D O C T O R A L DISSERTATION SERIES
X.
TITLE.

.

The

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Absorption Of

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M am ift Ki

And

The E f f e c t O f l i e k t ________

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Deter nnimdwh Of Vitamw V l h Fish

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UNIVERSITY.
DEGREE

L iv e r O ih

M tchiofah T!A &

Th.D.

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PUBLICATION NO

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DATE

UNIVERSITY MICROFILMS
U N I

ANN

ARBOR

MICHIGAN

_

I.

THE ULTRAVIOLET ABSORPTION OF VITAMIN K 1 AND THE
EFFECT OF LIGHT

II.

THE QUANTITATIVE CHROMATOGRAPHIC DETERMINATION OF
VITAMIN D IN FISH LIVER OILS

by
Frank Sargent Tomkins
A THESIS
Presented to the Graduate School of Michigan State College
Agriculture and Applied Science in Partial Fulfillment
of Requirements for the Degree of Doctor
of Philosophy

Department of Chemistry
Michigan State College
1942

ACKNOWLEDGMENT

The writer wishes to express his
appreciation to Dr. D. T. Ewing for his help
and guidance throughout this investigation; and
to Parke-Davis and Company, without whose assis­
tance the work would have been impossible.

THE ULTRAVIOLET ABSORPTION OF VITAMIN

AND THE EFFECT OF

LIGHT ON THE VITAMIN

The relationship between chemical structure and
ultraviolet absorption spectra is nowhere illustrated bet­
ter than in the study of vitamin K and related compounds.
One generalization which we presented in a previous publication

(1 )

, namely that the vitamin K absorption curve

presents a summation of the benzenoid and the quinoid
components of the disubstituted naphthoquinone molecule,
(2 )
recently has been extended by Morton and Sarlan
to the
anthraquinone series.

Because of the importance of this

aspect we have reinvestigated the structure of the ab-

.

sorption curve of the vitamin and have studied in detail
the influence of light and other factors such as the
presence of acetic acid which sometimes is added as a
stabilizer.

It was hoped also that the results of this

detailed study might clear up the controversy between
(3)(4)
(5)(6)
Karrer
and Doisy
regarding the absorption
coefficients of the pure vitamin.

Another object of our

work was to check the identity of the natural with the
synthetic vitamin.

2-

Apparatus and. Materials
The samples of vitamin K^t both natural and
synthetic, used in this Investigation were prepared by
Soisy and his associates at the Saint Louis University
School of Medicine.

They were examined with a Bausch

and Lomb medium quartz spectrograph and ultraviolet
sector photometer, with a Hilger No. H-698 hydrogen
discharge tube as the source of continuous ultraviolet
light.
The hexane used as the solvent in this study
was the Eastman "Practical” grade, which was purified
and redistilled.

The purification process consisted of

five to ten shakings with 10 $ fuming sulphuric acid, two
washings with 10 $ NagCOg solution, prolonged shaking
with 5$ KMn0^-10$NagC0g solution, fifteen to twenty
washings with distilled water, drying for twenty-four
hours over calcium oxide, and distillation twice over
freshly fused calcium chloride.

The purified hexane

boiled at 64.5-65.0°C* and its absorption spectrum between

X200 and X800

mfx

did not show the presence of impurities.

The source of irradiation for the study of the
effect of ultraviolet light on the vitamin consisted of
a General Electric mo. H-4 mercury arc lamp, a condensing
lens, a Cenco ultraviolet transmitting filter, and a
Cenco infra-red absorbing filter.

This combination

transmitted only the X365.5 and 366.3 myx lines of mercury.

3

Bausch and nomb lOrnm. absorption cells with
detachable quartz ends and monel metal fittings were
selected for this investigation.

The spectra were re­

corded on Eastman No. 40 plates, processed five minutes
in Eastman Developer Formula No. D-19.
Experimental Part
Specimens of the vitamin weighing between one
and two milligrams were dissolved in sufficient hexane to
give a 0.0025% concentration on the weight-volume basis.
Since in earlier work a trace of glacial acetic acid had
been added to all specimens of the vitamin for preserving
purposes and a question had arisen as to whether or not
the acetic acid was affecting the absorption and should
be removed, measurements were made both in the presence
and absence of acetic acid to disclose this effect if
present.

It was found that the presence of acetic acid

in amount equal to the weight of the vitamin had no
noticeable influence on the absorption curve.
In studying the effect of ultraviolet light on
the vitamin, the hexane solution was placed directly in
the absorption cell and the spectrum of the unirradiated
sample determined.

The cell containing the solution was

then exposed to the ultraviolet light generated as des­
cribed above, at a distance of 30 cm., for a definite
length of time, after which the cell was removed to the
spectrograph and the absorption spectrum again determined.

200

/00

240

280

Warn

FIG. 1

320

-L ength, m/i

ABSORPTION CURVE OF NATURAL
VITAMIN Kx IN HEXANE

360

4

Proceeding in this manner, a single sample served for a
complete ran, and errors involved in transferring volatile
hexane solutions from one container to another were
eliminated.
After samples of each solution were measured
they were placed in tightly stoppered flasks, weighed, and
stored in the dark.

These samples were re-run from time

to time to determine whether or not the vitamin decomposed
upon standing in the dark.
Figure 1 presents the detailed structure of the
absorption curve of the natural vitamin, and Figure 2 that
of the synthetic product.

It will be noticed that the

curves are essentially identical.
curves previously published

They differ from the

(1 )

in that they show a new

maximum at X239 mft and a minimum at A 240 mfi.

In both

cases the highest maximum is found at A 249 m fx and has
an extinction coefficient of 438.

The extinction co­

efficients of the many samples of vitamin

both na­

tural and synthetic, which we have evaluated during the
course of these investigations indicate that the
the pure vitamin is 435±5.

ot

This value is in good agree­

ment with that of 425, which we previously reported for
the synthetic product (Reference /IT page 356).

We feel

that this value more accurately represents the extinction
coefficient than that of 540 which we reported in the
same publication (Reference m

. page 350, Fig. 6 ).

400

300

to o

280

'Wave -Length,

FIG. 2.

320

360

rjyu

ABSORPTION CURVE OF SYNTHETIC
VITAMIN Kx IN HEXANE.

5-

It is known that vitamin

is affeeted by

light with loss of physiological activity and modifica­
tion of the absorption spectrum.

Ho systematic study

of the effect of light has been reported and little or
nothing is known concerning the chemical change that
occurs in the structure of the vitamin when exposed to
light.

Accordingly, we have made a study of the pro­

gressive changes that occur when hexane solutions of
the vitamin in a quartz container are exposed to ultra­
violet light over definite time intervals.
Figure 3 shows nine absorption curves for a
sample of natural vitamin Ki in hexane solution in the
absence of acetic acid which was exposed to A365.5 and
366.3 m/jL lines of mercury radiation, readings being
taken at 0, 15, 30, 45, 60, 90, 135, 195, and 255
minutes, respectively.

It will be noted that the ex­

posure produces a gradual lowering of the maxima at
AS39, 243, 249, 260, and 269 m^, and a less pronounced
decrease in the maximum at ^325 mp., indicating a
gradual decomposition of the vitamin.
Figure 4 shows a similar set of curves for a
sample of synthetic vitamin

containing a small amount

of acetic acid, determined under identical conditions as
those illustrated in Figure 3 .

The curves are essential­

ly the same except for a slight stabilizing effect of the

i

/c>"■

300

ki

lOO

230

320

W a r e -Le.ngf/i, mju

FIG. 3 ABSORPTION CURVES SHOWING THE
EFFECT OF ULTRAVIOLET RADIATIONS ON
NATURAL VITAMIN K, IN HEXANE

360

400

/cm .

300

/OO

240

320

360

Wave -Lenat,h, m u

FIG* k ABSORPTION CURVES SHOWING THE
EFFECT OF ULTRAVIOLET RADIATIONS ON
SYNTHETIC VITAMIN K-^ IN HEXANE

-6

aoetlo acid during the first few exposures.
In both of the above oases, the Initial effect
is most pronounced on the maxima at X260 and A 269 mpwhich we previously have shown to be associated with the
quinone structure (Reference j T f , page 350).

It follows,

therefore that the point of attack is through the quinone
grouping.

in connection with these figures it is interest­

ing to note that the nine eurves intersect at approximately
X277 mp, and that less definite iso-extinction coefficient
points occur at A230 mp. and AS05 nip.
J5io attempt was made to correct for the effect
of exposure of the sample to ultraviolet light from the
hydrogen discharge tube during the exposure of the plate,
because a series of runs made for the purpose showed that
the effect of this light was negligible.
(7)
MacCorquodale, Binkley, et al.

report that

vitamin Kg is unstable when exposed to light, and curves
were presented in our previous article showing the de­
terioration of the absorption curves of samples of
vitamins K-^ and K 2 when exposed to diffuse daylight.

In

order to determine, if possible, the wave lengths or
wave length of light causing this decomposition, samples
of vitamin K^ were exposed to various wave length regions
from infra-red to ultra-violet.

The results are shown

in the following table.

\

Source of
Radiation

Filters Used

He m a t Glower
N

If

Hone

n

ft
Hg Arc

Wratten
“A"
Wratten
»B»
"
Wratten
nQB
Cenco ultra­
violet transmit­
ting plus Cenco
infra-red absorb­
ing

Diffuse daylight

Hone

Effeot

3 hr s.

Zeiss R-30

Tungsten Filament
Lamp
it
n
n

Time

n

it

Slight decom­
position
Ho effect

45 min.

n

it

n

n

n

it

it

n

If

It

15 min.

Z

hr s.

Decomposition

Decomposition

These data show that light radiations between
^400 and 800 mfc have no appreciable effect on the vitamin.
The slight decomposition shown in the case of the Hernst
glower with no filter was probably due to the ultraviolet
light present in the radiation from the incandescent
filament of the glower.

Thus, the decomposition report­

ed earlier as being due to the effect of visible light
probably was due to the small amount of ultraviolet pres­
ent in diffuse daylight.
We reported previously that vitamin Kq. was un“
steble in hexane solution in the dark.

However, using

specially purified hexane, we can now report that vitamin

in dilute hexane solution is stable for periods up to
five months when stored in the dark at room temperature.

Discussion
Since js.arrer and Doisy first began publication
on vitamin K^ there has been a discrepancy between the
two laboratories concerning the correct value for
at
(8) l8m
A249 nut. in their first publication Dam et al.
gave
(5)
a value of 280 and McKee et al.
a value of 385. In
(3)(4)
subsequent publications Karrer
has claimed that
his vitamin preparation was pure and that 280 was the
correct value for the extinction coefficient.

In an

effort to discern the cause of the discrepancy, we have
made numerous measurements on both natural and synthetic
vitamin K^ samples which were prepared and supplied to
us by Doisy.

We find that the

(log EU = 4.29).

at X249 mfi. is 4353:5

This value is' in good agreement with

values reported by D. M. Bowen

(log Em - 4.24-4.27)

(in alcoholic solution) and T. J. Webb^9 ^(log
(alcoholic solution).

= 4.26)

We believe that this is the

correct value for either pure natural or synthetic vit­
amin K^ in hexane solution.
Of particular interest in connection with
the controversy between the two laboratories is the
1$
fact that both groups agree on the values for B-i£m
for
(1)(4)
vitamin K£
and for the diacetate of dihydro vitamin

(log E* a 4.i»)

(1)(4)

In view of our close agreement with other
laboratories it is Karrer's responsibility to explain
his low values for

for vitamin K^.

Prom a

structural point of view the chief difference between
vitamin

and vitamin Kg is the size of the side-chain

in the 3-position.

Since the side-chain in each ease is

aliphatic and contains no conjugated double bonds, the
absorption spectra for the two compounds would be ex­
pected to be quite similar*

If the absorption is due to

the naphthoquinone portion of the molecule and is not
influenced by the size of the aliphatic side-chain in
the 3-position, the

values for the two vitamins

should be inversely proportioned to their molecular
weights and the log %

values should be equal*

The

same reasoning holds true for the diacetates of vitamins
K^ and Kg.

Actually this is the case.

The log ^

values

for vitamins Kj_ and Kg are 4.27 and 4.29 respectively,
and the values for the corresponding diacetates are 4.93
and 4.93*

It is significant that the molar extinction

coefficients obtained for vitamins Zt and K 2 agree well
(9)
with values obtained by Tishler et al.
for three
crystalline 2 ,3-dial3cyl-1,4-naphthoquinones,

These

quantitative relationships are good evidence in support
of the correctness of our values.
A comparison of the absorption curves previous­
ly published by us

, as well as those now presented,

-1 0 (8 )

with that illustrated in the article by Dam, et al*
suggests that there might be a proportionate dis­
crepancy between the heights of the respective extinction
coefficients at X249 m p and X325 mp.
fact, the discrepancy is not serious*
was plotted as log
to enhance the

325 m

As a matter of
Karrer's curve

versus wave-length, which tends
maximum, and was compared in

this form with our curve which was plotted as
wave-length.

versus

This latter method shows better the fine

structure in the region X239 mp to X270 mp, but gives
a less pronounced maximum at X325 mp*
An examination of the absorption curves show­
ing the effect of ultraviolet light on solutions of
vitamin

gives a certain amount of information as to

the actual chemical change involved.

We hay’s stated

in the experimental part of this paper that the point of
attack probably is through the quinone grouping, and
may add that the X325 m p maximum which we previously
associated with the ring structure changes more slowly
than the rest of the curve.
2-Methyl-1,4-naphthoquinone upon exposure to
light for long periods of time is decolorized and
(1 0 )
forms a polymer of known structure
• It is possible
that a similar reaction occurs when vitamin
posed to light.

is ex­

-1 1 -

Summary
1.

A more careful examination of the absorp­

tion curve of vitamin

shows the presence of a new

maximum at A239 mp.*
2*

The

of pure vitamin E^ at A249 mjJ* is

3.

Vitamin E^ in hexane solution is stable

435±5.

upon standing in the dark at room temperature for as
long as five months.
4*

Vitamin E^ in hexane solution is decom­

posed rapidly by the action of ultraviolet light, while
visible and infra-red radiation have no effect.

The

point of attack probably is through the quinone group.
5.

The presence of acetic acid has no

noticeable effect on the absorption curve.
6.

on the basis of the absorption values re­

ported by Dam, Geiger, Glavind, Karrer, Rothschild, and
Solomon, their product appears to have been 60 to 80
per cent pure.
*********

12-

Ref erenoea
(1) Siring, 1. T. , Vandenbelt, J. M., and Kamm, Oliver,
J. Biol. Chem. 131, 345 (1939).
(2) Morton, R. A. and Sarlan, W. T., J. Ohem. Soo.
159 (1941).
(3) Karrer, P. and Geiger, A., Helv. ehim. Acta
22, 945 (1939).
(4) Karrer, P., Geiger, A., Legler, R . , Ruegger, A*,
and Solomon, H. , Helv. ehim. Aeta 22. 1464 (1939).
(5) McKee, R. W . , Binkley, S. B., MaoCorquodale, D. W . ,
Thayer, S. A., and Doisy, S. A#, J. Amer. Chem. Soo.
61. 1295 (1939).
(6 ) Binkley, S. B. , Mac Cor quo dale, D.. W . , Thayer, S. A.,
and Doisy, S. A . , J. Biol. Chem. 130, 219 (1939).
(7) MacCorquodale, D. W. , Binkley, S. B., McKee, R. W . ,
Thayer, S. A. , and Doisy, S. A . , Proc. Soc. Bxper.
Biol. & Med. 40. 482 (1939).
(8 ) Dam, H . , Geiger, A., Glavind, J., Karrer, P.,
Rothschild, S., and Solomon, H. , Helv. ehim. Acta
22, 310 (1939).
(9) Tishler, M . , Fieser, L. F . , and Wendler, N. 1.,
J. Amer. Chem. Soc. 62, 1982 (1940).
(10) Rev. Acad. Cienc. 31, 617 (1934).

-1 -

II
THE QUANTITATIVE CHROMATOGRAPHIC DETERMINATION OF
VITAMIN D IN FISH LIVER OILS
The quantitative determination of vitamin D
by chemical means has been the object of several inves(I)
tigations in recent years. Brockmann and Chen
report­
ed that vitamins Dg and Dg when treated with a solution
of antimony trichloride in chloroform give an orangeyellow color which soon reaches a maximum at X500 mjx,
and that the measurement of the absorption at this wave­
length gives a measure of the amount of vitamin present.
This procedure has been reinvestigated and
(2)
(a)
amplified by Marcussen
, Hield, Russell, and Zlmmerli ,
(4)
and Milas, Heggie, and Raynolds
. Ih each of these
reports it has been shown that the Brookmann and Chen
color reaction is applicable only where the vitamin is
present in the pure form or where interfering biological
materials have been reduced to very low concentrations.
The most troublesome of these interfering materials are
vitamin A, sterols, irradiation products of the sterols
other than vitamin D, and the tocopherols.

Among the

methods suggested for separation of such materials were
chromatographic adsorption, freezing out of sterols, and
the treatment of the sample with maleic anhydride for
the decomposition of vitamin A.

The method to be described here Involves an
improved chromatographic treatment to remove vitamin A,
the removal of interfering sterols by freezing and pre­
cipitation with digitonin, and the measurement of the
absorption at ^500 reproduced when the purified sample
is added to a solution of antimony triohloride in
chloroform containing a small amount of acetyl chloride.

Apparatus and Materials
The extinction coefficient measurements were
made on a Bausoh and Lomb polarization type visual
spectrophotometer.

The absorption cells were Bausch and

lomb one centimeter cells with glass spacers, quartz end
plates, and monel metal fittings.

Adsorption tubes for

the chromatographic procedure were made by sealing a
10 cm. length of 7 mm. pyrex tubing to the bottom of a
5/8nx 6 " pyrex test tube.
The SbClgCHClgCHgCOOl reagent was prepared
according to M e l d et al^3 ^, by dissolving 22g. of C.P.
antimony trichloride in purified chloroform, diluting
to 100 ml., and adding 2 ml. of redistilled acetyl chlor­
ide to the resulting solution.

The chloroform for this

reagent must be carefully purified and dried as follows:
Shake repeatedly with distilled water to remove the
alcohol, distill and discard the first quarter of the
distillate, reflux for two hours over P 2O 5 and filter.

3

The filtrate is shaken with activated carbon to remove
the small amount of phosgene formed during the refluxing
with P2°5* filtered and the filtrate redistilled.

The

purified chloroform should be stored in the dark.

This

product is relatively unstable and the purification
should be carried out on small lots.
The mixed solvent for the chromatographic pro­
cedure was prepared by adding 10 parts of C.P. anhydrous
ethyl ether to 50 parts (by volume) of commercial hexane
(Eastman Practical grade).
The ether was purified by washing repeatedly
with distilled water, distilling, and redistilling over
sodium.
The hexane was used as received.
The adsorbent was a fine grade of activated
Bentonite clay or "Superfiltrol".
Samples of fish liver oils and vitamin D
concentrate were supplied by Parke-Davis and Company.

Experimental
A sample of fish liver oil or concentrate to
contain 20,000 to 80,000 vitamin D units was weighed,
10 ml. of n/2 alcoholic KOH added, and the sample heated

for one hour on the steam bath.

The resulting solution

was cooled, 20 ml. of distilled water added, and the
solution extracted three times with 25 ml. portions of
ether.

The combined ether extracts were washed with

i

Dark blue--Yellowish greenV--Activated
bentonite
Cotton pad.

4- cm

Fig. 1. SCHEMATIC DIAGRAM SHOWING CHROMATOGRAPH
SET-UP AND APPEARANCE OF BANDS AT END OF RUN.

I

- 4-

distilled water In a separatory funnel until the wash­
ings gave no reaction with phenolphthalein, dried ever
anhydrous sodium sulfate, and filtered*
The filtrate was evaporated to dryness under
reduced pressure, the residue taken up in 10 ml* of
absolute methanol, and 5 ml. of the resulting solution
pipetted into a 15 ml. centrifuge tube*

The eentrlfuge

tube and metal holder were cooled to -15° C. in an
aoetone bath kept at this temperature by additions of
dry ice.

The cooled solution was centrifuged, the super­

natant liquid poured off, the preelpltate washed twiee
with 2 ml. portions of methanol— cooling and centrifuging
after each addition— and the liquid and washings trans­
ferred to a second 15 ml. centrifuge tube*
To the resulting 9 ml. of solution was added
1 ml. of distilled water and 2 ml. of 2# dlgitonin in

90# methanol solution and the mixture allowed to stand
at least two hours.

At the end of this time the mixture

was centrifuged and the precipitate washed twice with
2 ml. portions of 90# methanol; the washings were added
to the bulk of the separated liquid.

The solution was

then evaporated to dryness under reduced pressure and
the residue taken up in 5 ml. of the ether-hexane mixture.
The resulting solution was chromatographed as follows:
The adsorption tube was prepared by placing
a small cotton wad in the bottom of the tube and adding
the adsorbent to a depth of about 50mm. , tapping the tube

5

after each small addition to pack the adsorbent.

Slight

suetion (4 cm, Hg) was applied to the tube and 5 ml. of
the mixed solvent added to wet the adsorbent,

The 5 ml.

sample from the sterol separation was then added, followed
by 5 ml. of the solvent used to rinse the flash, and
finally 20 ml. of the solvent to develop the colored bands
formed in the adsorption tube.

£aoh addition of solvent

was made before the liquid from the previous addition had
quite disappeared, in order that drying out of the ad­
sorbent be prevented,

if the column becomes dry, further

addition of the solvent destroys the bands and the sample
is lost.

During the washing of the column with the last

20 ml. of solvent a very narrow and sometimes rather in­

distinct yellowish-green band appears at the lower edge
of the bine band formed by the vitamin A.

This band

provides a convenient reference point for the separation
of the column later and should be marked just before the
last of the wash solvent disappears.

When the last of

the liquid had gone through, the column was dried by
pulling air through it for five to ten minutes and the
upper portion of the column— to the mark previously m a d e removed with a bent spatula and discarded.
of the column contains all of the vitamin A.

This portion
The remain­

ing portion of the column was removed to a small erlenmeyer flask and the adsorption tube rinsed with 25 ml.
of anhydrous ether which was added to the same flask.
This ether-adsorbent mixture was shaken vigorously,

-6 -

allowed to stand until the adsorbent had settled, and the
supernatant liquid poured off through a coarse filter
into a second erlenmeyer.

This extraction was repeated

with three additional S5 ml. portions of ether and the
combined filtrate added to that from the adsorption pro­
cess.

This solution was evaporated to dryness under

reduced pressure, taken up in 10 ml. purified chloroform,
and 1 ml. of the resulting solution added to 10 ml* of
the antimony trichloride reagent.

The log IQ/l value of

the resulting Mixture at ^500 m^cwas determined, using
the Bausch and Lomb spectrophotometer, and the
value calculated.
The procedure may be interrupted after the
saponification and extraction, or after the extraction
of the lower portion of the adsorption column, when the
vitamin D active materials are in ether solution.

Such

a solution can stand overnight without measurable loss
in potency.

Data and Results
Table I shows the values obtained on a series
of seventeen fish liver oil and vitamin D concentrate
samples by the procedure described above.

The calculated

potency values are on the basis of sample ^44090 taken
as a standard.

In each case the

value given in the

table is the average value obtained from a set of two
duplicate samples.

Sample

Type

•“1 cm

Bio. potency
DUygm.

Cale. potency
DTT/gm.

44090 Vit. D Distillate

1.30

22,000

22,000

44080 Vit. D Distillate

1.74

31,000

29,500

47761 High vit. D Oil

1.01

15,000

17,100

41860 High vit. D Oil

.807

16,000

13,600

56021 High vit. D Oil

.670

13,000

11,300

55691 High vit. D Oil

.709

12,000

12,000

40090 High vit. D Oil

.912

20,000

15,400

18119 Bonita liver oil

1.09

36,000

18,400

55051 Vit. D Distillate

•859

18,500

14,600

44470 Tuna liver cone.

14.2

240,000

240,000

56961 Vit. D Distillate

1.05

20,000

17,800

57481 High vit. D Oil

•495

15,000

8,400

57951 Blue fin tuna oil

1.43

28,000

24,200

57971 Albacore liver oil

2.80

55,000

47,400

57991 Yellow fin tuna oil .946

3,000

16,000

58011 Bonita liver oil

3.20

65,000

54,000

57381 Tuna liver cone.

15.1

260,000

255,000

Table I
Sample #18119 was in very poor physical condition
— discolored and full of sediment and suspended partieles—
which may account for the very low result obtained in this
ease.

There is no obvious explanation for the low value

obtained for sample #57481 or the high value for #57991;
assuming that the biological value is correct as given.

-8

The values obtained by the chromatographic
procedure show good agreement with the biological values
— with the exception of the instances cited above— and
represent an appreciable saving in time and expense over
the biological assay method.

Discussion
Vitamin D containing biological materials such
as milk, butter, yeast, fish liver oils, etc., are ex­
ceedingly complex mixtures in which the vitamin D, although
it may be very active biologically, represents but a
minute fraction of the total amount of material present.
At the same time the vitamin "D" in the given material
may be any one of many vitamin D active substances, or a
combination of two or more such substances.
Fish liver oil, for example, is a mixture of
fatty acids, sterols, carotenoids, vitamin A, vitamin "D",
and in some instances tocopherols; in which, as it has
(5)
been shown by Brockmann and Busse
, the characteristic
antirachitic principle is vitamin D3 , the vitamin D$
being accompanied by varying quantities of vitamin Dg.
The problem of chemically determining the antirachitic
potency of such a sample then, offers two possibilities.
Either we must separate the vitamin D active principle
from the bulk of the material sufficiently well that the
interferences from these materials will be small, or we
must find a reaction specific to the vitamin D active

substance which will work in the presence of other bio­
logical materials and be sensitive to the small amount
of vitamin D present*

Since the first alternative seemed

to offer the most possibilities, it was along this line
that the work was directed.
The antimony trichloride color reactions,of
vitamins DQ and

are identical, as first stated by
2
ll)
13)
Brockmann and Chen
and confirmed by Hield et al
,

and the improved reagent described by the latter in the
same paper gives a greatly increased sensitivity to the
test.

The difficulty in the use of this reagent lies in

the fact that vitamin A, certain sterols, and fatty acids,
carotenoids, and tocopherols; all give color reactions
which interfere with the vitamin D color.

The vitamin A

color is particularly intense and troublesome.
(4) ""
Milas, Reggie, and Reynolds
describe a
method in which the non-saponifiable fraction of a fish
liver oil is heated with maleic anhydride in 1,4 dioxane
in order to destroy the vitamin A, carotenoids, and
possibly 7-dehydrocholesterol; after which the treated
non-saponifiable fraction is added to antimony trichloride
solution and the

at ^500 mp, taken as an indication

of the amount of vitamin D present.

Several samples were

run using this method'but consistent results could not
be obtained.

Upon further investigation it was found

that, contrary to the statement of the authors, maleie
anhydride in dioxane reacts with vitamin D under the

-1 0 -

eonditions recommended and results in the destruction of
part of the vitamin D as well as the vitamin A,

This

was tested by running samples of irradiated ergosterol
in corn oil, containing no vitamin A, with and without
the maleio anhydride treatment.
gave lower

The treated samples

values at >500 mjju with antimony tri­

chloride than did the untreated samples, as shown in the
accompanying Table II.
Sample No

Treatment

$
E1
lorn

As received

7.71

Non-saponifiable fraction
Milas procedure using fresh maleio
anhyd. and commercial dioxane

i*

Milas procedure with Eastman Kodak
Co. maleio anhyd. and commercial
dioxane
Milas procedure with fresh maleic
anhyd. and purified dioxane**
Heated 1 hr. with dioxane alone

8 .IE

S.79
3? 76
S. 59
7.5S
4.98

-tv

Heated 1 hr. with dioxane and .Eg
maleic anhyd.
Milas procedure

" d V

Milas procedure

3.50

" d b)

Milas procedure plus
heating after second
of KOH
Milas procedure plus
heating after second
of KOH

”( n b)

4. OS

15 min.
addition

4.87

15 min.
addition

3.63

Table II
* Sample #33809 is irradiated ergosterol in corn oil.
** The dioxane used in these tests was purified accord­
ing to: J. Amer. Chem. Soc. 58, SS64 (1936)

-1 1 -

These results show that in any case in which
the sample is heated with maleic anhydride, a breakdown
of the vitamin 3>g occurs; and that the breakdown is not
due to impurities in the dioxane.

The last four samples

were heated after the second addition of alcoholic KOH
to determine whether the breakdown product coaid be eonverted back to the free vitamin.

However, if the effeot

takes place it is very small--as shown by the very slight
increase in

value of the heated samples.

This

indicates that either the loss in vitamin Dg upon heat­
ing with maleic anhydride is not due to esterification
or that the vitamin Dg esters are not easily hydrolyzed.
Attention was next directed toward chromato­
graphic adsorption as a means of separating vitamin D
(2 )
from interfering materials, Marcus sen
reported that
an effective separation of vitamin A from vitamin D
could be made by adsorbing the vitamin A from a heptane
solution of the non-saponifiable fraction of a fish liver
oil.

Hydraffin K4 , an activated carbon, was used as the

adsorbent.

Since neither Hydraffin K4 nor heptane was

available, a series of tests were made using Norite A as
the adsorbent and hexane as the solvent, following the
Maroussen procedure in other respects.

It was found

that only a slight separation of the two vitamins was
possible by this method— both vitamins being very strong­
ly held by the Norite A.

Slightly better separation was

obtained when a small amount of ether was added to the

1.60

/.Z O

.80

450

550
Wdve-Leriy

650

th, rn

FIG. 2 ABSORPTION CURVE OF IRRADIATED
ERGOSTEROL IN CHLOROFORM TREATED WITH
SbCl^ REAGENT

1.60

UO

.40

450
W & v e -L e T ijth

,

650

750

ttj

FIG.
3 ABSORPTION CURVE OF CHOLESTEROL
IN CHLOROFORM TREATED WITH SbCl^ REAGENT

i

12

hexane, but there was still too slight a difference In
the adsorptive power of the Horite A, with respect to
the two vitamins, to make the separation complete*
Several other adsorbent-mixed solvent com­
binations were Investigated before the activated ben to nite-ether-hexane combination was found.

These Included

activated aluminum oxide, calcium hydroxide, dicalcium
phosphate, zinc carbonate, and bentonite; chloroform,
benzene, carbon tetrachloride, and hexane*

Of the ad­

sorbents, zinc carbonate, bentonite clay, and activated
bentonite were the only ones which gave positive results.
The action of the zinc carbonate was particularly
interesting because of the large number of sharply de­
fined bands which were visible in ultraviolet light after
the non-saponifiable fraction of a fish liver o il ha d
been run through a column of this material.

Ho sharp

separation of vitamins A and I) was obtained with this
adsorbent, but a more complete investigation of the bands
produced would make an interesting problem.

In each of

the above trials the adsorption column was cut into
sections, each section extracted with ether, the result­
ing solution evaporated to dryness and the residue
taken up in chloroform, and antimony trichloride solution
added.

A qualitative examination of the absorption

spectrum of the resulting solution, particularly in the
regions >620 and 500 m^i, gave an indication of the degree
of separation of vitamins A and D*

i

/. 60

M O

.60

.40

450

550

650

W&ve-Le-njth > m^J.

FIG.
k
ABSORPTION CURVE OF ADSORBATE
FROM CHROMATOGRAPH COLUMN IN CHLOROFORM
TREATED WITH SbCl^ REAGENT

750

8.0

6.0

4.0

2.0

360

FIG.

5 ABSORPTION CURVE OF IRRADIATED
ERGOSTEROL IN CORN OIL IN HEXANE

.80

.GO

AO

.ZO

320
280
W&ve-L enyth, mja.

FIG. 6 ABSORPTION CURVE OF ADSORBATE
FROM CHROMATOGRAPH COLUMN IN HEXANE

360

13-

The first positive separation was obtained
using bentonite as thb adsorbent and a hexane solution
of the saponified sample.

With this combination a blue

band was formed at the top of the adsorption eolumn
which when extracted and tested with antimony trichloride
solution, showed very strong absorption at A620 mj-i.
This was due to the presence of a large concentration of
vitamin A.

The lower portion of the column and the

filtrate, when tested in the same way, showed strong
absorption at ^500 mp and very slight absorption at
>620 mfi.

A better separation was obtained when a small

amount of ether was added to the hexane used as the sol­
vent.

This was to be expected since the adsorption pro­

cess had already shown the vitamin D to be less tenacious­
ly held by the bentonite, and consequently the de sorbing
action of the ether would be more effective toward this
vitamin.

The final modification of the adsorption

procedure was the substitution of activated bentonite
for bentonite as the adsorbent.

This is a finely divided

product which results in more uniform adsorption columns.
Figure 2 shows the absorption spectrum of
irradiated ergosterol (vitamin Dg), Figure 3 that of
commercial cholesterol, and Figure 4 that of the adsor—
bate from the chromatograph column-—all after treatment
with antimony trichloride solution.

It will be noted

that Figure 4 is the type of curve which would be expect­
ed to result from a mixture of the pure vitamin Dg and

14-

oholesterol.

This Is consistent with the statement by

earlier workers that sterols are among the Interfering
substances present in fish oils.,

Figure 5 and Figure 6

show the similarity between the ultraviolet absorption
curves of the adsorbate from a chromatograph column and
a solution of irradiated ergosterol in c o m oil, in hex­
ane.
Assuming that free sterols constitute the
greater part of the interfering materials left in such
an adsorbate, the next step was to find a means of re­
moving these sterols— either before or after the
chromatographic adsorption.

Freezing out of the sterols

from a methyl alcohol solution by cooling to -15° C. was
tried, but it was found that the removal by this means
was not complete.

The method finally adopted combined

the freezing process with a precipitation of the remain­
ing sterols with digit on in— before the adsorption.
Figure 7 is the absorption curve obtained from a sample
of the same oil as was used for Figure 4, after the
sterols had been removed by the above treatment.

It

shows a marked decrease in absorption in the region below
450 m , and more nearly matches the curve for the pure
vitamin.

It was this treatment that was incorporated

into the final procedure.

1.60

M O

L o*

t °/t

.40

550

6S0

750

W a v e - L e n y t A , m.

FIG.
7 ABSORPTION CURVE OF ADSORBATE
FROM CHROMATOGRAPH COLUMN IN CHLOROFORM
TREATED WITH SbClo REAGENT.
SAMPLE
TREATED WITH DIGITONIN

16

Summary
1*

A method fop the quant Itat lye determination

of vitamin D in fish liver oils has been presented.; in
which the oil is saponified, cooled and treated with
digitonin to remove sterols, chromatographed to separate
the vitamin A and D, and the vitamin D determined by
measuring the absorption at ^500 mjx produced when the
sample is added to a solution of antimony trichloride
in chloroform.
2*

A table is presented showing the results

obtained on a series of seventeen samples, using the
procedure outlined above.
*********

-1 6 -

References
(1)

Brookmann and Chen, Zelt. physiol. Chem.
241, 129-33 (1936)

(2)

Marcussen, Dansk. Tide, Farm, 13, 141-159 (1939)

(3)

Hield, Rassell, and Zimmerll, J. Biol. Chem.
136. 73-79 (1940)

(4)

Milas, Heggie, and Reynolds, J. Ind. Bng. Chem.
Anal* Ed. 13. £27-231 (1941)

(5)

Brookmann and Busse, Zelt. physiol. Chem.
256, 252 (1938)