B. C. ROY SARKAR AND K. C.
SEN
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a summary of the results obtained is given. It will be obvious from a perusal of the results given
in the Table that the biological value of the carotene in green fodders or that in an extract of green
fodder as determined by the Seshan-Sen chemical methods is almost identical with that of pure
carotene standardized by spectrographic analysis. In other words, it is possible to assay the biological
potency of different green fodders by the chemical method only, provided we allow the same margin
of error as is inherent in the biological technique. According to Coward (1933), the biological assay
of a test material with male rats over a period of 4 weeks after the depletion period may give rise
to an error of +19 or —16 per cent. Consequently, if the chemical methods of analysis is to be
standardized against a biological method, we cannot ordinarily expect a closer correlation than this.
As the standard carotene used for reference contained about 80 per cent ß-carotene and 20 per cent
α-carotene (chromatographic assay), the above results appear to show that a similar proportion might
exist in the carotene of the various fodder plants. It may, therefore, be assumed that the results
obtained by the Seshan-Sen method of carotene estimation are a fair index of the biologically active
carotene of fresh green fodder if pure carotene is used as a standard of reference and that ß-carotene
constitutes the major part of carotene in these plants.
(2) Chromatographic determination of the purity apparent carotene
A number of feeding stuffs, such as green fodders, dry roughaga, silage, etc., were studied. The
carotene was extracted according to the Seshan-Sen technique, the final petroleum ether solution
was passed through different adsorbents. Three different chromatographic materials were used,
namely, dicalcium phosphate [Moore, 1940; subsequently used by Bolton and Common, 1942;
Smith and Robb, 1943, and others], alumina (Brockmann) and Micron brand magnesia mixed with
equal parts of Hyflo Super-Col. 'Heavy' magnesia of the type used recently by Austin and Shipton
was not available in this country owing to war conditions. The results obtained in these experi-
ments were then compared with those obtained by using standard carotene samples subjected to
similar chromatographic analysis. The chromatography was done by first passing the solvent
through the column prepared according to the usual methods, then the carotene solution was poured
in before the top was dry, after which the chromatogram was developed with the same solvent.
Washing was continued till the filtrate was colourless. Two types of carotene extract were used
with dicalcium phosphate adsorbent, one being the petroleum ether extract before phase separation
was carried out (total carotenoids), and the other being the final solution after the phase separation
(apparent carotene). In both cases the solutions were dried over anhydrous sodium sulphate before
passing through the adsorbent. The results obtained are briefly as follows: When the carotene
from different fodder plants in petroleum ether was passed through either alumina or magnesia,
there was a loss of about 14-16 per cent. An almost similar result was obtained when the standard
carotene was also passed through the same adsorbents. Owing to the slowness of filtration through
the finely divided adsorbents, the ß-carotene part of the pigment was in contact with the adsorbent
for a long period, and probably some amount of destruction took place in these cases. Chromato-
graphy through dicalcium phosphate showed negligible loss in the case of the standard carotene.
The comparative results with the different materials are shown in Table II. The true carotene is
expressed as a percentage of apparent carotene as determined by the phase separation method in
all cases. It will be seen from the results given in Table II that a chromatographic analysis of the
apparent carotene obtained by the Seshan-Sen extraction method shows it to be 85-99 per cent
(average 94 per cent) true carotene in the case of green fodders as determined by Moores chromato-
graphic technique. There is practically no difference in the results when the chromatography is
carried out of the total carotenoids before phase separation or of the apparent carotene after phase
separation. In the case of dry roughages, silage or cattle faeces, the ordinary chemical extraction
method followed by phase separation with 90 per cent methyl alcohol does not give an idea of true
carotene, and an error is caused owing to the presence of non-carotene pigments which can be removed
by chromatography. In view of this fact the Seshan-Sen method of extraction of carotene has now
been combined with Moore's chromatographic method for the routine analysis of carotene in all
food materials (except where kryptoxanthon exists) without the necessity of phase separation with
methyl alcohol. The dicalcium phosphate adsorbent has been preferred to others because it can
be prepared easily and the filtration is very rapid. It is not however, suitable for separating lycopene.