UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Physico-chemical methods for vitamin D assay and a new spectrophotometer Whittemore, Thomas Edwin 1950

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1950_A8 W4 P4.pdf [ 2.9MB ]
Metadata
JSON: 831-1.0085395.json
JSON-LD: 831-1.0085395-ld.json
RDF/XML (Pretty): 831-1.0085395-rdf.xml
RDF/JSON: 831-1.0085395-rdf.json
Turtle: 831-1.0085395-turtle.txt
N-Triples: 831-1.0085395-rdf-ntriples.txt
Original Record: 831-1.0085395-source.json
Full Text
831-1.0085395-fulltext.txt
Citation
831-1.0085395.ris

Full Text

u  Lr  i  t°ir*  fi  7 fit  Ce>,fi • I  PHYSICO-CHEMICAL METHODS FOR VITAMIN D ASSAY AND A NEW  SPECTROPHOTOMETER  by Thomas Edwin  A Thesis the  Whittemore  submitted i n p a r t i a l  fulfilment of  requirements f o r the degree o f MASTER OF ARTS i n t h e department of PHYSICS  THE UNIVERSITY OF B R I T I S H April,  1950  ©LUMBIA  ABSTRACT The  design  and  construction of  spectrophotometer i s described. a wavelength r e s e t s p e c t r u m , and  accuracy  The  completed  20-30 A.  transmission  reading  s o u r c e makes an  instrument  o f 1 A o r l e s s over the  measures t r a n s m i s s i o n s  bandwidth of  a photoelectric  to 1 part  are  included.  accuracy  of 1 part  The  use  of  i n 10,000  visible  i n 50G  S u g g e s t i o n s f o r improved  has  with  accuracy  a  of  a controlled  theoretically  attainable. This  i n s t r u m e n t was  assay of low-potency o i l s of  the  that  not  with  oils. the  so  that vitamin  attempted.  r e a c t i o n by  authors  s p e c t r o p h o t o m e t e r on  I t showed t h a t t h i s r e a c t i o n was  no  showed  A preliminary i n -  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  a Beckman model D  D  Investigation  several  s u i t a b l e f o r t h i s purpose.  v e s t i g a t i o n of the out  c o u l d be  antimony t r i c h l o r i d e  i t was  constructed  several  carried typical  more s u i t a b l e t h a n  other. Because of  method was  the  instability  f o u n d whereby i n c r e a s e d  gave  of  The  feasi-  b l e method o f  assay f o r low-potency o i l s  concentration  of  D by  no  accuracy  increase  vitamin  these r e a c t i o n s  instrument  corresponding  the  i n accuracy  of  assay.  only  at present  chromatography.  involves  a  ACKNOWLEDGEMENT  T h i s work was c a r r i e d  out a t t h e B r i t i s h  C o l u m b i a R e s e a r c h C o u n c i l , V a n c o u v e r , B.C. The  author  Maddigan, d i r e c t o r the  funds  w i s h e s t o t h a n k D r . S. E .  o f t h e C o u n c i l , f o r making  available  and f a c i l i t i e s w h i c h made t h i s p r o j e c t  possible. The  author  i s indebted  t o D r . A.C. Young,  f o r m e r Head o f t h e P h y s i c s D i v i s i o n f o r many v a l u a b l e s u g g e s t i o n s , work, a n d f o r c o n s t a n t interest  incorporated i n this  h e l p and a d v i c e .  o f D r . A. M. C r o o k e r ,  the P h y s i c s Department  of the Council,  The h e l p f u l  a n d o t h e r members o f  of the U n i v e r s i t y of B r i t i s h  C o l u m b i a was a l s o a p p r e c i a t e d .  TABLE OF CONTENTS Page I.  INTRODUCTION A. B. G. D.  II.  P h y s i c s and V i t a m i n s • The S p e c t r o p h o t o m e t e r i n A s s a y Work The P r o b l e m o f V i t a m i n D A s s a y The P r o p o s e d P r o j e c t  ABSORPTION  • . . . .  1 4 4 6  SPECTROPHOTOMETRY  A. B.  Introduction The Laws o f A b s o r p t i o n  C.  P r e c i s i o n o f Measurement  7 8 10  III.  THE  IV.  A. Design P r i n c i p l e s B . The O p t i c a l S y s t e m G. M e c h a n i c a l D e s i g n D. The E l e c t r o n i c C i r c u i t s E. Performance VITAMIN D ASSAY  14 19 20 24 31  A. B. C.  34 36  D.  SPECTROPHOTOMETER  The P r o p e r t i e s o f V i t a m i n D Methods o f A s s a y . . . . . . . The C h e m i c a l C o m p o s i t i o n o f F i s h Liver Oils: 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  .  41 42  V.  CONCLUSION  49  VI.  BIBLIOGRAPHY  50  PLATES  Page I.  The S p e c t r o p h o t o m e t e r  Optical  II.  P l a n of Spectrophotometer  21  III.  R e g u l a t e d Power S u p p l y  24  IV.  Light  26  V.  The Main A m p l i f i e r .  VI.  (a) C a l i b r a t i o n C u r v e  Source Regulator....  System  19  28  32 (b) M e r c u r y Y e l l o w L i n e s  PHYSICO-CHEMICAL METHODS FOR  VITAMIN D ASSAY  AND A NEW  SPECTROPHOTOMETER  I.  A.  Physics  and  The  Vitamins  e x i s t e n c e o f what a r e now  d e m o n s t r a t e d as l o n g ago 1910,  INTRODUCTION  as 1881,  i t was  realization life  field, rapidly  until  o f the f u n d a m e n t a l importance o f the  processes  and  gave g r e a t  i n c r e a s e d from t h a t time  to p l a y a secondary r o l e  such instruments chemist  and  and  and  Their  vitamins  in  this  physiology  onward. a t l e a s t , be  i n t h i s work; t h a t o f  techniques  physiologist.  o f Funk  Impetus t o r e s e a r c h i n  knowledge o f v i t a m i n c h e m i s t r y  was  after  r e c e i v e d much a t t e n t i o n .  P h y s i c i s t s must, f o r t h e p r e s e n t tent  not  when t h e d i s t i n g u i s h e d s t u d i e s o f H o p k i n s and  were p u b l i s h e d , t h a t v i t a m i n s  all  but  known as v i t a m i n s  as may  be  o f use  supplying  t o the  However, s u c h r a p i d p r o g r e s s  b e e n made d u r i n g t h e p a s t f o r t y y e a r s  con-  would have been  bioas  has  impossible  2 without instruments such as the modern spectrophotometer and polarimeter, and further advances i n instrument design, or the application of new physical methods to t h i s f i e l d are of prime importance.  For example, the application of high-vacuum 1  techniques by Hickman  and others resulted i n the "molecular  s t i l l " , and improved techniques i n i n f r a - r e d 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 i n quantities of the order of a few micrograms per gram to carry out t h e i r work as catalysts and bring about profound physiological changes.  In  f a c t one of the most sensitive assay methods makes use of t h i s property, and involves the observation of laboratory animals under c a r e f u l l y controlled conditions and d i e t .  Although wide-  l y used, i t has disadvantages which cannot wholly be overcome. The wide v a r i a t i o n i n response of i n d i v i d u a l 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 i n each t e s t .  A single assay by t h i s method  involves weeks or months of work, and i s thus expensive and slow.  For t h i s reason physical and chemical methods are pre-  f e r a b l e , even i f less accurate, i n routine assay work. Many physical methods have been applied, but only a 2 few have found general use. According to Loofbourow, these 1 2  Hickman, K.C.D.,"Adventures i n Vacuum Chemistry", "Science i n Progress". 4th ser. pp.205-248, Yale Univ.Press, New Haven, 1945. Loofbourow, J.R., "Physical I d e n t i f i c a t i o n of Vitamins and Harmones", "Vitamins and Hormones". Vol.1, pp.109155, Academic Press, New York, 1943.  are a b s o r p t i o n spectrophotometry  (visible  and u l t r a v i o l e t ) ,  colorimetry, f l u o r e s c e n c e spectrophotometry, Of t h e s e , a b s o r p t i o n s p e c t r o p h o t o m e t r y  and f l u o r i m e t r y .  i s t h e 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 t h u s h a v e a characteristic  a b s o r p t i o n spectrum  i n the v i s i b l e  o r near  ultraviolet* If t o be u s e d arise.  a q u a n t i t a t i v e measurement o f s u c h a s p e c t r u m i s  t o o b t a i n an a s s a y , a number o f c o m p l i c a t i o n s may  Briefly 1.  these a r e :  Other  substances  a b s o r b i n g i n t h e same  spectral  r e g i o n may be p r e s e n t i n c o n c e n t r a t i o n s many t i m e s of  that  the v i t a m i n . 2.  hold,  F o r l a r g e organic molecules Beer's  l a w may n o t  a n d a b s o r p t i o n may d e p e n d o n t h e s o l v e n t ,  t h e pH, t o a m a r k e d 3.  and on  degree.  Photochemical  d e g r a d a t i o n o f t h e 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 "colorimetrie  from the use o f a chemical  t e s t " f o r t h e v i t a m i n , and c a r e f u l  of the a b s o r p t i o n spectrum  measurement  o f t h e r e a c t i o n c o m p l e x so f o r m e d .  However, t h e s e c o m p l e x e s o f t e n t e n d t o b e u n s t a b l e , a n d t h e i r a b s o r p t i o n s p e c t r a may v a r y w i t h t i m e , t e m p e r a t u r e ination.  I f sufficient  and i l l u m -  improvement w i t h r e g a r d t o i t e m 1 i s  o b t a i n e d i n a d d i t i o n t o t h e e l i m i n a t i o n o f i t e m 3, o f t h e s e f a c t o r s may make t h i s m e t h o d  practicable.  control  4 B.  The  Spectrophotometer  The accuracy  Work  v a l u e o f an a s s a y p r o c e d u r e  and r e p r o d u c i b i l i t y  w i t h w h i c h t h e y c a n be try  i n Assay  depends u p o n  of i t s r e s u l t s ,  obtained.  and  the  Thus p h o t o e l e c t r i c  the  speed photome-  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  faster,  and m i n i m i z e s  ment s u c h Ltd.  as t h e  be  suitable  spectrum  Spectrographic  "Spekker" spectrophotometer  gives results  may  personal errors.  of f a i r  accuracy  and  o f Adam H i l g e r  repreducibility,  i f a p e r m a n e n t r e c o r d o f t h e whole  i s required.  Photoelectric  equip-  and  absorption  spectrophotometers  such  as t h e Beckman M o d e l D have c o m p a r a b l e o r b e t t e r c h a r a c t e r i s tics  combined w i t h g r e a t e r f l e x i b i l i t y .  obtained d i r e c t l y from  the instrument,  wavelength o n l y , or over interest. may  Density readings and may  be made a t  A l s o , d a t a on t i m e - d e p e n d e n t c h e m i c a l r e a c t i o n s instruments.  T h e s e a d v a n t a g e s h a v e made t h e p h o t o e l e c t r i c photometer s t a n d a r d equipment  be  and  a s s a y work.  The  done depends l a r g e l y on  meter.  An  one  any r a n g e o f w a v e l e n g t h s w h i c h i s o f  be o b t a i n e d w i t h t h e s e  s e a r c h and  are  i n laboratories  engaged i n r e -  a c c u r a c y w i t h w h i c h s u c h work the performance o f the  instrument which gives r a p i d  d e n s i t y r e a d i n g s , and  spectro-  and  spectrophoto-  accurate  t r a n s m i t s o n l y a narrow  can  wavelength  spectral  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 T h e r e now  dure f o r almost  exists  Assay  a satisfactory rapid  a l l t h e more common v i t a m i n s .  assay For  proce-  some,  such  a s v i t a m i n A,  e o l o r i m e t r i c methods a r e  o t h e r s , s u c h as fluorimetric  the B v i t a m i n s ,  these  s a t i s f a c t o r y , while have been r e p l a c e d  or m i c r o b i o l o g i c a l techniques.  The  for  by  outstanding 3  e x c e p t i o n h e r e i s v i t a m i n D, p o i n t s out sterols  and  that comparatively  chemically similar  f o r several reasons.  large quantities of  to vitamin D  w h e r e v e r t h i s v i t a m i n i s f o u n d , and from i t . in liver  V i t a m i n A, oils,  inactive  generally  c a n n o t e a s i l y be  present separated  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  and h e l p s  b a n d o f v i t a m i n D.  are  Morton  t o mask t h e r e l a t i v e l y weak a b s o r p t i o n 4  Williams  vitamin D content  a s c r i b e s the  on  the  of tissues largely  It  i s t r u e t h a t t h e v i t a m i n c a n be  by  the  lack of  information  to economic  produced q u i t e  reasons.  inexpensively  i r r a d i a t i o n of stilt able m a t e r i a l s with u l t r a v i o l e t  n e v e r t h e l e s s much work h a s i r r a d i a t e d foods, and u n t i l  this  the problem o f  vitamin concentrates,  i s s o l v e d the  A review At the present  b e e n done on  assay  and  of v i t a m i n D assay  t i m e none i s c o m p l e t e l y  assaying  "fortified"  o f t i s s u e s must  light,  oils,  wait.  methods i s g i v e n i n I V satisfactory,  2.  although 5  the p r e l i m i n a r y r e s u l t s  o f the  seem p r o m i s i n g .  t i m e t h i s p r o j e c t was  At  the  technique  o f DeWitt  and  Sullivan  commenced, J u l y ,  .1946, t h e e o l o r i m e t r i c r e a c t i o n s u g g e s t e d b y S o b e l , Mayer and 3 M o r t o n , R.A., " A b s o r p t i o n S p e c t r a o f V i t a m i n s and Hormones", 2nd e d . p . 37, Adam H i l g e r L t d . , L o n d o n , 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 t h e V i t a m i n C o n t e n t o f T i s s u e s " , " V i t a m i n s and Hormones". V o l I , pp. 229247, A c a d e m i c P r e s s , New Y o r k , 1943.  5  D e W i t t , J.B. Ed.,  and 18,  S u l l i v a n , M.X., 117, 1946.  Ind. Eng.  Chem,, A n a l y t i c a l  6  6 Kramer  had n o t 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 o f t h e v i t a m i n D c o m p l e x f o r m e d 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 b e t w e e n t h e vitamin  a n d some o f t h e i n t e r f e r i n g  r e c o r d o f any a t t e m p t t o u s e t h i s  s t e r o l s was c l a i m e d .  t e s t on n a t u r a l o i l s  No  was  available.  D.  The P r o p o s e d P r o j e c t The  p r o p o s e d p r o j e c t h a d two aims i n v i e w .  These  were as f o l l o w s : 1. tant  ,  The s p e c t r o p h o t o m e t e r h a s become a v e r y  instrument,  widely  work o f a l l k i n d s .  used i n chemical  By m o d i f y i n g  what i t was b e l i e v e d t h a t accuracy able  and s t a b i l i t y  and b i o l o g i c a l  the usual  an i n s t r u m e n t  c o u l d be b u i l t  impor-  design  some-  o f improved  at a cost  t o t h a t o f the commercial instruments  compar-  available at  present. 2.  When c o m p l e t e d t h i s  investigate  6  Sobel,  instrument  the problem o f vitamin D  A . E . , M a y e r , A.M., Analytical  Ed.,  c o u l d be u s e d t o  assay.  a n d K r a m e r , B., I n d . E n g . Chem.  17,  160,  1945..  7  II.  A.  ABSORPTION SPECTROPHOTOMETRY  Introduction When m o n o c h r o m a t i c 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 u p o n one f a c e filled  with  o f a plane  parallel  c e l l , or cuvette,  a s o l u t i o n , i t may be d i s p o s e d  o f i n any o f t h e  f o l l o w i n g ways: 1.  by r e f l e c t i o n  2.  by Rayleigh  at the various i n t e r f a c e s .  s c a t t e r i n g a n d Raman e f f e c t  i n the  solution. 3.  by absorption  and c o n v e r s i o n  change, e t c . , o r r e e m i s s i o n 4. b y t r a n s m i s s i o n In the absorption ratio the  Ijx , where I  To  t f  through c e l l  and s o l u t i o n .  spectrophotometry o f s o l u t i o n s the  i s the i n t e n s i t y  i n c i d e n t u p o n , a n d Ix  i n t e n s i t y e m e r g e n t f r o m x cms. o f s o l u t i o n , i s t o be This ratio  t r a n s m i s s i o n , T, o f x cms. o f s o l u t i o n .  losses of intensity  a t t r i b u t a b l e to the c e l l  cell  solvent  t o that o f a "blank", alone.  or i d e n t i c a l  i s called  Correction for itself  s o l v e n t may be made b y c o m p a r i n g t h e t r a n s m i s s i o n and  chemical  as f l u o r e s c e n c e ;  m e a s u r e d as a f u n c t i o n o f w a v e l e n g t h . the  to heat,  cell  or to the  of solution containing  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 c a l l e d  11  colorimetry".  This  term i s also applied to the measurement of absorption bands of the "complexes" formed i n a chemical "eolorimetric" reaction, and since t h i s i s generally done by colorimetry as defined above, no d i s t i n c t i o n 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 w e l l . B.  The Laws of Absorption When monochromatic l i g h t traverses a homogeneous  absorbing medium, the l i g h t transmitted by each succeeding layer of a given thickness i s a constant f r a c t i o n of the l i g h t incident upon i t .  Thus the fundamental d i f f e r e n t i a l  of absorption spectrophotometry i s where  -» d I  _  &  equation  I  i s the "absorption c o e f f i c i e n t " of the medium. Integration between 1=0 and l=x gives  Lambert's Law:  l  or  x  log^T  = i 2  3  01  0  JL **"  In a s o l u t i o n , absorption varies with concentration of the solute.  For many solutes absorption i s proportional  to concentration over a wide range. Law:  oc = ^3 c  This i s known as Beer's  where ^3 i s the "absorption co-  e f f i c i e n t " per u n i t concentration  c .  9  These laws may be combined as In  Ioq  or  K  .If =  K C ^>  i s generally c a l l e d the "extinction c o e f f i c i e n t " , and i f  the concentration c i s i n percent (meaning grams of solute per 100 ml. of solution) icm~  Q ( m % ) * X ( m cm.]  may be given instead. The expression  ^ 5( - j 0<  *  2  0  s  c a l l e d the "den-  s i t y " D. The "molecular extinction c o e f f i c i e n t " logarithm are sometimes used.  £  or i t s  I t i s the same asKabove when  c i s given i n moles per l i t e r . i f a number of substances with extinction c o e f f i cients  K,,Ki,---, K  are present as a mixture i n the  n  absorption c e l l , with concentrations  c, , c , . , - - . c  respective-  n  l y , and i f there i s no chemical i n t e r a c t i o n , the resultant density w i l l be D  = I oq  la I->c  =,  "X ( K,C,+-K Ci x  t  • - • +  K  n  C  n  )  For s t r i c t l y monochromatic l i g h t traversing a homogeneous medium Lambert's Law i s exact.  Deviations from Beer's  Law, on the other hand, are quite common, e s p e c i a l l y when the solution contains large and asymmetrical molecules.  They occur  because the nature of the solvent, i t s pH, and the concentrat i o n of absorbing material may influence the molecules of the  10 absorbing material.  This  i s p a r t i c u l a r l y true  molecules formed i n chemical e o l o r i m e t r i c Law  should not  G.  P r e c i s i o n of In  the  be  a s s a y work t h e  ( I f B e e r ' s law =  complex  reactions,  and  Beer's  verification.  L a m b e r t - B e e r Law  c when t h e  i s obeyed).  A~D  i s used to  o p t i c a l density Then  C  A C  where  error  i n the  find  been measured  "D  c  i n density  fractional error  D has  = -p^  i s the  e r r o r A "E>  w h i c h r e s u l t s f r o m an The  the  Measurement  concentration  AC  assumed w i t h o u t  of  and  in  concentration  determination. estimation  of  con-  c  centration density  i s equal to  the  p h o t o g r a p h i c method, i f the  d e t e r m i n e d v i s u a l l y , has estimates that & D  an  o f H .03 ±  can AC  Discussing 8 Loofbourow  be -  &~£>  A I  ±  4_D  the  match-points  i n d e p e n d e n t o f D, D of  expected.  •  assumes t h a t  difference  a  average d e n s i t y  C  able  the  determination. The  error  A ~D i n  fractional error  1.5  This -  1  with a gives  .02  an or  and  are Loofbourow  probably accuracy  of  2$.  T>  p r e c i s i o n of  the  i t i s l i m i t e d by  in light  photoelectric the  i n t e n s i t y , and  minimum  that  A  method, determin-  I  5!  L o o f b o u r o w , J.R., "Physical I d e n t i f i c a t i o n of Vitamins Hormones", " V i t a m i n s and Hormones". V o l I , p . 121, A c a d e m i c P r e s s , New Y o r k , 1943.  8  I b i d . p. 122.,  is  and  11  independent o f the i n t e n s i t y I .  I f t h i s i s true  tion error  as f o l l o w s ;  ±. &S  c a n be d e r i v e d  the concentra-  c  D  * I o ql01£  =  loc,  cLT> a. i x dP - -  ( 0  I  -  o  l o g  l  0  .  I *  - . 1 o g ,„ A. I oq  ix ' JL , ^ I x  l 0  1* i The tration will  AC  - f  AT?  -  . H3H3.fc  f r a c t i o n a l error i n density, be l e a s t when  I  locj  x  )o  4 Ixl  fl)  and thus i n concen-  Is.  i s a maximum, i . e .  when  I oc This of  J i 0  la  =  assumption that  P  =• - 4-3 i+3  the error  l£ i s v a l i d o n l y f o r the s i m p l e s t  i n s t r u m e n t , s u c h as t h e c o l o r i m e t e r the  A IX  s  independent  type o f p h o t o e l e c t r i c t y p e . >:CIt: i m p l i e s  that  a c c u r a c y i s l i m i t e d s i m p l y by the s i z e o f the s m a l l e s t  scale  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 better  ments o t h e r f a c t o r s d e t e r m i n e t h i s l i m i t . sidered but  ^  T h e s e w i l l be c o n -  i n more d e t a i l u n d e r " D e s i g n P r i n c i p l e s " P a r t  a r e l i s t e d b e l o w w i t h t h e optimum d e n s i t y  fractional  concentration 1.  The  instru-  photographic  & l  f o r minimum  e r r o r computed f o r e a c h :  Random f l u c t u a t i o n s i n l i g h t  resultant  III, 1,  x  i s proportional  and v i s u a l m e t h o d s .  o f D, a n d t h e g r e a t e s t  possible  source i n t e n s i t y . to  Thus a n  U  as i n t h e  i s independent  D s h o u l d be u s e d .  12  2.  Random variations i n gain of amplifier. A l  x  i s proportional to IQ Ix  . « stant.  -  i -y^:  a  where a, i s the proportionality con-  Gn substitution i n equation ( 1 ) , the condition that  the denominator have i t s maximum value gives E  3.  =  =  .717  Shot e f f e c t i n the phototube. This i s the ultimate l i m i t a t i o n on the photo-  e l e c t r i c type of instrument.  I t i s caused by the random  manner i n which electrons leave the photocathode, and i s proportional to the square root of the current through the phototube . For high-vacuum phototubes t h i s current i s l i n e a r l y dependent upon the i n t e n s i t y of illumination of the cathode. Thus  where I i s the photoelectric current, and  &  2  , a_ are con3  stants. Combining t h i s equation and equation (1) as before, J) -  the optimum density i s  4.  1 1^%-^  =  .6686  Thermal a g i t a t i o n "noise" i n r e s i s t o r s , a I-* i s independent of  5.  T  x  ,  D = .'+3 43  thus  Inexact repositioning of the absorption c e l l i n  the l i g h t beam:  Zl I  x  be as large as possible.  i s proportional to I  x  , and D should  A w e l l designed  and  have i t s p r e c i s i o n l i m i t e d b y rather  than  any  constructed instrument shot e f f e c t  o f the o t h e r f a c t o r s .  t h e optimum o p t i c a l d e n s i t y D o f t h e is  about  .87,  corresponding  t o 10%  i n the  For  such  would  phototube an  instrument  s o l u t i o n b e i n g measured  - 15%  transmission.  14  III.  THE SPECTROPHOTOMETER  A. Design P r i n c i p l e s The f i r s t step i n the design of a spectrophotometer for  a c e r t a i n job i s an evaluation of the factors which w i l l  limit i t s efficiency.  In addition, ease of operation and f l e x -  i b i l i t y must be weighed against construction d i f f i c u l t y , c o s t  v  and a v a i l a b i l i t y of materials, and a s a t i s f a c t o r y compromise reached.  Such considerations indicated that an instrument  capable of measuring the i n t e n s i t y of the transmitted l i g h t within 1 part i n 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 v i s i b l e region of the spectrum), was a reasonable  goal.  A photoelectric spectrophotometer consists of a l i g h t source giving a continuous spectrum, provision f o r passing some part of t h i s l i g h t to the entrance s l i t of a spectrometer, and a phototube with an i n d i c a t i n g device. This receives the part of the spectrum under observation from the e x i t s l i t of the spectrometer, and indicates i t s i n t e n s i t y . The absorption c e l l containing.the  solution under observation,  and a "blank" c e l l may be placed i n the l i g h t path either  15  before  or after  i t passes through the spectrometer,  m e t h o d o f c o m p a r i n g t h e t r a n s m i s s i o n o f t h e sample e i t h e r by i n t e r c h a n g i n g ••splitting"  the l i g h t  If  them i n t h e l i g h t  path,  are simply  interchanged  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  error i n the transmission readings. b a t t e r y o p e r a t i o n o f the source  incorporated.  manually,  will  The u s u a l  and b l a n k ,  o r by  beam b e t w e e n them, must be  the c e l l s  a n d some  varia-  introduce expedient  i s not s a t i s f a c t o r y ,  an of  since  random v a r i a t i o n s o f t h e 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  voltage  v a r i a t i o n s on the o r d e r  storage  battery. The  source the  i  F  =  a.' iff  (a'  A i  be  filament power o f  F  a  constant)  t h e change i n i n t e n s i t y  H  A and  L  CL L-  Assuming a constant 1 millivolt  = _  5 a.' i  &1  F  5 A i  F  F  f i l a m e n t r e s i s t a n c e , then,  o r 1 p a r t i n 6000 a c r o s s  p r o d u c e a change i n s o u r c e in  a 6 volt  through i t .  a s m a l l change i n c u r r e n t  will  across  p r o p o r t i o n a l to the f i f t h  passing |__  for  of m i l l i v o l t s  luminous i n t e n s i t y , L, o f a tungsten  i s approximately  current,  conditions, leading to  a change o f  a 6 volt battery  i n t e n s i t y on the o r d e r  1000, m a k i n g m e a s u r e m e n t s t o 1 p a r t i n 10,000 Usually  would  of 1 part impossible.  the s o l u t i o n t o t h i s problem i s sought  t h r o u g h some method o f s p l i t t i n g  the l i g h t  beam, o r o f p a s s i n g  16 it  a l t e r n a t e l y t h r o u g h t h e sample  s a t i s f a c t o r y o f these  i s probably  and b l a n k c e l l s . the r o c k i n g m i r r o r  used i n the Hardy-General E l e c t r i c but in  this  involve  recording  involves mechanical complications  an e x p e n s i v e  instrument.  method  spectrophotometer,  only practicable  P o s s i b l e b e a m - s p l i t t i n g methods  the use o f a W o l l a s t o n  to give c i r c u l a r l y  The most  prism with  polarized light,  quarter  wave p l a t e s  or half-silvered  mirrors.  T h e s e « ± i have s e v e r a l d i s a d v a n t a g e s i n common. 1.  B o t h sample  of the l i g h t  source,  and b l a n k must since i n t e n s i t y  the  same i n e a c h p a r t o f i t .  use  o f an i n t e g r a t i n g s p h e r e . 2.  placed the  spectrometer.  ized, and  The a b s o r p t i o n  i n the l i g h t  especially  this  oells  with  slit of  prism  polar-  i s used,  the wavelength  setting  troublesome i f  F u r t h e r m o r e , i f t h e ab-  p a r t o f the i n c i d e n t l i g h t  the phototube w i l l  as t o t h e t r a n s m i t t e d l i g h t . errors, particularly  the e x i t  This i s particularly active.  must be  t h e n be p a r t i a l l y  i f a P e l l i n - B r o c a type  sorbing substance reemits  in  i t leaves  p o l a r i z a t i o n may v a r y  fluorescence  v a r i a t i o n s are not  and b e a m - s p l i t t e r  The beam w i l l  sample i s o p t i c a l l y  region  T h i s may n e c e s s i t a t e t h e  beam a f t e r  of the spectrometer. the  " s e e " t h e same  respond to t h i s  as w e l l  This can l e a d to large  i f the phototube  i s more s e n s i t i v e  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 p h o t o t u b e s and a b a l a n c i n g  necessary.  as  c i r c u i t are  17 A n o t h e r s o l u t i o n t o t h i s p r o b l e m may be f o u n d through not  c o n t r o l o f the source  intensity.  this d i d  a p p e a r t o have b e e n t r i e d b e f o r e , p r o b a b l y b e c a u s e o f t h e  difficulty  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  g e n e r a l l y r e q u i r e , i t appeared use  Although  were made o f t h e l i g h t  simple  interchange  of cells  t o be p r a c t i c a b l e  available.  sources i f efficient  T h i s would a l l o w a  to give accurate transmission  readings. To make maximum u s e o f t h e l i g h t the o p t i c a l  the source,  s y s t e m was d e s i g n e d w i t h l a r g e a p e r t u r e , a n d  corrected lenses. tungsten wire  A Mazda t y p e  851 K b u l b w i t h a s t r a i g h t  f i l a m e n t was u s e d  as the l i g h t  mounted w i t h i t s f i l a m e n t v e r t i c a l , filament  from  c o u l d be made t o f a l l  source, and  s o t h a t an image o f t h e  along the entrance  slit  of the  spectrometer. The  minimum l i g h t  intensity  a t the phototube  c a n be m e a s u r e d w i t h t h e d e s i r e d d e g r e e o f a c c u r a c y upon t h e s i z e o f " s h o t " e f f e c t developed fier, due  and t h e r m a l  i n t h e p h o t o c e l l and the f i r s t  a n d o n random v a r i a t i o n s  which  depends  agitation  voltages  stage o f the ampli-  i n i t s voltage supply.  "Drift"  t o slow c h a n g e s i n s u p p l y v o l t a g e i s n o t o f p r i m a r y im-  p o r t a n c e , b u t must be m i n i m i z e d .  Ordinary  "B" b a t t e r i e s , o r  a w e l l - r e g u l a t e d power s u p p l y make s a t i s f a c t o r y  voltage  sources. When s m a l l c u r r e n t s ( e . g . 10"" amps.) must be measured  a c c u r a t e l y , p r e c a u t i o n s must be t a k e n  currents  small.  Ordinary bakelite  tube  t o keep  leakage  b a s e s were f o u n d t o  18 be  very poor  i n this respect.  b a s e must be and  used,  i t may  clamp t h e t u b e  Scattered light  C o a t i n g the g l a s s w i t h  h i g h h u m i d i t i e s were i s a problem  photometer, s i n c e t r a n s m i t t e d l i g h t v a r y by  most u n f a v o u r a b l e  i n this  type  intensities  layer  at  of spectrodifferent In  the  s t r o n g a b s o r p t i o n band  A n g s t r o m s i n w i d t h , n o t more t h a n 1 p a r t i n 10 be  sili-  encountered.  a f a c t o r o f more t h a n t e n .  case o f a s i n g l e  o t h e r w a v e l e n g t h s may  of  base,  the f o r m a t i o n o f a m o i s t u r e  a d v i s a b l e i n ease  w a v e l e n g t h s may  type  be n e c e s s a r y t o remove t h e  i n position.  cone compound t o p r e v e n t w o u l d be  I f tubes w i t h t h i s  7  of l i g h t  s c a t t e r e d so as t o r e a c h t h e  10 of  photo-  5 tube  f o r a c c u r a c y t o 1 p a r t i n 10  ment.  In p r a c t i c e ,  possibly, constant  such extreme c a s e s w i l l  d e v i a t i o n type used The  spectrometer,  i n order to minimize  instrument, wavelength  selector.  on  In this  exit  slits.  be  s u f f i c i e n t l y high that  be  decreased  on  the  However, a  scattered  light.  stability  the mechanical  may  of  The  adjustment  d i s p e r s i o n o f the p r i s m  the entrance  slit  the  wavelength  b y r o t a t i o n o f t h e p r i s m , and simultaneous  be  the  accuracy of  type of spectrometer,  t r a l bandwidth i s c o n t r o l l e d by and  (except,  r a t h e r than the L i t t r o w  the mechanical  and p a r t i c u l a r l y  s e l e c t i o n i s accomplished  unevenly  not occur  accuracy w i t h which a c e r t a i n wavelength  depends g e n e r a l l y on  entrance  t r a n s m i s s i o n measure-  i n compounds c o n t a i n i n g r a r e e a r t h s ) .  t y p e , was  reset  i n the  specof  the  should  width need  never  t o t h e p o i n t where t h e c o l l i m a t o r l e n s i s v e r y  illuminated.  Thus the d i s p e r s i o n n e c e s s a r y  geometry o f the o p t i c a l  system.  depends  THE SPECTROPHOTOMETER OPTICAL SYSTEM  19 Finally, must be  the e n t i r e  e n c l o s e d by  electrical  light  p a t h o f the  a l i g h t - t i g h t h o u s i n g , and  s h i e l d i n g must be  instrument suitable  provided f o r phototubes  and  their  amplifiers.  B.  The  Optical The  System  p l a n o f the o p t i c a l  m e t e r i s shown i n p l a t e wire filament passes  of the  through the  I.  L i g h t from  source S  the spectrometer entrance  of  the f i l a m e n t  there.  o f the  slit  spectrophoto-  the v e r t i c a l  i s c o l l i m a t e d by  absorption c e l l A  at  the c o l l i m a t o r  system  straight-  the l e n s L  and i s b r o u g h t  ,  1 to a focus  s  b y L , f o r m i n g an image 1 2 L i g h t passing through t h i s s l i t f i l l s  , t h e n p a s s e s t h r o u g h t h e p r i s m P and t h e 3 lens L w h i c h f o r m a s p e c t r u m a t t h e e x i t s l i t s . The p h o t o 4 2 tube E i s p l a c e d f a r enough b e h i n d s so t h a t l i g h t p a s s i n g 2 2 t h r o u g h t h e s l i t a l m o s t c o v e r s i t s c a t h o d e a r e a . The c o n t r o l phototube  E  lens L  keeps the  s o u r c e i n t e n s i t y c o n s t a n t by  correcting  1 any  i n t e n s i t y changes t h r o u g h  regulator  a D.  C.  amplifier  and c u r r e n t  circuit. Components o f t h e o p t i c a l 1.  Crooker.  The  system  a r e as  follows:  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  T h e y were made b y C a n d i a n  Dr.  Arsenals Ltd., Leaside,  Ont. L  and L  a r e t y p e G J 1-2 2 B.P.L. 4.96", O.D. 1.260"  achromats,  E.F.L.  5.14",  1  L  i s a t y p e DW  1-2  achromat w i t h E.F.L.  3 B . F . L . 10.78", O.D.  1.986"  11.02",  20  L  i s a t y p e OK 1-2 a c h r o m a t w i t h E . F . L . 7.61", 4  B . F . L . 7.31", O.D. 2.05" 2.  T h e 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 b y  Canadian  Arsenals Ltd.  I t i s o f E.D.F. 653/336  40m.m. h i g h , w i t h an e n t r a n c e f a c e o f 108 m.m. f a c e o f 68 m.m. 3.  length, a l l angles  cms. 4.  i n diameter The s o u r c e  lamp, r e q u i r i n g 5. mum  5"- 0  a r e Cenco t y p e i m i A  cms. i n l e n g t h and3-5  w i t h o p e n t o p a n d wover p l a t e .  i s a Mazda t y p e 851 K  galvanometer  .5 amps a t 4-5 v o l t s .  The p h o t o t u b e s  sensitivity  glass,  and e x i t  - o .  The a b s o r p t i o n c e l l s A a n d A  cuvettes, o f pyrex  glass,  a t about  a r e R.C.A. t y p e 9 2 9 , h a v i n g  maxi-  5000 A n g s t r o m s w i t h t h e s o u r c e  above. C.  Mechanical  The  Design  o p t i c a l p a r t s a r e mounted o n a t r i a n g u l a r  made b y w e l d i n g t h r e e p i e c e s o f 4" wide s t e e l and m a c h i n i n g 8" f r o m  the f l a t  a r i s e s from  the f a c t  stock i s allowed f a i r l y  fitting  it.  T h i s forms a very  heavy base, b u t has s e v e r a l disadvantages.  c h i e f o f these  The  T h i s base i s supported  the p r i s m t a b l e b e a r i n g and c a t h o d e - f o l l o w e r  c o u l d be mounted b e n e a t h  moderately  channel  together  t h e t a b l e - t o p b y t h r e e mahogany l e g s , one a t e a c h  c o r n e r , so t h a t circuit  upper s u r f a c e .  channel  base  and w e l d i n g ,  that extruded  rigid, The  steel  wide t o l e r a n c e s i n manufacture.  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 t o g i v e a s u i t a b l y f l a t a f t e r welding  does l i t t l e  surface.  Planing this  surface  t o i m p r o v e i t , as t h e b a s e w i l l  o u t o f shape i n t h e p l a n e r , and r e t u r n t o 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, fact  t h a t t h e b a s e was  fitting job..  not f l a t  o f t h e p a r t s w h i c h mounted on i t , a v e r y  A l s o , the uneven  of  making  of  this  housing.  A more s a t i s f a c t o r y  base  sides of  welding.  Lenses L  and L 3  a r e mounted i n 3" x 4" p i e c e s o f j?" 4  A hole of s u f f i c i e n t  out, l e a v i n g a l / l 6  held  hand-  time-consuming  s o r t c o u l d be made by f i n i s h i n g t h e o u t e r  brass p l a t e .  w  size  to take  f l a n g e a r o u n d one  the l e n s  side.  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 b r a s s  s l o t t e d t o c l e a r two # 8 machine  s t e e l base.  Thus these  l e n s e s may  wire.  and L  1  which  screws to h o l d i t t o the be moved w h i l e  ment i s b e i n g f o c u s s e d , and t h e n f i r m l y f i x e d Lenses L  was  The l e n s i s  E a c h p l a t e i s s w e a t e d t o a b a s e o f 3/8" b r a s s is  the  s i d e s o f t h e f l a n g e s add t o t h e p r o b l e m  a light-tight  the f l a n g e s b e f o r e  cut  necessitated careful  bend  the  instru-  i n position.  a r e mounted i n a s i m i l a r manner, b u t a r e f i x e d  2  permanently i n p o s i t i o n . Each s l i t of  i s mounted on a f i x e d v e r t i c a l  ^ " b r a s s by means o f a b r a s s c o l l a r  allows each s l i t  partition  and s e t s c r e w .  This  t o be moved d u r i n g f o c u s s i n g .  The p r i s m i s mounted on a " t a b l e " o f £ "  aluminum,  b e i n g h e l d i n p l a c e b y a clamp c o n s i s t i n g o f a t o p p l a t e o f brass with a rubber  c u s h i o n , and t h r e e v e r t i c a l  prism table, roughly c i r c u l a r  rods.  i n s h a p e , h a s an 11"  arm  The  22 extending t a n g e n t i a l l y from through  the c o l l i m a t o r  i t s circumference.  T h i s arm  ( L ) l e n s mount, and r e a c h e s  passes  almost  to  3 the  slit  s , where t h e m i c r o m e t e r w a v e l e n g t h s e l e c t o r i s  1  located. to take  the  s u p p o r t i n g s h a f t which runs  base to the b e a r i n g b e n e a t h . ball  i s a I"  Near the c e n t e r o f t h e p r i s m t a b l e  through  the  D e t a i l of t h i s double  b e a r i n g i s shown i n p l a t e E .  t a n t , as t h e r e p r o d u c i b i l i t y  hole  tapped  steel stressed  This bearing i s very  o f the wavelength s e t t i n g  impordepends  on i t . Wavelength s e l e c t i o n a Starrett #  depth  6 machine  annealed  i s accomplished  gauge m i c r o m e t e r h e l d t o t h e  s t e e l base  by  s c r e w s ( b o t h ends o f t h e m i c r o m e t e r b l o c k were  and  drilled).  The  m i c r o m e t e r s p i n d l e works a g a i n s t  a short piece of polished s i l v e r to  b y means o f  the p r i s m  table  arm  near  the micrometer s p i n d l e by  steel  i t s end.  r o d which i s clamped The  a small h e l i c a l  arm  i s held against  s p r i n g at the  micro-  meter. The  absorption c e l l s  c r a d l e s mounted on carriage  done f r o m To  s i d e by  side i n brass  a c a r r i a g e o f l / 8 " square  c a n be moved a l o n g r a i l s  to interchange t o be  rest  the c e l l s .  of the  steel rod.  This  same m a t e r i a l so  A b r a s s r o d and b e a r i n g a l l o w s  as this  the o u t s i d e .  simplify  c o n s t r u c t i o n and  reduce  light  leakage  t h e s t e e l b a s e i s c o v e r e d by a b r a s s p l a t e between the source and l e n s L • The b r a s s end p l a t e A s u p p o r t s t h e s o u r c e 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 f e e d - t h r o u g h i n s u l a t o r , t h e l a t t e r p a i r handling connections  t o the phototube E  .  1  The  23 source  i s m o u n t e d i n a b r a s s c o l l a r w h i c h may be shimmed so  as t o b r i n g t h e f i l a m e n t side o f the end p l a t e fitted rical post  t o make t h i s  into  the c o r r e c t  position.  The o u t -  i s c o v e r e d b y a p i e c e o f 3/4 " p l y w o o d  end o f the i n s t r u m e n t l i g h t p r o o f .  connections t o the source d i r e c t l y behind i t ,  a r e made t h r o u g h  Elect-  a binding  and t h e g r o u n d e d o u t e r c o n d u c t o r 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  t h e s o u r c e i n an o c t a l  , i s mounted a b o u t 3" f r o m 1 socket h e l d j u s t o f f the s t e e l base b y  b r 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 t h e c o n d e n s e r , C , a r e mounted i n t h e s p a c e b e t w e e n S a n d L . 1 T h i s p a r t o f t h e i n s t r u m e n t i s e n c l o s e d b y a c o v e r o f 22 gauge s h e e t b r a s s w h i c h s h i e l d s  the phototube,  1  and keeps  light  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  slit  was o r i g i n a l l y mounted i n an o c t a l  socket  held  j u s t beyond the end o f the s t e e l base by a b r a s s p l a t e .  Later  it  was f o u n d n e c e s s a r y t o remove i t s b a s e ,  clamped  in  a horizontal position  It  i s about  its  a g a i n s t t h e end w a l l o f i t s h o u s i n g .  2" f r o m t h e e x i t  photocathode  a n d i t i s now  slit  o f the spectrometer.  i s approximately square,  i t s mounting  Since posi-  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  t h e a d j a c e n t mahogany l e g i n s i d e  a brass shield tothe  cathode-follower  i n p u t stage o f the a m p l i f i e r .  its  i s the center conductor o f a short piece of  ^  M  photocathode  c o a x i a l c a b l e , the outer conductor  The l e a d  from  o f which i s grounded.  HAMMOND  27H  REGULATED  POWER PLATE  SUPPLY I To Fact  Rxq«  2H  24 The E  l i g h t - p r o o f c o v e r between l e n s L  i s made f r o m p i e c e s o f 3/4"  f i rfive-ply.  and p h o t o t u b e 1 The s i d e s a r e  2 b o l t e d to the  s t e e l b a s e , and f a s t e n e d t o a l l f i x e d  p a r t i t i o n s w i t h machine screws.  The  removed t o a depth o f about l / 8 the base o r a p a r t i t i o n , The  i n g the  absorption c e l l s .  and  top i s i n three  c o u l d be h i n g e d  f a c e o f the plywood  was  where i t i s i n c o n t a c t  M  so t h a t t h e s e  tight.  vertical  j o i n t s w o u l d be  s e c t i o n s , the T h i s p a r t may  to f a c i l i t a t e  light-  s m a l l e s t one be  changing  removed o f the  with  cover-  easily,  absorption  cells. The  entire  i n s i d e o f the  dull black with a non-reflecting shellac  in  D.  Electronic  The  instrument  i s painted  " p a i n t " of lampblack  The  Circuits Voltage Regulated  Power S u p p l y  T h i s power s u p p l y i s o f c o n v e n t i o n a l  D.G.  t h a t the g a i n o f the 6SJ7 stage  amplifier  .1  s u p p l i e s 330  and r e g u l a t o r , 330 volts  minimize  10  .  (Plate 10 design  and  m)  balanced  to o b t a i n a s t a -  I t s c a l c u l a t e d impedance i s l e s s  ohm. It  105  i s low,  s t a g e s w i t h 6SH753 a r e u s e d 5  b i l i z a t i o n r a t i o n o f 10 than  and  acetone.  1.  except  a  volts  volts  a t 6 m.a.  t o t h e p h o t o t u b e and ripple  a t 50 m.a.  to the l i g h t  source  to the main a m p l i f i e r ,  cathode-follower  stage.  the h e a t e r o f the 6SJ7 i s s u p p l i e d f r o m  M.I.T. R a d i a t i o n L a b . S e r i e s , V o l 21, P a r t 3, McGraw H i l l , New Y o r k , 1948.  and  To a 6  p.556,  25 v o l t storage battery, and the time constant of the regulator c i r c u i t i s kept small.  I t i s b u i l t on a Hammond 12" x 17" x  3" chassis with the regulator as f a r 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 i n 10,000 under normal conditions of load and l i n e voltage v a r i a t i o n . Since the amplifiers which i t supplies are a l l of the "balanced" type, c a l c u l a t i o n shows that regul a t i o n to t h i s degree i s s u f f i c i e n t to keep concentration errors r e s u l t i n g from supply voltage variations well below those r e s u l t i n g from shot e f f e c t i n the phototube. Tests were c a r r i e d out on the completed power supply with a Dumont 5" oscilloscope and showed: a.  At a load current of 30 m.a. the l i n e voltage  could be varied between 105 and 125 v o l t s , with the output voltage, as seen on the oscilloscope at f u l l gain, remaining  apparently constant at 340 v o l t s . b.  No trace of r i p p l e appeared at f u l l gain on the  oscilloscope within the control range. c.  There was some tendency to o s c i l l a t e at high  frequencies (above 10 Kc) beyond the extremes of the cont r o l range, but none i n t h i s range. d.  At a l i n e 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, dependent*  which are somewhat i n t e r -  ! 6SL7  LIGHT SOURCE  6SL7  6SL7  REGULATOR  PLATE K SOURCE I^TEHSITY CONVT/NNT TO I '  10,000  Ta Fa.cc Pfr<>« 2,1a  26  2.  The  The  a m p l i f i e r and  Hammond 8"  x 12"  L i g h t Source Regulator  x  3"  regulator circuit  c h a s s i s , and  b a l a n c e d a m p l i f i e r (6SL7S;) w i t h 10  4  , and  a 6L6  t y p e 929  a voltage  phototube E  ( a b o u t 3")  current flowing o f one  from the  through the  light 7 10  t r i o d e s e c t i o n o f the  volts.  The  a balancing  g r i d o f the voltage  ohm  first  other  f r o m a 22^  a  a  three-stage  g a i n o f more t h a n  regulator.  i s mounted t h e  correct  c  2  distance  i s on  c o n s i s t s of  t r i o d e connected current  The  ( P l a t e ED  s o u r c e S> so resistor  t h a t the  keeps the  a m p l i f i e r stage at  photogrid  22  triode section i s supplied volt  with  battery.  AWV  i l l  ,. - 1 i  !  GAIN  >  \o  H  1 Figure tor.  A brief  1  shows a s i m p l i f i e d  a n a l y s i s of  circuit  i t s operation,  of  the  b a s e d on  regula-  this  circuit,  follows. The  fractional  change i n s o u r c e b r i g h t n e s s ,  w h i c h r e s u l t s f r o m a change be  found.  d  V  in battery  voltage  , must  27  The change i n c u r r e n t will by  be t h e d i f f e r e n c e b e t w e e n t h e change i n c u r r e n t A L  the b a t t e r y ,  current  A L  -  s  as b e f o r e  be e q u a l 10  A L  Ak.  A L  -  B  =  supplied  •  P  (1)  P  (2)  5 AL^  • I*  L  since  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 t o the f r a c t i o n a l  change i n t h e v o l t a g e  e across  7  ohm  resistor  -  AL  for  A i  the 6L6,  A_e  = <j A  p  (3)  =  m  ^  Q  Ae  where G i s t h e a m p l i f i e r g a i n g- t h e t r a n s e o n d u c t a n c e o f t h e 6L6 e, t h e g r i d v o l t a g e o f t h e 6 L 6 . . from (1) from (2)  and (3) *  A  AL If  AL  s  =  AL  A i  s  -  Ai  -  L  |  +  t h e change i n b a t t e r y  and  from  _A_L  _  for  a battery voltage  V  A\/  =  >0l  v  o  l  t  A  ^  "R A L  12 « s  A£ s)  al w  10,000  s '  A\/  is -  B  5 Ak. =  Q  Gl§£)  voltage  -  ^  - g  B  w  A V  For  %  -  B  S  (2)  ,  5  , and t h e c o m p e n s a t i n g change i n p l a t e  B  through the r e g u l a t o r tube,  A L  and  A L  through the source,  = volts 10  Z7 A L 5 A i  B  fi  «  also the  THE  MAIN  A MPLI FIER  PLATE TUBES  TYPE  6SL7GT  T o Fa.ce ?a.<t* 2 8  28 Thus v a r i a t i o n s c o n t r i b u t e much l e s s than shot e f f e c t The feed-back  i n light  source i n t e n s i t y  to the f r a c t i o n a l  should  concentration error  does.  amplifier  conditions,  i n this  c i r c u i t works under heavy  a n d t h e .05 m . f . d . c o n d e n s e r  G  was 1  found necessary t o decrease seconds  i t s time  c o n s t a n t t o about  .5  and p r e v e n t o s c i l l a t i o n which o t h e r w i s e r e s u l t s  the l a g i n response of the l i g h t 3.  o f t h e lamp t o c u r r e n t c h a n g e s .  source c o n t r o l  i s now  The Main A m p l i f i e r  This circuit from the measuring  amplifies  from  Operation  satisfactory.  (Plate%) the photoelectric current  . The c a t h o d e - f o l l o w e r i n p u t 2 s t a g e and b a l a n c i n g n e t w o r k a r e i n s i d e a Hammond 4" x 8" x 2" c h a s s i s mounted b e n e a t h t h e s p e c t r o p h o t o m e t e r b a s e n e a r E , 2  while the amplifier  phototube, E  itself  i s o n a s e p a r a t e 8" x 1 2 " x 3"  chassis. Before t h i s  a m p l i f i e r was d e s i g n e d , t h e f o l l o w i n g  e s t i m a t e s were made: 1). The approximate  photoelectric  current expected  from E . 2 Light slit is  reaching the c o l l i m a t o r l e n s ,  of width equal t o the diameter -2  2 x 10  a s s u m i n g a 1 cm  o f the source  filament,  lumens. Prom t h e r e s p o n s e  c u r v e f o r an S-4 p h o t o c a t h o d e  2.6 7 0 ° K t u n g s t e n s o u r c e , and t h e a v e r a g e amps p e r lumen) f o r t y p e 929 p h o t o t u b e s book) , t h e maximum c u r r e n t e x p e c t e d ,  and  c u r r e n t (45 m i c r o ( s e e R.G.A. Tube Hand-  a t 5000 A n g s t r o m s i s o n  29 -9 the  order  o f 10  amps/Angstrom.  Thus w i t h a bandwidth o f -8 10 A n g s t r o m s and no a b s o r p t i o n , a b o u t 10 amps i s e x p e c t e d -9 a t 5000 A n g s t r o m s , a n d a b o u t 10 stroms.  W i t h an a b s o r b i n g  amps a t 4000 o r 6000 Ang-  s o l u t i o n o f optimum  density,  -10 a b o u t 10 the  amps w i l l be o b t a i n e d  n e a r t h e extreme ends o f  visible  s p e c t r u m , t h u s t o measure t h e d e n s i t y t o 1 p a r t -14 \ i n 10,000, c h a n g e s o f 10 amps must be m e a s u r a b l e . 2).  The m a g n i t u d e o f i n t e r f e r i n g a).  The s i m p l e  effects.  shot-effect formula  fey a p p l i c a t i o n o f C a m p b e l l ' s T h e o r e m t o t h e c i r c u i t f o r phototubes. I  _  This  Ij e  g i v e s , f o r t h e r.m.s. s h o t  where i  i s valid  effect  current,  i s the p h o t o e l e c t r i c current, e the  J xnc  i  electronic shunting  c h a r g e , R t h e 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 capacitance. 9 F o r R = 10 ohms, C = 50 u u f , a n d , t h e maximum -8  expected current  i - 10  amps t h i s  Maximum r.m.s. s h o t F o r minimum i = 10 rise  derived  effect  amps,  i  s  ~  gives: current  i  '0  s  ^  1  0  ture.  T  In this  amplifier,  i  and T t h e a b s o l u t e  This figure represents noise before  level,  and c o n s i d e r a b l e  i - 7 x IO"' " 4  T  the lowest  amps  amps.  t o a bc )u •r r e nT th e r mia l fo lfu r.m.s. c t u a t i o vnasl uie n t h ie _r e=s i s/Ht a nKcTe R  where K i s B o l t z m a n n ' s c o n s t a n t  '  temperaamps.  attainable  experimentation  s o m e t h i n g c l o s e t o i t c a n be a c h i e v e d .  give  thermal  may be n e c e s s a r y In particular,  30  semi-conductors  s u c h as t h e r e s i s t o r R v a r y w i d e l y i n n o i s e  characteristics, c u r r e n t s may  especially  a t l o w f r e q u e n c i e s , and l e a k a g e  produce c o n s i d e r a b l e n o i s e .  quality resistance,  and c a r e f u l  The u s e o f a h i g h -  insulation  s h o u l d keep  thermal  "noise" l e s s than shot e f f e c t  " n o i s e " o y e r most o f t h e s p e c -  trum.  i s not completely  in  The p r e s e n t i n s t r u m e n t  satisfactory  this respect. A b a l a n c e d D.C.  amplifier  circuit  a v o l t a g e g a i n v a r i a b l e b e t w e e n 1,000 of the output  i s used to o b t a i n  and 60,000  at the g r i d s  tube.  T h u s t h e computed minimum o b s e r v a b l e -14 -5 c u r r e n t change i n t h e p h o t o t u b e o f 10 amps, o r 10 volts on t h e c a t h o d e - f o l l o w e r g r i d s , w i l l change o f .6 v o l t s o n t h e o u t p u t output meter. of the output can pass  However,  result  on t h e  t h i s m e t e r , l o c a t e d between the  t u b e , i s so a r r a n g e d t h a t n o t more t h a n 1  t h r o u g h i t u n d e r any c o n d i t i o n s .  as a n u l l  theoretical  g r i d s , o r 2.4 m.a.  cathodes m.a.  It's sensitivity  d e c r e a s e s 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 , only  in a  and i t i s i n t e n d e d  indicator.  In p r a c t i c e ,  i t was f o u n d t h a t  and 10,000 was most s a t i s f a c t o r y .  a g a i n between 5,000  The a m p l i f i e r  n o t be v a r i e d d u r i n g u s e , as t h e s l i g h t  gain should  a s asymmetry o f t h e  two s e c t i o n s o f t h e g a i n c o n t r o l c a u s e 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 m a r k e d  "potentiometer" i n Plate  c o n n e c t e d t o an e x t e r n a l c a l i b r a t e d  was  p o t e n t i o m e t e r so t h a t i t  was v a r i a b l e b e t w e e n 11 v o l t s  and 22 v o l t s .  r e a d i n g s were t a k e n f r o m  potentiometer.  this  ¥  Transmission  31  Difficulties follower  and  were e n c o u n t e r e d when t h e  phototube c i r c u i t s  from leakage currents  i n the  t i o n leakage  places,  and  i n other  p h o t o c e l l and  was  moved.  Considerable  still  rebuilt,  not  and  the  These r e s u l t e d  6SL7 b a s e ,  which v a r i e d with  objectionably high resistor noise.  circuit  was  were t e s t e d .  base o f  the  The  r e p l a c e d by (Noballoy)  cireult a 6J6,  c o u l d be  re-  i t s operation  one  should  ohm  and  use  result  the  follow: 6SL7  resistor  used at  a l l insulation,  glass surfaces  circuit  r e b u i l t , with  s u b s t i t u t e d f o r the  compound on  the  expectations.  a h i g h - q u a l i t y 10  C a r e f u l c l e a n i n g of  humidity,  p h o t o t u b e was  S u g g e s t i o n s f o r improvement o f t h i s 1.  the  insula-  This part of  improvement r e s u l t e d , b u t  i n agreement w i t h  cathode-  in  present. of  silicone  satisfactory  operation. or plier  2.  The  tube.  eliminate shot  E.  p h o t o t u b e c o u l d be  r e p l a c e d by  Three or f o u r stages  a l l leakage  effect noise  by  a  photomulti-  of multiplication  t r o u b l e s , and  will  increase  should  the  a small f a c t o r only.  Performance The  i n g from the The  optical source source  mercury lamp. silvered mirror  s y s t e m was  and  working toward the  was  T h i s was placed  a l i g n e d and  then  r e p l a c e d by  a c c o m p l i s h e d by i n f r o n t of 1  . 1  use  focussed,  start-  phototube. a 100  watt  of a plane,  G.E. front-  Pi_ATE JZLb * MERCURY YELLOW LINES To  Face  Page  32  32  The  calibration  g r a p h p l a t e "EE a and t h e o u r v e  i n g the mercury y e l l o w l i n e s , the l a t t e r , volt  mm.). this  p l a t e "ZT b , were o b t a i n e d .  In  v o l t a g e s i n d i c a t e d were r e a d f r o m a 20,000 ohm p e r  voltmeter across  follower.  show-  Both s l i t s  the cathode r e s i s t o r o f the cathode were v e r y n a r r o w ( w i d t h s l e s s  than  .01  The r e s o l v i n g power o f t h e i n s t r u m e n t , e s t i m a t e d f r o m g r a p h , i s 1000. Accuracy w i t h which  l i m i t e d by the s i z e  a w a v e l e n g t h may  be r e s e t i s  o f t h e s c a l e d i v i s i o n s on the m i c r o m e t e r .  With care, i t i s p o s s i b l e  to reset  t o .1 o f t h e s m a l l e s t  s i o n , o r one t e n - t h o u s a n d t h o f an i n c h .  divi-  This corresponds to  .25A a t 4,000 A, a n d t o 1A a t 6,000 A . 3 D i s p e r s i o n at the e x i t  slit,s  , i s 9 x 10  A / i n .or  2 360 A/mm.  i n t h e n e i g h b o u r h o o d o f 5,000 A. This  s p e c t r o p h o t o m e t e r was  t r a n s m i s s i o n t o one p a r t culties  i n t e n thousand.  w i t h the cathode f o l l o w e r  measurement t o one p a r t  o f 20-30 A.  the d e c r e a s e i n s e n s i t i v i t y  i s a l l that  o f the phototube  o  5  Voi-TS  GrR.H>  I  i  o  Fi  .  i  L  s  g.n  i s possible  I T  o  i  due t o  and cathode  i s raised  s  diffi-  previously,  This i s partly  as t h e 6SL7 g r i d p o t e n t i a l  I  Because o f the  c i r c u i t mentioned  i n f i v e hundred  at p r e s e n t , w i t h bandwidth  lower c i r c u i t  d e s i g n e d t o measure t h e  (fig.  folII-').  33 This i s probably cuit.  I f these  as s u g g e s t e d , appears  the r e s u l t  currents i n this  a r e e l i m i n a t e d , and r e s i s t o r n o i s e  a much c l o s e r  approach t o the d e s i g n  cir-  decreased value  possible. V i t a m i n D assay  ment.  o f leakage  was n o t a t t e m p t e d  A preliminary investigation  reaction,  o f the Sobel  made w i t h a Beokman m o d e l D  showed t h i s r e a c t i o n instrument i n the next  accuracy.• chapter.  t o be t o o u n s t a b l e The r e s u l t s  with this  instru-  colorimetric  spectrophotometer, t o warrant  of this  increased  investigation  appear  34  IV.  A.  VITAMIN D ASSAY  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  D activity.  These i n c l u d e i r r a d i a t e d  sterols  with  vitamin  ergosterol or calci-  ferol  (vitamin D ), i r r a d i a t e d 7-dehydrocholesterol (vitamin 2 B )., a n d o t h e r s s u c h a s 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 ) w h i c h have lower p o t e n c y and appear t o o c c u r i n 4 11 nature to a very l i m i t e d extent. I r r a d i a t i o n o f the s t e r o l precursors with u l t r a - v i o l e t l i g h t i s necessary f o r vitamin B a c t i v i t y , and p r o b a b l y has t h e e f f e c t bond i n t h e s t e r o l m o l e c u l e . D i s never diation  complete,  into  buted  a  double  The t r a n s f o r m a t i o n t o v i t a m i n  as t h e v i t a m i n i t s e l f  other products,  Although  o f opening  i s changed b y i r r a 12  some o f w h i c h a r e q u i t e  i t s precursor sterols  are widely  i n nature, the vitamin occurs i n very small  toxic. distri-  quantities  11  B i l l s , C. E . , M a s s i n g a l e , O.N., H i c k m a n , K . C D . , a n d G r a y , 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. 1 2 6 , 2 4 1 , 1 9 3 8 .  12  M o r t o n , R. A., " A b s o r p t i o n S p e c t r a o f V i t a m i n s a n d H o r mones", 2nd e d i t i o n , p . 3 5 , Adam H i l g e r L t d . , L o n d o n 1942.  35 e x c e p t i n some l i v e r fish.  oils, particularly  However, t h e e c o n o m i c i m p o r t a n c e  those of of f i s h  a source o f v i t a m i n D i s d e c l i n i n g , because v i t a m i n produced  by  i r r a d i a t i o n has  pensive,  be  used  and may  certain liver  the  oils  "synthetic"  become r e l a t i v e l y  to " f o r t i f y "  oils  as  inex-  w h i c h a r e low i n  potency. The  two  i m p o r t a n t D v i t a m i n s (D  similar i n physical about for  and c h e m i c a l p r o p e r t i e s ,  equal i n a n t i r a c h i t i c  most a n i m a l s  potency  and f o r humans.  g e n e r a l l y much l e s s  and D ) a r e 2 3  effective  (ability  T3x  ('CALC^EROIJ)  to cure  than v i t a m i n D  to cure r i c k e t s  chitic  VITAMIN  13  D  be  rickets) is 2 vitamins  3  sunlight  d u r i n g t h e 1 9 t h c e n t u r y , and t h e  p r o p e r t y o f i r r a d i a t e d f o o d s was  to  . Both 3 and many o t h e r in crystalline  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 used  appear  For birds vitamin D  are i n s o l u b l e i n w a t e r , b u t s o l u b l e i n f a t s organic solvents. Although not very stable 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 .  VITAMIN  and  quite  were antira-  s t u d i e d by Hess  and  H u b e r , W., and B a r l o w , O.W., "The C h e m i c a l 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 i t a m i n s 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  S t e e n b o c k i n 1919.  The a n t i r a c h i t i c  D i n 1922, and p r e p a r e d 1927. but  At f i r s t  vitamin  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  e r g o s t e r o l was  i n 1934 i t was f o u n d  f a c t o r was named  t h o u g h t t o be t h e o n l y  t h a t t h e r e must be o t h e r  precursor,  substances 14  with vitamin D a c t i v i t y , obtained  when i r r a d i a t e d .  I n 1936  the pure v i t a m i n B from tunny l i v e r  Brockmann  o i l , , a n d showed  it  t o be v i t a m i n D . The p r e s e n c e o f o t h e r D v i t a m i n s i n 3 15 some f i s h l i v e r o i l s h a d b e e n s u g g e s t e d b y B i l l s to explain 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 oils.  B.  Methods o f A s s a y  The p o t e n c y 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 specified i n International Units  (abbr.  I.U.)  I n t e r n a t i o n a l U n i t i s b a s e d on comparison w i t h standard  p e r gram. an  international  solution of i r r a d i a t e d ergosterol i n o l i v e  biological  assay method i n w h i c h the c a l c i f i c a t i o n  The  o i l by a o f the 16  bones o f r a t s produced by s t a n d a r d For both D  and D 2  and unknown i s m e a s u r e d .  , one I.U. i s e q u i v a l e n t  to  approximately  3  14  B r o c k m a n n , H., " I s o l a t i o n o f V i t a m i n D f r o m Tunny L i v e r O i l " , Z P h y s i o l . Chem., 241, 104, 1936.  15  Bills,  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 t h e 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 , a n d Cox, L o n d o n , 1 9 4 7 .  C.E., M a s s i n g a l e ,  O.N.,  and Imboden, W., S c i e n c e , 80, 596, 1934.  37 one-fortieth of  a microgram o f the pure v i t a m i n .  o f an o i l w h i c h i s t o be specified  used i n the  The  feeding of chickens  i n e i t h e r B . S . I , o r A.O.A.C. u n i t s , b o t h  are based on b i o l o g i c a l The  assay  with  By  these  ultraviolet  vitamin D precursors  and  accurate  which  to w i t h i n  methods p o t e n c i e s  o f 5 I.U./g. c a n be m e a s u r e d a l m o s t 4 6 c l e s (10 - 10 I.U./g.) The  of  is  chicks.  b i o l o g i c a l methods a r e  when p r o p e r l y u s e d .  potency  as e a s i l y  absorption  on  the  as h i g h  s p e c t r a o f the  t h e i r photochemical  15% order  poten-  various  transformation  products  have b e e n r e p o r t e d i n numerous p a p e r s , t h e r e s u l t s 17 o f w h i c h r e summarized b y M o r t o n . The D v i t a m i n s a r e a  similar  i n absorption, each having  a s i n g l e broad  i t s maximum a b o u t 2650 A n g s t r o m s and E  1%  band  a b o u t 500.  with A  solu-  lcm t i o n o f p u r e v i t a m i n i n some n o n - a b s o r b i n g s o l v e n t s u c h ether  c a n be  assayed  a t 2650 A n g s t r o m s . cal  degradation The  as  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 T h i s must be  o f the  extensive  done r a p i d l y ,  vitamin w i l l literature  take  as  photochemi-  place.  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 r e v i e w e d up t o 1941 b y M o r t o n . 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 C h e n p r o v e d most s a t i s f a c t o r y . T h i s i s the  17  M o r t o n , R.A., " A b s o r p t i o n S p e c t r a o f V i t a m i n s and Hormones", 2nd e d i t i o n , pp. 30-37, Adam H i l g e r L t d . , L o n d o n 1942.  18  I b i d . , p.  19  Broekmann, H., and C h e n , Y.H., Z . P h y s i o l . Chem., 241, 129,  37. 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 it  has  has  proven v e r y s a t i s f a c t o r y .  o n l y about  one-fiftieth  vitamin A i n this measure.  test,  and  i n v i t a m i n A a s s a y where Unit f o r unit, vitamin D  the a b s o r p t i o n c o e f f i c i e n t thus  C o n s i d e r a b l e work h a s  of  i s much more d i f f i c u l t b e e n done on t h i s  to  reaction, 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 t h e r e a g e n t , w h i c h may be i m p r o v e d by t h e 21 addition of acetyl chloride, and o f t h e i n f l u e n c e o f t h e solvent.  These r e s u l t e d  i n the t e c h n i q u e used by DeWitt  and  22 Sullivan  who  used  i n ethylene chloride stroms. pure  As  a m o d i f i e d antimony t r i c h l o r i d e  and m e a s u r e d t h e a b s o r p t i o n a t 5000 Ang-  i n the u l t r a v i o l e t  method, low  concentrations of  v i t a m i n D c a n e a s i l y be m e a s u r e d , w i t h t h e  advantage  that photochemical T h e s e two  directly  a c t i o n does n o t  additional  occur.  methods a r e r e n d e r e d u s e l e s s when  t o l o w o r medium p o t e n c y  liver  oils  other  substances which absorb  vitamin.  reagent  or " f o r t i f i e d "  oils,  oils,  because  s u c h as n a t u r a l  fish  o f the i n t e r f e r e n c e as  the  S a p o n i f i c a t i o n o f t h e o i l removes t h e f a t s ,  and  c e n t r a t e s t h e v i t a m i n up fiable fraction.  the  applied  same w a v e l e n g t h s  t o t e n t i m e s o r more i n t h e  con-  unsaponi-  However, t h e 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  S h a n t z , 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 I n d . E n g . Chem. ( A n a l y t i c a l E d ) , 16, 179, 1944.  21  N e i l d , C f c H . , R u s s e l l , W.G., and Z i m m e r l i , A., photometric Determination of Vitamins D B i o l . C h e m . , 1 3 6 , 7 3 , 1940.  22  of  2  D",  "The S p e c t r o and D3", J .  D e W i t t , 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", I n d . and E n g . 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 obscure i t s absorption band.  still  This i s e s p e c i a l l y true of  u l t r a v i o l e t absorption, and u l t r a v i o l e t measurement has not been used i n vitamin D assay recently except on solutions of almost pure vitamin.  The antimony t r i c h l o r i d e 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 t h i s method were made by a 23 number of workers.  Milas, Heggie, and Reynolds  using a  Hardy-General E l e c t r i c recording spectrophotometer studied t h i s problem.  They t r i e d to make empirical corrections f o r  vitamin A and s t e r o l 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 f o r the elimination of vitamin A and the s t e r o l s , could assay o i l s of potency greater than 5,000 I.U./g. F i n a l l y , DeWitt 25 and S u l l i v a n  used an improved chromatographic technique  similar to that of Broekmann to separate vitamin A and sterols from the vitamin D i n the unsaponifiable f r a c t i o n .  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 f o r the Quantitative Estimation of Vitamin D", Ind. Eng. Chem. ( A n a l y t i c a l Ed.) 13, 227, 1941.  24  Ewing, D. T., Kingsley, G. V., Emmett, A. D. and Brown,R.A., "Physical-Chemical Method f o r the Determination of Vitamin i n F i s h L i v e r O i l s " , Ind. Eng. Chem. ( A n a l y t i c a l Ed.), 15, 301, 1943. .  25  DeWitt, J . B., and S u l l i v a n , M. X.,loc. c i t .  40 to capsules and to t a b l e t s , y i e l d i n g r e s u l t s i n agreement with those obtained by bioassay. A new eolorimetric reaction f o r 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 i n t h e i r pure form were used i n t h i s work.  I t was found that very good discrimination  between vitamins and sterols could be obtained.  Although  the absorption c o e f f i c i e n t s were not given i n t h i s paper, they were apparently very small i n a l l cases.  The  authors  did not give any information on the a p p l i c a b i l i t y of t h i s 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 S u l l i v a n might r e s u l t . Estimation of vitamin D i n i r r a d i a t e d ergosterol -2 by the u l t r a v i o l e t and antimony t r i c h l o r i d e methods has been 27 discussed by Ewing, Poweil, Brown, and Emmett. P i r l o t and 28 Rouir used the Sobel reagent successfully f o r t h i s purpose, 26 Sobel, A. E., Mayer, A.M., and Kramer, B., "New G o l o r i metric Reaction of Vitamins D„ and D* and Their Provitamins", Ind. Eng. Chem., ( a n a l y t i c a l Ed.)17,160,1945. 27  Ewing, D.T., Powell, M.J., Brown, R.A., and Emmett, A.D., "The Determination of Vitamin D by Two Physical Chemical Methods," A n a l y t i c a l Chemistry, 20, 317, 1948. 2  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 C.  anhydride.  The Chemical Composition of F i s h Liver O i l s F i s h l i v e r o i l s vary widely i n 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 f a t t y 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 s t e r o l s , hydrocarbons, and alcohols.  In  eodliver o i l , cholesterol,C H 0H,makes up about h a l f the 27 45 unsaponifiable portion, while i n other o i l s unsaturated hydrocarbons such as squalene, C H , may predominate. V i t a 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 f a c t the unsaponifiable portion of eodliver 21 41 2  29  Hickman, K.C.D., "Adventures i n Vacuum Chemistry ,' Science i n Progress, 4th ser. p.p. 205-248, Yale University Press, New Haven, 1945.  30  Drummond, J.C., and H i l d i t c h , T.P., "The Relative Values of Cod Liver O i l s from Various Sources", HM Stationary O f f i c e , London, 1930.  31  1  , "The Composition of L i v e r O i l s of the Basking Shark and Spiny Shark", (South A f r i c a n F i s h Products XXVII), J.S.C.I., 67,104, 1948.  42 oil  i s estimated  to contain l e s s  than  .05% v i t a m i n D i n  general. A l t h o u g h v i t a m i n A and t h e s t e r o l s considered  r e s p o n s i b l e f o r most o f t h e i n t e r f e r i n g  because o f t h e i r is  reason  compounds metric  D»  are g e n e r a l l y  chemical  to suspect will  similarity  that the other  show a b s o r p t i o n ,  absorption  to the vitamin, unsaturated  a n d may  take  there  organic  part i n eolori-  reactions.  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  Reaction  An i n v e s t i g a t i o n o f t h e 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 D spectrophotometer.  c a r r i e d out with  a Beckman m o d e l  The a b s o r p t i o n maximum a t 32  A n g s t r o m s r e p o r t e d b y S o b e l , Mayer and K r a m e r v i t a m i n D was  of primary i n t e r e s t ,  although  p l o t t e d f r o m 3000-10,000 A n g s t r o m s i n some As a p r e l i m i n a r y , t h e a b s o r p t i o n w h o l e c o d l i v e r o i l was  6250-6500 as due t o  curves cases.  spectrum o f a  examined ( s e e f i g . I ) .  I t s absorp-  t i o n i s q u i t e low a r o u n d 6000 A n g s t r o m s , and t h e r e t o be a p o s s i b i l i t y  that vitamin D concentrations  w h o l e o i l c o u l d be m e a s u r e d d i r e c t l y of  the o i l d i d not i n t e r f e r e with  were  i f the other  seemed i n the components  t h e v i t a m i n D - G.D.H.  reaction.  32  S o b e l , A . E . , Mayer A.M.,  and K r a m e r , B . , l o p . c i t .  43  Eastman Kodak p r a c t i c a l grade glycerol hydrin was i t was  obtained, and since i t was  s l i g h t l y brown i n color  r e d i s t i l l e d i n vacuo before being used, as suggested  by Sobel.  The d i s t i l l a t e appeared to be colourless and  used i n a l l the following work. prepared by adding .225 (CP.)  1,3-dichlor-  The  activated reagent  was was  g. (about 1%) of acetyl chloride  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 This increased  noticed.  slowly (see f i g . Ila) but was not severe  enough to i n t e r f e r e seriously with the tests which followed. This reagent was stored i n darkness,0°C that the discoloration was  I t was  assumed  due to impurities i n the glycerol  dichlorhydrin, because the addition of 1% acetyl chloride to a portion of the o r i g i n a l u n d i s t i l l e d reagent resulted i n the r a p i d 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 t h e g l y c e r o l probably eliminate t h i s  The concentrate  oils  6  (10  used  dichlorhydrin  would  completely,  i n these  t e s t s were a v i t a m i n D  I.U./g.) a n d a t y p i c a l  3 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 a n d 1500 I.U./g. v i t a m i n A according to l a b e l First  specifications.  the procedure  o f S o b e l was f o l l o w e d a n d t h e  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 t i m e was s t u d i e d u s i n g t h e vitamin D  3  concentrate  the  concentrate  this  i n a 1 cm. c e l l ,  added f r o m  (see f i g . I I I ) .  A .5$ s o l u t i o n o f  i n c h l o r o f o r m was p r e p a r e d .  3b 2 c c . o f  1 c c . o f a c t i v a t e d G-.D.H. r e a g e n t was  a p i p e t t e , and t h e whole t h o r o u g h l y mixed.  comparison c e l l  A  c o n t a i n i n g 2 c c . o f pure c h l o r o f o r m and 1 c c .  o f G.D.H. r e a g e n t  had p r e v i o u s l y been prepared.  were t a k e n  as r a p i d l y  as p o s s i b l e ,  the c u r v e s  shown ( s e e t a b l e I ) .  Readings  and i n t e r p o l a t e d t o o b t a i n  The t e m p e r a t u r e  o f t h e absorp  t i o n c e l l s was b e t w e e n 27 G a n d 2 8 ° C , a n d i t was e s t i m a t e d a  TABLE I  Wavelenth  Time f r o m mixing min.  Percent Transmission  Correction f o r 15 m i n . curve  Estimated transmission at 15 m i n s .  800  23  79  +  8.0  87  700  10  78  -  5.0  73  Dens:  .06 .137 1  675  12  7 1 3  -  4.0  650  13  67|  -  2.5  65  .187  640  13^ 2  64  -  2.0  62  .207  630  14  -  1.3  625  1 142  6li 2 1 6li 2  -  0.6  620  15  610  0  15i  6li 2 61  +  0.6  600  16  >s|  +  1.0  580  17  65  +  2.0  540  24  62|  +  9.0  520  25  59l  510  26  500  2  490  27  450  4  .157  2  .217  61  .215  4  .210 .210  64|  .192 .170  -i  .145  +10.0  69|  .157  61  +11.0  72  .142  62  +11.5  .134  62  +12.0  73^ 2 74  6lJ  +12.5  74  .130  .130  46 that  the a b s o r p t i o n  I.U.  of vitamin  '  jbo  cell  contained  b e t w e e n 9,000 a n d 10,000  D.  *o*»  '  '  Fi q- m These c u r v e s at  clearly  ™ «  '  So*>  _____  show t h e v i t a m i n D maximum  6250 A n g s t r o m s , i n a d d i t i o n t o a weak maximum a t a b o u t  5000 A n g s t r o m s , p r o b a b l y due t o 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 )• T h e y 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 f r o m some o t h e r the  spectrum.  with  source, This  e s p e c i a l l y toward the v i o l e t  absorption  appears to i n c r e a s e  end o f  greatly  t i m e , as d o e s t h a t due t o t h e v i t a m i n . N e x t , t h e same p r o c e d u r e 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 chloroform, fig. the  I V ) . Although only absorption  tuents to  and a d d i n g 2 c c . o f t h e G.D.H. r e a g e n t ( s e e  cell,  a b o u t 90 I.u". o f v i t a m i n D was i n  strong  absorption  o f t h e o i l was o b s e r v e d .  due t o o t h e r  consti-  The a b s o r p t i o n maximum due  v i t a m i n A a t 5250 A n g s t r o m s was v i s i b l e .  Prom t h e s t r o n g  47 and that  r a p i d l y changing absorption  o b s e r v e d , i t was  t h i s method c o u l d n o t be a p p l i e d d i r e c t l y  concluded  to this  feeding o i l .  •  U  LY  F.q  ! .  I  A p o r t i o n o f t h e o i l was t h e n i n g the procedure o f DeWitt unsaponifiable of chloroform the  saponified, follow-  and S u l l i v a n ,  and t h e n e u t r a l ,  was a d d e d t o 2 c c . o f t h e cell.  The v e r y  G.D.H. r e a g e n t i n  strong absoption  ( s e e f i g . V) s u g g e s t e d t h a t t h e i n t e r f e r i n g  oil.  )  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 1c c .  absorption  mained w i t h  .  ...  1 _  the vitamin  D i l u t i o n with  substances r e -  i n the unsaponifiable  chloroform  observed  portion of the  to l / l O concentration  after  m i x i n g showed t h a t B e e r ' s l a w d i d n o t h o l d . D i l u t i o n o f the unsaponified with  chloroform  previous  to mixing,  portion of the o i l  so t h a t t h e u n s a p o n i f i a b l e  p o r t i o n o f .3 g . o f t h e o i l i n 1 c c . o f c h l o r o f o r m with  2 c c . o f t h e G.D.H. r e a g e n t ,  gave s i m i l a r  was m i x e d  results.  48  i  •  •  •  i  i i  i  F.cj The in  the  strong  as  reagent  ratios,  the  the  i  1000  U  this  r e a c t i o n on  assay of  greater d e t a i l  any  r e p o r t by  t h a n the  potency o i l s  but  and  r a p i d changes  suggested the  solvent  Campbell  use  of  Dr.  suited  oils.  treats this  above d i s c u s s i o n .  to  well  very high potency 33  reaction  Campbell  than the f e e d i n g o i l (P5995),  f o u n d t h a t d e v i a t i o n s f r o m B e e r ' s Law  were s e r i o u s .  a second v i t a m i n D a b s o r p t i o n  He  absorption  t h a n the  and also  maximum  4000 A n g s t r o m s w h i c h , u n d e r c e r t a i n c o n d i t i o n s , gave  somewhat s t r o n g e r  33  i • i i  s u g g e s t s t h a t t h i s method i s n o t  vitamin D  used h i g h e r  at  i  I n t e r f e r i n g absorption  dependence o f  A recent in  i  s p e c t r u m w h i c h were o b s e r v e d w i t h f e e d i n g o i l P5995,  as w e l l  to  .  7000  009  a  6250 A n g s t r o m s .  C a m p b e l l , 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 ", A n a l y t i c a l C h e m i s t r y , 20, 766, 1948. 3  49  V.  CONCLUSION  A p h o t o e l e c t r i c spectrophotometer, using source  o f c o n t r o l l e d i n t e n s i t y has  wavelength r e s e t accuracy s p e c t r u m , and bandwidth of The v i t a m i n D has  o f 1 A o r l e s s over the to 1 p a r t  been i n v e s t i g a t e d w i t h found unsuitable  typical  Tests  were c o n d u c t e d w i t h  oils  f r a c t i o n i n the  o f low  f o r the  i n v e s t i g a t i o n of the  stability  B e e r ' s Law  was  little.  A c o l o r i m e t r i c r e a c t i o n was present,  accompanied the  besides  not  shown t o  take  v i t a m i n D.  v i t a m i n D i n the  where t h e i r r e l a t i v e l y  the  of  of  unsaponifiable  this reaction  place with other  Some o f t h e s e  unsaponifiable  strong  assay  obeyed i n t h i s r e a c t i o n .  absorption  weak a b s o r p t i o n maxima o f v i t a m i n D  sub-  substances  f r a c t i o n of  completely ( E, * = 80 * I cm 1  the  obat  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  thoroughly  i n v e s t i g a t e d by  for  assay of low-potency o i l s ,  direct  o f the  spectro-  would improve  ^  6250 A)  a  c a s e o f one o i l .  results  the  with  vitamin D assay  whole o i l , a n d w i t h  accuracy  scured  i n 500,  p o t e n c y ( i . e . b e l o w 10,000 I . U . / g . ) .  instrument  oil,  visible  a Beckman M o d e l D  showed t h a t i n c r e a s e d  stances  a  A.  three  very  I t has  glycerol-dichlorhydrin colorimetric reaction for  p h o t o m e t e r , and  An  light  been c o n s t r u c t e d .  measures t r a n s m i s s i o n s 20-30  a  v i t a m i n D by  necessary.  others,  chromatographic  and  a s s a y m e t h o d has  been  i s considered u n r e l i a b l e preliminary  separation  concentration  appears to  be  50  VI.  BIBLIOGRAPHY  BOOKS B l o o r , W. R,, B i o c h e m i s t r y o f t h e P a t t y A c i d s , R e i n h o l d Pub. C o r p . , New Y o r k , 1 9 4 3 . B r o d e , W. R., ; C o m i c a l lf\c.  Spectroscopy.  John  Wiley a n d Sons  , 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 t h e V i t a m i n s , 2nd e d . , B a i l l i e r e , T i n d a l l , a n d Cox, L o n d o n , 1 9 4 7 . 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 a n d 2 1 , McGraw H i l l , New Y o r k , 1948. Sawyer, R. A., E x p e r i m e n t a l S p e c t r o s c o p y , P r e n t i c e - H a l l I n c . , New i o r k , 1946.  ARTICLES H i c k m a n , K.C.D., " A d v e n t u r e s i n Vacuum C h e m i s t r y " , S c i e n c e i n P r o 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. L o o f b o u r o w , 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 V i t a m i n s a n d Hormones," V i t a m i n s and Hormones, V o l . I , A c a d e m i c ' P r e s s , New : Y o r k , 1 9 4 3 . 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 t h e V i t a m i n C o n t e n t o f T i s s u e s , " V i t a m i n s a n d Hormones, V o l . I , A c a d e m i c P r e s s , New Y o r k , 1 9 4 3 . PAPERS Bills,  C. E . , M a s s i n g a l e , 0. N., H i c k m a n , K.C.D., a n d G r a y , E . leB., "A New V i t a m i n D i n C o d l i v e r O i l " , B i o . Chem., 126, 2 4 1 , 1938.  Bills,  C. E . , M a s s i n g a l e , O.N.,, a n d Imboden, W., 596, 1934.  S c i e n c e , 50,  Broekmann, H., " I s o l a t i o n o f V i t a m i n D f r o m T u n a L i v e r Z. P h y s i o l . Chem., 241, 1 0 4 , 1 9 3 6 .  Oil",  Broekmann, H., a n d Chen, Y. H., Z. P h y s i o l . Chem., 241, 1 2 9 , 1936. C a l d w e l l , M. H., a n d Hughes, J . S . , "Changes i n A b s o r p t i o n S p e c t r a Due t o A g i n g o f t h e C a r r - P r i c e R e a c t i o n M i x t u r e w i t h V i t a m i n A a n d Common C a r o t e n o i d s " , J . B i o l . , Chem., 166, 5 6 5 , 1946.  51 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 " , A n a l y t i c a l C h e m i s t r y , 20, 766, 1948. 3  "The  C o m p o s i t i o n o f L i v e r O i l s o f t h e B a s k i n g S h a r k and S p i n y Shark", (South A f r i c a n P i s h Products XXVII), J o u r n a l o f S o u t h A f r i c a n C h e m i c a l I n d u s t r y , 6 7 , 104, 1948.  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 Proc e d u r e f o r t h e Q u a n t i t a t i v e E s t i m a t i o n o f V i t a m i n D" I n d . E n g . Chem., A n a l y t i c a l E d . , 1 8 , 117, 1 9 4 6 .  Drummond, J . C , a n d H i l d i t c h , T.P., "The R e l a t i v e V a l u e s o f Cod L i v e r O i l s f r o m V a r i o u s S o u r c e s " , H. M. S t a t i o n a r y O f f i c e , London, 1930. E w i n g , D. T., K i n g s l e y , G. V., Emmett, A. D., a n d Brown, R. A. " P h y s i c a l - C h e m i c a l Method f o r t h e D e t e r m i n a t i o n o f V i t a m i n D i n P i s h L i v e r O i l s , " I n d . E n g . Chem., A n a l y t i c a l E d . , 1 5 , 301, 1 9 4 3 . E w i n g , D. T., P o w e l l , E . J . , Brown, R. A., a n d -Emmett, » A . D . , "The D e t e r m i n a t i o n o f V i t a m i n Dp b y Two P h y s i c a l C h e m i c a l M e t h o d s " , A n a l y t i c a l C h e m i s t r y , 20, 317, 1949. H u b e r , W.,  and B a r l o w , O.W., "The C h e m i c a l a n d B i o l o g i c a l S t a b i l i t y of C r y s t a l l i n e Vitamins 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., 1 4 9 , 1 2 5 , 1943. 2  3  M i l a s , R. A., H e g g i e , R., and R a y n o l d s , J.A., " S p e c t r o p h o t o m e t r i c Method f o r t h e Q u a n t i t a t i v e E s t i m a t i o n o f V i t a m i n D", I n d . E n g . Chem., A n a l y t i c a l E d . , 1 3 , 227, 1 9 4 1 . N i e l d , G. H., R u s s e l l , W. C., a n d Z i m m e r l i , A., "The S p e c t r o photometric Determination of Vitamins D and D " , J . B i o l . Chem., 1 3 5 , ,73, 1 9 4 0 . 2  3  S h a n t z , E . M., " A n t i m o n y 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", I n d . E n g . Chem., n a l y t i e a l E d . , 1 6 , 1 7 , 1944. A  S o b e l , A. E . , M a y e r , A. M., a n d Kramer, B., "New C o l o r i m e t r i c Reaction of Vitamin D and D~ and T h e i r P r o - v i t a m i n s I n d . E n g . Chem., A n a l y t i c a l E d . , 1 7 , 160, 1 9 4 5 . 0  S o b e l , A. E . , a n d W e r b i n , A n a l y t i c a l  C h e m i s t r y , 1 9 , 107, 1947.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0085395/manifest

Comment

Related Items