PHYSICO-CHEMICAL METHODS FO R VITAMIN D ASSAY AND A …

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PHYSICO-CHEMICAL METHODS FOR VITAMIN D ASSAY

AND

A NEW SPECTROPHOTOMETER

by

Thomas Edwin Whittemore

A T h e s i s submitted i n p a r t i a l f u l f i l m e n t o f

the requirements f o r the degree of

MASTER OF ARTS

i n the department

o f

PHYSICS

THE UNIVERSITY OF BRITISH ©LUMBIA

A p r i l , 1950

ABSTRACT

The design and c o n s t r u c t i o n o f a p h o t o e l e c t r i c

spectrophotometer i s d e s c r i b e d . The completed instrument has

a wavelength r e s e t accuracy of 1 A or l e s s over the v i s i b l e

spectrum, and measures t r a n s m i s s i o n s to 1 p a r t i n 50G w i t h a

bandwidth o f 20-30 A. Suggestions f o r improved accuracy o f

t r a n s m i s s i o n r e a d i n g are i n c l u d e d . The use of a c o n t r o l l e d

source makes an accuracy of 1 p a r t i n 10,000 t h e o r e t i c a l l y

a t t a i n a b l e .

T h i s instrument was c o n s t r u c t e d so t h a t v i t a m i n D

assay of low-potency o i l s c o u l d be attempted. I n v e s t i g a t i o n

of the antimony t r i c h l o r i d e r e a c t i o n by s e v e r a l authors showed

t h a t i t was not s u i t a b l e f o r t h i s purpose. A p r e l i m i n a r y i n

v e s t i g a t i o n of the g l y c e r o l d i c h l o r h y d r i n r e a c t i o n was c a r r i e d

out w i t h a Beckman model D spectrophotometer on s e v e r a l t y p i c a l

o i l s . I t showed t h a t t h i s r e a c t i o n was no more s u i t a b l e than

the o t h e r .

Because of the i n s t a b i l i t y of these r e a c t i o n s no

method was found whereby i n c r e a s e d instrument accuracy gave a

corresponding i n c r e a s e i n accuracy of assay. The o n l y f e a s i

b l e method of assay f o r low-potency o i l s at p r e s e n t i n v o l v e s

c o n c e n t r a t i o n of the v i t a m i n D by chromatography.

ACKNOWLEDGEMENT

T h i s work was c a r r i e d out at the B r i t i s h

Columbia Research C o u n c i l , Vancouver, B.C.

The author wishes t o thank Dr. S. E.

Maddigan, d i r e c t o r of the C o u n c i l , f o r making a v a i l a b l e

the funds and f a c i l i t i e s which made t h i s p r o j e c t

p o s s i b l e .

The author i s indebted t o Dr. A.C. Young,

former Head of the P h y s i c s D i v i s i o n of the C o u n c i l ,

f o r many v a l u a b l e suggestions, i n c o r p o r a t e d i n t h i s

work, and f o r constant help and a d v i c e . The h e l p f u l

i n t e r e s t of Dr. A. M. Crooker, and other members of

the P h y s i c s Department of the U n i v e r s i t y of B r i t i s h

Columbia was a l s o a p p r e c i a t e d .

TABLE OF CONTENTS

Page

I . INTRODUCTION

A. P h y s i c s and Vitamins • • 1 B. The Spectrophotometer i n Assay Work . . . . 4 G. The Problem of V i t a m i n D Assay 4 D. The Proposed P r o j e c t 6

I I . ABSORPTION SPECTROPHOTOMETRY

A. I n t r o d u c t i o n 7 B. The Laws of A b s o r p t i o n 8

C. P r e c i s i o n o f Measurement 10

I I I . THE SPECTROPHOTOMETER

A. Design P r i n c i p l e s 14 B. The O p t i c a l System 19 G. Mechanical Design 20 D. The E l e c t r o n i c C i r c u i t s 24 E . Performance 31

IV. VITAMIN D ASSAY

A. The P r o p e r t i e s o f V i t a m i n D 34 B. Methods of Assay . . . . . . . 36 C. The Chemical Composition o f F i s h

L i v e r O i l s : . 41 D. The G l y c e r o l D i c h l o r h y d r i n C o l o r i -

me t r i e R e a c t i o n 42

V. CONCLUSION 49

VI. BIBLIOGRAPHY 50

PLATES

Page

I . The Spectrophotometer O p t i c a l System 19

I I . P l a n of Spectrophotometer 21

I I I . Regulated Power Supply 24

IV. L i g h t Source R e g u l a t o r . . . . 26

V. The Main A m p l i f i e r . 28

V I . (a) C a l i b r a t i o n Curve 32

(b) Mercury Y e l l o w L i n e s

PHYSICO-CHEMICAL METHODS FOR VITAMIN D ASSAY

AND

A NEW SPECTROPHOTOMETER

I . INTRODUCTION

A. P h y s i c s and Vitamins

The e x i s t e n c e o f what are now known as v i t a m i n s was

demonstrated as l o n g ago as 1881, but i t was not u n t i l a f t e r

1910, when the d i s t i n g u i s h e d s t u d i e s of Hopkins and of Funk

were p u b l i s h e d , t h a t v i t a m i n s r e c e i v e d much a t t e n t i o n . T h e i r

r e a l i z a t i o n of the fundamental importance o f the v i t a m i n s i n

a l l l i f e processes gave great Impetus to r e s e a r c h i n t h i s

f i e l d , and knowledge of v i t a m i n chemistry and p h y s i o l o g y

r a p i d l y i n c r e a s e d from t h a t time onward.

P h y s i c i s t s must, f o r the p r e s e n t at l e a s t , be con

t e n t to p l a y a secondary r o l e i n t h i s work; t h a t o f s u p p l y i n g

such instruments and techniques as may be of use to the b i o

chemist and p h y s i o l o g i s t . However, such r a p i d progress as has

been made d u r i n g the p a s t f o r t y years would have been i m p o s s i b l e

2 without instruments such as the modern spectrophotometer and polarimeter, and further advances in instrument design, or the application of new physical methods to this f i e l d are of prime importance. For example, the application of high-vacuum

1 techniques by Hickman and others resulted in the "molecular s t i l l " , and improved techniques in infra-red spectroscopy are beginning to prove valuable.

A fundamental problem of vitamin research i s that of assaying samples of foodstuffs or tissues. This i s d i f f i c u l t , since vitamins need only be present in quantities of the order of a few micrograms per gram to carry out their work as catalysts and bring about profound physiological changes. In fact one of the most sensitive assay methods makes use of this property, and involves the observation of laboratory animals under carefully controlled conditions and diet. Although widely used, i t has disadvantages which cannot wholly be overcome. The wide variation in response of individual animals makes i t necessary to apply s t a t i s t i c a l methods, and to use a-large number of animals in each test. A single assay by this method involves weeks or months of work, and i s thus expensive and slow. For this reason physical and chemical methods are preferable, even i f less accurate, in routine assay work.

Many physical methods have been applied, but only a 2

few have found general use. According to Loofbourow, these 1 Hickman, K.C.D.,"Adventures in Vacuum Chemistry", "Science

in Progress". 4th ser. pp.205-248, Yale Univ.Press, New Haven, 1945.

2 Loofbourow, J.R., "Physical Identification of Vitamins and Harmones", "Vitamins and Hormones". Vol.1, pp.109-155, Academic Press, New York, 1943.

are a b s o r p t i o n spectrophotometry ( v i s i b l e and u l t r a v i o l e t ) ,

c o l o r i m e t r y , f l u o r e s c e n c e spectrophotometry, and f l u o r i m e t r y .

Of t h e s e , a b s o r p t i o n spectrophotometry i s the most important,

s i n c e most v i t a m i n s c o n t a i n u n s a t u r a t e d l i n k a g e s and thus have

a c h a r a c t e r i s t i c a b s o r p t i o n spectrum i n the v i s i b l e or near

u l t r a v i o l e t *

I f a q u a n t i t a t i v e measurement of such a spectrum i s

to be used to o b t a i n an assay, a number o f c o m p l i c a t i o n s may

a r i s e . B r i e f l y these a r e :

1. Other substances a b s o r b i n g i n the same s p e c t r a l

r e g i o n may be presen t i n c o n c e n t r a t i o n s many times t h a t

o f the v i t a m i n .

2. F o r l a r g e o r g a n i c molecules Beer's law may not

h o l d , and a b s o r p t i o n may depend on the s o l v e n t , and on

the pH, to a marked degree.

3. Photochemical d e g r a d a t i o n o f the v i t a m i n may

take p l a c e .

Some b e n e f i t s may r e s u l t from the use of a chemical

" c o l o r i m e t r i e t e s t " f o r the v i t a m i n , and c a r e f u l measurement

of the a b s o r p t i o n spectrum o f the r e a c t i o n complex so formed.

However, these complexes o f t e n tend to be u n s t a b l e , and t h e i r

a b s o r p t i o n s p e c t r a may va r y w i t h time, temperature and i l l u m

i n a t i o n . I f s u f f i c i e n t improvement w i t h r e g a r d to item 1 i s

ob t a i n e d i n a d d i t i o n to the e l i m i n a t i o n o f item 3, c o n t r o l

of these f a c t o r s may make t h i s method p r a c t i c a b l e .

4

B. The Spectrophotometer i n Assay Work

The value o f an assay procedure depends upon the

accuracy and r e p r o d u c i b i l i t y of i t s r e s u l t s , and the speed

w i t h which they can be o b t a i n e d . Thus p h o t o e l e c t r i c photome

t r y i s g e n e r a l l y p r e f e r a b l e to v i s u a l photometry s i n c e i t i s

f a s t e r , and minimizes p e r s o n a l e r r o r s . S p e c t r o g r a p h i c equip

ment such as the "Spekker" spectrophotometer o f Adam H i l g e r

L t d . g i v e s r e s u l t s o f f a i r accuracy and r e p r e d u c i b i l i t y , and

may be s u i t a b l e i f a permanent r e c o r d of the whole a b s o r p t i o n

spectrum i s r e q u i r e d . P h o t o e l e c t r i c spectrophotometers such

as the Beckman Model D have comparable o r b e t t e r c h a r a c t e r i s

t i c s combined w i t h g r e a t e r f l e x i b i l i t y . D e n s i t y r e a d i n g s are

o b t a i n e d d i r e c t l y from the instrument, and may be made at one

wavelength o n l y , or over any range of wavelengths which i s o f

i n t e r e s t . A l s o , data on time-dependent chemical r e a c t i o n s

may be o b t a i n e d w i t h these i n s t r u m e n t s .

These advantages have made the p h o t o e l e c t r i c s p e c t r o

photometer standard equipment i n l a b o r a t o r i e s engaged i n r e

s e a r c h and assay work. The accuracy w i t h which such work can

be done depends l a r g e l y on the performance of the spectrophoto

meter. An instrument which g i v e s r a p i d and accurate wavelength

and d e n s i t y r e a d i n g s , and t r a n s m i t s o n l y a narrow s p e c t r a l

band, i s d e s i r a b l e .

G. The Problem o f V i t a m i n D Assay

There now e x i s t s a s a t i s f a c t o r y r a p i d assay proce

dure f o r almost a l l the more common v i t a m i n s . F o r some, such

as v i t a m i n A, e o l o r i m e t r i c methods are s a t i s f a c t o r y , while f o r o t h e r s , such as the B v i t a m i n s , these have been r e p l a c e d by

f l u o r i m e t r i c or m i c r o b i o l o g i c a l t e c h n i q u e s . The o u t s t a n d i n g 3

e x c e p t i o n here i s v i t a m i n D, and f o r s e v e r a l reasons. Morton

p o i n t s out t h a t comparatively l a r g e q u a n t i t i e s o f i n a c t i v e

s t e r o l s c h e m i c a l l y s i m i l a r to v i t a m i n D are g e n e r a l l y p r e s e n t

wherever t h i s v i t a m i n i s found, and cannot e a s i l y be separated

from i t . V i t a m i n A, a l s o , g e n e r a l l y accompanies i t , e s p e c i a l l y

i n l i v e r o i l s , and h e l p s to mask the r e l a t i v e l y weak a b s o r p t i o n 4

band o f v i t a m i n D. W i l l i a m s a s c r i b e s the l a c k o f i n f o r m a t i o n

on the v i t a m i n D content o f t i s s u e s l a r g e l y to economic reasons.

I t i s t r u e t h a t the v i t a m i n can be produced q u i t e i n e x p e n s i v e l y

by the i r r a d i a t i o n of s t i l t able m a t e r i a l s w i t h u l t r a v i o l e t l i g h t ,

n e v e r t h e l e s s much work has been done on the problem o f a s s a y i n g

i r r a d i a t e d f o o d s , v i t a m i n c o n c e n t r a t e s , and " f o r t i f i e d " o i l s ,

and u n t i l t h i s i s s o l v e d the assay o f t i s s u e s must wa i t .

A review of v i t a m i n D assay methods i s g i v e n i n IV 2.

At the present time none i s completely s a t i s f a c t o r y , although 5

the p r e l i m i n a r y r e s u l t s of the technique o f DeWitt and S u l l i v a n

seem p r o m i s i n g . At the time t h i s p r o j e c t was commenced, J u l y ,

.1946, the e o l o r i m e t r i c r e a c t i o n suggested by S o b e l , Mayer and 3 Morton, R.A., "Absorption S p e c t r a of Vitamins and Hormones",

2nd ed. p. 37, Adam H i l g e r L t d . , London, 1942.

4 W i l l i a m s , R.J., "The S i g n i f i c a n c e o f the V i t a m i n Content o f T i s s u e s " , "Vitamins and Hormones". V o l I , pp. 229-247, Academic P r e s s , New York, 1943.

5 DeWitt, J.B. and S u l l i v a n , M.X., Ind. Eng. Chem,, A n a l y t i c a l Ed., 18, 117, 1946.

6

6 Kramer had not been e x t e n s i v e l y i n v e s t i g a t e d . Although the

a b s o r p t i o n band of the v i t a m i n D complex formed i n t h i s r e

a c t i o n was r e l a t i v e l y weak, good d i s c r i m i n a t i o n between the

v i t a m i n and some of the i n t e r f e r i n g s t e r o l s was claimed. No

r e c o r d o f any attempt to use t h i s t e s t on n a t u r a l o i l s was

a v a i l a b l e .

D. The Proposed P r o j e c t

The proposed p r o j e c t had two aims i n view. These

were as f o l l o w s : ,

1. The spectrophotometer has become a very impor

t a n t instrument, w i d e l y used i n chemical and b i o l o g i c a l

work of a l l k i n d s . By m o d i f y i n g the u s u a l d e s i g n some

what i t was b e l i e v e d t h a t an instrument o f improved

accuracy and s t a b i l i t y c o u l d be b u i l t at a c o s t compar

able to t h a t o f the commercial instruments a v a i l a b l e at

pr e s e n t .

2. When completed t h i s instrument c o u l d be used to

i n v e s t i g a t e the problem o f v i t a m i n D assay.

6 S o b e l , A.E., Mayer, A.M., and Kramer, B., Ind. Eng. Chem. A n a l y t i c a l Ed., 17, 160, 1 9 4 5 . .

7

I I . ABSORPTION SPECTROPHOTOMETRY

A. I n t r o d u c t i o n

When monochromatic r a d i a t i o n i s p e r p e n d i c u l a r l y i n

c i d e n t upon one f a c e of a plane p a r a l l e l c e l l , o r c u v e t t e ,

f i l l e d w i t h a s o l u t i o n , i t may be di s p o s e d o f i n any o f the

f o l l o w i n g ways:

1. by r e f l e c t i o n at the v a r i o u s i n t e r f a c e s .

2. by R a y l e i g h s c a t t e r i n g and Raman e f f e c t i n the

s o l u t i o n .

3 . by a b s o r p t i o n and c o n v e r s i o n to heat, chemical

change, e t c . , or r e e m i s s i o n as f l u o r e s c e n c e ;

4. by t r a n s m i s s i o n through c e l l and s o l u t i o n .

In the a b s o r p t i o n spectrophotometry o f s o l u t i o n s the

r a t i o Ijx , where I t f i s the i n t e n s i t y i n c i d e n t upon, and Ix To

the i n t e n s i t y emergent from x cms. of s o l u t i o n , i s to be

measured as a f u n c t i o n o f wavelength. T h i s r a t i o i s c a l l e d

the t r a n s m i s s i o n , T, o f x cms. of s o l u t i o n . C o r r e c t i o n f o r

l o s s e s o f i n t e n s i t y a t t r i b u t a b l e to the c e l l i t s e l f or to the

s o l v e n t may be made by comparing the t r a n s m i s s i o n o f s o l u t i o n

and c e l l to t h a t o f a "blank", or i d e n t i c a l c e l l c o n t a i n i n g

s o l v e n t alone.

3 The measurement of I x f o p a band of wavelengths,

To

often isolated by f i l t e r s , i s called 11 colorimetry". This term i s also applied to the measurement of absorption bands of the "complexes" formed in a chemical "eolorimetric" reaction, and since this i s generally done by colorimetry as defined above, no distinction need be made. However, where necessary i t w i l l be assumed that a eolorimetric reaction may be investigated by absorption spectrophotometry as well.

B . The Laws of Absorption

When monochromatic light traverses a homogeneous absorbing medium, the light transmitted by each succeeding layer of a given thickness i s a constant fraction of the light incident upon i t . Thus the fundamental differential equation of absorption spectrophotometry i s -» d I _ & I

where i s the "absorption coefficient" of the medium. Integration between 1=0 and l=x gives

Lambert's Law: l x = i 0 JL

or log^T 2 3 0 1 **"

In a solution, absorption varies with concentration

of the solute. For many solutes absorption i s proportional

to concentration over a wide range. This i s known as Beer's

Law: oc = 3 c where ^3 i s the "absorption co

efficient" per unit concentration c .

9

These laws may be combined as

I n or I o q .If = K C >

K i s generally called the "extinction coefficient", and i f the concentration c i s in percent (meaning grams of solute per 100 ml. of solution)

icm~ Q ( m % ) * X ( m cm.]

may be given instead.

The expression ^0<5(0 - j 2 * s called the "density" D.

The "molecular extinction coefficient" £ or i t s logarithm are sometimes used. It i s the same asKabove when c i s given in moles per l i t e r .

i f a number of substances with extinction coefficients K , , K i , - - - , K n are present as a mixture i n the absorption c e l l , with concentrations c, , c,.,--. c n respectivel y , and i f there i s no chemical interaction, the resultant density w i l l be

D = I oq l a =, " X ( K , C , + - K x C i t • - • + K n C n ) I->c

For s t r i c t l y monochromatic light traversing a homo

geneous medium Lambert's Law i s exact. Deviations from Beer's

Law, on the other hand, are quite common, especially when the

solution contains large and asymmetrical molecules. They occur

because the nature of the solvent, i t s pH, and the concentra

tion of absorbing material may influence the molecules of the

1 0

absorbing m a t e r i a l . T h i s i s p a r t i c u l a r l y true of the complex

molecules formed i n chemical e o l o r i m e t r i c r e a c t i o n s , and Beer's

Law should not be assumed without v e r i f i c a t i o n .

G. P r e c i s i o n o f Measurement

In assay work the Lambert-Beer Law i s used to f i n d

the c o n c e n t r a t i o n c when the o p t i c a l d e n s i t y D has been measured

( I f Beer's law i s obeyed). Then C = -p^c "D and

AC = A ~ D where A C i s the e r r o r i n c o n c e n t r a t i o n

which r e s u l t s from an e r r o r A "E> i n d e n s i t y d e t e r m i n a t i o n .

The f r a c t i o n a l e r r o r i n the e s t i m a t i o n o f con-c

c e n t r a t i o n i s equal to the f r a c t i o n a l e r r o r A ~D i n the

d e n s i t y d e t e r m i n a t i o n .

The photographic method, i f the match-points are

determined v i s u a l l y , has a &~£> independent of D, and Loofbourow

estimates t h a t an average d e n s i t y D of 1 . 5 w i t h a probably

e r r o r & D o f H .03 can be expected. T h i s g i v e s an accuracy of

± AC - ± 4_D - 1 . 0 2 or 2$. C • T>

D i s c u s s i n g the p r e c i s i o n o f the p h o t o e l e c t r i c method, 8

Loofbourow assumes t h a t i t i s l i m i t e d by the minimum determin

able d i f f e r e n c e A I i n l i g h t i n t e n s i t y , and t h a t A I i s

5! Loofbourow, J.R., " P h y s i c a l I d e n t i f i c a t i o n of V itamins and Hormones", "Vitamins and Hormones". V o l I , p. 121, Academic P r e s s , New York, 1943.

8 I b i d . p. 122.,

1 1

independent o f the i n t e n s i t y I . I f t h i s i s t r u e the concentra

t i o n e r r o r ±. &S can be d e r i v e d as f o l l o w s ; c

D * I o ql01£ = l o c , ( 0 I o - l o g l 0 I * .

cLT> - - . 1 o g ,„ A. a. i x i x '

d P - - I oq l 0 JL , ^ I x

1 *

i AC - f AT? - . H3H3.fc 4 I x l f l )

The f r a c t i o n a l e r r o r i n d e n s i t y , and thus i n concen

t r a t i o n w i l l be l e a s t when I x locj ) o Is. i s a maximum, i . e .

when

I o c J i 0 l a = P =• - 4-3 i+3

T h i s assumption t h a t the e r r o r A I X ^ s independent

of l£ i s v a l i d o n l y f o r the s i m p l e s t type of p h o t o e l e c t r i c

instrument, such as the c o l o r i m e t e r type. >:CIt: i m p l i e s t h a t

the accuracy i s l i m i t e d simply by the s i z e o f the s m a l l e s t

s c a l e d i v i s i o n which i s d i s t i n g u i s h a b l e . In b e t t e r i n s t r u

ments o t h e r f a c t o r s determine t h i s l i m i t . These w i l l be con

s i d e r e d i n more d e t a i l under "Design P r i n c i p l e s " P a r t I I I , 1 ,

but are l i s t e d below w i t h the optimum d e n s i t y f o r minimum

f r a c t i o n a l c o n c e n t r a t i o n e r r o r computed f o r each:

1 . Random f l u c t u a t i o n s i n l i g h t source i n t e n s i t y .

The r e s u l t a n t & l x i s p r o p o r t i o n a l to U as i n the

photographic and v i s u a l methods. Thus a n i s independent

of D, and the g r e a t e s t p o s s i b l e D should be used.

12

2 . Random variations in gain of amplifier. A l x i s proportional to IQ

I x

. « - a i -y : where a, i s the proportionality con-stant. Gn substitution in equation ( 1 ) , the condition that the denominator have i t s maximum value gives

E = = .717

3. Shot effect in the phototube. This i s the ultimate limitation on the photo

electric type of instrument. It i s caused by the random manner in which electrons leave the photocathode, and i s proportional to the square root of the current through the phototube .

For high-vacuum phototubes this current i s linearly dependent upon the intensity of illumination of the cathode. Thus

where I i s the photoelectric current, and &2 , a_3 are constants.

Combining this equation and equation (1) as before, the optimum density i s J) - 1 1^%- = . 6 6 8 6

4. Thermal agitation "noise" in resistors, a I-* i s independent of T x , thus D = .'+3 43

5. Inexact repositioning of the absorption c e l l i n the light beam: Zl I x i s proportional to I x , and D should be as large as possible.

A w e l l designed and c o n s t r u c t e d instrument would

have i t s p r e c i s i o n l i m i t e d by shot e f f e c t i n the phototube

r a t h e r than any of the o t h e r f a c t o r s . F o r such an instrument

the optimum o p t i c a l d e n s i t y D of the s o l u t i o n b e i n g measured

i s about .87, c o r r e s p o n d i n g to 10% - 15% t r a n s m i s s i o n .

14

III. THE SPECTROPHOTOMETER

A. Design Principles

The f i r s t step in the design of a spectrophotometer for a certain job i s an evaluation of the factors which w i l l limit i t s efficiency. In addition, ease of operation and flexi b i l i t y must be weighed against construction d i f f i c u l t y , cost v

and availability of materials, and a satisfactory compromise reached. Such considerations indicated that an instrument capable of measuring the intensity of the transmitted light within 1 part in 10,000, with a spectral bandwidth of 10 Angstroms, and a reset accuracy of better than t. 5 Angstroms over i t s useful range (the visible region of the spectrum), was a reasonable goal.

A photoelectric spectrophotometer consists of a light source giving a continuous spectrum, provision for passing some part of this light to the entrance s l i t of a spectrometer, and a phototube with an indicating device. This receives the part of the spectrum under observation from the exit s l i t of the spectrometer, and indicates i t s intensity. The absorption c e l l containing.the solution under observation, and a "blank" c e l l may be placed in the light path either

15

b e f o r e o r a f t e r i t passes through the spectrometer, and some

method of comparing the t r a n s m i s s i o n o f the sample and bl a n k ,

e i t h e r by i n t e r c h a n g i n g them i n the l i g h t path, o r by

• • s p l i t t i n g " the l i g h t beam between them, must be i n c o r p o r a t e d .

I f the c e l l s are simply interchanged manually, v a r i a

t i o n s i n the i n t e n s i t y o f the l i g h t source w i l l i n t r o d u c e an

e r r o r i n the t r a n s m i s s i o n r e a d i n g s . The u s u a l expedient o f

b a t t e r y o p e r a t i o n o f the source i s not s a t i s f a c t o r y , s i n c e

random v a r i a t i o n s o f the i n t e r n a l r e s i s t a n c e o f b a t t e r i e s

occur, p a r t i c u l a r l y under h i g h c u r r e n t c o n d i t i o n s , l e a d i n g to

volta g e v a r i a t i o n s on the order o f m i l l i v o l t s a c r o s s a 6 v o l t

storage b a t t e r y .

The luminous i n t e n s i t y , L, o f a tungsten f i l a m e n t

source i s approximately p r o p o r t i o n a l to the f i f t h power o f

the c u r r e n t , i F p a s s i n g through i t .

|__ = a.' iff ( a ' a c o n s t a n t )

f o r a small change i n c u r r e n t A i F the change i n i n t e n s i t y

w i l l be H

A L = 5 a.' i F & 1 F

and CL L- _ 5 A i F

Assuming a constant f i l a m e n t r e s i s t a n c e , then, a change o f

1 m i l l i v o l t o r 1 p a r t i n 6000 across a 6 v o l t b a t t e r y would

produce a change i n source i n t e n s i t y on the order o f 1 p a r t

i n 1000, making measurements to 1 p a r t i n 10,000 i m p o s s i b l e .

U s u a l l y the s o l u t i o n to t h i s problem i s sought

through some method of s p l i t t i n g the l i g h t beam, or of p a s s i n g

16

i t a l t e r n a t e l y through the sample and blank c e l l s . The most

s a t i s f a c t o r y o f these i s probably the r o c k i n g m i r r o r method

used i n the Hardy-General E l e c t r i c r e c o r d i n g spectrophotometer,

but t h i s i n v o l v e s mechanical c o m p l i c a t i o n s o n l y p r a c t i c a b l e

i n an expensive instrument. P o s s i b l e b e a m - s p l i t t i n g methods

i n v o l v e the use o f a W o l l a s t o n p r i s m w i t h q u a r t e r wave p l a t e s

to give c i r c u l a r l y p o l a r i z e d l i g h t , o r h a l f - s i l v e r e d m i r r o r s .

These «±i have s e v e r a l disadvantages i n common.

1. Both sample and bl a n k must "see" the same r e g i o n

of the l i g h t source, s i n c e i n t e n s i t y v a r i a t i o n s are not

the same i n each p a r t o f i t . T h i s may n e c e s s i t a t e the

use o f an i n t e g r a t i n g sphere.

2. The a b s o r p t i o n o e l l s and b e a m - s p l i t t e r must be

p l a c e d i n the l i g h t beam a f t e r i t l e a v e s the e x i t s l i t o f

the spectrometer. The beam w i l l then be p a r t i a l l y p o l a r

i z e d , e s p e c i a l l y i f a P e l l i n - B r o c a type p r i s m i s used,

and t h i s p o l a r i z a t i o n may vary w i t h the wavelength s e t t i n g

o f the spectrometer. T h i s i s p a r t i c u l a r l y troublesome i f

the sample i s o p t i c a l l y a c t i v e . Furthermore, i f the ab

s o r b i n g substance r e e m i t s p a r t o f the i n c i d e n t l i g h t as

f l u o r e s c e n c e the phototube w i l l respond to t h i s as w e l l

as to the t r a n s m i t t e d l i g h t . T h i s can l e a d to l a r g e

e r r o r s , p a r t i c u l a r l y i f the phototube i s more s e n s i t i v e

i n the s p e c t r a l r e g i o n o f the f l u o r e s c e n c e , which i s

o f t e n the case.

3. Two phototubes and a b a l a n c i n g c i r c u i t are

nece s s a r y .

17

Another s o l u t i o n to t h i s problem may be found

through c o n t r o l of the source i n t e n s i t y . Although t h i s d i d

not appear to have been t r i e d b e f o r e , p r o b a b l y because o f the

d i f f i c u l t y o f c o n t r o l l i n g the h i g h c u r r e n t s which sources

g e n e r a l l y r e q u i r e , i t appeared to be p r a c t i c a b l e i f e f f i c i e n t

use were made of the l i g h t a v a i l a b l e . T h i s would allow a

simple interchange o f c e l l s to give accurate t r a n s m i s s i o n

r e a d i n g s .

To make maximum use o f the l i g h t from the source,

the o p t i c a l system was designed w i t h l a r g e a p e r t u r e , and

c o r r e c t e d l e n s e s . A Mazda type 851 K bulb w i t h a s t r a i g h t

tungsten wire f i l a m e n t was used as the l i g h t source, and

mounted w i t h i t s f i l a m e n t v e r t i c a l , so t h a t an image of the

f i l a m e n t c o u l d be made to f a l l a l o n g the entrance s l i t of the

spectrometer.

The minimum l i g h t i n t e n s i t y at the phototube which

can be measured w i t h the d e s i r e d degree o f accuracy depends

upon the s i z e o f "shot" e f f e c t and thermal a g i t a t i o n v o l t a g e s

developed i n the p h o t o c e l l and the f i r s t stage o f the ampli

f i e r , and on random v a r i a t i o n s i n i t s v o l t a g e supply. " D r i f t "

due to slow changes i n supply v o l t a g e i s not of primary im

portance, but must be minimized. O r d i n a r y "B" b a t t e r i e s , or

a w e l l - r e g u l a t e d power supply make s a t i s f a c t o r y v o l t a g e

s o u r c e s .

When small c u r r e n t s ( e . g . 10"" amps.) must be mea

sured a c c u r a t e l y , p r e c a u t i o n s must be taken to keep leakage

c u r r e n t s s m a l l . O r d i n a r y b a k e l i t e tube bases were found to

18

be very poor i n t h i s r e s p e c t . I f tubes w i t h t h i s type o f

base must be used, i t may be n e c e s s a r y to remove the base,

and clamp the tube i n p o s i t i o n . C o a t i n g the g l a s s w i t h s i l i

cone compound to prevent the f o r m a t i o n of a moisture l a y e r

would be a d v i s a b l e i n ease h i g h h u m i d i t i e s were encountered.

S c a t t e r e d l i g h t i s a problem i n t h i s type of s p e c t r o

photometer, s i n c e t r a n s m i t t e d l i g h t i n t e n s i t i e s at d i f f e r e n t

wavelengths may vary by a f a c t o r o f more than t e n . In the

most unfavourable case o f a s i n g l e s t r o n g a b s o r p t i o n band 10 7

Angstroms i n width, not more than 1 p a r t i n 10 of l i g h t o f o t h e r wavelengths may be s c a t t e r e d so as to r e a c h the photo-

5 tube f o r accuracy to 1 p a r t i n 10 i n the t r a n s m i s s i o n measure

ment. In p r a c t i c e , such extreme cases w i l l not occur ( e x c e p t ,

p o s s i b l y , i n compounds c o n t a i n i n g r a r e e a r t h s ) . However, a

constant d e v i a t i o n type spectrometer, r a t h e r than the L i t t r o w

type, was used i n order to minimize s c a t t e r e d l i g h t .

The accuracy w i t h which a c e r t a i n wavelength may be

r e s e t depends g e n e r a l l y on the mechanical s t a b i l i t y of the

instrument, and p a r t i c u l a r l y on the mechanical accuracy o f the

wavelength s e l e c t o r . I n t h i s type of spectrometer, wavelength

s e l e c t i o n i s accomplished by r o t a t i o n of the prism, and spec

t r a l bandwidth i s c o n t r o l l e d by simultaneous adjustment of the

entrance and e x i t s l i t s . The d i s p e r s i o n o f the p r i s m should

be s u f f i c i e n t l y h i g h t h a t the entrance s l i t width need never

be decreased to the p o i n t where the c o l l i m a t o r l e n s i s very

unevenly i l l u m i n a t e d . Thus the d i s p e r s i o n necessary depends

on the geometry o f the o p t i c a l system.

THE SPECTROPHOTOMETER OPTICAL SYSTEM

19

F i n a l l y , the e n t i r e l i g h t path o f the instrument

must be e n c l o s e d by a l i g h t - t i g h t housing, and s u i t a b l e

e l e c t r i c a l s h i e l d i n g must be p r o v i d e d f o r phototubes and t h e i r

a m p l i f i e r s .

B. The O p t i c a l System

The p l a n of the o p t i c a l system o f the spectrophoto

meter i s shown i n p l a t e I . L i g h t from the v e r t i c a l s t r a i g h t -

wire f i l a m e n t of the source S i s c o l l i m a t e d by the l e n s L , 1

passes through the a b s o r p t i o n c e l l A and i s brought to a f o c u s

at the spectrometer entrance s l i t s by L , f o r m i n g an image 1 2

of the f i l a m e n t t h e r e . L i g h t p a s s i n g through t h i s s l i t f i l l s

the c o l l i m a t o r l e n s L , then passes through the p r i s m P and the 3

l e n s L which form a spectrum at the e x i t s l i t s . The photo-4 2

tube E i s p l a c e d f a r enough behind s so t h a t l i g h t p a s s i n g 2 2

through the s l i t almost covers i t s cathode area. The c o n t r o l

phototube E keeps the source i n t e n s i t y constant by c o r r e c t i n g 1

any i n t e n s i t y changes through a D. C. a m p l i f i e r and c u r r e n t

r e g u l a t o r c i r c u i t .

Components o f the o p t i c a l system are as f o l l o w s :

1. The f o u r l e n s e s k i n d l y were s u p p l i e d by Dr.

Crooker. They were made by Candian A r s e n a l s L t d . , L e a s i d e ,

Ont. L and L are type GJ 1-2 achromats, E.F.L. 5.14", 1 2

B.P.L. 4.96", O.D. 1.260"

L i s a type DW 1-2 achromat w i t h E.F.L. 11.02", 3

B.F.L. 10.78", O.D. 1.986"

20

L i s a type OK 1-2 achromat w i t h E.F.L. 7.61", 4

B.F.L. 7.31", O.D. 2.05"

2. The l a r g e P e l l i n B r o c a p r i s m P was a l s o made by

Canadian A r s e n a l s L t d . I t i s of E.D.F. 653/336 g l a s s ,

40m.m. h i g h , w i t h an entrance f a c e o f 108 m.m. and e x i t

f a c e of 68 m.m. l e n g t h , a l l angles - o .

3. The a b s o r p t i o n c e l l s A and A are Cenco type i m i A

c u v e t t e s , o f pyrex g l a s s , 5"- 0 cms. i n l e n g t h and3-5

cms. i n diameter w i t h open top and wover p l a t e .

4. The source i s a Mazda type 851 K galvanometer

lamp, r e q u i r i n g .5 amps at 4-5 v o l t s .

5. The phototubes are R.C.A. type 929, having maxi

mum s e n s i t i v i t y at about 5000 Angstroms w i t h the source

above.

C. Mechanical Design

The o p t i c a l p a r t s are mounted on a t r i a n g u l a r base

made by welding three p i e c e s o f 4" wide s t e e l channel t o g e t h e r

and machining the f l a t upper s u r f a c e . T h i s base i s supported

8" from the t a b l e - t o p by three mahogany l e g s , one at each

c o r n e r , so t h a t the p r i s m t a b l e b e a r i n g and ca t h o d e - f o l l o w e r

c i r c u i t c o u l d be mounted beneath i t . T h i s forms a very r i g i d ,

moderately heavy base, but has s e v e r a l disadvantages. The

c h i e f o f these a r i s e s from the f a c t t h a t extruded s t e e l

channel stock i s allowed f a i r l y wide t o l e r a n c e s i n manufacture.

The f i t t i n g and welding, t h e r e f o r e , cannot be done a c c u r a t e l y

PLATE II - PLAN or SPECTROPHOTOMETER To FACE P^ce 21

21 enough to give a s u i t a b l y f l a t s u r f a c e . P l a n i n g t h i s s u r f a c e

a f t e r w elding does l i t t l e to improve i t , as the base w i l l bend

out o f shape i n the p l a n e r , and r e t u r n to i t s o r i g i n a l shape

a f t e r t h i s o p e r a t i o n i s complete., In the present case, the

f a c t t h a t the base was not f l a t n e c e s s i t a t e d c a r e f u l hand-

f i t t i n g o f the p a r t s which mounted on i t , a very time-consuming

job.. A l s o , the uneven s i d e s o f the f l a n g e s add to the problem

of making a l i g h t - t i g h t h ousing. A more s a t i s f a c t o r y base

of t h i s s o r t c o u l d be made by f i n i s h i n g the outer s i d e s o f

the f l a n g e s before welding.

Lenses L and L are mounted i n 3" x 4" p i e c e s of j?" 3 4

brass p l a t e . A h o l e o f s u f f i c i e n t s i z e to take the l e n s was

cut out, l e a v i n g a l / l 6 w f l a n g e around one s i d e . The l e n s i s

h e l d a g a i n s t t h i s f l a n g e by a r e t a i n i n g r i n g o f bra s s wire.

Each p l a t e i s sweated to a base of 3/8" b r a s s which

i s s l o t t e d to c l e a r two # 8 machine screws to h o l d i t to the

s t e e l base. Thus these l e n s e s may be moved while the i n s t r u

ment i s b e i n g f o c u s s e d , and then f i r m l y f i x e d i n p o s i t i o n .

Lenses L and L are mounted i n a s i m i l a r manner, but are f i x e d 1 2

permanently i n p o s i t i o n .

Each s l i t i s mounted on a f i x e d v e r t i c a l p a r t i t i o n

of ^" b r a s s by means of a b r a s s c o l l a r and s e t screw. T h i s

allows each s l i t to be moved d u r i n g f o c u s s i n g .

The pr i s m i s mounted on a " t a b l e " of £ " aluminum,

b e i n g h e l d i n p l a c e by a clamp c o n s i s t i n g o f a top p l a t e of

brass w i t h a rubber c u s h i o n , and three v e r t i c a l r o d s . The

pri s m t a b l e , r o u g h l y c i r c u l a r i n shape, has an 11" arm

22

extending t a n g e n t i a l l y from i t s c i r c u m f e r e n c e . T h i s arm passes

through the c o l l i m a t o r (L ) l e n s mount, and reaches almost to 3

the s l i t s , where the micrometer wavelength s e l e c t o r i s 1 l o c a t e d . Near the c e n t e r of the p r i s m t a b l e i s a I" h o l e tapped

to take the s u p p o r t i n g s h a f t which runs through the s t e e l

base to the b e a r i n g beneath. D e t a i l of t h i s double s t r e s s e d

b a l l b e a r i n g i s shown i n p l a t e E . T h i s b e a r i n g i s very impor

t a n t , as the r e p r o d u c i b i l i t y of the wavelength s e t t i n g depends

on i t .

Wavelength s e l e c t i o n i s accomplished by means o f

a S t a r r e t t depth gauge micrometer h e l d to the s t e e l base by

# 6 machine screws (both ends o f the micrometer b l o c k were

annealed and d r i l l e d ) . The micrometer s p i n d l e works a g a i n s t

a s h o r t p i e c e of p o l i s h e d s i l v e r s t e e l r o d which i s clamped

to the prism t a b l e arm near i t s end. The arm i s h e l d a g a i n s t

the micrometer s p i n d l e by a s m a l l h e l i c a l s p r i n g at the micro-

meter.

The a b s o r p t i o n c e l l s r e s t s i d e by s i d e i n b r a s s

c r a d l e s mounted on a c a r r i a g e o f l / 8 " square s t e e l r o d . T h i s

c a r r i a g e can be moved al o n g r a i l s of the same m a t e r i a l so as

to interchange the c e l l s . A b r a s s r o d and b e a r i n g allows t h i s

to be done from the o u t s i d e .

To s i m p l i f y c o n s t r u c t i o n and reduce l i g h t leakage

the s t e e l base i s covered by a b r a s s p l a t e between the source and l e n s L • The brass end p l a t e A supports the source 5 ,

2 a G-R c o a x i a l c a b l e s o c k e t , and a feed-through i n s u l a t o r , the

l a t t e r p a i r h a n d l i n g connections to the phototube E . The 1

23

source i s mounted i n a b r a s s c o l l a r which may be shimmed so

as t o b r i n g the f i l a m e n t i n t o the c o r r e c t p o s i t i o n . The out

s i d e o f the end p l a t e i s covered by a p i e c e o f 3/4 " plywood

f i t t e d to make t h i s end o f the instrument l i g h t p r o o f . E l e c t

r i c a l connections to the source are made through a b i n d i n g

post d i r e c t l y behind i t , and the grounded o u t e r conductor o f

the c o a x i a l c a b l e .

The r e g u l a t o r phototube, E , i s mounted about 3" from 1

the source i n an o c t a l socket h e l d j u s t o f f the s t e e l base by

br a s s mounting b l o c k s . I t s v o l t a g e d i v i d e r , R ; R , and 1 2

the condenser, C ,are mounted i n the space between S and L . 1 1

T h i s p a r t o f the instrument i s e n c l o s e d by a cover o f 22

gauge sheet b r a s s which s h i e l d s the phototube, and keeps l i g h t

out.

The measuring phototube, E ,which r e c e i v e s l i g h t from 2

the e x i t s l i t was o r i g i n a l l y mounted i n an o c t a l socket h e l d

j u s t beyond the end of the s t e e l base by a b r a s s p l a t e . L a t e r

i t was found necessary to remove i t s base, and i t i s now clamped

i n a h o r i z o n t a l p o s i t i o n a g a i n s t the end w a l l o f i t s housing.

I t i s about 2" from the e x i t s l i t o f the spectrometer. S i n c e

i t s photocathode i s approximately square, i t s mounting p o s i

t i o n i s not important.

W i r i n g from the socket o f t h i s phototube, E , runs 2

through the adjacent mahogany l e g i n s i d e a b r a s s s h i e l d t o the

cat h o d e - f o l l o w e r i n p u t stage o f the a m p l i f i e r . The l e a d from

i t s photocathode i s the c e n t e r conductor o f a short p i e c e of

^ M c o a x i a l c a b l e , the out e r conductor o f which i s grounded.

HAMMOND 27H

R E G U L A T E D POWER SUPPLY

PLATE I To Fact Rxq« 2H

24

The l i g h t - p r o o f cover between l e n s L and phototube 1

E i s made from p i e c e s of 3/4" f i r f i v e - p l y . The s i d e s are 2

b o l t e d to the s t e e l base, and f a s t e n e d to a l l f i x e d v e r t i c a l

p a r t i t i o n s w i t h machine screws. The f a c e o f the plywood was

removed to a depth o f about l / 8 M where i t i s i n c o n t a c t w i t h

the base o r a p a r t i t i o n , so t h a t these j o i n t s would be l i g h t -

t i g h t . The top i s i n three s e c t i o n s , the s m a l l e s t one cover

i n g the a b s o r p t i o n c e l l s . T h i s p a r t may be removed e a s i l y ,

and c o u l d be hinged to f a c i l i t a t e changing of the a b s o r p t i o n

c e l l s .

The e n t i r e i n s i d e o f the instrument i s p a i n t e d a

d u l l b l a c k w i t h a n o n - r e f l e c t i n g " p a i n t " of lampblack and

s h e l l a c i n acetone.

D. The E l e c t r o n i c C i r c u i t s

1. The Voltage Regulated Power Supply ( P l a t e m) 10

T h i s power supply i s of c o n v e n t i o n a l d e s i g n

except t h a t the g a i n o f the 6SJ7 stage i s low, and balanced

D.G. a m p l i f i e r stages w i t h 6SH753 are used to o b t a i n a s t a -5

b i l i z a t i o n r a t i o n of 10 . I t s c a l c u l a t e d impedance i s l e s s

than .1 ohm.

I t s u p p l i e s 330 v o l t s a t 50 m.a. to the l i g h t source

and r e g u l a t o r , 330 v o l t s at 6 m.a. to the main a m p l i f i e r , and

105 v o l t s to the phototube and c a t h o d e - f o l l o w e r stage. To

minimize r i p p l e the h e a t e r o f the 6SJ7 i s s u p p l i e d from a 6

10 M.I.T. R a d i a t i o n Lab. S e r i e s , V o l 21, P a r t 3, p.556, McGraw H i l l , New York, 1948.

25 volt storage battery, and the time constant of the regulator ci r c u i t i s kept small. It i s built on a Hammond 12" x 17" x 3" chassis with the regulator as far away from the power transformer and 110 volt A.C. wiring as possible.

This power supply i s designed to give a voltage constant to 1 part in 10,000 under normal conditions of load and line voltage variation. Since the amplifiers which i t supplies are a l l of the "balanced" type, calculation shows that regulation to this degree i s sufficient to keep concentration errors resulting from supply voltage variations well below those resulting from shot effect in the phototube.

Tests were carried out on the completed power supply with a Dumont 5" oscilloscope and showed:

a. At a load current of 30 m.a. the line voltage could be varied between 105 and 125 volts, with the output voltage, as seen on the oscilloscope at f u l l gain, remaining apparently constant at 340 volts.

b. No trace of ripple appeared at f u l l gain on the oscilloscope within the control range.

c. There was some tendency to oscillate at high frequencies (above 10 Kc) beyond the extremes of the contr o l range, but none in this range.

d. At a line voltage of 115, regulation commenced at about 15 m.a. and continued beyond 50 m.a.

The output voltage and range of control can be varied by means of the three potentiometers, which are somewhat interdependent*

! 6SL7 6SL7 6SL7

LIGHT SOURCE REGULATOR PLATE K SOURCE I^TEHSITY CONVT/NNT TO I 10,000

' Ta Fa.cc Pfr<>« 2,1a

http://Fa.cc

26

2. The L i g h t Source Regu l a t o r ( P l a t e ED

The a m p l i f i e r and r e g u l a t o r c i r c u i t i s on a

Hammond 8" x 12" x 3" c h a s s i s , and c o n s i s t s of a three-stage

balanced a m p l i f i e r (6SL7S;) w i t h a v o l t a g e g a i n o f more than 4

10 , and a 6L6 t r i o d e connected c u r r e n t r e g u l a t o r .

The type 929 phototube E i s mounted the c o r r e c t 2 c

d i s t a n c e (about 3") from the l i g h t source S> so t h a t the photo-7

c u r r e n t f l o w i n g through the 10 ohm r e s i s t o r keeps the g r i d

o f one t r i o d e s e c t i o n o f the f i r s t a m p l i f i e r stage a t 22

v o l t s . The g r i d o f the o t h e r t r i o d e s e c t i o n i s s u p p l i e d w i t h

a b a l a n c i n g v o l t a g e from a 22^ v o l t b a t t e r y .

i l l

A W V

,. - 1 i

! G A I N > \oH

1

F i g u r e 1 shows a s i m p l i f i e d c i r c u i t o f the r e g u l a

t o r . A b r i e f a n a l y s i s of i t s o p e r a t i o n , based on t h i s c i r c u i t ,

f o l l o w s .

The f r a c t i o n a l change i n source b r i g h t n e s s , ,

which r e s u l t s from a change

be found.

d V i n b a t t e r y v o l t a g e must

27

The change i n c u r r e n t through the source, A L 5 ,

w i l l be the d i f f e r e n c e between the change i n c u r r e n t s u p p l i e d

by the b a t t e r y , A L B , and the compensating change i n p l a t e

c u r r e n t through the r e g u l a t o r tube, A L P •

A L s - A L B - A L P ( 1 )

as b e f o r e A k . = 5 AL^ ( 2 ) L • I*

and s i n c e the p h o t o e l e c t r i c response i s l i n e a r , t h i s w i l l a l s o

be equal to the f r a c t i o n a l change i n the v o l t a g e e across the 7

1 0 ohm r e s i s t o r

A L - A_e ( 3 )

f o r the 6L6, A i p = <jm A = ^ Q Ae

where G i s the a m p l i f i e r g a i n g- the transeonductance o f the 6L6 e, the g r i d v o l t a g e of the 6L6. .

from ( 1 ) A L s = A L B - ^ Q A £

from ( 2 ) and ( 3 ) A i s - A i B - g w als)

A L* - | + % Gl§£) 10,000 AL S

w s '

I f the change i n b a t t e r y v o l t a g e i s A \ /

A V - "R A L B - Z 7 A Lfi

and from ( 2 ) _A_L _ 5 Ak. = 5 A i B «

f o r a b a t t e r y v o l t a g e V = 1 2 v o l t s

F o r A\/ = >0l v o l t A ^ « s 10

THE MAIN PLATE

A MPLI FIER

T U B E S T Y P E 6SL7GT T o Fa.ce ?a.<t* 2 8

http://Fa.ce

28

Thus v a r i a t i o n s i n l i g h t source i n t e n s i t y should

c o n t r i b u t e much l e s s to the f r a c t i o n a l c o n c e n t r a t i o n e r r o r

than shot e f f e c t does.

The a m p l i f i e r i n t h i s c i r c u i t works under heavy

feed-back c o n d i t i o n s , and the .05 m.f.d. condenser G was 1

found necessary to decrease i t s time constant to about .5

seconds and prevent o s c i l l a t i o n which otherwise r e s u l t s from

the l a g i n response o f the lamp to c u r r e n t changes. O p e r a t i o n

of the l i g h t source c o n t r o l i s now s a t i s f a c t o r y .

3. The Main A m p l i f i e r ( P l a t e % )

T h i s c i r c u i t a m p l i f i e s the p h o t o e l e c t r i c c u r r e n t

from the measuring phototube, E . The c a t h o d e - f o l l o w e r i n p u t 2

stage and b a l a n c i n g network are i n s i d e a Hammond 4" x 8" x 2" c h a s s i s mounted beneath the spectrophotometer base near E ,

2

whi l e the a m p l i f i e r i t s e l f i s on a separate 8" x 12" x 3"

c h a s s i s .

B efore t h i s a m p l i f i e r was designed, the f o l l o w i n g

estimates were made: 1 ) . The approximate p h o t o e l e c t r i c c u r r e n t expected

from E . 2

L i g h t r e a c h i n g the c o l l i m a t o r l e n s , assuming a 1 cm

s l i t of width equal to the diameter o f the source f i l a m e n t , -2

i s 2 x 10 lumens.

Prom the response curve f o r an S-4 photocathode and

2.6 70°K tungsten source, and the average c u r r e n t (45 micro-

amps per lumen) f o r type 929 phototubes (see R.G.A. Tube Hand

book) , the maximum c u r r e n t expected, at 5000 Angstroms i s on

29

-9 the order o f 10 amps/Angstrom. Thus w i t h a bandwidth of

-8 10 Angstroms and no a b s o r p t i o n , about 10 amps i s expected

-9 at 5000 Angstroms, and about 10 amps at 4000 or 6000 Angstroms. With an absorbing s o l u t i o n o f optimum d e n s i t y ,

-10 about 10 amps w i l l be o b t a i n e d near the extreme ends of the v i s i b l e spectrum, thus to measure the d e n s i t y to 1 p a r t

-14 \ i n 10,000, changes o f 10 amps must be measurable.

2 ) . The magnitude o f i n t e r f e r i n g e f f e c t s .

a ) . The simple s h o t - e f f e c t f o r m u l a d e r i v e d

fey a p p l i c a t i o n o f Campbell's Theorem to the c i r c u i t i s v a l i d

f o r phototubes. T h i s g i v e s , f o r the r.m.s. shot e f f e c t c u r r e n t ,

I _ Ij e where i i s the p h o t o e l e c t r i c c u r r e n t , e the

i J xnc

e l e c t r o n i c charge, R the s e r i e s r e s i s t a n c e , and 0 i t s e f f e c t i v e

shunting c a p a c i t a n c e . 9

F o r R = 10 ohms, C = 50 u u f , and, the maximum -8

expected c u r r e n t i - 10 amps t h i s g i v e s :

Maximum r.m.s. shot e f f e c t c u r r e n t i s 1 0 ' amps

Fo r minimum i = 10 amps, i s ~ '0 amps.

b ) • Thermal f l u c t u a t i o n s i n the r e s i s t a n c e R gi v e r i s e to a c u r r e n t i T o f r.m.s. value i _ = /H- K T

where K i s Boltzmann's constant and T the absolute tempera

t u r e . In t h i s a m p l i f i e r , i T i - 7 x I O " ' 4 " amps.

T h i s f i g u r e r e p r e s e n t s the lowest a t t a i n a b l e thermal

n o i s e l e v e l , and c o n s i d e r a b l e e x p e r i m e n t a t i o n may be necessary

be f o r e something c l o s e to i t can be achieved. In p a r t i c u l a r ,

30

semi-conductors such as the r e s i s t o r R vary widely i n n o i s e

c h a r a c t e r i s t i c s , e s p e c i a l l y at low f r e q u e n c i e s , and leakage

c u r r e n t s may produce c o n s i d e r a b l e n o i s e . The use of a h i g h -

q u a l i t y r e s i s t a n c e , and c a r e f u l i n s u l a t i o n should keep thermal

" n o i s e " l e s s than shot e f f e c t " n o i s e " oyer most o f the spec

trum. The present instrument i s not completely s a t i s f a c t o r y

i n t h i s r e s p e c t .

A b a l a n c e d D.C. a m p l i f i e r c i r c u i t i s used to o b t a i n

a v o l t a g e g a i n v a r i a b l e between 1,000 and 60,000 at the g r i d s

of the output tube. Thus the computed minimum observable -14 -5

c u r r e n t change i n the phototube o f 10 amps, or 10 v o l t s

on the ca t h o d e - f o l l o w e r g r i d s , w i l l r e s u l t i n a t h e o r e t i c a l

change of .6 v o l t s on the output g r i d s , o r 2.4 m.a. on the

output meter. However, t h i s meter, l o c a t e d between the cathodes

of the output tube, i s so arranged t h a t not more than 1 m.a.

can pass through i t under any c o n d i t i o n s . I t ' s s e n s i t i v i t y

decreases as i t ' s s c a l e r e a d i n g i n c r e a s e s , and i t i s i n t e n d e d

o n l y as a n u l l i n d i c a t o r .

In p r a c t i c e , i t was found t h a t a g a i n between 5,000

and 10,000 was most s a t i s f a c t o r y . The a m p l i f i e r g a i n should

not be v a r i e d d u r i n g use, as the s l i g h t as asymmetry of the

two s e c t i o n s of the g a i n c o n t r o l cause some v a r i a t i o n i n the

p o s i t i o n o f the n u l l .

The l e a d marked "potentiometer" i n P l a t e ¥ was

connected to an e x t e r n a l c a l i b r a t e d potentiometer so t h a t i t

was v a r i a b l e between 11 v o l t s and 22 v o l t s . T r a n s m i s s i o n

readings were taken from t h i s p otentiometer.

31

D i f f i c u l t i e s were encountered when the cathode-

f o l l o w e r and phototube c i r c u i t s were t e s t e d . These r e s u l t e d

from leakage c u r r e n t s i n the p h o t o c e l l and 6SL7 base, i n s u l a

t i o n leakage i n o t h e r p l a c e s , which v a r i e d w i t h the h u m i d i t y ,

and o b j e c t i o n a b l y h i g h r e s i s t o r n o i s e . T h i s p a r t o f the

c i r c u i t was r e b u i l t , and the base of the phototube was r e

moved. Con s i d e r a b l e improvement r e s u l t e d , but i t s o p e r a t i o n

was s t i l l not i n agreement w i t h e x p e c t a t i o n s .

Suggestions f o r improvement of t h i s c i r c u i t f o l l o w :

1. The c i r e u l t c o u l d be r e b u i l t , w i t h the 6SL7

r e p l a c e d by a 6J6, a h i g h - q u a l i t y 10 ohm r e s i s t o r

(Noballoy) s u b s t i t u t e d f o r the one used at p r e s e n t .

C a r e f u l c l e a n i n g of a l l i n s u l a t i o n , and use of s i l i c o n e

compound on g l a s s s u r f a c e s should r e s u l t i n s a t i s f a c t o r y

o p e r a t i o n .

or 2. The phototube c o u l d be r e p l a c e d by a photomulti-

p l i e r tube. Three o r f o u r stages o f m u l t i p l i c a t i o n should

e l i m i n a t e a l l leakage t r o u b l e s , and w i l l i n c r e a s e the

shot e f f e c t n o i s e by a s m a l l f a c t o r o n l y .

E . Performance

The o p t i c a l system was a l i g n e d and f o c u s s e d , s t a r t

i n g from the source and working toward the phototube.

The source was t h e n r e p l a c e d by a 100 watt G.E.

mercury lamp. T h i s was accomplished by use o f a p l a n e , f r o n t -

s i l v e r e d m i r r o r p l a c e d i n f r o n t o f 1 . 1

Pi_ATE JZLb * MERCURY YELLOW LINES To F a c e Page 3 2

32

The c a l i b r a t i o n graph p l a t e "EE a and the ourve show

i n g the mercury y e l l o w l i n e s , p l a t e "ZT b, were o b t a i n e d . In

the l a t t e r , v o l t a g e s i n d i c a t e d were r e a d from a 20,000 ohm per

v o l t v o ltmeter across the cathode r e s i s t o r o f the cathode

f o l l o w e r . Both s l i t s were very narrow (widths l e s s than .01

mm.). The r e s o l v i n g power o f the instrument, estimated from

t h i s graph, i s 1000.

Accuracy w i t h which a wavelength may be r e s e t i s

l i m i t e d by the s i z e o f the s c a l e d i v i s i o n s on the micrometer.

With c a r e , i t i s p o s s i b l e to r e s e t to .1 of the s m a l l e s t d i v i

s i o n , o r one ten-thousandth of an i n c h . T h i s corresponds to

.25A a t 4,000 A, and to 1A a t 6,000 A. 3

D i s p e r s i o n at the e x i t s l i t , s , i s 9 x 10 A / i n . o r 2

360 A/mm. i n the neighbourhood o f 5,000 A.

T h i s spectrophotometer was designed to measure the

t r a n s m i s s i o n to one p a r t i n t e n thousand. Because of the d i f f i

c u l t i e s w i t h the cathode f o l l o w e r c i r c u i t mentioned p r e v i o u s l y ,

measurement to one p a r t i n f i v e hundred i s a l l t h a t i s p o s s i b l e

at p r e s e n t , w i t h bandwidth of 20-30 A. T h i s i s p a r t l y due to

the decrease i n s e n s i t i v i t y o f the phototube and cathode f o l

lower c i r c u i t as the 6SL7 g r i d p o t e n t i a l i s r a i s e d ( f i g . II-').

GrR.H> V o i - T S I I . L I

o 5 i o i s T o i s

Fi g . n

33

T h i s i s probably the r e s u l t o f leakage c u r r e n t s i n t h i s c i r

c u i t . I f these are e l i m i n a t e d , and r e s i s t o r n o i s e d e c r e a s e d

as suggested, a much c l o s e r approach to the de s i g n value

appears p o s s i b l e .

V i t a m i n D assay was not attempted w i t h t h i s i n s t r u

ment. A p r e l i m i n a r y i n v e s t i g a t i o n o f the Sob e l c o l o r i m e t r i c

r e a c t i o n , made w i t h a Beokman model D spectrophotometer,

showed t h i s r e a c t i o n to be too u n s t a b l e to warrant i n c r e a s e d

instrument accuracy.• The r e s u l t s o f t h i s i n v e s t i g a t i o n appear

i n the next c h a p t e r .

34

IV. VITAMIN D ASSAY

A. The P r o p e r t i e s o f V i t a m i n D

There are s e v e r a l i r r a d i a t e d s t e r o l s w i t h v i t a m i n

D a c t i v i t y . These i n c l u d e i r r a d i a t e d e r g o s t e r o l o r c a l c i

f e r o l ( v i t a m i n D ) , i r r a d i a t e d 7 - d e h y d r o c h o l e s t e r o l ( v i t a m i n 2

B )., and ot h e r s such as i r r a d i a t e d 2 2 - d i h y d r o e r g o s t e r o l 3-

( v i t a m i n D ) which have lower potency and appear to occur i n 4 11

nature to a very l i m i t e d e x t e n t . I r r a d i a t i o n o f the s t e r o l

p r e c u r s o r s w i t h u l t r a - v i o l e t l i g h t i s nece s s a r y f o r v i t a m i n

B a c t i v i t y , and pro b a b l y has the e f f e c t o f opening a double

bond i n the s t e r o l molecule. The t r a n s f o r m a t i o n to v i t a m i n

D i s never complete, as the v i t a m i n i t s e l f i s changed by i r r a -12

d i a t i o n i n t o o t h e r p r o d u c t s , some of which are q u i t e t o x i c .

Although i t s p r e c u r s o r s t e r o l s are wid e l y d i s t r i

buted i n nature, the v i t a m i n occurs i n very small q u a n t i t i e s

11 B i l l s , C. E., Ma s s i n g a l e , O.N., Hickman, K.CD., and Gray, E . l e B . , "A New V i t a m i n D i n D o d l l v e r O i l " , J . B i o l . Chem. 126, 241, 1938.

12 Morton, R. A., "Ab s o r p t i o n S p e c t r a of Vitamins and Hormones", 2nd e d i t i o n , p. 35, Adam H i l g e r L t d . , London 1942.

35

except i n some l i v e r o i l s , p a r t i c u l a r l y those o f c e r t a i n

f i s h . However, the economic importance of f i s h l i v e r o i l s as

a source of v i t a m i n D i s d e c l i n i n g , because the " s y n t h e t i c "

v i t a m i n produced by i r r a d i a t i o n has become r e l a t i v e l y i n e x

pensive, and may be used to " f o r t i f y " o i l s which are low i n

potency.

The two important D v i t a m i n s (D and D ) are q u i t e 2 3

s i m i l a r i n p h y s i c a l and chemical p r o p e r t i e s , and appear to be

about equal i n a n t i r a c h i t i c potency ( a b i l i t y to cure r i c k e t s )

f o r most animals and f o r humans. F o r b i r d s v i t a m i n D i s 2

g e n e r a l l y much l e s s e f f e c t i v e than v i t a m i n D . Both v i t a m i n s 3

are i n s o l u b l e i n water, but s o l u b l e i n f a t s and many o t h e r o r g a n i c s o l v e n t s . Although not very s t a b l e i n c r y s t a l l i n e

13 form, they are q u i t e s t a b l e i n s o l u t i o n .

V I T A M I N T3 x ( 'CALC^EROIJ) V I T A M I N D 3

H i s t o r i c a l l y , b o t h c o d l i v e r o i l and s u n l i g h t were

used to cure r i c k e t s d u r i n g the 19th c e n t u r y , and the a n t i r a

c h i t i c p r o p e r t y of i r r a d i a t e d foods was s t u d i e d by Hess and

13 Huber, W., and Barlow, O.W., "The Chemical and B i o l o g i c a l S t a b i l i t y o f C r y s t a l l i n e V itamins D„ and D- and T h e i r D e r i v a t i v e s " , J . B i o l . Chem., 149, 125, ° 1943.

36

Steenbock i n 1919. The a n t i r a c h i t i c f a c t o r was named v i t a m i n

D i n 1922, and prepared by i r r a d i a t i o n o f e r g o s t e r o l about

1927. At f i r s t e r g o s t e r o l was thought to be the o n l y p r e c u r s o r ,

but i n 1934 i t was found t h a t there must be ot h e r substances 14

w i t h v i t a m i n D a c t i v i t y , when i r r a d i a t e d . I n 1936 Brockmann

o b t a i n e d the pure v i t a m i n B from tunny l i v e r o i l , , and showed

i t t o be v i t a m i n D . The presence of other D vi t a m i n s i n 3 15

some f i s h l i v e r o i l s had been suggested by B i l l s to e x p l a i n the v a r i a t i o n s i n r a t / c h i c k e f f i c a c y r a t i o , between d i f f e r e n t

o i l s .

B. Methods of Assay

The potency o f a v i t a m i n D p r e p a r a t i o n i s u s u a l l y

s p e c i f i e d i n I n t e r n a t i o n a l U n i t s (abbr. I.U.) per gram. The

I n t e r n a t i o n a l U n i t i s based on comparison w i t h an i n t e r n a t i o n a l

standard s o l u t i o n of i r r a d i a t e d e r g o s t e r o l i n o l i v e o i l by a

b i o l o g i c a l assay method i n which the c a l c i f i c a t i o n o f the 16

bones o f r a t s produced by standard and unknown i s measured.

F o r both D and D , one I.U. i s e q u i v a l e n t to approximately 2 3

14 Brockmann, H., " I s o l a t i o n o f V i t a m i n D from Tunny L i v e r O i l " , Z P h y s i o l . Chem., 241, 104, 1936.

15 B i l l s , C.E., Massingale, O.N., and Imboden, W., S c i e n c e , 80, 596, 1934.

16 Coward, K.H., "The B i o l o g i c a l S t a n d a r d i z a t i o n o f the V i t a m i n s " , 2nd e d i t i o n , B a i l l i e r e , T i n d a l l , and Cox, London, 1947.

37

o n e - f o r t i e t h o f a microgram o f the pure v i t a m i n . The potency

of an o i l which i s to be used i n the f e e d i n g o f chickens i s

s p e c i f i e d i n e i t h e r B.S.I, or A.O.A.C. u n i t s , b o t h o f which

are based on b i o l o g i c a l assay w i t h c h i c k s .

The b i o l o g i c a l methods are accurate to w i t h i n 15%

when p r o p e r l y used. By these methods p o t e n c i e s on the o r d e r

of 5 I.U./g. can be measured almost as e a s i l y as h i g h poten-4 6

c l e s (10 - 10 I.U./g.)

The u l t r a v i o l e t a b s o r p t i o n s p e c t r a of the v a r i o u s

v i t a m i n D p r e c u r s o r s and t h e i r photochemical t r a n s f o r m a t i o n

products have been r e p o r t e d i n numerous papers, the r e s u l t s 17

of which a r e summarized by Morton. The D v i t a m i n s are

s i m i l a r i n a b s o r p t i o n , each h a v i n g a s i n g l e broad band w i t h 1%

i t s maximum about 2650 Angstroms and E about 500. A s o l u -lcm

t i o n of pure v i t a m i n i n some non-absorbing s o l v e n t such as

ether can be assayed a c c u r a t e l y by measuring i t s a b s o r p t i o n

at 2650 Angstroms. T h i s must be done r a p i d l y , as photochemi

c a l d e g r a d a t i o n of the v i t a m i n w i l l take p l a c e .

The e x t e n s i v e l i t e r a t u r e on c o l o r i m e t r i c assay 18

methods f o r v i t a m i n D i s reviewed up to 1941 by Morton . Of s e v e r a l c o l o r t e s t s suggested, the antimony t r i c h l o r i d e t e s t

19 o f Broekmann and Chen proved most s a t i s f a c t o r y . T h i s i s the

17 Morton, R.A., " A b s o r p t i o n S p e c t r a o f Vitamins and Hormones", 2nd e d i t i o n , pp. 30-37, Adam H i l g e r L t d . , London 1942.

18 I b i d . , p. 37.

19 Broekmann, H., and Chen, Y.H., Z . P h y s i o l . Chem., 241, 129, 1936.

38

f a m i l i a r C a r r - P r i c e r e a c t i o n used i n v i t a m i n A assay where

i t has proven v e r y s a t i s f a c t o r y . U n i t f o r u n i t , v i t a m i n D

has o n l y about o n e - f i f t i e t h the a b s o r p t i o n c o e f f i c i e n t o f

v i t a m i n A i n t h i s t e s t , and thus i s much more d i f f i c u l t to

measure. C o n s i d e r a b l e work has been done on t h i s r e a c t i o n , 20

i n v o l v i n g s t u d i e s o f i t s v a r i a t i o n w i t h temperature and time,

o f the s t a b i l i t y o f the reagent, which may be improved by the 21

a d d i t i o n o f a c e t y l c h l o r i d e , and of the i n f l u e n c e o f the

s o l v e n t . These r e s u l t e d i n the technique used by DeWitt and 22

S u l l i v a n who used a m o d i f i e d antimony t r i c h l o r i d e reagent

i n ethylene c h l o r i d e and measured the a b s o r p t i o n at 5000 Ang

stroms. As i n the u l t r a v i o l e t method, low c o n c e n t r a t i o n s o f

pure v i t a m i n D can e a s i l y be measured, w i t h the a d d i t i o n a l

advantage t h a t photochemical a c t i o n does not occur.

These two methods are rendered u s e l e s s when a p p l i e d

d i r e c t l y to low o r medium potency o i l s , such as n a t u r a l f i s h

l i v e r o i l s or " f o r t i f i e d " o i l s , because o f the i n t e r f e r e n c e o f

o t h e r substances which absorb the same wavelengths as the

v i t a m i n . S a p o n i f i c a t i o n o f the o i l removes the f a t s , and con

c e n t r a t e s the v i t a m i n up to t e n times or more i n the unsaponi-

f i a b l e f r a c t i o n . However, the o t h e r s t e r o l s p r e s e n t , i n a d d i t i o n

20 Shantz, E.M., "Antimony T r i c h l o r i d e R e a c t i o n o f V i t a m i n D", Ind. Eng. Chem. ( A n a l y t i c a l E d ) , 16, 179, 1944.

21 N e i l d , CfcH.,Russell, W.G., and Zimmerli, A., "The S p e c t r o -photometric Determination o f Vitamins D 2 and D3", J . B i o l . Chem.,136, 73, 1940.

22 DeWitt, J.B., and S u l l i v a n , M.X., "A S p e c t r o p h o t o m e t r y Procedure f o r the Q u a n t i t a t i v e E s t i m a t i o n of Vitamins D", Ind. and Eng. Chem. ( A n a l y t i c a l Ed.) 18, 117, 1946.

39

to the vitamin A accompany the vitamin D there, and s t i l l obscure i t s absorption band. This i s especially true of ultraviolet absorption, and ultraviolet measurement has not been used in vitamin D assay recently except on solutions of almost pure vitamin. The antimony trichloride method gives better discrimination, and o i l s containing more than 10,000 I.U./g. may be assayed with f a i r accuracy. Attempts to measure o i l s of lower potency by this method were made by a

23 number of workers. Milas, Heggie, and Reynolds using a Hardy-General Electri c recording spectrophotometer studied this problem. They tried to make empirical corrections for vitamin A and sterol interference, and to eliminate vitamin A with maleic anhydride, but met with l i t t l e success. Ewing,

24 Kingsley, Emmett and Brown with Improved chemical methods for the elimination of vitamin A and the sterols, could assay o i l s of potency greater than 5,000 I.U./g. Finally, DeWitt

25 and Sullivan used an improved chromatographic technique similar to that of Broekmann to separate vitamin A and sterols from the vitamin D in the unsaponifiable fraction. This was successfully applied to low potency o i l s (around 100 I.U./g.),

23 Milas, N.A., Heggie, R. and Raynolds, J.A., "Spectroscopic Method for the Quantitative Estimation of Vitamin D", Ind. Eng. Chem. (Analytical Ed.) 13, 227, 1941.

24 Ewing, D. T., Kingsley, G. V., Emmett, A. D. and Brown,R.A., "Physical-Chemical Method for the Determination of Vitamin in Fish Liver Oils", Ind. Eng. Chem. (Analytical Ed.), 15, 301, 1943. .

25 DeWitt, J. B., and Sullivan, M. X.,loc. c i t .

40

to capsules and to tablets, yielding results in agreement with those obtained by bioassay.

A new eolorimetric reaction for vitamin D assay 26

was suggested by Sobel, Mayer, and Kramer . They used as a reagent glycerol 1,3 - dichlorhydrin with 1% acetyl chloride added, and chloroform as a solvent. Pure vitamins D and D ,

2 3 and some of the related sterols in their pure form were used in this work. It was found that very good discrimination between vitamins and sterols could be obtained. Although the absorption coefficients were not given in this paper, they were apparently very small i n a l l cases. The authors did not give any information on the applicability of this method to the assay of natural o i l s and vitamin preparations. This appeared well worth investigating, as a simpler procedure than that of DeWitt and Sullivan might result.

Estimation of vitamin D in irradiated ergosterol -2

by the ultraviolet and antimony trichloride methods has been 27

discussed by Ewing, Poweil, Brown, and Emmett. Pirlot and 28

Rouir used the Sobel reagent successfully for this purpose, 26 Sobel, A. E., Mayer, A.M., and Kramer, B., "New Golori-metric Reaction of Vitamins D„ and D* and Their Provitamins", Ind. Eng. Chem., (analytical Ed.)17,160,1945.

27 Ewing, D.T., Powell, M.J., Brown, R.A., and Emmett, A.D., "The Determination of Vitamin D 2 by Two Physical Chemical Methods," Analytical Chemistry, 20, 317, 1948.

28 P i r l o t , V., and Rouir, S.T., "The Determination of Vitamin Do i n Products of Irradiated Ergosterol", Bui. Soc. Chim. Beiges, 56, 296, 1947.

41

but found i t necessary to remove taohysterol f i r s t , which they did with citraconic anhydride.

C. The Chemical Composition of Fish Liver Oils

Fish l i v e r o i l s vary widely in composition with species and to a lesser extent with environment. Complete analyses are d i f f i c u l t , and few appear to have been done,

29,30,31 however a general picture obtained from several sources i s as follows.

The saponifiable portion of the o i l consists of combined fatty acids and i s of l i t t l e interest as i t w i l l generally be removed before a vitamin assay i s attempted.

The unsaponifiable portion, which comprises around 1% of the l i v e r o i l s of Gadidae (e.g. Cod) and 10$ to 50$ of those of Elasmobranchii (e.g. Dogfish) i s a complex mixture containing sterols, hydrocarbons, and alcohols. In eodliver o i l , cholesterol,C H 0H,makes up about half the

27 45 unsaponifiable portion, while in other o i l s unsaturated hydrocarbons such as squalene, C H , may predominate. Vita-

30 50 mins A, D, and E are present i n very small quantities along with other complex alcohols such as sebrachyl alcohol , 8 H 0 OH, i n fact the unsaponifiable portion of eodliver 21 41 2

29 Hickman, K.C.D., "Adventures in Vacuum Chemistry1,' Science in Progress, 4th ser. p.p. 205-248, Yale University Press, New Haven, 1945.

30 Drummond, J.C., and Hilditch, T.P., "The Relative Values of Cod Liver Oils from Various Sources", HM Stationary Office, London, 1930.

31 , "The Composition of Liver Oils of the Basking Shark and Spiny Shark", (South African Fish Products XXVII), J.S.C.I., 67,104, 1948.

42

o i l i s es t i m a t e d to c o n t a i n l e s s than .05% v i t a m i n D i n

g e n e r a l .

Although v i t a m i n A and the s t e r o l s are g e n e r a l l y

c o n s i d e r e d r e s p o n s i b l e f o r most o f the i n t e r f e r i n g a b s o r p t i o n

because o f t h e i r chemical s i m i l a r i t y to the v i t a m i n , there

i s reason to suspect t h a t the ot h e r u n s a t u r a t e d o r g a n i c

compounds w i l l show a b s o r p t i o n , and may take p a r t i n e o l o r i

m e t r i c r e a c t i o n s .

D» The G l y c e r o l D i c h l o r h y d r l n C o l o r i m e t r l c R e a c t i o n

An i n v e s t i g a t i o n of the G l y c e r o l D i c h l o r h y d r l n

(G.D.H.) c o l o r r e a c t i o n was c a r r i e d out w i t h a Beckman model

D spectrophotometer. The a b s o r p t i o n maximum at 6250-6500 32

Angstroms r e p o r t e d by S o b e l , Mayer and Kramer as due to

v i t a m i n D was of primary i n t e r e s t , although curves were

p l o t t e d from 3000-10,000 Angstroms i n some c a s e s .

As a p r e l i m i n a r y , the a b s o r p t i o n spectrum o f a

whole c o d l i v e r o i l was examined (see f i g . I ) . I t s absorp

t i o n i s q u i t e low around 6000 Angstroms, and there seemed

to be a p o s s i b i l i t y t h a t v i t a m i n D c o n c e n t r a t i o n s i n the

whole o i l c o u l d be measured d i r e c t l y i f the other components

o f the o i l d i d not i n t e r f e r e w i t h the v i t a m i n D - G.D.H.

r e a c t i o n .

32 S o b e l , A.E., Mayer A.M., and Kramer, B.,l o p . c i t .

43

Eastman Kodak practical grade glycerol 1,3-dichlor-hydrin was obtained, and since i t was slightly brown in color i t was r e d i s t i l l e d in vacuo before being used, as suggested by Sobel. The d i s t i l l a t e appeared to be colourless and was used in a l l the following work. The activated reagent was prepared by adding .225 g. (about 1%) of acetyl chloride (CP.) to 20 c.c. of the r e d i s t i l l e d glycerol dichlorhydrin. After approximately one hour some discolouration was noticed. This increased slowly (see f i g . Ila) but was not severe enough to interfere seriously with the tests which followed. This reagent was stored in darkness,0°C It was assumed that the discoloration was due to impurities i n the glycerol dichlorhydrin, because the addition of 1% acetyl chloride to a portion of the original undistilled reagent resulted in the rapid development of color (see f i g . l i b ) . Repeated

44 vacuum d i s t i l l a t i o n s o f the g l y c e r o l d i c h l o r h y d r i n would

probably e l i m i n a t e t h i s completely,

The o i l s used i n these t e s t s were a v i t a m i n D 6 3

concentrate (10 I.U./g.) and a t y p i c a l f e e d i n g o i l (P5995)

c o n t a i n i n g 400 I.U./g. v i t a m i n D and 1500 I.U./g. v i t a m i n A

ac c o r d i n g to l a b e l s p e c i f i c a t i o n s .

F i r s t the procedure o f Sobel was f o l l o w e d and the

v a r i a t i o n o f a b s o r p t i o n w i t h time was s t u d i e d u s i n g the

v i t a m i n D conc e n t r a t e (see f i g . I I I ) . A .5$ s o l u t i o n o f 3

the concentrate i n c h l o r o f o r m was prepared. 3b 2 c c . of

t h i s i n a 1 cm. c e l l , 1 c c . o f a c t i v a t e d G-.D.H. reagent was

added from a p i p e t t e , and the whole thoroughly mixed. A

comparison c e l l c o n t a i n i n g 2 c c . of pure c h l o r o f o r m and 1 c c .

of G.D.H. reagent had p r e v i o u s l y been prepared. Readings

were taken as r a p i d l y as p o s s i b l e , and i n t e r p o l a t e d to o b t a i n

the curves shown (see t a b l e I ) . The temperature o f the absorp

t i o n c e l l s was between 27 aG and 28°C, and i t was est i m a t e d

TABLE I

Wave- Time from Percent C o r r e c t i o n E s t i m -l e n t h mixing Transmis- f o r 15 min. ated t r a n s -

min. s i o n curve m i s s i o n at 15 mins.

Dens:

800 23 79 + 8.0 87 .06

700 10 78 - 5.0 73 .137

675 12 7 31 - 4.0 1

.157

650 13 67| - 2.5 65 .187

640 13^ 2 64 - 2.0 62 .207

630 14 6 l i - 1.3 .217 2 2 .217 1 1 625 14- 6 l i - 0.6 61 .215 2 2

4 .215

620 15 6 l i 2

0 4 .210

610 1 5 i 61 + 0.6 .210

600 16 >s| + 1.0 64| .192

580 17 65 + 2.0 .170

540 24 62| + 9.0 - i .145

520 25 59l +10.0 69| .157

510 26 61 +11.0 72 .142

500 24 62 +11.5 73^ 2

.134

490 27 62 +12.0 74 .130

450 6 l J +12.5 74 .130

46

t h a t the a b s o r p t i o n c e l l c o n t a i n e d between 9,000 and 10,000

I.U. o f v i t a m i n D.

' jbo ' *o*» ' ™ « ' S o * >

Fi q- m _____ These curves c l e a r l y show the v i t a m i n D maximum

at 6250 Angstroms, i n a d d i t i o n to a weak maximum at about

5000 Angstroms, probably due to 7 - d e h y d r o c h o l e s t e r o l ( p r o v i t a m i n D )• They a l s o show c o n s i d e r a b l e a b s o r p t i o n

3

from some other source, e s p e c i a l l y toward the v i o l e t end o f

the spectrum. T h i s a b s o r p t i o n appears to i n c r e a s e g r e a t l y

w i t h time, as does t h a t due to the v i t a m i n .

Next, the same procedure was f o l l o w e d u s i n g a

s o l u t i o n o f .220 gms. o f whole f e e d i n g o i l (P5995) i n 1 c c .

of c h l o r o f o r m , and adding 2 c c . o f the G.D.H. reagent (see

f i g . I V ) . Although o n l y about 90 I.u". o f v i t a m i n D was i n

the a b s o r p t i o n c e l l , s t r o n g a b s o r p t i o n due to oth e r c o n s t i

tuents o f the o i l was observed. The a b s o r p t i o n maximum due

to v i t a m i n A at 5250 Angstroms was v i s i b l e . Prom the s t r o n g

47

and r a p i d l y changing a b s o r p t i o n observed, i t was concluded

t h a t t h i s method c o u l d not be a p p l i e d d i r e c t l y to t h i s

f e e d i n g o i l . •

! F .q LY I U . 1 _ . . . . )

A p o r t i o n o f the o i l was then s a p o n i f i e d , f o l l o w

i n g the procedure of DeWitt and S u l l i v a n , and the n e u t r a l ,

u n s a p o n i f i a b l e p o r t i o n from 3 g o f o i l d i s s o l v e d i n 1 c c .

of c h l o r o f o r m was added to 2 c c . o f t h e G.D.H. reagent i n

the a b s o r p t i o n c e l l . The very s t r o n g a b s o p t i o n observed

(see f i g . V) suggested t h a t the i n t e r f e r i n g substances r e

mained w i t h the v i t a m i n i n the u n s a p o n i f i a b l e p o r t i o n o f the

o i l . D i l u t i o n w i t h c h l o r o f o r m t o l / l O c o n c e n t r a t i o n a f t e r

m i x i n g showed t h a t Beer's law d i d not h o l d .

D i l u t i o n o f the u n s a p o n i f i e d p o r t i o n of the o i l

w i t h c h l o r o f o r m p r e v i o u s to mixing, so th a t the unsaponif iable

p o r t i o n o f .3 g. of the o i l i n 1 c c . o f c h l o r o f o r m was mixed

w i t h 2 c c . o f the G.D.H. reagent, gave s i m i l a r r e s u l t s .

48

i • • • i i i i . i i i • i i i

009 7000 1000

F . c j U

The s t r o n g I n t e r f e r i n g a b s o r p t i o n and r a p i d changes

i n the spectrum which were observed w i t h f e e d i n g o i l P5995,

as w e l l as the dependence o f t h i s r e a c t i o n on s o l v e n t to

reagent r a t i o s , suggests t h a t t h i s method i s not w e l l s u i t e d

to the v i t a m i n D assay of any but very h i g h potency o i l s . 33

A r e c e n t r e p o r t by Campbell t r e a t s t h i s r e a c t i o n

i n g r e a t e r d e t a i l than the above d i s c u s s i o n . Dr. Campbell

used h i g h e r potency o i l s than the f e e d i n g o i l (P5995), and

found t h a t d e v i a t i o n s from Beer's Law were s e r i o u s . He a l s o

suggested the use of a second v i t a m i n D a b s o r p t i o n maximum

at 4000 Angstroms which, under c e r t a i n c o n d i t i o n s , gave a

somewhat s t r o n g e r a b s o r p t i o n than the 6250 Angstroms.

33 Campbell, J.A., " M o d i f i e d G l y c e r o l D i c h l o r h y d r l n R e a c t i o n f o r V i t a m i n D 3", A n a l y t i c a l Chemistry, 20, 766, 1948.

49

V. CONCLUSION

A p h o t o e l e c t r i c spectrophotometer, u s i n g a l i g h t

source o f c o n t r o l l e d i n t e n s i t y has been c o n s t r u c t e d . I t has a

wavelength r e s e t accuracy o f 1 A o r l e s s over the v i s i b l e

spectrum, and measures t r a n s m i s s i o n s to 1 p a r t i n 500, w i t h a

bandwidth of 20-30 A.

The g l y c e r o l - d i c h l o r h y d r i n c o l o r i m e t r i c r e a c t i o n f o r

v i t a m i n D has been i n v e s t i g a t e d w i t h a Beckman Model D s p e c t r o

photometer, and found u n s u i t a b l e f o r the v i t a m i n D assay of

three t y p i c a l o i l s o f low potency ( i . e . below 10,000 I.U./g.).

T e s t s were conducted w i t h whole o i l , a n d w i t h the u n s a p o n i f i a b l e

f r a c t i o n i n the case of one o i l .

An i n v e s t i g a t i o n of the s t a b i l i t y o f t h i s r e a c t i o n

showed that i n c r e a s e d instrument accuracy would improve assay

r e s u l t s very l i t t l e . Beer's Law was not obeyed i n t h i s r e a c t i o n .

A c o l o r i m e t r i c r e a c t i o n was shown to take p l a c e w i t h o t h e r sub

stances p r e s e n t , b e s i d e s v i t a m i n D. Some of these substances

accompanied the v i t a m i n D i n the u n s a p o n i f i a b l e f r a c t i o n of the

o i l , where t h e i r r e l a t i v e l y s t r o n g a b s o r p t i o n completely ob

scured the weak a b s o r p t i o n maxima of v i t a m i n D ( E,1* = 80 a t ^ * I cm

6250 A) i n the f e e d i n g o i l samples i n v e s t i g a t e d .

As the antimony t r i c h l o r i d e assay method has been

thoroughly i n v e s t i g a t e d by o t h e r s , and i s c o n s i d e r e d u n r e l i a b l e

f o r d i r e c t assay o f low-potency o i l s , p r e l i m i n a r y c o n c e n t r a t i o n

of the v i t a m i n D by chromatographic s e p a r a t i o n appears to be

necessary.

50

V I . BIBLIOGRAPHY

BOOKS

B l o o r , W. R,, B i o c h e m i s t r y o f the P a t t y A c i d s , R e i n h o l d Pub. Corp., New York, 1943.

Brode, W. R., ; C o m i c a l Spectroscopy. John Wiley and Sons lf\c. , N . Y. , 1 9 3 5 .

Coward, K. H., The B i o l o g i c a l S t a n d a r d i z a t i o n o f the V i t a m i n s , 2nd ed., B a i l l i e r e , T i n d a l l , and Cox, London, 1947.

M.I.T. R a d i a t i o n L a b o r a t o r y S e r i e s , V o l s . 18 and 21, McGraw H i l l , New York, 1948.

Sawyer, R. A., Exp e r i m e n t a l Spectroscopy, P r e n t i c e - H a l l Inc., New i o r k , 1946.

ARTICLES

Hickman, K.C.D., "Adventures i n Vacuum Chemistry", Science i n Pro g r e s s , 4 t h s e r . , Y a l e U n i v e r s i t y P r e s s , New Haven, 1945.

Loofbourow, J . R., " P h y s i c a l I d e n t i f i c a t i o n o f Vitamins and Hormones," Vitamins and Hormones, V o l . I , Academic 'Press, New :York, 1943.

W i l l i a m s , R. J . , "The S i g n i f i c a n c e o f the V i t a m i n Content o f T i s s u e s , " Vitamins and Hormones, V o l . I , Academic P r e s s , New York, 1943.

PAPERS

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