A. Karmen, J. Assoc. Ofl Agric. Chem., 1964, 47, 15. 5. P. G. W. Scott, Process Biochem., 1967, 16. 6. Methods of Analysis of Oils and Fats, BS 684: 1958, British Standards Institute, London, 1958. 7. R. Bassette and M. Keeney, J. Assoc. Ofl Agric. Chem., 1956, 39, 469. 8. G. H. Walker and M. S. Green, J. Assoc. Publ. Anal., 1965, 3, 87. 9. A. R. Phillips and B. J. Sanders, ibid., 1968, 6, 89. IO. C. Bishoo. R. F. Glascock, E. M. Newell and V. A. Welch, J: Lipid Res., 1971, 12, 777. A.