THE ULTRAVIOLET IRRADIATION OP CALCIFEROL
IN VARIOUS SOLVENTS

By
Robert Yates

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
DOCTOR OP PHILOSOPHY
Department of Chemistry

1952

ACKNOWLEDGMENT

The author wishes to express his
sincere appreciation to Dr. D. T. E w i n g
for his encouragement and valuable guidance
throughout this investigation.
Grateful acknowledgment i s also
made to the Graduate Council for t h e grant
of a fellowship which aided in t h e c o m p l e t i o n
of this work.

##•##

THE ULTRAVIOLET IRRADIATION OF CALCIFEROL
IN VARIOUS SOLVENTS

By
Robert Yates

AN ABSTRACT

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemistry
Year

Approved ___________ ,

1952

f

Robert Yates

THESIS ABSTRACT

The irradiation of calciferol has been studied to some
extent previously but the reactions involved and their rela ­
tionships have not been completely elucidated.

This inves­

tigation was undertaken to study these reactions and their
relationships and to determine the effects of wavelength of
irradiating energy and solvent on the reactions.
The irradiations were conducted in ethanol, n-hexane and
a mixture of n-hexane and ethyl ether using several lines of
the m e r c u r y spectrum.

The absorption spectra of the irradiated

solutions were m easured at various time intervals and the com­
positions of the mixtures were estimated on the basis of
absorption spectra data.
Evidence was obtained for the presence of a substance
which has a spectrum similar to protachysterol w h i c h has not
previously been reported in the irradiated calciferol solu­
tions.

This substance appeared to be formed simultaneously

wi t h toxisterol and the suprasterols and to react photochem­
ically after its formation.
The nature of the solvent appeared to have no effect on
these reactions.

The wavelength of irradiating light appeared

to have no effect on the course of the reactions, although the
rate of conversion of calciferol varied to some extent.

TABLE OF CONTENTS

PAGE
INTRODUCTION ........................................

1

EXPERIMENTAL ........................................

5

Chemicals and Equipment .........................

5

Validity of Beer's Law as Applied to Calciferol .

6

Irradiation Procedure ...........................

7

Results ..........................................

7

DIS C U S S I O N ............................................ 19
SUMMARY................................................ 29
REFERENCES

30

1

INTRODUCTION
The conversion of ergosterol into an antlrachitically
active substance by means of ultraviolet light has been
studied rather extensively for a number of years.

Our pres­

ent knowledge of the process is largely the result of the
investigations of Windaus.

In 1930 » Windaus, G-aede, Kflser,

and Stein (1) announced the isolation of two crystalline
irradiation products which had no antirachitic activity and
which showed no ultraviolet absorption at wave lengths
greater than 250 mu.

These compounds were named suprasterols

I and II because they were formed by over-irradiation of
ergosterol.

Some time later, Askew and co-workers (2) and

Windaus and co-workers (3 ) announced the isolation of active
crystalline products by quite different methods.

These pro­

ducts later proved to be different, although both were found
to contain the true vitamin D.

The product Isolated by

Windaus and co-workers (4*, 5> 6), called vitamin

was

shown to be an addition compound of the vitamin, now called
vitamin D£, and an Isomeric alcohol, called lumlsterol.

In

1932, Windaus, von Werder and Lftttringhaus (7) obtained
tachysterol from the irradiation mixture.

Its high reacti­

vity with oxygen led Windaus to believe tachysterol preceded
calciferol in the sequence of reactions, since the presence

2

of oxygen in the process reduces the yield of vitamin.
Dimroth (8) found that on short irradiation both ergosterol
and lumisterol yield a substance resembling tachysterol in
its absorption spectrum.

In 1932, Windaus, Lflttringhaus

and Busse (9) isolated toxisterol as an oil and concluded
that it was an Irradiation product of the vitamin Itself
because vitamin preparations which were over-irradiated had
a higher toxic factor than those which were not.

Windaus

and Auhagen (10) obtained evidence for the existence of
another substance, which they called "protachysterol," in
irradiated ergosterol solutions.

This substance appeared

to be transformed both thermally and photochemically into a
compound resembling tachysterol in its absorption spectrum.
Windaus and co-workers (7) concluded that the following se­
quence of reactions was involved in the Irradiation of
ergosterol.
^Suprasterol I
Ergosterol->Lumlsterol-^Tachysterol->Vitamin D-»Suprasterol II
\
Toxisterol
The Importance of the vitamin and its formation from
ergosterol caused the primary interest in the problem to cen­
ter around the early stages of the process, with the consequence
that only minor stress was placed on the decomposition of the
vitamin Itself.

The futility of the experiments conducted

3

by Windaus and Auhagen (11) and by Windaus, Busse, and
Weidllch (17) also deferred interest in this phase of the
process.
In 1943, Dimroth and Stockstrom (1 3 ) attempted to study
more carefully the photo-conversion of the vitamin into
toxisterol and the suprasterols.

They irradiated an ethanol

solution of a synthetic model (I) which contained the same
nuclear arrangement of unsaturation as the vitamin (II).
They concluded that the decomposition of calciferol followed
the sequence
Calciferol — > Toxisterol — > Suprasterols
and proposed structure III as that representing toxisterol.

I

n

m

Recently Green (14) studied the Irradiation of calci­
ferol in ether and benzene using the direct antimony tri­
chloride method for determining concentrations of calciferol.
Throughout these experiments he observed the absorption
spectra of the mixtures, but found no evidence for the pres­
ence of toxisterol.

However, he obtained from irradiated

solutions another material which he called suprasterol III.

4

The factors governing its formation were unknown as indicated
by the varying amounts formed under similar conditions*
Green also observed in the irradiation of ergosterol in
ether and benzene that maximum potency of the product was
obtained after about the same length of time in both ether
and benzene, this maximum potency being about one and onehalf times as great in ether as in benzene.

Bills, Honeywell

and Gox (15 ) had previously observed a solvent effect in the
irradiation of ergosterol in ether, cyclohexane and ethanol.
The maximum potency was reached in ethanol more rapidly than
in cyclohexane, and most slowly in ether.

The maximum po­

tency was greater in ether than in cyclohexane or ethanol.
Green also observed a solvent effect in the Irradiation
of calciferol in benzene and ether, the rate of destruction
being slower in benzene than in ether, which may be due to
the ultraviolet absorption properties of benzene.
The reactions which occur when ergosterol is irradiated
have been s h o w to be very complex.

They include the forma­

tion and subsequent decomposition of calciferol.

The latter

has been studied to some extent but the reactions which
occur when calciferol is irradiated have not been completely
elaborated.

The purpose of this investigation was to study

the relationship between the reactions which occur when cal­
ciferol is Irradiated with ultraviolet light and to determine
what effects, if any, solvent and activating wave length have
on these reactions.

EXPERIMENTAL
Ohemloals and Equipment
1*

The calciferol used in this investigation was ob­

tained as "Deltaxin" from Sterwin Chemicals, Inc., New York
City, New York.

This product was purified by recrystalliza­

tion from ethanol and stored at about -20° C. under carbon
dioxide.
The value of E

1 cm.) at 265 mu of the purified

calciferol (Figure 1) was found to be 480 in ethanol and
474 in hexane.

No change in the absorption spectrum was ob­

served over a period of several months when the calciferol
was stored as described.
2.

n-Hexane, obtained as "Skellysolve B a from the

Skelly Oil Company, 3711 California Avenue, Chicago, Illinois,
was purified by the method of Mair and White (16), using a
chromatographic column (4 cm. in diameter by 75 cm* In
length) of silica gel which had been activated by heating
at 250 ° for 24 hours.
3»

Anhydrous ethyl ether was obtained in purified

state by distilling the commercial anhydrous ethyl ether
(C. P.) from a mixture of anhydrous sodium sulfite (C. P.)
and sodium hydroxide immediately prior to use.
4.

Commercial absolute ethanol was purified by first

removing the aldehydes with silver nitrate and sodium

~

i
o

300

—

rH

H

200

100

260
280
WAVE LENGTH, MILLIKICRONS
FIGURE 1
ABSORPTION SPECTRA OF CALCIFEROL IN
(1) ETHANOL AND (2) HEXANE

hydroxide (l?) and then dehydrating with amalgamated
aluminum foil (18).
5.

The absorption spectra were obtained using a

Beckman Quartz Spectrophotometer, Model DU, with a hydrogen
discharge lamp source.

Figure 2 is a diagram of one of the

absorption cells used.

These were composed of the elements

of cells designed for use with the Bausch and Lorab Sector
Photometer.

Each cell consisted of two optically flat

quartz discs separated bj' a 0.5 cm. glass spacing ring and
held in place by a brass holder.

The cell3 were mounted in

a special brass cell carriage for use with the Beckman
spectrophotometer.
6.

The Bausch and Lorab Grating Monochrometer, cata­

logue number 33-86-40, equipped with a Hanovia Quartz Alpine
Sun Burner, type S-100, was used as a source of monochro­
matic radiation.

The monochrometer has an equivalent aper­

ture of f/4.4, a focal length of 250 mm. and linear dispersion
of 66 A. per mm.

The grating has a ruled surface of 50 x 50

mm., containing 600 grooves per mm., blazed for first order
in the range 200-400 mu.
The Validity of Beer*8 Law as Applied to Oaloiferol
Five solutions of calciferol in n-hexane containing
2.09, 1.67, 1.26, 0.84 and 0.42 mg./lOO ml., and five solu­
tions of calciferol in ethanol containing 2.00, 1.60, 1.20,
0.80 and 0.40 mg./lOO ml. were prepared and their absorption
spectra were measured over the wave length range 2 3 0 -3 0 0 mu.

FISURS 2
DIASRAM OF A330RP7IOH CELL
1.
?.
3.
k.

0.5^ cm. CLASS SHASIUQ RIEC
QUARTZ DISKS
TEFLON PRESSURE RIN5S
BRA3S HOLDER

1

I om.

1

7

It was found that the measured extinctions of the solutions
varied directly with concentration at wave lengths 230 mu,
250 mu, 265 mu and 280 mu as shown in Figure 3 and Figure
Irradiation Procedure
A standard irradiation procedure was devised after some
preliminary experimentation to determine the most practical
arrangement.

This general procedure consisted of placing

the absorption cell containing the solution to be irradiated
at the point of focus of the monochromator beam.

After the

desired time interval, the absorption spectrum of the solu­
tion was measured and the cell was replaced in the monochro­
mator beam for the next time interval.
It was found most convenient to conduct all Irradiations
with entrance and exit slit widths of 2.5 mm., the exit beam
thus having an effective band width of 16.5 mu.

The approxi­

mate irradiation times at which the absorption spectra were
measured were 0 , 1 0 , 2 0 , 3 5 , 6 0 , 9 5 , 1^5 and 210 minutes.
The irradiations were carried out in ethanol, n-hexane
and a mixture of ethyl ether and n-hexane (one part by volume
ether to 19 parts hexane).

Monochromatic light of wave

lengths 24-83, 2537, 2 6 5 4 , 2753, 29 67 and 3132 A. were used
for each solvent system.

All initial concentrations of cal­

ciferol were of the order of 2 mg./lOO ml.
Results
The absorption spectra of the irradiated solutions of
calciferol in ethanol are shown in Figures 5-10, those for

i

s
o
M
EH

g

H
X
w

0.300

Eh

0.200

0.100

0

O.hO
0.80
1.20
1.60
CONCENTRATION, mg./lOO ml.

2.00

FIGURE 3
RELATIONSHIP BETWEEN EXTINCTION AND
CONCENTRATION FOR CALCIFEROL IN HEXANE AT
(1 ) 230 mu, (2 ) 250 mu, (3 ) 26 5 mu, (L) 280 mu.

o.;oo

g

0 .3 0 0

0 .2 0 0

0 .1 0 0

0
0

0.80

1 .2 0

1.60

2.00

CONCENTRATION, mg./lOO ml.
FIGURE 4
RELATIONSHIP BETWEEN EXTINCTION AND
CONCENTRATION FOR CALCIFEROL IN ETHANOL AT
(1 ) 2 3 O mu, (2) 230 mu, (3 ) 2 6 5 mu, (*0 280 mu.

8

the irradiations in hexane in Figures 11-16, and those for
the irradiations in hexane-ether mixture in Figures 17-22.
A number of these experiments were repeated and the
results were found to agree generally although variations
in the rates of decrease of calciferol concentrations were
observed.

These variations were attributed to variations in

the intensity of the irradiating light.
The calculated percentages of toxisterol, calciferol,
protachysterol and combined suprasterols for the various
Irradiations conducted are recorded in Tables I-XVIII along
with the E (l$, 1 cm.) values upon which these calculations
are based.
The method of calculation of the composition of the
irradiation mixtures will be discussed later.

21*0

260
280
WAVE LENGTH, mu

30°

FIGURE 5
IRRADIATION IN ETHANOL WITH Hg 2^83 A. LINE
0 MINUTES
(1)
(2) 10 MINUTES
(3) 20 MINUTES
35 MINUTES
W

15) 60 MINUTES
(6)
95 MINUTES
(7) 19'5 MINUTES
(8) 210 MINUTES

cm

■300

pH

W 200

100

280
260
WAVE LENGTH, mu
FIGURE 6
IRRADIATION IN ETHANOL WITH Hg 253? A. LINE
(1)
(2)
(3)
(4)

0 MINUTES
10MINUTES
22 MINUTES
3? MINUTES

(5) 60 MINUTES
(6 ) 95 MINUTES
(7) 145 MINUTES
(3) 205 MINUTES

200

100

260

300

280

WAVE LENGTH, rau
FIGURE 7 IRRADIATION IN ETHANOL WITH Hg 2654 A. LINE
(1 ) 0 MINUTES
1 2 ) 13 MINUTES
(3) 20 MINUTES
(4) 35 MINUTES

(5) 60 MINUTES
(6 ) 97 MINUTES
(7) 145 MINUTES
(8 ) 210 MINUTES

I

300

200

100

0
280

260

300

WAVE LENGTH, mu
FIGURE 8
IRRADIATION IN ETHANOL WITH Hg 2753 A. LINE
(1) 0 MINUTES
(2) 11 MINUTES
(3 ) 20 MINUTES
W
35 MINUTES

60 MINUTES
(5)
16)
95 MINUTES
17) 145 MINUTES
18) 210 MINUTES

cm

-300

1—
1

200

100

240

280
260
WAVE LENGTH, mu

300

FIGURE 9
IRRADIATION IN ETHANOL WITH Hg 2967 A. LINE
(1 ) 0 MINUTES
(2 ) 10 MINUTES
13) 20 MINUTES
W
35 MINUTES

15) 60 MINUTES
(6 ) 113 MINUTE 8
(?) 147 MINUTES
(8 ) 210 MINUTES

rH

xoo

260
280
WAVE LENGTH, mu

300

FIGURE 10
IRRADIATION IN ETHANOL WITH Hg 31J2 A. LINE
(1 ) 0 KINUTES
(2 ) 10 MINUTES
(3) 20 KINUTES
(A) 35 MINUTES

(5) 60 MINUTES
(6 ) 95 MINUTES
(?) 165 MINUTES
(8 ) 216 MINUTES

cm

H
W 200

100

260

280

300

WAVE LENGTH, mu
FIGURE 11
IHHADIATIGH IK HEXANE WITH Hg 2683 A. LINE
(1) 0 MINUTES
(2) 10 MINUTES
(3) 23 MINUTES
(A) 35 MINUTES

(5) 60 MINUTES
(6 ) 95 MINUTES
(?) 165 MINUTES
(8 ) 210 MINUTES

cm

300

h 200

100

2*t0

280
260
WAVE L ENGTH, mu
FIGURE 12

IRRADIATION IN HEXANE WITH Hg 2537 A. LINE
(1) 0
MINUTES
(2) 10
MINUTES
(3) 20
MINUTES
(it) 35 MINUTES

(5) 65 MINUTES
(6 ) 105 MINUTES
(7) 160 MINUTES

cm

*300

r—f

200

100

240

280
260
WAVE LENGTH, mu
FIGURE 13

IRRADIATION IN HEXANE WITH Hg 2654 A. LINE
(1) 0 KINUTES
(2) 10 MINUTES
(3) 20 MINUTES
(4) 35 MINUTES

(5) 60 MINUTES
(6) 95 KINUTES
(7) 1^5 KINUTES

2 A0

300

260
280
WAVE LENGTH, mu
FIGUHE 1A

IRRADIATION IN HEXANE WITH Hg 2753 A. LINE
(1) 0
(2) 10
(3) 20
(A) 35

MINUTES
MINUTES
KINUTES
MINUTES

(5)
(6 )
(7)
(8 )

60
97
1^5
213

MINUTES
MINUTES
MINUTES
MINUTES

(l£,

1 OH.)

400

200

100

240

260

280
WAVE LENGTH, mu

300

FIGURE 15
IRRADIATION IN HEXANE WITH Hg 296? A. LINE
(1) 0 MINUTES
(2) 13 KINUTES
(3) 20 KINUTES
(4) 35 KINUTES

(5) 62 MINUTES
(6 ) 95 KINUTES
(7) 145 MINUTES
(8 ) 210 MINUTES

cm

•3 0 0
-7

w.
rH

100

280
260
WAVE LENGTH, mu
FIGURE 16
IRRADIATION IN HEXANE WITH lig 3112 A. LINE
(1 ) 0 MINUTES
(2 ) 10 MINUTES
(3) 20 MINUTES
(6 ) 60 MINUTES

(5) 60 MINUTES
(6 ) 96 MINUTES
(7) 165 MINUTES
(8 ) 210 MINUTES

cm

400

300

u 200

100

0
260
280
WAVE LENGTH, mu

300

FIGURE 17
IRRADIATION IN HEXANE-ETHER WITH Hg 24 83 A. LINE
11) 0 MINUTES
(2) 11 MINUTES
(3) 20 MINUTES
(4) 35 MINUTES

(5) 60 KINUTES
(6 ) 90 MINUTES
(7) 145 MINUTES
(8 ) 210 KINUTES

2 30 0

100

260
280
WAVE LENGTH, nm

300

FIGURE 16
IRRADIATION IN HEXANE-ETHER WITH Hg 253? A. LINE
(1 ) 0 MINUTES
(2 ) 10 MINUTES
(3) 21 MINUTES
(4) 36 MINUTES

55 MINUTES
(5)
(6 ) 96 MINUTES
(?) 150 MINUTES
(8 ) 218 KINUTES

400

2 300

100

260

230
WAVS LENGTH, mu

300

FIGURE 19
IRRADIATION IN HBXANE-ETHER WITH Hg 2654 A. LINE
(1 ) 0 MINUTES
(2 ) 10 MINUTES
(3) 20 KINUTES
(4) 35 MINUTES

(5) 66 KINUTES
(6 ) 93 MINUTES
(7) 144 MINUTES
(3) 210 MINUTES

rH

rH

w 200

100

260
230
WAVE LEMOTH, rau

300

FIOURE 20
IRRADIATION IN KHXANE-ETH1CR 'WITH Hg 2753 A. LINE
(1) 0 MINUTES
(2) 10 KINUTES
(3) 20 KINUTE3
(A) 75 MINUTES

(5) 65 MINUTES
(6 ) 112 MINUTES
(7) 150 MINUTES
(3) 210 KINUTES

cm.

400

300

M 200

100

260

230

300

WAVS LENGTH, rau
FIGURE 21
IRRADIATION IN HEXANE-ETHER WITH Hg 2967 A. LINE
(1 ) 0 MINUTES
(2 ) 10 MINUTES
(3) 21 MINUTES
(4) 35 MINUTES

(5) 62 MINUTES
(6 ) 100 MINUTES
17) 145 KINUTES
(3) 205 MINUTES

a 300

w 200

100

0
260

230

300

WAVS LENGTH, mu
FIGURE 22
IRRADIATION IN HEXANE-ETHER WITH Kg 3132 A. LIN'S
(1) 0 MINUTES
(2) 10 MINUTES
(3) 20 MINUTES
(6 ) 35 MINUTES

(5) 60 MINUTES
(6 ) 100 KINUTE3
(7) 1^5 MINUTES
(8 ) 210 MINUTE3

TABLE I
IRRADIATION IN ETHANOL WITH Hg 2483 A. LINE
Initial concentration:

t

0
10
20
35
60
95
145
210

t

Ei

e2

*3

E4

406
361
316
274
229
187
149
130

480
417
353
295
231
174
130
109

341
304
266
231
193
151
121
102

248
240
231
223
209
194
176
163

2.07 mg./lOO ml.

T

D

P

3
7
11
14
17
19
18
17

97
79
60
44
26
15
5
4

0
6
13
19
25
24
25
22

S
9
32
55
73
94
108
114
112

= Irradiation time

Ei = E M ,

1 cm.) at 250 mu

T = % concentration of toxisterol

e2 = E W ,

1 cm.) at 265 mu

D = % concentration of calciferol

E-j

E W ,

1 cm.) at 280 mu

P = % concentration of protachysterol

E W ,

1 cm,) at 230 mu

S a % concentration of suprasterols

TABLE II
IRRADIATION IN ETHANOL WITH Hg 2537 A. LINE
Initial concentration: 2.08 mg./lOO ml.

t

Ei

e2

0
10
22
37
60
95
145
205

404
359
304
270
222
177
142
121

480
417
348
289
220
157
111
86

348
304
260
230
184
139
100
78

S

E4

T

D

P

250
240
229
220
208
192
176
162

4
6
8
14
18
20
20
19

93
79
62
41
24
11
5
3

6
6
11
22
24
24
19
16

10
33
59
76
99
115
124
124

S

TABLE III
IRRADIATION IN ETHANOL WITH Hg 2654 A. LINE
Initial concentration: 2.08 mg./lOO ml.

t

0
13
20
35
60
97
145
210

Ei

E2

E3

E^

T

D

P

409
316
282
232
185
148
127
112

480
362
323
249
179
122
84
57

349
277
252
20 5
156
111
77
50

248
220
210
196
186
175
170
164

6
9
9
13
17
19
22
23

92
60
50
31
15
6
2
1

6
17
19
24
25
22
16
10

7
38
48
71
99
116
131
139

TABLE IV
IRRADIATION IN ETHANOL WITH Hg 2753 A. LINE
Initial concentration: 2.08 mg./lOO ml.

t

Ei

E2

0
11
20
35
60
95
145
210

407
362
329
296
244
197
166
145

480
425
383
327
258
189
139
101

h

346
311
287
253
208
162
123
89

E4

251
236
228
219
218
207
203
202

T

D

P

5
5
7
12
14
18
20
23

94
80
68
52
35
18
9
5

4
8
12
18
22
24
22
16

10
24
38
54
92
115
138
159

S

S

TABLE V
IRRADIATION IN ETHANOL WITH Hg 296? A. LINE
Initial concentration: 2.08 mg./lOO ml.

t

0
10
20

P
60
113
147
210

Ei

e2

E3

408
372
338
300
248
178
163
129

480
446
408
356
295
203
168
114

341
322
302
272
230
170
139
96

Ei}.
248
236
225
210
193
170
166
156

T

D

P

4
2
3'
5
5

97
88
76
60
47
24
20
11

0
4
10
16
17
22
17
14

8

11
14

9
15
22
32
48
72
85
103

TABLE VI
IRRADIATION IN ETHANOL WITH Hg 3132 A. LINE
Initial concentration: 2.08 mg./lOO ml.

t

Ei

0
10
20
35
60
95
165
216

422
400
392
368
334
303
244
219

480
454
444
415
378
339
266
234

E3

E4

T

D

P

337
320
314
298
276
252
202
181

266
2 57
253
243
231
220
199
190

8
8
8
9
9
10
10
11

99
93
90
81
71
60
44
36

-3
_9
-1
3
7
10
11
13

S

26
31
32
44
53
69
76

TABLE VII
IRRADIATION IN ETHANOL WITH Hg 2483 A. LINE
Initial concentration: 2.18 mg./lOO ml.

t
0
10
23
35
60
95
145
210

E1

E2

E3

E4

T

D

P

397
36O
316
285
234
192
156
129

474
421
360
316
246
186
139
104

327
303
272
248
207
167
129
96

236
232
227
222
212
197
184
166

2
6
10
12
16
18
18
18

100
80
60
47
28
14
7
5

3
13
21
26
31
32
28
20

S
-6
16
41
59
84
100
103
103

TABLE VIII
IRRADIATION IN HEXANE WITH Hg 2537 A. LINE
Initial concentration: 2.18 mg./lOO ml.

t

El

e2

E3

e4

T

0
10
20
35
65
105
160

400
346
307
259
198
160
125

474
396
339
271
181
124
80

325
282
247
206
146
103
65

239
231
224
215
203
190
174

2
8
11
14
19
21
21

D

P

S

2
10
13
17
19
16
9

0
30
5.2
76
112
128
138

D

P

S

98
76
57
45
22
12
6

4
10
14
14
16
12
6

6
34
63
83
117
141
157

100
77
62
44
23
12
8

TABLE IX
IRRADIATION IN HEXANE WITH Hg 2654 A. LINE
Initial concentration: 2.18 mg./lOO ml.

t
0
10
20

P

60
95
145

Ei
403
345
293
254
190
153
126

=2

*3

474
392
319
266
172
110
66

328
279
234
199
137
89
51

T
246
234
224
217
204
196
186

4
8
12
14
18
22
24

TABLE X
IRRADIATION IN HEXANE WITH Hg 2753 A. LINE
Initial concentration: 2.18 mg./lOO ml.

t

0
10
20
35
60
97
145
213

Ei
397
359
326
280
227
177
141
119

474
426
384
320
248
174
119
79

E3

e4

T

D

P

328
302
277
241
204
146
106
71

235
230
220
210
196
182
177
171

2
4
5
8
12
15
18
20

99
84
73
54
31
19
8
4

4
9
12
18
28
22
20
14

-6
14
24
47
68
94
120
133

3

3

TABLE XI
IRRADIATION IN HEXANE WITH Hg 2967 A. LINE
Initial concentration: 2.18 mg./lOO ml.

t

0
13
20
35
62
95
145
210

Ei

Eg

E3

E^

T

D

P

396
353
338
303
253
208
1 65
129

474
430
408
363
299
238
177
125

333
308
296
270
228
187
143
103

236
226
221
210
196
181
174
164

2
1
3
4
5
7
9
11

96
84
77
64
50
36
24
• 15

8
12
14
18
19
20
18
14

-6
8
14
25
45
62
87
106

TABLE XXX
IRRADIATION IN HEXANE WITH Hg 3132 A. LINE
Initial concentration: 2.18 mg./lOO ml.

t

%

0
10
20
40
60
96
145
210

403
389
376
352
327
290
238
195

474
458
441
414
385
340
276
2 21

*3

e4

T

D

321
311
303
286
268
240
198
161

240
234
229
218
214
199
177
163

3
3
4
3
3
4
4
5

103
99
93
86
79
68
53
41

P

-2
-1
2
3
4
7
8
8

S

-1
1
6
8
20
29
39
54

TABLE XIII
IRRADIATION IN HEXANE-ETHER WITH Hg 2483 A. LINE
Initial concentration: 2.07 mg./lOO ml.

t

0
11
20

P
60
90
145
210

Ei

E2

402
356
323
279
231
190
147
118

474
414
367
302
228
174
121
89

%

%

328
293
265
230
183
145
105
76

240
235
231
223
210
200
182
165

T
4
6
8
13
18
19
19
17

D
99
82
69
49
30
19
10
7

P
4
9
12
20
23
22
18
13

S
-1
25
46
69
86
113
122
124

TABLE XIV
IRRADIATION IN HEXANE-ETHER WITH Hg 2537 A. LINE
Initial concentration: 2.07 mg./lOO ml.

t

Ei

e2

E3

32/*

T

0
10
21
36
55
96
150
218

401
332
280
226
190
145
114
90

474
378
302
228
173
110
72
47

325
270
226
180
142
93
58
35

240
229
218
207
199
183
166
151

3
8
13
16
19
20
19
17

D

100
73
51
32
20
10
7
5

P

S

2
10
16
20
20
15
8
3

0
36
64
91
112
129
135
135

P

S

TABLE XV
IRRADIATION IN HEXANE-ETHER WITH Hg 2654 A. LINE
Initial concentration: 2.07 mg./lOO ml.

t
0
10
20
35
66
98
144
210

E1

e2

E3

402
326
271
220
165
138
118
102

474
374
305
229
144
99
67
42

325
273
230
184
123
87
58
34

Eij,
242
226
214
202
190
183
177
167

T
3
8
9
14
19
21
22
22

D
100
69
51
31
13
6
3
2

2
14
18
22
20
16
11
6

2
36
60
85
117
133
147
150

TABLE XVI
IRRADIATION IN HEXANE-ETHER WITH Hg 2753 A. LINE
Initial concentration: 2.07 mg./100 ml.

t

Ei

E2

E3

E/j,

T

0
10
20
35
65
112
150
210

400
356
316
264
198
146
126
110

474
415
362
296
204
125
93
63

325
292
262
223
164
108
82
55

243
230
219
20 7
190
178
171
168

2
5
7
9
13
17
19
20

D
101
83
68
5.0
27
11
6
3

P

S

2
7
12
17
20
19
16
11

2
18
86
114
127
139

P

S

TABLE XVII
IRRADIATION IN HEXANE-ETHER WITH Hg 29 67 A. LINE
Initial concentration: 2.07 mg./lOO ml.

t
0
10
21
35
62
100
145
205

Ei

E2

e3

e^

T

400
370
336
300
241
183
140
105

474
438
398
352
280
204
147
99

326
306
283
254
208
159
118
81

241
223
218
208
187
164
154
144

3
3
4
5
6
8
9
10

D
100
89
78
67
50
31
20
12

2
6
9
11
14
16
14
11

0
0
16
30
47
62
84
99

TABLE XVIII
IRRADIATION IN HEXANE-ETHER WITH Hg.3132 A..LINE
Initial concentration:

t
0
10
20

P
60
100
145
210

El
402
388
376
361
329
289
254
210

2.07 mg./lOO ml.

%

*3

E4

T

D

474
463
447
425
391
342
297
243

324
315
309
297
276
246
216
181

242
237
232
225
213
200
187
172

3
1
2
4
3
4
4
5

101
100
93
87
78

66
55

43

P
1
0
4
6
8
10
11
12

S
1
5
6
9
15
29
39
51

oo

19

DISCUSSION
The absorption spectra of the products of the irradia­
tion of ergosterol, with the exception of that for protachysterol, are quite characteristic.

The measurement of the

absorption spectra of irradiated calciferol solutions is a
convenient method for determining the composition of the mix­
tures obtained.

The validity of the results of this method

are dependent upon the applicability of Beer's Law to the
mixtures obtained.

The relationship of concentration to ex­

tinction for solutions of pure calciferol in hexane and
ethanol has been determined and the results indicated that
Beer's Law could be applied over the concentration range 0.4
- 2 . 0 mg./lOO ml. and over the wave length range 230-300 mu.
On the basis of these results and the observations of others
in the cases of other sterols, it was assumed that mixtures
obtained by the irradiation of calciferol would also follow
Beer's Law.
Careful observation of the absorption spectra of irradi­
ated calciferol solutions indicated that as the irradiation
proceeded the rate of decrease of the extinction at 280 mu
was not proportional to, but was somewhat less than propor­
tional to, the rate of decrease of extinction at 265 mu.
Because of the nature of the absorption spectra of toxisterol

i

20

and the suprasterols it would be expected that the rate of
decrease of the extinction at 280 mu would be slightly greater
than the rate of decrease of extinction at 265 mu.

Therefore,

a logical conclusion was that a substance other than toxisterol and the suprasterols was being formed and that the
spectrum of this substance had a higher extinction at 280 mu
than at 265 mu.
It was obvious that a spectrophotometric analysis of the
mixtures must include a determination of the concentration of
this component.

In order to accomplish this the absorption

spectrum of the substance must be known.

The sterols most

likely to be associated with calciferol in the irradiation
mixtures were those arising from the irradiation of ergosterol.

Of these sterols, those which satisfied the condition

that the extinction at 280 mu be greater than that at 265 mu
were ergosterol, lumlsterol, taohysterol and protachysterol,
and perhaps others which have not yet been isolated.

In ad­

dition, there was the condition that the absorption spectrum
calculated on the basis of the relative concentrations of
the various substances must agree with the experimentally
determined absorption spectrum.

The closest agreement be­

tween observed and calculated absorption spectra in the range
250-300 mu was obtained using in the calculations the absorp­
tion spectrum of protachysterol as that representing the
substance being formed.

21

Additional evidence that the compound being formed was
protachysterol was the observation that in the final stages
of the irradiation there appeared a slight Inflection in the
absorption spectrum at 280 mu, which may have been caused by
the formation of a trace of tachysterol.
Theoretically, the analysis of the mixtures can be ac­
complished by solving a series of simultaneous equations.
Inasmuch as the absorption spectra of suprasterol I and suprasterol II are identical, a spectrophotometric analysis
cannot differentiate between them.

The analysis would be

expected to yield the concentrations of (1 ) the combined su­
prasterols (I and II),

(2) suprasterol III, (3 ) toxisterol,

(^) calciferol and (5) protachysterol.

This would require

the solution of five simultaneous equations.

It was found,

however, that no more than three equations could be utilized.
The reason for this was that the measurements upon which
these calculations were based contained only three signifi­
cant figures.

Upon completing the necessary arithmetical

manipulations of fourth or fifth order determinants the
number of significant figures in the calculated concentra­
tions was reduced to one or zero.
It is evident from the absorption spectra shown in
Figure 23 that small variations in concentrations in toxi­
sterol, calciferol and protachysterol have a marked effect
on the absorption spectra of the mixtures in the range

a 300
o
rH

rH

zoo

100

260

280

WAVE LENGTH, mu
FIGURE 23
ABSORPTION SPECTRA OF (1) TOXISTEROL,
(2) SUPRASTEROLS I & II, (3) SUPRASTEROL III
AND W
PROTACHYSTEROL.

I

22

2 5 0 -2 8 0 mu, while changes in the concentrations of supraste­
rol I and suprasterol II have practically no effect at wave
lengths of 250 mu and greater.

Changes in suprasterol III

concentrations affect, to a limited extent, the absorption
in the range 250-280 mu.

Therefore, the three equations

which gave the best approximation to the concentrations of
toxisterol, calciferol and protachysterol were those involv­
ing the extinctions at 250, 265 and 280 mu.

These three

equations were:
a]_T 4 b]_D t

c^P s

a£T + b£D + C£P = E2
a^T «t b^D +

C3P = E3

where T is the fraction of calciferol which has
been converted to toxisterol;
D is the fraction of calciferol which has not
reacted;
P is the fraction of calciferol which has been
converted to protachysterol;
a^,

and a^ are E (1$, 1 cm.) values of toxi­
sterol at 2 5 0 , 265 and 280 mu, respectively;

b]_, X>2 and

are the E (1$, 1 cm.) values of

calciferol at 2 5 0 , 265 and 280 mu, respectively;
c1 , C2

and C3 are the E (1$, 1 cm.)values

of pro­

tachysterol at 2 5 0 , 265 and 280 mu,respectively;

23

E2 an<3- E3 are the E (1% t 1 cm.) values of the
irradiation mixture at 2 5 0 , 265 and 280 mu,
respectively.
These three equations can be solved by the method of
determinants to give the three expressions for T, D and P,
which are, upon substitution of the values for the a ‘s,
b*s and c ‘s:
for ethanol solutions:
T * 0.00323 Ei - 0.00381 E2 -* 0.00162 E3
D =-0.000733 Ei * 0.00721 E2 - 0.00642 E3
P = 0.000284 Ei - 0.00630 E 2 -* 0.00852 E 3
for hexane solutions:
T = 0.00324 Ex - 0.00376 E2 * 0.00157 E3
D = -0.000582 Ei -» O.OO6 7 I E2 - 0.00598 B 3
P = 0.000196 Ei - 0.00550 E2 * 0.00783 Ej
The extinction at 230 mu of the Irradiated solutions
would be the sum of the extinctions of the constituents
present.

Since the concentrations of toxisterol, calciferol

and protachysterol had been calculated, the contribution of
each of these constituents to the extinction coefficient at

230 mu could be calculated.
Subtracting these contributions from the measured ex­
tinction and dividing the result by the E (l^, 1 cm.) value
of the suprasterols yielded the approximate concentration
of the combined suprasterols*

This was expressed in the form

24

of an equation as follows:

E^ - (k]_T + k£D 4 k3P)
kzj,
where S is the fraction of calciferol which has
been converted to suprasterols;
E^ is the E (1 %, 1 cm.) of the mixture at 230 mu;
T, D and P are concentrations of toxisterol, cal­
ciferol and protachysterol, respectively;
ki, k£, k^ and k^ are the E (1%, 1 cm.) values of
toxisterol, calciferol, protachysterol and
suprasterols (I, II and III), respectively,
at 230 mu.
Upon substitution of the values of k]_, k£, £3 and k^ this
equation became:
S = 0.0115

- (1.12 T 4 2.85 D * 2.15 P).

Using these equations the approximate percentages of
calciferol, toxisterol, protachysterol and the combined
suprasterols have been calculated for the irradiations con­
ducted in ethanol, hexane and hexane-ether mixture, and are
recorded In Tables I-XVTII.
Figures 24 to 41 represent the variation of concentra­
tions of the constituents with time under the various
conditions of the irradiation.
One of the most outstanding characteristics observed in
these figures is the apparent variation of concentration of
combined suprasterols with time.

The values of these

140

120

100

20

0

160
120
80
IRRADIATION TIME, MINUTES

200

FIGURE 24
IRRADIATION IN ETHANOL WITH Hg 2483 A. LINE

120

100

80

60

ko

20
0
0

160

120

80

200

IRRADIATION TIME, MINUTES
FIGURE 25
IRRADIATION IN ETHANOL WITH Hg 2537 A. LINE
©

= D;

O

= P;

•

= T;

©

= S

120

CONCENTRATION,

100

80
60

40
20
0
0

40

160
80
120
IRRADIATION TIKE, KINUTES

200

FIGURE 26
IRRADIATION IN ETHANOL WITH Hg 2654 A. LINE

140

120

100

80
60

40

20
0
0

40

160
80
120
IRRADIATION TIKE, KINUTE3

200

FIGURE 27
IRRADIATION IN ETHANOL WITH Hg 2753 A. LINE
©

= D;

O

= P;

•

» T;

©

= 3

120

100

80

o

60

40

20
0
0

120

200
160
SO
IRRADIATION TIKE, KIMUTES
FIGURE 28
IRRADIATION IK ETHANOL WITH Hg 2967 A. LIKE
©

= D;

O

= P;

•

= 7;

O

= S

120

100

80
6o

20
0
0

AO

80

120

160

200

IRRADIATION’ TIKE, MINUTES
FIGURE 29
IRRADIATION IN ETHANOL WITH H g 3122 A. LINE
©

= D;

O

= P;

0

= T;

© = S

120

80

20
0
0

40

80
120
160
200
IRRADIATION TIME, MINUTES
FIGURE 30

IRRADIATION IN HEXANE WITH Hg 2483 A - Lit®
©

= D

0

= P

•

= T

©

= S

COHCECTRATION, £

CONCENTRATION, %

140

120

100

o 60

20

0

40

160
200
80
120
IRRADIATION TINE, MINUTES
FIGURE 33

IRRADIATION IN HEXANE WITH Hg 2753 A. LIKE
« = D;

O

= P;

•

= T;

0 = 3

120

CONCENTRATION

100

80
60

20
0
0

200
160
80
120
IRRADIATION TIME, MINUTES
FIGURE 3 6
IRRADIATION IN' HEXANE WITH Hg 2967 A. LINE

120

100

o 60

20

0

6-0

160
200
80
120
IRRADIATION TIME, MINUTES
FIGURE 35

IRRADIATION IK HEXANE WITH Hg 3132 A. LINE
©

= D;

0

= P;

• = T ;

©

= S

120

100

80

60

20
0
80

160

120

200

IRRADIATION TIME, MINUTES
FIGURE 36
IRRADIATION IN HEXANE-ETKER WITH Hg 2A83 A. LINE
© = D ;

0 = P ;

•

= T;

©

= S

I

120

CONCENTRATION

100

60

20
0
0

80

160

120

200

IRRADIATION TIKE, MINUTES
FIOURE 37
IRRADIATION IN HEXANE-ETHER WITH Hg 2537 A. LINE
©

- D;

O

= P;

•

= T;

©

= S

120

CONCENTRATION

100

80

60

20
0
0

80

120

160

200

IRRADIATION TIKE, MINUTES
FIGURE 38
IRRADIATION IN HEXANE-ETEER WITH Hg Z b 5 k A. LINE

120
W.100

80

20
0
0

40

80
120
160
IRRADIATION TIME, MINUTES

200

FIGURE 39
IRRADIATION IN HEXANE-ETKER WITH Hg 2753 A. LINE
©

= D;

O = P;

•

= T;

© = S

120

CONCENTRATION

100

80

60
40
20
0
0

40

120
160
80
IRRADIATION TIKE, MINUTES

200

FI CURE 40
IRRADIATION IN HEXANE-ETKER WITH Hg 2967 A. LINE

120

100

g 60

20

0

160
200
120
80
IRRADIATION TIME, MINUTES

40

FIGURE 41
IRRADIATION IN HEXANE-ETHER WITH Hg 3132 A. LINE
© = D ;

0

= P;

•

= T;

©

= S

concentrations were much greater than was possible.

For

example, in Figure 27 the calculated concentration of supra­
sterols after an irradiation time of 210 minutes was 159 $,
which was an absurd value.

The method of calculation of the

suprasterol concentrations was inadequate to obtain accurate
values.
It was noted that the changes in suprasterol concentra­
tions were similar to those of toxisterol concentration.

For

example, in Figure 24 the rate of change of toxisterol con­
centration was nearly constant after 95 minutes and the rate
of change of suprasterol concentration was low, while in
Figure 27 the toxisterol concentration was changing slowly
at all times and the suprasterol concentration was changing
rapidly.

This effect was observed in every case, suggesting

that the large errors in calculated suprasterol concentra­
tions were caused by the inadequacy of the equations used,
particularly with regard to toxisterol.

This was probably

partly due to the presence of suprasterol III in the irradi­
ation mixture, which would contribute to some extent to the
absorption at 250 mu, a contribution which has been neglected
in the calculations of composition.
Eecause of the very low E (1$, 1 cm.) values of the su­
prasterols at 230 mu, the calculated concentrations which
were based upon the measured extinction at 2 3 0 mu were very
sensitive to small errors in absorption measurements in that

26

region.

Unfortunately, the reproducibility of extinction

values in the region 220-240 mu was much less than would
be desirable.
As a result of the inaccuracies Involved in the mea­
surements and the Inadequacies of the treatment, only
qualitative conclusions regarding the suprasterols and
toxisterol could be expressed.
The formation of suprasterols was the principal re­
action which oecured on irradiation of calciferol with
ultraviolet light of wave lengths between 2483 and- 3132 A.
The formation of toxisterol accompanied this reaction but
to a much lesser extent.

There was no evidence to support

a conclusion that the formation of toxisterol preceded the
formation of suprasterols.
In Figures 24-41 the concentration of protachysterol
Increased rapidly early in the irradiation, reached a maxi­
mum value and finally decreased when the concentration of
calciferol had reached rather low values.

This indicated

that the formation of protachysterol from calciferol was a
process which occured simultaneously with the formation of
toxisterol and suprasterols, with subsequent decomposition,
probably to the suprasterols.

There was no evidence that

the formation of protachysterol preceded the formation of
toxisterol or the suprasterols, but the possibility that it
did was not excluded.

f l

2?

The rate of change of calciferol concentration, as
shown in Figures 2 k - k \ t appeared to be exponential.

In an

effort to discover the order of the reaction the proposition
was made that the reaction would follow the general rate
expression:
- M

» k (D)n ,
clt
where (D ) is the concentration of calciferol;
k is the rate constant for the reaction under
the conditions of the irradiation;
n is the order of the reaction.
Taking logarithms of both sides of the equation there re­
sulted :
In

= In k + n In (D).
dt
As a good approximation the rate, - d(D), was replaced by
dt
- A ( D ) / A t and the concentration,

(D), was replaced by the

average concentration, '(d) , in the time interval, A t .

A

plot of In - A ( D ) / A t as a function of In CdT would be
linear, if n was constant.

Figure H-2 shows this plot using

data from several different irradiation experiments.

In no

case was this plot linear, indicating the variance of n
throughout the reaction.

This result was in agreement with

the conclusion of Green (l*f) that the reaction was of chang­
ing order.
On the basis of the evidence obtained in this investi­
gation the photoreaction of calciferol has been considered
a combination of three simultaneous reactions, one of which

2.56

MINUTE

1.28

% PER

3.32

A (D)/At,

3.16

3.08

0.02

0.01

1

2

6

8

16

AVERAGE CONCENTRATION OF D, %
FIGURE 62
VARIATION OF REACTION RATE WITH CONCENTRATION
OF CALCIFEROL IRRADIATED IN ETHANOL WITH
(1) Hg 2537 A. (2) Hg 2753 A.
(3) Hg 2967 A.

28

led to the formation of a substance resembling protaohysterol which underwent further reaction, probably to form
one or more of the suprasterols.

This has been represented

as follows:
Suprasterols
*
Calciferol;-— > Protachysterol
Toxisterol.
The reaction resulted from the irradiation of calci­
ferol with light of wave lengths between 2483 and 3132 A . ,
although the reaction produced by light of wave lengths
greater than 2967 A. was much slower than that produced by
light of shorter wave lengths.

The very slow rate of reac­

tion produced by the Hg 3132 A. line may have been the reason
why Bowden and Snow (19) did not observe a change in the ab­
sorption spectrum after irradiation of calciferol with light
of that wave length.
There was no apparent variation of the reaction as a
result of the influence of solvent when the irradiation was
performed in ethanol, hexane or a mixture of hexane and
ether.

29

SUMMARY
Solutions of calciferol in ethanol, n-hexane and a
mixture of n-hexane and ethyl ether were irradiated with
monochromatic light of wave lengths 2483, 253 7 , 2654, 2753,
2967 and 3132 A.

The absorption spectra of the solutions

were measured at various times during the irradiations*
Absorption spectra measurements indicated the presence
of a substance resembling protaehysterol in its absorption
spectrum*
The composition of the irradiation mixtures with re­
spect to (1) calciferol,

(2) toxisterol, (3 ) protaehysterol

and (4) combined suprasterols was estimated on the basis of
absorption spectra measurements*
Upon irradiation, calciferol was converted into toxi­
sterol, protaehysterol and suprasterols I, II and III; the
protaehysterol thus formed was converted photochemically to
one or more of the suprasterols.
The photoreaotion of calciferol was of changing order
with respect to time.
The photoreaction of calciferol was brought about by
light of wave lengths in the range 2483-3132 A . , although
the effectiveness of light of the longer wave lengths was
somewhat less than that of shorter wave lengths.

The reaction

was apparently the same in each of the three solvents used.

30

REFERENCES
1. Windaus, A., et al., Ann., 4 8 3 . 17 (1930).
2. Askew, F. A., et al., Proc. Roy. Soc. (London). B 107.
76, 91 (193077
3. Windaus, A., Lftttringhaus, A., and Deppe, M., Ann.,
48 9 , 252 (1 9 3 1 ); Windaus, A., Proc. Roy. Soc. (London),
B 108. 568 (193D.
4. Windaus,

A., and Linsert, 0., Ann.,

489, 269(1931)*

5. Windaus,

A., et al., Ann., 492. 226 (1932).

6. Windaus, A., Dlthmar, K ., and Fernholz, E . , Ann., 493,
259 (1932).
7« Windaus, A., von Werder, F., and Lftttringhaus, A., Ann.,
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C. A.,
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f l

31

15- Bills, 0. E., Honeywell, E. M . , and Cox, W. M., Jr.,
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