A GENERAL METHOD FOR DETECTION AND RECORDING OF COMPONENT BANDS
IN CHROMATOGRAPHY WITH LIQUID ELUENTS

By

Lo M ich a el C arson

A THESIS

S u b m itte d t o t h e S ch o o l f o r Advanced G rad u ate S tu d ie s
o f M ichigan S t a t e U n i v e r s i t y o f A g r ic u ltu r e and
A p p lied S c ie n c e i n p a r t i a l f u l f i l l m e n t o f
th e re q u ire m e n ts f o r th e d e g re e o f
DOCTOR OF PHILOSOPHY
D epartm ent o f C h em istry

1959

ProQuest Number: 10008632

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ACKNOWLEDGMENT
I w ish t o acknow ledge t h e a s s i s t a n c e and in v a lu a b le
c o u n s e l o f P r o f e s s o r James C* S te r n b e r g , and t o e x p re s s
my s i n c e r e a p p r e c i a t i o n f o r h i s p a tie n c e and en co u ra g e m e n t<>
Acknowledgment i s a l s o due t h e R e se a rc h C o r p o r a tio n
and N a tio n a l I n s t i t u t e s o f H e a lth f o r t h e i r g r a n ts
s u p p o r tin g t h i s w o rk .

To R uth

VITA

Lo M ic h a e l C arson was b o rn O cto b er 5* 1932 i n Johnson C ity ,
T en n essee.,

He s p e n t f o u r y e a r s a t E a s t T en n essee S t a t e C o lle g e w here

h e r e c e iv e d h i s Bo So and A.' Bo d e g re e s i n 195U°
He a tte n d e d M ich ig an S t a t e U n iv e r s ity 1955--1959 w here h i s m ajo r
was P h y s i c a l C h e m istry and m in o rs w ere M athem atics and P h y s ic s „
He was a G rad u ate T e a c h in g A s s i s t a n t 19$h - 1958 and a G rad u ate R e se a rc h
A s s i s t a n t 1958-1959 °
He e x p e c ts t o co m p lete h i s g r a d u a te work a t M ichigan S t a t e
U n i v e r s i t y and j o i n th e s t a f f a t C e n tr a l M ichigan U n iv e r s ity o

A GENERAL METHOD FOR DETECTION AND RECORDING OF COMPONENT BANDS
IN CHROMATOGRAPHY WITH LIQUID ELUENTS

By
Lo M ich ael C arson

AN ABSTRACT

S u b m itte d t o t h e S c h o o l f o r Advanced G rad u ate S tu d ie s
o f M ich ig an S t a t e U n i v e r s ity o f A g r ic u ltu r e and
A p p lie d S c ie n c e i r i p a r t i a l f u l f i l l m e n t o f
t h e re q u ire m e n ts f o r t h e d e g re e o f
DOCTOR OF PHILOSOPHY
D ep artm en t o f C h em istry
Year 1959

A pproved

ABSTRACT

A net^ g e n e r a l method h a s b een d ev elo p e d f o r d e t e c t i n g and r e c o r d in g
t h e p r e s e n c e o f s o l u t e b an d s i n th e e f f l u e n t s tre a m of a l i q u i d - e l u e n t
c h ro m a to g ra p h ic colum n0

S in c e t h i s method i s b a s e d upon changes i n v a p o r

p r e s s u r e w h ich o cc u r i n th e s o lv e n t due t o th e p re s e n c e o f a s o l u t e b a n d ,
i t s a p p l i c a b i l i t y i s n o t l i m i te d to s p e c i f i c c l a s s e s o f s u b s ta n c e s .
The d e t e c t o r d e v elo p ed u t i l i z e s th e te m p e ra tu re d i f f e r e n c e w hich i s
e s t a b l i s h e d b etw een a w ick b a th e d w ith t h e e f f l u e n t of a ch ro m a to g ra p h ic
colum n and a r e f e r e n c e w ick b a th e d w ith p u re s o lv e n tj th e w icks a r e
e n c lo s e d i n a th e r m o s ta te d chamber t h a t i s s a tu r a t e d w ith s o lv e n t v ap or*
The te m p e r a tu re change i s d e te c te d th ro u g h th e d i f f e r e n c e i n r e s i s t a n c e
o f a p a i r o f t h e r m i s t o r s , one o f w hich i s i n c o n ta c t w ith th e w ick b a th e d
w ith p u re s o l v e n t , w h ile th e o th e r i s i n c o n ta c t w ith t h e w ick b a th e d
w ith t h e e f f l u e n t s o lu tio n *

The th e r m is to r s c o n s t i t u t e tw o arms of a

W h eatsto n e b r id g e , w hich can be b a la n c e d when b o th o f t h e th e r m is to r s a r e
i n c o n ta c t w ith w ick s b a th e d w ith p u re s o lv e n t*

Any u n b a la n c e i n th e

b r id g e w hich o c c u rs as a r e s u l t o f s o lu t e b an d s p a s s in g i s c o n tin u o u s ly
re c o rd e d on a r e c o r d in g p o te n tio m e te r*
D e te c to r re s p o n s e i s a c o l l i g a t i v e p r o p e r ty o f th e s o l u t i o n p a s s in g
o v e r th e t h e r m i s t o r s * A m a th e m a tic a l tr e a tm e n t h a s b e e n d e v elo p ed and
p r e d i c t s a l i n e a r d ep en d en ce o f d e t e c t o r re s p o n s e on mole f r a c t i o n o f
s o lu te *

The m a th e m a tic a l a n a l y s is i s b ased on a c o n d itio n o f th e rm a l

b a la n c e , and r e q u i r e s s u i t a b l e a p p ro x im a tio n s , b a se d on th e s m a ll m ag n itu d e

vi

o f th e o b serv ed changes* f o r d e r i v a t i o n o f th e l i n e a r r e la tio n s h ip ®

The

k i n e t i c s o f th e s o lv e n t v a p o r i z a t i o n and c o n d e n s a tio n p ro c e s s e s * as
in f lu e n c e d by th e p r e s e n c e o f s o lu te * a r e p o s tu l a t e d t o p l a y th e m ajo r
r o l e i n d e t e c t o r response®
F a r th e r m a th e m a tic a l a n a l y s is o f th e c o n c e n tr a tio n p r o f i l e f u r n is h e s
a r e l a t i o n s h i p b etw een th e c o n c e n tr a tio n g iv in g th e o b serv ed r e s p o n s e and
t h e i n i t i a l sam ple c o n c e n tra tio n ®
The e v o lu tio n o f th e d e t e c t o r and i t s r e l a t i o n s h i p t o o th e r means of
c h ro m a to g ra p h ic d e te c tio n * and t o r e l a t e d m e a su rin g te c h n iq u e s * a r e
describ ed ®

Q u a l i t a t i v e r e s u l t s o b ta in e d i n d i f f e r e n t s o lv e n t- s o r b e n t

sy stem s f o r a v a r i e t y o f s o l u t e s a r e p re s e n te d * t h e re s p o n s e t o v o l a t i l e
and n o n - v o l a t i l e s o lv e n t s i s d iscu ssed ®

P a r tic u la r a tte n tio n has been

d i r e c t e d to w ard s th e a n a l y s is o f th e e r g o s t e r o l i r r a d i a t i o n m ixture®
Some q u a n t i t a t i v e r e s u l t s a r e p r e s e n te d and i n d i c a t e th e n a tu r e and
s e n s i t i v i t y o f th e response®

A t 33

»■w ith a sam ple column flo w r a t e o f

5> ml®/day* a r e f e r e n c e column flo w r a t e o f 2 ml®/day* c o tt o n th r e a d w ic k s ,
n o m in al 2000-ohm th e r m i s t o r s w ith a te m p e ra tu re c o e f f i c i e n t o f U®5 pen
c e n t p e r d eg re e* f ix e d b r id g e r e s i s t a n c e s o f 2.000 ohms* and an a p p lie d
b r id g e v o l t a g e of 6 v o l t s * th e d e t e c t o r h a s a s e n s i t i v i t y o f 1 8 8 ®5 mv*/M®*
-.3

o r t h e c a p a c ity t o d e t e c t s o l u t e i n 0®01 ml® o f 10 M® s o lu tio n s * t h i s
Q
„9
c o rre s p o n d s t o d e t e c t i o n o f 10 -1 0
m oles of m a te ria l®
The new method sh o u ld complement gas chrom ato g rap h y as an a n a l y t i c a l
method* w ith its ^ m o s t im p o rta n t a p p l i c a t i o n s i n a r e a s w here g as ch ro m ato g rap h y
i s w e a k e s t— a n a l y s i s o f h i g h - b o i l i n g and th e r m a lly - u n s ta b l e components®
v ii

I t sh o u ld b e o f p a r t i c u l a r v a lu e i n b io c h e m ic a l* n a t u r a l p ro d u c ts * and
p h a r m a c e u tic a l work®

P h y s ic o -c h e m ic a l a p p l i c a t i o n s o f t h e d e t e c t o r

in c lu d e i t s u s e i n d e te r m in in g m o le c u la r w e ig h ts* e v a lu a ti n g a c t i v i t y
e f f e c t s * s tu d y in g m o le c u la r com plexing and d i s s o c i a t i o n * and o b ta in in g
fu n d a m e n ta l in fo r m a tio n on th e n a tu r e o f th e c h ro m a to g ra p h ic p r o c e s s *

v iii

TABLE OF CONTENTS

Page
L , INTRODUCTION

.

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o

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«

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o

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o

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d

o

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o

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0

o

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A* G en eral o e o a o o o & e o e o c o a e o e f t o o f t e e e o p o e e c t o e o o o t
B . C om parison o f Gas and L iq u id Chrom atography,
C . P r e v io u s D e te c to r s f o r L iq u id Chromatography
I I . THE PRESENT INVESTIGATION
A.
B.
C.
D.

.

0

.

.

0

.

.

.

0

.

.

o

*

1
1
3

c t o o t p f o a e * *
6 6 9 0 0 6 6 0 0 9

o e o o a

9

0 o o

o

7

d - O 6 0 O O Q e 0 O O Q O O O O O O - O O C G O O O 0 O 4 > O C O « C C O 0 O

G en eral D e s c r ip t io n o f th e T ech n iq u e.
R e la te d T e c h n i q u e s o . « . . . . . o o , e . . . o o o . c , o » o . . * . o .
E v o lu tio n o f th e P r e s e n t D e t e c t o r . . . . . . . . . ............ ..
Components o f th e P r e s e n t C hrom atographic A pparatus . . . . . .
1 o Columns o . . o o o . o . o o . o o o o . . . o o o . . . 0 0 o a o 0 o o o * o o o o o • e . o
2 . W icks
3« T h erm isto rs
h * S o lv e n t Flow C o n tr o l and Out-Gas D e v ic e
5* Sam ple I n j e c t i o n
6 . T h erm o sta tin g and Tem perature C o n t r o l . . . . .
7 . W heatston e B r id g e •
8 . T reatm ent o f S o lv e n t and P r e p a r a tio n o f Columns
^ o P u r i f i c a t i o n o f R eagen ts and P r e p a r a tio n ., o f
SolU tlO nS . o o o . . . . . . . o o o o o . . . » » » • . a . o . e . o o
O O O O O O Q O O O e O O O O O O O O C O O O O O O O O O O O O C O O O O m C O Q O O O C O

OOOOOOOOOCOOOGOCOOGOOOOOOOOOOGOOOOOt OOOO
0 0 0 0 0 6 0 0 0 0 0

0 0 0 0 6 0

6 0 0 0 6 0 6 6 6 0 0 0 9

O

0 6 0 O 0 0 O 6 O C O O O O

*0 6 6 0 6 0

0 0 6 6 0 0 6 9 0 0 9 9 0 6 0 0 0 6 6 0 0 9 9 0 6 6 6 0 6 0

6 6 6

I I I . QUALITATIVE APPLICATIONS OF THE DETECTOR..
Gener a l c . . . o . . . . . . . o o o . . . . 0 0 0 0 . 0 0 0 0 0 . . . . . . . . . . . o o o .
W ater ( S o l v e n t ) - C e l l u l o s e (A d so r b e n t) S y s t e m . . . . . . .
B u ta n o l S a tu r a te d w ith W a te r -C e llu lo s e S y s t e m . . . . . .
C ycloh exan e (9 5 p er c e n t , v . / v . ) and A ceto n e (5 p er c e n t ,
v . / v . ) (S o lv e n t)-A lu m in a ( S o r b e n t ) . . .
E. The E r g o s t e r o l I r r a d ia t io n Mi x t u r e . . . . . .

6 6 0 6

0

0 0 6 6 0 6 0 9

Ao
B.
C.
D.

OC G0 6 0 0 0 6 0 0 9

00

0 6

9 6

0 6 0 6

0 0 0 0 0

Ge n e r a l . . . . . . . . . . . . . . . . . .
E l e c t r i c a l B a la n c e C o n d itio n
T h erm istor T em perature— R e s is ta n c e R e la t io n s h ip s . . . . . . . . .
Thermal B a la n c e C o n d itio n s . . . . . . . . . . . . . . o . . . . . . . . . . .
1 . R a tes o f Heat I n p u t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 o R a tes o f H eat R e m o v a l o . « o . a , . , . o o c o o * « o . o e . . . . . o o o .
3 ° The B a la n c e C o n d itio n s a o . e o o . o . . . e . . . . « . . a . a . . . a . o « o
, 6 6 6 6 0 6 0 0 0 0 6 0 0 6 0 0 9 9 0 0 6 0 6 0 9 6 0 6 6

c o n tin u e d

ix

7

8
11

16
16
17
20
25
25
28
29
32
33
35

A.
Bo
C.
D.

IV . MATHEMATICAL ANALYSIS OF DETECTOR RESPONSE

I

35
35
36
38
UO
k3

53
53
55
56
56
hi
58

TABLE OF CONTENTS - C o n tin u ed
Page
E« D e r iv a tio n o f R e s p o n s e -C o n c e n tra tio n R e la tio n s h ip s f o r
I d e a l S o lu tio n s o f N o n -V o la tile S o l u t e s . . . . . . . ...................
Fo C o n c e n tr a tio n P r o f i l e o f S o lu te B a n d s • • • • • • • • • • • .....................
G. F a c to r s C au sin g N o n -L in ear R e sp o n se
................

h9

61
6?

V. 'QUANTITATIVE CORRELATIONS WITH THEORY.. . . . . . . . . . . . . . . . . . . . . . . . . .

70

A. G e n e r a l
'..................................
B . L arg e Sam ple Volume, F l a tt e n e d P eak s (C « C0 ) . . . . . . . . . . . . .
C . S m a ll Sam ple Volumes (C < C0
....o...............

70
71
73

VI O DISCUSSION . . 00000000. . . . 00. . . . . . . . o. oooo. o®. OOO... O* 0. 0. . . . . . . . .

76

A. The P re -P e a k E f f e c t . . . . . . o . . o. oo. . oo. . . o. oo. . . . . . . . o. . . . . .
B . U ltim a te S e n s i t i v i t y o f t h e D e t e c t o r . . . . . . . . o . . . . . . . . . . . . .
C o S u g g e s tio n s f o r F u r th e r W o r k . . . . . . o . o . o . . o o . . . . o . . . . o . . o . o

76
80
82

V II . SUMMARY. 000. . 0.0 OOOOOOOOOOO.O. . . . o . . 00. O. . . . . . ocoo. 00. . 0. . . . . . 0 .

85

BIBLIOGRAPHY . o o . o o o o o o o . o o o o . c o o o o . a o . e o o o . . . . o o o . o . . e o o . o . o e o o e a o . . .

88

APP ENDICES oooo. o. o 0000. 0. 0. . . 0. 000. 0. 0. 0. 0000*000. .0 00. 0 . 0.0 . . . 00. 04.

89

X

LIST OF FIGURES
FIGURE

Page

lo Columns shown mounted i n th e r m o s ta tin g j a c k e t above
d e t e c t o n chamberoa*a**aaoaaacoaooo»oa9aaoaa«oo£>aaaoaoaaoaa»<>*<

18

2«> D e t e c t o r d e s ig n u s in g th e r m is to r s s e a le d i n g la s s e n v e lo p e s <>o,

22

3o D e t e c t o r d e s ig n f o r g la s s - b e a d

23

lie O u t-g a s d e v ic e * lo c a te d i n s o lv e n t l i n e b etw een r e s e r v o i r and
sam p le i n j e c t i o n p o r ta H eat o f lam p d i s p l a c e s d is s o lv e d a i r 5
w hich i s v e n te d th ro u g h s to p c o c k and d r y in g tu b e.

26

%a A rrangem ent f o r sam ple in je c tio n *

2?

6« B rid g e c i r c u i t a *******............

31

7 o R e p r e s e n ta tiv e chrom atogram o f e r g o s t e r o l i r r a d i a t i o n m ix tu re *

U2

8a G a u ssia n s p re a d of c o n c e n tr a tio n f o r sam ple i n j e c t i o n s of
V a ry in g s i z e a**************. *, *********. **. ********. *********«

62

9«. D ependence o f re s p o n s e on c o n c e n tr a tio n f o r s o lu t i o n s o f
b ip h e n y l i n c y c lo h e x a n e (9S%» v / v )*=*■=*a c e to n e ($%? v /v ) s o lv e n t*

72

10a R e p r e s e n ta tiv e chrom atogram show ing p re -p e a k e f f e c t* * *••«**« a a

77

xi

1

I * INTRODUCTION

A . G en eral
The ad v an ce o f th e d evelopm ent o f a c c u r a t e , r a p i d , and g e n e r a l
te c h n iq u e s o f s e p a r a t i o n and a n a ly s is i s one o f t h e b e s t y a r d s t i c k s f o r
c h e m ic a l p r o g r e s s .

D i f f e r e n t i a l m ig r a tio n i s one o f th e m ost u s e f u l

g e n e r a l t o o l s o f s e p a r a t i o n , in c lu d in g w ith i n i t s scope t h e te c h n iq u e s
o f e l e c t r o p h o r e s i s , m u ltip le p a r t i t i o n i n g , mass s p e c tro s c o p y , th e rm a l
and g r a v i t a t i o n a l d i f f u s i o n , d i f f e r e n t i a l s e d im e n ta tio n , and ch ro m ato g rap h y .
B . C om parison o f Gas and L iq u id C hrom atography
G hroroatography i s one o f th e m ost w id e ly a p p li c a b le m ethods o f
d i f f e r e n t i a l m ig r a tio n , s in c e i t i s p o t e n t i a l l y u s e f u l f o r s e p a r a t i o n o f
a l l s o lu b le s u b s ta n c e s .

C hrom atography i s l i m i t e d , how ever * b y t h e f a c t

t h a t each c l a s s o f s e p a r a t i o n s r e q u i r e s a d i f f e r e n t s o r b e n t- s o lv e n t
sy ste m and d e t e c t o r .

I n l i q u i d ch ro m ato g rap h y , t h e s o lv e n t may ra n g e

from lo w - b o ilin g o rg a n ic s u b s ta n c e s to s o lu ti o n s o f fu s e d s a l t s , and t h e
s o r b e n t may ra n g e from p a p e r s t r i p s o r s t r i n g s t o packed colum ns.

The

s o lv e n t i n v a p o r ch ro m ato g rap h y does n o t v a r y as much, s in c e i t i s n o t
ch o sen so much f o r e l u t i n g power a s f o r i t s a p p l i c a b i l i t y t o t h e d e t e c t o r
ch o sen ; ho w ev er, t h e s o r b e n t i s s e le c te d on th e b a s i s o f i t s p h y s ic a l
and c h e m ic a l p r o p e r t i e s , i n p a r t i c u l a r i t s v a p o r p r e s s u r e , th e r m a l
s t a b i l i t y , and a f f i n i t y f o r th e com ponents t o be s e p a r a t e d .

The la c k o f

g e n e r a l i t y o f an y one s o lv e n t - s o r b e n t sy stem i s a lm o st e q u iv a le n t f o r

2

l i q u i d and v a p o r ch ro m ato g rap h y j how ever, t h e d e t e c t o r s a v a i l a b l e f o r
v a p o r c h ro m ato g rap h y a r e much more g e n e r a l th a n th o s e r e q u ir e d f o r l i q u i d
chrom atographyo

T h is f a c t h a s g r e a t l y in flu e n c e d th e r a p id d ev elo p m en t

o f v a p o r ch ro m ato g rap h y , w h ich h a s underg o n e s p e c t a c u l a r grow th o f a p p l i ­
c a t i o n d u r in g t h e p a s t f o u r y e a r s .
Gas ch ro m ato g rap h y , w ith i t s v a r io u s g e n e r a l , s e n s i t i v e , r a p i d ,
c o n v e n ie n t, and r e l a t i v e l y in e x p e n s iv e d e t e c t o r s , h a s become a s ta n d a rd
bench and l i n e te c h n iq u e f o r a n a l y s is and p r o d u c tio n c o n tr o l*

Vapor

ch ro m ato g rap h y i s , h o w ev er, encum bered w ith two m ajo r l i m i t a t i o n s :

it

can b e a p p lie d o n ly t o t h o s e s u b s ta n c e s w hich h av e a p p r e c ia b le v a p o r
p r e s s u r e s b elo w 300~U0 0 °G, and i t does n o t a lo n e g iv e a d e q u a te q u a l i t a t i v e
in fo r m a tio n a b o u t t h e s u b s ta n c e s s e p a r a te d ,

Even though v a p o r chroma­

to g ra p h y i s l i m i t e d , t h e r e c e n t tr e n d h as been t o ex ten d i t beyond i t s
n a t u r a l c o n v e n ie n t ra n g e b e c a u s e o f t h e v e r s a t i l i t y o f i t s d e t e c t i n g
d e v ic e s .

S in c e many o r g a n ic m o le c u le s , e s p e c i a l l y th o s e o f b i o l o g i c a l

im p o rta n c e , a r e n o t th e r m a lly s t a b l e , and s in c e m ost s u i t a b l e s o r b e n ts
h a v e a p p r e c ia b le v a p o r p r e s s u r e a t e le v a te d te m p e r a tu r e s , t h i s e x te n s io n
o f gas ch ro m ato g rap h y h a s a lm o st re a c h e d i t s p r a c t i c a l l i m i t .
S o lu b le s u b s ta n c e s w ith low v a p o r p r e s s u r e may, how ever, b e s e p a r a te d
u s in g l i q u i d c h ro m ato g rap h y .

The tim e -h o n o re d te c h n iq u e f o r f o llo w in g

t h e dev elo p m en t o f a l i q u i d column chrom atogram h a s b een t o c o l l e c t th e
e f f l u e n t s tre a m in e q u a l f r a c t i o n s and a n a ly z e each f r a c t i o n i n d i v i d u a l l y ,
e i t h e r c h e m ic a lly o r p h y s ic a lly *
consum ing.

T his p ro c e s s i s v e r y te d io u s and tim e

To a c h ie v e a p a r a l l e l and com plem entary s t a t u s t o t h e r a p i d l y -

d e v e lo p in g f i e l d o f v a p o r ch ro m ato g rap h y , th e f i e l d o f l i q u i d ch ro m ato g rap h y

3

now r e q u i r e s a d e t e c t o r w hich w i l l be g e n e r a l t o a l a r g e number o f s o lv e n t
sy ste m s and s o l u t e s , resp o n d s e n s i t i v e l y t o sma3-l, d i l u t e s a m p le s , g iv e
p r e d i c t a b l e r e s p o n s e ( p r e f e r a b l y l i n e a r r e s p o n s e ) t o sam ples o v e r a w ide
ra n g e o f c o n c e n t r a t i o n , b e sim p le t o c o n s tr u c t and e a s y t o o p e r a t e , and
r e q u i r e l i t t l e a t t e n t i o n d u r in g th e dev elo p m en t o f a chrom atogram .
T h is s tu d y h a s b ee n co n cern ed w ith an a tte m p t t o d e v e lo p a d e t e c t o r
f u l f i l l i n g m ost o f t h e s e r e q u ir e m e n ts .

B e fo re d e s c r i b in g th e p r e s e n t

d e t e c t o r , how ever, p r e v io u s a p p ro a c h e s t o t h e problem w i l l b e re v ie w e d .
C . P re v io u s D e te c to r s f o r L iq u id C hrom atography
Kenyon &t a l . (1 ) h av e d ev elo p ed a d e t e c t o r w hich u t i l i z e s v a r i a t i o n
i n a b s o r p tio n o f u l t r a v i o l e t l i g h t .

The e lu e n t s tre a m from a c h ro m a to g ra p h ic

column i s allo w e d t o d r a i n i n t o a q u a r t z , u l t r a v i o l e t - a b s o r p t i o n c e l l .
A l i g h t so u rc e and p h o t o c e l l a r e a rra n g e d so t h a t l i g h t i s r e f l e c t e d from
t h e s u r f a c e o f th e l i q u i d i n th e c e l l a s i t i s f i l l e d from t h e colum n.
W ien th e e f flu e r rtr f i l l s t h e c e l l , th e p h o to c e ll a c t u a t e s th e s c a n mechanism
f o r a r e c o r d in g s p e c tro p h o to m e te r .

A f te r t h e a b s o r p tio n sp e c tru m o f th e

e f f l u e n t h a s b ee n r e c o r d e d , t h e a b s o r p tio n c e l l i s pumped o u t, and t h e
s p e c tro p h o to m e te r i s r e s e t a u t o m a ti c a lly t o i t s i n i t i a l w a v e le n g th .

T hus,

t h e a b s o r p tio n s p e c t r a o f t h e s o lu t e bands a r e re c o rd e d i n t e r m i t t e n t l y
d u r in g t h e d ev elo p m en t o f t h e chrom atogram , a llo w in g th e o b s e rv e r t o
d e te rm in e t h e c o n c e n tr a tio n and t h e c o m p o sitio n o f each s o l u t e b a n d .

T h is

te c h n iq u e i s v e r y a c c u r a t e , b u t i s l i m i t e d to s u b s ta n c e s w hich a b so rb i n
t h e u l t r a v i o l e t and t o s o lv e n t s w hich a r e t r a n s p a r e n t ; in a d d i t i o n t o t h i s
l i m i t a t i o n , t h e a p p a r a tu s i s v e r y e x p e n s iv e t o c o n s tr u c t and d a t a
i n t e r p r e t a t i o n i s o f te n in v o lv e d and t e d i o u s .

u

Baumann and B la e d e l (2 ) have a d a p te d h ig h - f r e q u e n c y co n d u ctan ce
m easurem ents t o th e d e t e c t i o n o f s o l u t e b an d s In l i q u i d ch ro m a to g ra p h y .
I n c h ro m a to g ra p h ic p r o c e s s e s w here l a r g e changes i n c o n d u c ta n c e o c c u r i n
t h e r e g io n o f t h e s o l u t e b a n d s , i t i s p o s s i b l e t o d e t e c t t h e i r p re s e n c e
th ro u g h h ig h - f r e q u e n c y c o n d u ctan ce m e asu rem en ts.

These m easurem ents a llo w

t h e d e t e c t i o n o f c h ro m a to g ra p h ic b an d s w ith o u t t h e u s e o f i n t e r n a l
e le c tro d e s .

T h is te c h n iq u e can l o c a t e bands b e f o r e th e y a r e e lu te d from

t h e colum n; h ow ever, i t i s v e r y l i m i t e d i n sco p e and s e n s i t i v i t y .
C la x to n ( 3 ) d e t e c t s s o l u t e bands, u t i l i z i n g t h e h e a t ev o lv ed accompany­
in g t h e a d s o r p tio n o f g a s e s and l i q u i d s on s o r b e n t s u r f a c e s .

A net heat

e v o l u t i o n may a l s o r e s u l t from s o l u t e d is p la c e m e n t o f t h e ad so rb e d s o lv e n t
a t th e su rfa c e of th e s o rb e n t.

D e te c tio n o f t h i s n e t h e a t o f a d s o r p tio n

w ith a s u i t a b l e te m p e r a tu r e - s e n s in g d e v ic e makes p o s s i b l e t h e u s e o f t h i s
phenomenon t o f o llo w th e d ev elopm ent o f a chrom atogram .

The m ajo r p ro b ­

lem s in v o lv e d i n t h e d ev elo p m ent o f t h i s te c h n iq u e a r e c o r r e l a t i o n s o f
t h e e x te n t o f te m p e r a tu re r i s e w ith t h e n a tu r e and t h e c o n c e n tr a tio n o f
th e s o lu te b a n d s.

T h is ap p ro a c h seems a p p lic a b le o n ly t o th o s e s o lv e n t -

s o r b e n t sy stem s w hich h av e a s u f f i c i e n t l y h ig h s u r f a c e e n e rg y .
Spackm an, S t e i n , and Moore (U) have d ev elo p e d a s p e c i f i c a p p l i c a t i o n
o f a b s o r p tio n s p e c tro p h o to m e tr y f o r t h e d e t e c t i o n o f amino a c id s w hich
h av e b e e n s e p a r a te d c h ro m a to g ra p h ic a H y .

The amino a c id s a r e s e p a r a te d

b y io n -e x c h a n g e colum ns w hich a r e e lu te d w ith a s o lv e n t o f c o n tin u o u s ly
v a r y in g pH.

The e f f l u e n t i s t r e a t e d c o n tin u o u s ly w ith a s ta n d a rd n i n -

h y d r in r e a g e n t , a llo w e d t o r e a c t , and p a s se d th ro u g h a r e c o r d in g s p e c t r o ­
p h o to m e te r.

The c h a r a c t e r i s t i c n irih y d rin a b s o r p tio n i s o b serv ed a t 570 n^z

5

and UJ4O mp, w h ich p e r m its c o n v e n ie n t q u a n t i t a t i v e a n a l y s is o f am ino
a c id s o

T h is t e c h n iq u e , w h ich was a lm o st 10 y e a r s i n d ev e lo p m e n t, i s so

much more c o n v e n ie n t th a n p r e v io u s ly u sed m ethods t h a t i t i s u sed to d a y
'a lm o s t t o t h e e x c lu s io n o f t h e e a r l i e r m eth o d s.
S c h u l t z , Bodraann, and Cantow (5 ) h av e d ev elo p ed a d i f f e r e n t i a l
r e f r a c t o m e t e r w h ich h a s b e e n m o d ifie d b y t h e P h o en ix P r e c i s i o n I n s tr u m e n t
Company ( 6 ) , f o r c o n tin u o u s ly m o n ito rin g t h e e f f l u e n t o f a c h ro m a to g ra p h ic
colum n.

The d i f f e r e n t i a l r e f r a c to m e te r i s p o t e n t i a l l y t h e m ost v e r s a t i l e

te c h n iq u e t o d a te f o r r e c o r d in g th e developm ent o f a chrom atogram , s in c e
r e f r a c t i v e in d e x i s a g e n e r a l p h y s ic a l p r o p e r t y and i t can b e re c o rd e d
c o n tin u o u s ly .

The te c h n iq u e i s n o t c o m p le te ly g e n e r a l f o r a l l s o l u t e s ,

b u t i t i s v e r y s e n s i t i v e f o r th o s e s o lu t e s w hich have a r e f r a c t i v e in d e x
w hich i s a p p r e c ia b ly d i f f e r e n t from t h a t o f th e s o lv e n t u s e d .
r e f r a c t o m e t r y i s s e n s i t i v e t o 1 .5 x 10

_6

D iffe re n tia l

r e f r a c t i v e - i n d e x u n i t s o r 1 .8 x 10

m illig ra m s p e r m i l l i l i t e r o f NaCl i n w a t e r .

The te c h n iq u e i s a s fo llo w s *

The r e f r a c t i v e - i n d e x p ris m i s d iv id e d d ia g o n a lly i n t o two co m p artm en ts.
One o f t h e com partm ents c o n ta in s t h e p u re s o lv e n t , and th e o t h e r h o ld s
a p o r t i o n o f t h e e f f l u e n t s tr e a m , w hich i s c o n tin u o u s ly flo w in g th ro u g h i t .
A p a i r o f p h o t o c e l l s i s a rra n g e d so a s t o d e t e c t th e d e f l e c t i o n o f a
m onochrom atic beam w hich p a s s e s th ro u g h th e r e f r a c t i v e in d e x c e l l .

The

beam d e f l e c t i o n d e t e c t e d b y t h e p h o to c e lls a c tu a t e s a r e c o r d e r p en and
a m echanism w hich moves t h e c e l l so a s t o b a la n c e th e beam a g a in .

The

c h a r t - d r i v e r a t e on t h e r e c o r d e r i s c o n tr o ll e d b y a m a ss -b a la n c e mechan­
ism , so t h a t t h e c h a r t i s d r iv e n a c c o rd in g t o th e r a t e o f flo w o f t h e
s o lv e n t.

T h is m ethod i s t h e m ost g e n e r a l t h a t h a s b e e n d is c u s s e d so f a r ;

_5

6

h o w ev er, i t i s l i m i t e d t o l a r g e p r e p a r a t i v e columns s in c e t h e dead volume
o f t h e p ris m i s to o l a r g e f o r u s e w ith s m a ll a n a l y t i c a l colum ns.

7

I I . THE PRESENT INVESTIGATION
A. G e n e ra l D e s c r ip ti o n o f t h e T echnique
A new te c h n iq u e h a s b e e n d e v elo p ed f o r d e t e c t i n g th e p r e s e n c e o f
s o l u t e b an d s i n t h e e f f l u e n t s tre a m o f a l i q u i d c h ro m ato g rap h , u t i l i z i n g
a p h y s ic a l p r o p e r t y ^ d i f f e r e n t from an y o f th o s e d is c u s s e d i n t h e p re c e d ­
in g s e c t i o n - The d e t e c t o r s e n s e s a d i f f e r e n c e i n te m p e r a tu re e s ta b l i s h e d
b etw e en a s o l u t i o n and i t s s o lv e n t i n a s o lv e n t - v a p o r - s a t u r a t e d cham ber.
T h is te m p e r a tu re d i f f e r e n c e a r i s e s b e c a u s e o f t h e d i f f e r e n c e i n t h e
r a t e s o f e v a p o r a tio n from t h e s o lv e n t and th e s o l u t i o n .
T h is method u t i l i z e s t h e m easurem ent, b y means o f t h e r m i s t o r s , o f
t h e te m p e r a tu re d i f f e r e n c e w hich i s e s ta b l i s h e d b etw een a c e l l u l o s e w ick
s a t u r a t e d w ith t h e p u re e l u t i n g s o lv e n t , and a s i m i l a r w ick w hich i s
C o n tin u o u s ly b a th e d w ith th e l i q u i d e f f l u e n t from t h e c h ro m a to g ra p h ic
column* b o th w ic k s a r e e n c lo s e d i n a th e r m o s ta te d cham ber s a tu r a t e d w ith
s o lv e n t v a p o r .

The th e r m i s t o r s c o n s t i t u t e two arm s o f a c o n v e n tio n a l

"W heatstone b r i d g e , w hich can be b a la n c e d when b o th t h e w ick s a r e b a th e d
w ith p u re s o lv e n t* th e te m p e r a tu re e f f e c t o f a s o l u t e p ro d u c e s an
u n b a la n c e w hich can b e m e a su re d .
The te m p e r a tu r e s se n se d by th e th e r m is to r s a r e p r i m a r i l y d ep en d e n t
upon th e c o m p e titio n o f t h e f o llo w in g p r o c e s s e s !
1 . H eat i n p u t .
a . E le c tr ic a l h e a tin g .
b . H eat e v o lv e d b y c o n d e n s a tio n o f s o lv e n t on t h e w ic k .

8

2 o H eat rem o v al *
a . T herm al c o n d u c tio n o f h e a t t o t h e l i q u i d f lo w in g th ro u g h
t h e w ick*
bo H eat a b so rb e d i n v a p o r i z a t i o n o f th e s o lv e n t ( a n d , p o s s i b l y ,
v o l a t i l e s o l u t e s ) from t h e w ick*
c» H eat l o s s e s th ro u g h c o n d u c tio n , c o n v e c tio n o r r a d i a t i o n t o
th e i n t e r i o r o f t h e th e r m o s ta te d cham ber.
S in c e t h e e l e c t r i c a l h e a t i n g o f t h e t h e r m i s t o r s , th e th e r m a l c o n d u c ti v i ty
o f t h e w ic k s , and th e c o n d e n s a tio n o f s o lv e n t a r e n o t a p p r e c ia b ly a l t e r e d
b y th e p r e s e n c e o f t h e s o l u t e band on th e w ic k , th e r e l a t i v e r a t e s o f
v a p o r i z a t i o n a t t h e two t h e r m is t o r s m ust l a r g e l y d e te rm in e t h e th e r m a l
u n b a la n ce *

The change i n th e v a p o r p r e s s u r e o f t h e s o lv e n t due t o th e

p re s e n c e o f t h e s o l u t e band i s th u s i n d i r e c t l y d e te c te d by th e t h e r m i s t o r ,
w hich shows a n in c r e a s e i n te m p e r a tu re f o r n o n - v o l a t i l e s o l u t e s , and a
d e c r e a s e i n te m p e r a tu re f o r c e r t a i n v o l a t i l e s o lu te s *

The in c r e a s e i n

te m p e r a tu re o f t h e t h e r m i s t o r i s t h e r e s u l t o f an in c r e a s e i n th e n e t r a t e
o f r e l e a s e o f th e h e a t o f c o n d e n s a tio n b y t h e s o lv e n t c o n d e n sin g on t h e
s u r f a c e o f t h e w ick ( o r a d e c r e a s e i n th e h e a t l o s s cau sed b y v a p o r i z a t i o n
o f t h e s o l v e n t ) , and o f an i n c r e a s e o f e l e c t r i c a l h e a tin g cau sed b y t h e
lo w e rin g o f t h e r e s i s t a n c e o f th e t h e r m i s t o r .
B * R e la te d T echniques
A lthough t h i s te c h n iq u e h a s n e v e r p r e v io u s ly b e e n u sed a s a chrom ato­
g r a p h ic d e t e c t o r , r e l a t e d th e rm o m e tric m ethods have b e e n u sed b y s e v e r a l
w o rk e rs i n th e l a s t d ecad e f o r th e m easurem ent o f m o le c u la r w e ig h t and

9

t h e e s ti m a t i o n o f o sm o tic c o e f f i c i e n t s .

The l i q u i d ch ro m ato g rap h y

d e t e c t o r was d e v e lo p e d c o m p le te ly in d e p e n d e n tly o f t h e s e th e rm o m e tric
m ethods! h o w ev er, t h e r e i s s t r i k i n g s i m i l a r i t y betw een th e s e te c h n iq u e s
and t h e new l i q u i d ch ro m ato g rap h y d e t e c t o r .
B a ld e s (7 ) i s c r e d i t e d w ith t h e f i r s t s u c c e s s f u l a p p l i c a t i o n o f
th e rm o m e tric m ethods f o r th,e d e te r m in a tio n o f m o le c u la r w e ig h t.

H is

a p p a r a tu s c o n s is te d o f a p a i r o f th erm o c o u p le s e n c lo s e d i n a solv en t™ v ap o rs a t u r a t e d chambero

One th erm o c o u p le had a d ro p o f s o lv e n t suspended upon

i t , and t h e o t h e r had a d ro p of s o l u t i o n .

The te m p e ra tu re d i f f e r e n c e was

o b serv ed b y a g a lv a n o m e te r d e f l e c t i o n due t o t h e d i f f e r e n c e i n p o t e n t i a l
from t h e th e rm o c o u p le s .

T h is method was found t o b e much f a s t e r th a n

o sm o tic p r e s s u r e o r i s o te n is c o p y f o r d e te rm in in g m o le c u la r w e ig h t.

The

o b serv ed te m p e r a tu re r i s e was l e s s th a n was e x p e c te d , b e c a u s e o f h e a t
d i s s i p a t i o n , so t h a t t h e method co u ld h o t b e u se d f o r m e a su rin g m o le c u la r
w e ig h ts w ith o u t c a l i b r a t i o n .

However, th e m ajor l i m i t a t i o n o f t h i s

te c h n iq u e was t h e low s e n s i t i v i t y of th e th e rm o c o u p le s .
T a y lo r and H a ll ( 8 ) , and B rad y , H u ff, and McBain (9 ) c o n c u r r e n tly
im proved t h i s te c h n iq u e b y s u b s t i t u t i n g t h e r m is to r s f o r t h e th e rm o c o u p le s ,
s u b s t a n t i a l l y i n c r e a s i n g th e s e n s i t i v i t y o f th e d e v ic e .

The new a r ra n g e ­

m ent c o n s is te d o f a p a i r o f t h e r m i s t o r s suspended i n a th e r m o s ta te d
cham ber s a t u r a t e d w ith s o lv e n t v a p o r .

The t h e r m i s to r s c o n s ti t u t e d two

arms o f a W h ea tsto n e b r id g e , s u p p lie d w ith an a l t e r n a t i n g p o t e n t i a l b y an
o s c illa to r.
o s c illo s c o p e .

The b r id g e u n b a la n c e was a m p lifie d and o b serv ed on an
The r e s i s t a n c e change i n th e v a r i a b l e p a r t o f t h e b r id g e

was u sed a s a m e a su re o f t h e te m p e r a tu re d i f f e r e n c e b etw een t h e two

10

t h e r m i s t o r s <> The te m p e r a tu re d i f f e r e n c e was c o r r e l a t e d w ith t h e m o le c u la r
w e ig h t o
M i l l e r and S t o l t e n (1 0 ) d e s ig n e d an a p p a r a tu s w hich ap p ro a c h e s t h e
th e rm o d y n a m ic a lly -e x p e c te d te m p e r a tu re r i s e more n e a r ly th a n th e p r e v io u s
d e s ig n s o

T h is te c h n iq u e In v o lv e d p la c in g t h e th e r m is to r s i n s m a ll p o o ls

o f m e rc u ry , w hich w ere co v ered by th e s o lv e n t and sam ple s o lu tio n s®
A h ig h e r te m p e r a tu re r i s e was o b se rv e d f o r t h e s o l u t i o n , b u t i t to o k lo n g e r
f o r e q u ilib r iu m t o b e e s t a b l i s h e d , more sam ple was r e q u ir e d th a n f o r t h e
d rop m eth o d , and t h e c e l l had t o b e removed and c le a n e d a f t e r each
d e te r m in a tio n *
Iy e n g a r ( 1 1 ) , and K u lk a r n i ( 1 2 ) , h av e r e p o r te d s i m i l a r d e v ic e s i n
o t h e r j o u r n a l s , b u t t h e i r m o d if ic a tio n s d id n o t s u b s t a n t i a l l y a l t e r t h e
n a t u r e o f t h e technique®
H ig u c h i e t a l . (1 3 )* hy u s in g an e v a c u a te d chamber and an in g e n io u s
m a g n e t i c - s t i r r i n g d e v ic e , h av e su cceeded i n d e s ig n in g , f o r a c t i v i t y co­
e f f i c i e n t m easurem ent, an a p p a r a tu s w hich re a c h e s 80-90 p e r c e n t o f t h e
t h e o r e t i c a l te m p e r a tu re r i s e , s in c e s t i r r i n g o f t h e s o lv e n t and o f t h e
s o l u t i o n re d u c e s th e s u r f a c e en erg y r e q u ir e d f o r th e mass exchange w hich
p ro d u c e s t h e te m p e r a tu re rise ®

The e v a c u a te d chamber e f f e c t i v e l y re d u c e s

t h e tim e r e q u ir e d f o r th e maximum te m p e ra tu re change t o b e o b s e rv e d , and
i t b u f f e r s te m p e r a tu r e changes from th e th e r m o s ta te d b a t h so as t o d e c r e a s e
th e r m a l i n s t a b i l i t y ®
The s t r i k i n g s i m i l a r i t y betw een th e s e v a r ie d m easurem ents and t h e
new d e t e c t o r f o r l i q u i d ch ro m ato g rap h y i s in p a r t d e c e iv in g , s in c e th e
o b j e c t i n t h e th e rm o m e tric m easurem ents d e s c rib e d was o b s e r v a tio n o f t h e

11

maximum p o s s i b l e te m p e ra tu re d i f f e r e n c e betw een a s o lv e n t and a s o l u t i o n
w hich can b e o b ta in e d th e rm o d y n a m ic a lly , w h ile th e o b j e c t i n th e d e s ig n o f
a

l i q u i d ch ro m ato g rap h y d e t e c t o r i s t o o b ta in t h e maximum p o s s i b l e

s e n s i t i v i t y f o r a g iv e n c o n c e n tr a tio n o f sam p le.

The w ick and t h e l a r g e

b r id g e v o l t a g e w hich a r e u se d w ith t h e c h ro m a to g ra p h ic d e t e c t o r a r e d e v ic e s
t o i n c r e a s e t h i s te m p e r a tu re change above th e th e rm o d y n a m ic a lly e x p e c te d
v a l u e , s in c e t h e w ick in c r e a s e s th e s u r f a c e a r e a and a l t e r s th e s u r f a c e
e n e rg y , and t h e l a r g e d e t e c t o r v o lta g e c a u se s h e a tin g ^ w hich in c r e a s e s th e
te m p e r a tu re d i f f e r e n c e b etw een th e t h e r m is to r s , i n th e p re s e n c e o f a s o l u t e
b a n d ,th r o u g h i n c r e a s i n g e v a p o r a tio n r a t e s .
G. E v o lu tio n o f t h e P r e s e n t D e te c to r
S in c e m ost o f t h e tim e s p e n t in th e developm ent o f t h e p r e s e n t l i q u i d
ch ro m ato g rap h y d e t e c t o r in v o lv e d e x p lo r a to r y i n v e s t i g a t i o n , t h e c o u rs e o f
t h e d ev elo p m en t o f t h e a p p a r a tu s w i l l b e t r a c e d b r i e f l y from i t s c o n c e p tio n
to i t s p re se n t s ta te .
The f i r s t d e t e c t o r was d e s ig n e d t o d e t e c t th e p r o g r e s s o f t h e d e v e lo p ­
m ent o f a chrom atogram on a s t r i n g colum n.

The c h o ic e o f th e r m i s t o r s a s

t h e s e n s in g e le m e n ts , and t h e u s e o f v a p o r p r e s s u r e a s t h e p r o p e r ty
m easured w ere th e r e s u l t o f e l im in a ti o n o f t h e o th e r p o s s i b l e means o f
d e t e c t i o n o f s o l u t e b an d s l o c a te d on a s t r i n g colum n.

A d e te c to r f o r

s t r i n g ch ro m ato g rap h y sh o u ld n o t d i s t u r b t h e chrom atogram , b u t m ust be
a b le t o l o c a t e t h e s o l u t e b an d s and i n d i c a t e t h e i r r e l a t i v e c o n c e n tr a tio n s .
All o f t h e commonly u se d te c h n iq u e s (1U) w ere c o n s id e r e d , b u t none was
found t h a t co u ld b e a d a p te d t o t h i s p u r p o s e .

A l l o f t h e e x i s t i n g te c h n iq u e s

12

w ere e i t h e r to o s p e c i f i c f o r one p a r t i c u l a r k in d o f s o l u t e , o r to o i n - ’
s e n s i t i v e t o s m a ll q u a n t i t i e s o f so lu te* .

The m ost p ro m is in g o f th e

te c h n iq u e s c o n s id e re d was d i f f e r e n t i a l r e f r a c t o m etiy , b u t i t r e q u ir e d
e x p e n s iv e and c o m p lic a te d in s tr u m e n ta tio n * and i t co u ld b e u sed o n ly b y
e l u t i n g t h e b an d s o f f th e s t r i n g *
One p h y s ic a l p r o p e r ty re m a in in g t o be c o n s id e re d was v a p o r p r e s s u r e .
The te c h n iq u e s commonly u sed f o r m easu rin g v a p o r p r e s s u r e w ere u n s a t i s ­
f a c t o r y f o r m ea su rin g t h e p re s e n c e o f a s o l u t e band on s t r i n g , s in c e th e y
w ould r e q u i r e i n t e r r u p t i o n o f th e developm ent o f th e chrom atogram .

The

id e a f o r u s e o f th e r m i s t o r s a s d e t e c t o r s o f v a p o r p r e s s u r e was t h e r e s u l t
o f t h e o b s e r v a tio n t h a t c o lo re d s o lu t e b an d s on a s t r i n g su sp en d ed i n a
v a p o r - s a t u r a t e d chamber a p p e a re d t o s p re a d more th a n m ight b e e x p e c te d
from t h e n a t u r a l s p re a d in g due t o t h e a d s o r p tio n o f th e com ponents on th e
s trin g .

I f p a r t o f t h e o b serv ed s p re a d in g w ere due t o th e n e t c o n d e n s a tio n

o f s o lv e n t v a p o r from t h e s a t u r a t e d atm o sp h ere s u rro u n d in g t h e s t r i n g ,
t h e n th e r e g i o n o f th e s t r i n g o ccu p ied b y th e s o l u t e band s h o u ld be
s l i g h t l y w arm er th a n t h e cham ber.

The p ro b le m , th e n , was t o f i n d th e

p r o p e r cham ber c o n d itio n s and t h e p r o p e r d e t e c t o r f o r t h i s te m p e r a tu re
ris e .

T h e rm is to r and th e rm o c o u p le s e n s in g d e v ic e s w ere c o n s id e r e d , and

t h e t h e r m i s t o r was ch o sen b e c a u s e of i t s much g r e a t e r s e n s i t i v i t y .
A sim p le t h e r m i s t o r d e t e c t o r c i r c u i t was d e s ig n e d , c o n s i s t i n g o f two
t h e r m i s t o r s as o p p o sin g arras o f a W h eatsto n e b r id g e .

The th e r m i s t o r s

w ere clam ped o n to g. s t r i n g su sp en d ed on a w ir e fram e w i t h i n a b e l l j a r
s a t u r a t e d w ith w a t e r .

The s o lv e n t (w a te r) was s u p p lie d t o th e s t r i n g

b y c a p i l l a r y s ip h o n from a b e a k e r a t t h e to p o f t h e s t r i n g .

The u n b a la n c e

13

o f t h e b r id g e was o b serv ed w ith a g alv a n o m e te r <> The s i g n a l o b serv ed on
t h e g a lv a n o m e te r was c o m p le te ly in c o n c lu s iv e u n t i l t h e b e l l j a r was
co v e re d w ith a l a r g e , i n s u l a t i n g c l o t h .

The c lo t h s e rv e d b o th a s a

te m p e r a tu r e b a f f l e a n d , a s a l i g h t s h i e l d .

The sam p les w ere p la c e d on

t h e s t r i n g w ith a lo n g c a p i l l a r y and m e d ic in e - d ro p p e r .

T h is d e t e c t o r

a rra n g e m e n t gave a b a r e ly - o b s e r v a b le re s p o n s e t o a d ro p o f s a t u r a t e d N aC l.
T h is r e s u l t , a lth o u g h n o t e n t i r e l y s a t i s f y i n g , was p o s i t i v e enough t o
i n d i c a t e t h a t a d e t e c t o r m ig h t be d e s ig n e d u s in g t h i s b a s ic p r i n c i p l e .
The d e t e c t o r , s t r i n g , and s o lv e n t r e s e r v o i r w ere removed from th e
b e l l j a r and p la c e d i n t o a s i l v e r e d Dewar f l a s k co v ered w ith a co rk
a rra n g e d w ith a h o l e s u i t a b l e f o r sam ple i n j e c t i o n .

T h is sy stem a f f o r d e d

a r e a s o n a b ly a d i a b a t i c c o n ta in e r f o r th e d e t e c t o r ; how ever, i t had s e v e r a l
u n d e s ir a b l e f e a t u r e s .

S a m p le s, when i n j e c t e d , w ould d i s t u r b th e e q u ilib r iu m

o f t h e d e t e c t o r f o r h o u r s ; t h e system r e q u ir e d h o u rs t o s t a b i l i z e , b u t was
e a s i l y d i s t u r b e d w i t h room te m p e r a tu re f l u c t u a t i o n s , and sam ple i n j e c t i o n
was a t b e s t u n c e r t a i n and u n p r e d ic ta b le .
I n s p i t e o f t h e s e d i f f i c u l t i e s , t h e d e t e c t o r showed p ro m ise i n th e
s e n s i t i v i t y o f i t s re s p o n s e and i n i t s a p p a re n t l i n e a r i t y w it h i n t h e l i m i t s
o f sam ple i n j e c t i o n .

T a b le I i s r e p r e s e n t a t i v e o f t h e r e s p o n s e w hich was

o b ta in e d f o r s u c c e s s iv e d i l u t i o n s o f a s a t u r a t e d NaCl s o l u t i o n .

The

in c r e a s i n g d e v i a t i o n fro m l i n e a r i t y can b e e x p la in e d b y t h e f a c t t h a t t h e
flo w r a t e d e c re a s e d d u r in g th e tim e th e sam ples w ere r u n .

The flo w r a t e

was o b se rv e d t o d e c r e a s e w ith o u t a p p a r e n t re a s o n , even w ith a c o n s ta n th ead d e v i c e .

The s o lv e n t flo w on s t r i n g d e c r e a s e d o v e r a p e r io d o f

s e v e r a l w eeks and f i n a l l y s to p p e d .

A d d itio n o f d e te r g e n ts o r a lc o h o ls

iu

TABLE I
REPRESENTATIVE RESPONSE FOR EARLY DETECTOR

S u c c e s s iv e D i l u t i o n s
Of S a tu r a te d NaCl

R e c o rd e r R esponse
( Ce n tim e te r - m in u te s )

0 .1

28U
30.2

0 .0 1

3*2

Q .001

w ould r e s t o r e t h e flo w t e m p o r a r ily , b u t t h e s o lv e n t flo w w ould d e c r e a s e
a g a in u n t i l f i n a l l y i t c o u ld n o t b e r e s t o r e d .

T h is c h a r a c t e r i s t i c o f

s t r i n g columns f o rc e d abandonm ent o f t h e i r u s e w ith a d e t e c t o r .

The

d e t e c t o r was a l s o re c o g n iz e d a s b e in g p o t e n t i a l l y more u s e f u l f o r column
o r p a p e r ch ro m ato g rap h y th a n s t r i n g , b e c a u s e s e p a r a tio n s on s t r i n g w ere
v e ry lim ite d .
Up t o t h i s s ta g e i n t h e developm ent o f a d e t e c t o r , b o th o f th e
t h e r m i s t o r s had b e e n l o c a te d on th e same s t r i n g .

T h is arra n g e m e n t was

a d o p te d t o g iv e an i n i t i a l d e f l e c t i o n c o rre s p o n d in g to th e t o t a l sa m p le ,
w hich was i n j e c t e d j u s t above th e to p t h e r m i s t o r , and a second p o s i t i v e
peak o r s e r i e s o f p e a k s c o rre s p o n d in g t o th e s o lu t e ban d s s e p a r a te d b y th e
colum n.

The to p t h e r m i s t o r was u s e l e s s e x c e p t f o r a b a la n c e e le m e n t,

s i n c e sam p le i n j e c t i o n d is t u r b e d th e e q u ilib r iu m d u r in g t h e tim e t h a t
t h e sam ple p a s s e d i t .

The t h e r m i s t o r was t h e r e f o r e s u b s e q u e n tly p la c e d

on a s e p a r a t e s t r i n g , w hich a c te d a s a r e f e r e n c e w ick o n ly and more
e f f e c t i v e l y s e rv e d a s a b a la n c e e le m e n t.

15

S in c e t h e s t r i n g had t o be abandoned as a colum n, th e em phasis o f
t h e p r o j e c t was s h i f t e d t o d e s ig n in g a d e t e c t o r f o r l i q u i d column chroma­
to g ra p h y •

The s t r i n g d e t e c t o r d id n o t r e q u i r e much m o d if ic a tio n f o r

a d a p t a t i o n t o colum n ch ro m ato g rap h y , s in c e th e s t r i n g s w ere s t i l l u sed as
w ic k s t o c a r i y t h e e f f l u e n t o f t h e colum ns ov er t h e t h e r m i s t o r s .

Two

c h ro m a to g ra p h ic columns w ere mounted o v e r th e d e t e c t o r by b o r in g h o le s
i n t h e cork c o v e rin g th e Dewar f l a s k and a t t a c h i n g t h e s t r i n g s t o th e
b o tto m s o f t h e co lu m n s.

The s i g n a l from t h i s d e t e c t o r was found t o b e v e r y

u n s a t i s f a c t o r y b e c a u se o f a g r e a t l y in c r e a s e d n o is e l e v e l .

The s o u rc e s o f

d i f f i c u l t y w ere i d e n t i f i e d as b e in g a s s o c ia te d w ith th e flo w r a t e and
t h e te m p e r a tu re f l u c t a t i o n o f th e s o l v e n t .

The w hole a p p a r a tu s was moved

t o a c o n s ta n t—te m p e r a tu re room and th e s o lv e n t p r e s s u r e was c o n tr o lle d
w ith a C a r t e s ia n m a n o s ta t.

These changes im proved th e s t a b i l i t y o f th e

d e t e c t o r , b u t t h e o n - o f f r e g u l a t i o n o f t h e room te m p e ra tu re was o b serv ed
a s a s in e wave on th e back g ro und t r a c e .
The back g ro u n d was s t a b i l i z e d b y th e r m o s ta tin g t h e d e t e c t o r cham ber
and th e columns w ith w a te r from a c o n s ta n t te m p e ra tu re b a th c o n t r o l l e d t o
± GoGG80C.

The colum ns and t h e d e t e c t o r w ere immersed i n t o t h e b a th ,

p r o t e c t e d by a p l a s t i c b a g .

I t was o b serv ed t h a t t h e sy stem e q u i l i b r a t e d

much more r e a d i l y when th e Dewar f l a s k around th e d e t e c t o r was r e p la c e d
b y a g la s s c o n t a i n e r co v ered w ith aluminum f o i l .

B e tte r e q u ilib ra tio n

w ith t h e b a th te m p e r a tu re was o b ta in e d by u s in g a c o n d e n se r j a c k e t around
th e co lu m n s.

The p l a s t i c b ag was e v e n tu a l ly r e p la c e d w ith a lo n g g la s s

c y l i n d e r w e ig h te d w ith le a d and i n s u la te d around t h e to p w ith g l a s s w o o l.

16

T hese s te p s f i n a l l y s u f f i c i e n t l y s t a b i l i z e d th e d e t e c t o r t h a t a
s u i t a b l e b ack g ro u n d was o b ta in e d .,

However, t h e d e t e c t o r s e n s i t i v i t y had

d e c re a s e d so much as t o make t h e d e t e c t o r a lm o st u s e l e s s , s in c e i t was
found t h a t l a r g e c o n c e n tr a te d sam ples w ere r e q u ir e d t o o b t a in a p p r e c ia b le
s ig n a ls .

T h is low ered s e n s i t i v i t y was f i n a l l y t r a c e d t o th e l a r g e s o lv e n t

flo w r a t e s usedo

The colum ns (8 nun. i . d . ) r e q u ir e d flo w r a t e s o f s e v e r a l

m il l i l i t e r s p e r h o u r t o e l u t e sam ples w it h i n a r e a s o n a b le le n g th o f tim e .
T hese la r g e - d i a m e t e r colum ns w ere r e p la c e d by c a p i l l a r y colum ns (2 mm. i . d . ) ,
so t h a t t h e flo w r a t e co u ld be red u ced t o l e s s th a n 10 m i l l i l i t e r s p e r
day.

Sam ple s i z e s w ere re d u c e d t o a fesw h u n d re d th s o f a m i l l i l i t e r o f

s o lu tio n .
T hese chan g es b r in g t h e d e t e c t o r up t o e s s e n t i a l l y i t s p r e s e n t fo rm .
The n a tu r e and p r o p e r t i e s o f each of t h e i n d i v i d u a l com ponents o f t h e
p r e s e n t c h ro m a to g ra p h ic sy stem w i l l now b e c o n s id e re d i n more d e t a i l .
Do Components o f th e P r e s e n t C hrom atographic A p p a ra tu s
1 . Columns
F u n d a m e n ta lly t h e d ia m e te r and le n g th , o f t h e colum n employed w ith
t h i s d e te c to r a re n o t lim ite d .

S in c e th e flo w r a t e o f s o lv e n t i n t o t h e

d e t e c t o r m ust b e l i m i t e d , h ow ever, th e columns m ust be o f s m a ll d ia m e te r
i f a l l o f th e e l u e n t s tre a m i s t o flo w th ro u g h th e d e t e c t o r .

I f u s e of

l a r g e colum ns i s d e s i r e d , a d e v ic e f o r d i v e r t i n g a p a r t o f t h e e f f l u e n t
s tre a m m ust b e em ployed.

The s m a ll c a p i l l a r y colum ns employed i n th e

d ev elo p m en t o f t h e d e t e c t o r a r e v e r y c o n v e n ie n t f o r a n a l y t i c a l p u r p o s e s ,
s in c e th e y r e q u i r e o n ly v e r y s m a ll am ounts o f sam p le.

These s m a ll colum ns

17

a r e d i f f i c u l t t o p a c k , b u t i f h a n d le d p r o p e r ly th e y may b e u sed i n d e f i n i t e l y
w ith l i t t l e

change i n p r o p e r t i e s .

The colum ns packed i n 2 mm® i . d t h i c k - w a l l e d c a p i l l a r y tu b in g w ere
p la c e d j u s t above th e d e t e c t o r chamber a s shown i n F ig u re 1®

The b o tto m

o f t h e column e x te n d s j u s t below th e co rk co v e r i n s i d e o f th e d e t e c t o r
cham ber, so t h a t t h e w ick d o es n o t to u c h th e cork®

The th e r m o s ta tin g

j a c k e t m ust b e lo n g enough so t h a t t h e s o lv e n t p a s s in g down th e columns
becom es e q u i l i b r a t e d w ith th e b a th te m p e r a tu r e .

The in ta k e f o r th e th erm o -

s t a t i n g j a c k e t s h o u ld b e l o c a t e d a t t h e r e g u l a t o r f o r th e b a t h , so t h a t
t h e j a c k e t te m p e r a tu re does n o t v a r y a p p r e c ia b ly .
The colum n p a c k in g i s , o f c o u r s e , s e l e c t e d f o r th e p a r t i c u l a r ty p e
o f s e p a r a t i o n w hich i s t o b e made.
2 . M icks
G o tto n s t r i n g o r th r e a d w ick s have b e e n used th ro u g h o u t m ost o f t h i s
w o rk .

The s i z e and th e weave o f th e w ick a r e d i c t a t e d b y th e flo w r a t e

o f t h e s o l v e n t , th e d e s ig n o f th e d e t e c t o r , and, th e v o l a t i l i t y o f th e
s o lv e n t.

Maximum s e n s i t i v i t y i s a t t a i n e d w ith minimum w ick s i z e and

maximum s u r f a c e a r e a .

The w ick m ust b e l a r g e enough, i n r e l a t i o n t o t h e

flo w r a t e , f o r th e s o lv e n t t o flo w w ith o u t fo rm in g d r o p l e t s o r s u r g e s , and
t o c o v e r a s much o f t h e t h e r m i s t o r s u r f a c e a s p o s s ib le ^ how ever, s in c e th e
s e n s i t i v i t y i s d e c re a s e d as w ick s i z e i s in c r e a s e d , t h e optimum s e n s i t i v i t y
i s o b ta in e d w ith t h e minimum w ick s i z e w hich w i l l g iv e a smooth s o lv e n t
f lo w .
The i d e a l s i t u a t i o n m ig h t b e t o have th e d e t e c t o r t h e r m i s t o r i n
c o n ta c t w ith a l a r g e - a r e a ,u n i - m o l e c u l a r l a y e r o f s o l v e n t .

T h is sh o u ld

18

FIGURE 1 .

COLUMNS SHOWN MOUNTED IN THERMOSTATING JACKET
ABOVE DETECTOR CHAMBER.

19

a llo w co m p le te e q u i l i b r a t i o n o f s o lv e n t w ith th e v a p o r p h a se and sh o u ld
g iv e t h e maximum te m p e r a tu re change f o r a g iv e n amount o f s o lu te *

I t is

i m p r a c t i c a l t o a c h ie v e t h i s s i t u a t i o n e x p e r im e n ta lly f o r two re a so n s*
I* t h e flo w r a t e w ould n e c e s s a r i l y b e to o s m a ll, and
2 . t h e t h e r m i s t o r te m p e ra tu re i s s l i g h t l y above t h e te m p e r a tu re
o f th e s a t u r a t e d cham berj t h i s in c r e a s e d te m p e ra tu re would
ca u se t h e s o lv e n t t o d r y and cau se e r r a t i c flow *
An a tte m p t was made t o c o n s tr u c t a d e t e c t o r t o a p p ro ach t h i s t t id e a l11
s i t u a t i o n a s n e a r l y a s p o s s ib le *

The w ick was e lim in a te d , and t h e s o lv e n t

flo w ed down t h e t h e r m i s t o r i n a s te a d y (th o u g h n o t u n im o le c u la r) s tre a m .
T h is d e t e c t o r a rra n g e m e n t was s e n s i t i v e , b u t i t was n o t s t a b l e , b e c a u s e
t h e s o lv e n t s tre a m was e r r a t i c .

The p re s e n c e of th e w ick seems t o

s t a b i l i z e th e s o lv e n t flo w th ro u g h t h e d e t e c t o r , and i t p o s s i b l y g iv e s
added s t a b i l i z a t i o n b e c a u s e ,o f i t s l a r g e r h e a t c a p a c i t y .
The p r e s e n c e o f a c o tto n w ick was a t one tim e f e a r e d t o i n t e r f e r e
w ith t h e s e p a r a t i o n s on alu m in a and ca u se undue s p re a d in g o f component
bands.

A g la s s - w o o l w ick was s u b s t i t u t e d f o r th e c e l l u l o s e w ic k , b u t i t

d id n o t p ro v e as s a t i s f a c t o i y , p o s s i b ly b e c a u s e t h e l a r g e r s u r f a c e a r e a
o f t h e c o t t o n w ick p e r m its much more r a p id c o n d e n s a tio n and v a p o r i z a t i o n
o f t h e s o lv e n t v a p o r , g iv in g g r e a t e r re s p o n s e t o th e s o l u t e b a n d s .

The

flo w c h a r a c t e r i s t i c s may a l s o h av e b e e n l e s s s t a b l e w ith t h e g la s s - w o o l
w ic k .
The m o st d e s i r a b l e l o c a t i o n of t h e th e r m is to r s on th e w ick seems t o
b e 3-U c e n tim e te r s from t h e mouth o f t h e colum n.

P lacem en t o f th e

t h e r m i s t o r to o n e a r t h e colum n d oes n o t a llo w tim e f o r e q u i l i b r a t i o n o f

20

t h e s o lv e n t w ith t h e cham ber a tm o s p h e re « I f th e t h e r m is to r s a r e p la c e d
t o o f a r fro m t h e columns, t h e s o lu t e b an d s may b e s p re a d , due t o t h e n e t
c o n d e n s a tio n o f th e s o lv e n t on t h e w ick a t th e s o l u t e band®
The end o f t h e w ick m ust b e to u c h in g th e b o tto m o f t h e d e t e c t o r
cham ber o r some a u x i l i a r y c o n t a i n e r , s in c e any d rop f o rm a tio n on t h e w ick
d i s t u r b s t h e flctw r a t e o f t h e s o lv e n t a c r o s s t h e t h e r m i s t o r , c a u s in g
b ack g ro u n d i n s t a b i l i t y „ M ien s ip h o n s w ere u sed t o e x h a u s t t h e d e t e c t o r
cham ber o f e x c e s s s o l v e n t , th e y had t o b e a rra n g e d so t h a t no d ro p s would
form even on th e e x t e r n a l o u t l e t , s in c e t h i s a l s o was found t o d i s t u r b
t h e d e te c to r o
The optimum flo w r a t e f o r a g iv e n w ick may b e found b y p l a c in g th e
w ick i n a t r a n s p a r e n t cham ber s a tu r a t e d w ith s o lv e n t v a p o rj th e s o lv e n t
flo w i s th e n d e c re a s e d down th e w ick u n t i l t h e w ick c o n tin u o u s ly a p p e a rs
t o b e s l i g h t l y damp w ith s o l v e n t *
3* T h e rm is to rs
The r e s i s t a n c e o f a t h e r m i s t o r a s a f u n c t io n o f te m p e r a tu re may b e
e x p re s s e d b y th e e q u a tio n ,
r - r 0 e -b <T- T°>
W here r i s t h e r e s i s t a n c e i n ohms, r 0 i s th e r e s i s t a n c e a t te n p e r a t u r e T0,
b i s t h e te m p e r a tu re c o e f f i c i e n t , and T i s t h e te m p e r a tu r e .
The t h e r m i s t o r s s e l e c t e d f o r a d e t e c t o r sh o u ld b e a s n e a r l y m atched
a s p o s s i b l e , t o m inim ize t h e i n s t a b i l i t y caused b y th e f l u c t u a t i o n s i n
t h e am b ien t te m p e r a tu re o f t h e th e r ra o s ta te d cham ber and o f t h e flo w in g
s o lv e tito

The v a lu e s o f b sh o u ld b e w i t h i n one p e r c e n t and th e v a lu e s

o f r 0 w i t h i n f i v e p e r c e n t f o r th e two t h e r m i s t o r s .

The t h e r m i s to r s u sed

had a r e s i s t a n c e o f betw een 2 ,0 0 0 and U,000 ohms a t th e te m p e r a tu re o f
th e t h e r m o s t a t .

H ig h er r e s i s t a n c e t h e r m is to r s a r e l e s s s e n s i t i v e , and

lo w er r e s i s t a n c e th e r m i s t o r s ca u se to o much e l e c t r i c a l h e a tin g a t t h e
v o l t a g e s r e q u ir e d f o r s e n s i t i v e o p e r a t io n .

S in c e o v e r h e a tin g o f t h e

t h e r m i s t o r w i l l c a u s e i t s r e s i s t a n c e t o d ro p , w hich in c r e a s e s th e c u r r e n t
and c a u s e s s t i l l more h e a t i n g , lo w er r e s i s t a n c e th e r m i s t o r s wi.11 b e more
i n c l i n e d t o d e g e n e r a te and b u rn o u t.
Two d e s ig n s o f t h e r m i s t o r s have b e e n u s e d , d ep en d in g on th e s o lv e n t
and w ick d e s i r e d .
a . T h e rm is to r s e a le d i n t h e m iddle o f a g la s s en v e lo p e ( s e e F ig u re 2 ) .
T h is ty p e o f elem en t was u sed i n n o n - c o r r o s iv e , n o n -c o n d u c tin g s o lv e n t s .
I t s m ajo r a d v a n ta g e i s t h e f a c t t h a t t h e w ick can b e a tta c h e d to. th e
t h e r m i s t o r v e r y s e c u r e l y , w ith a l a r g e s u r f a c e a r e a o f t h e w ick sp re a d
t h i n l y over th e s u rfa c e of th e th e rm is to r.

T h is a rran g em en t i s v e r y c lo s e

t o t h e i d e a l th e r m is to r - w ic k r e l a t i o n s h i p , w ith a maximum exposed s u r f a c e
a r e a o f w ick i n c o n ta c t w ith th e t h e r m i s t o r .

The te c h n iq u e f o r s e c u r in g

t h e t h e r m i s t o r t o t h e w ick i s t o u n r a v e l t h e w ick up t o t h e th e r m i s t o r
and a llo w i t t o rew in d around th e g la s s e n v e lo p e .

The s tr a n d s o f th e

w ick a r e s p re a d o v e r t h e s u r f a c e o f t h e t h e r m is to r and t i e d w ith a f i n e ,
c o a r s e th r e a d , so t h a t s l i g h t j a r r i n g w i l l n o t a l t e r th e j u x t a - p o s i t i o n .
C are m ust b e e x e r c is e d t o p r e v e n t c o n s t r i c t i o n o f t h e w ick by th e t h r e a d .
b . G lass p ro b e t h e r m i s t o r ( th e r m is t o r s e a le d i n th e end o f a g la s s
b e a d - - s e e F ig u re 3 )«

The t h e r m is t o r s f o r t h i s d e t e c t o r w ere s u p p o rte d b y

a wooden l a t h , w hich was su sp en d ed i n th e th e r m o s ta te d cham ber from th e

22

FIGURE 2 .

DETECTOR DESIGN USING THERMISTORS SEALED
IN GLASS ENVELOPES.

23

ih ebm isto bs

f ig u r e

3

DBTBGTOR

DESIGN FOR GUSS-BSAD

2h

co rk co v e r 0

The w ick was u n ra v e le d and re-w ound around t h e bead o f th e

t h e r m i s t o r , t o g iv e a maximum exposed w ick s u r f a c e i n c o n ta c t w ith th e
t h e r m i s t o r <, P ro b e t h e r m i s t o r s a r e more s e n s i t i v e i n t h e i r re s p o n s e th a n
a r e g la s s - e n v e lo p e t h e r m i s t o r s , s in c e t h e p ro b e th e r m i s t o r s a r e i n c l o s e r
c o n ta c t w ith t h e i r t h i n n e r g l a s s s u p p o r t s .

The t h e r m i s t o r s s h o u ld b e

i n c l i n e d s l i g h t l y upward t o p r e v e n t any im p u r iti e s d is s o lv e d from th e
wooden l a t h from coming i n c o n ta c t w ith th e w ick*

I f t h e w ick i s im p ro p e rly

s e c u re d t o t h e t h e r m i s t o r , t h e s o lv e n t may ru n backw ard down th e th e r m i s t o r
and s h o r t th e c o n n e c tin g le a d s .
U. S o lv e n t Flow C o n tr o l and O ut-G as D evice
S o lv e n t flo w m ust b e c o n tr o ll e d v e r y c a r e f u l l y i n o r d e r t o m a in ta in
th e s t a b i l i t y r e q u i r e d f o r q u a n t i t a t i v e d e t e c t i o n o f s m a ll s a m p le s.

Changes

i n flo w r a t e o f l e s s th a n 0*1 m i l l i l i t e r s a day may c a u s e f u l l - s c a l e
d e fle c tio n .

A tte m p ts t o c o n t r o l s o lv e n t flo w w ith gas p r e s s u r e and a

C a r t e s i a n - d i v e r m a n o sta t

w ere n o t s u c c e s s f u l , s in c e th e p r e s s u r e c o n t r o l

was n o t s u f f i c i e n t l y s e n s i t i v e ,

"Where p o s s i b l e , g r a v i t y f u r n is h e s th e

m ost c o n s ta n t s o u rc e o f s o lv e n t p r e s s u r e .

The s o lv e n t su p p ly f o r t h e

colum ns was a 3 - l i t e r , ro u n d -b o tto m ed f l a s k , w hich was s u p p o rte d b y an
a d ju s ta b le la b o ra to ry -ja c k .

B ecau se o f t h e la r g e r e s e r v o i r volum e and th e

slow f l o w - r a t e o f s o l v e n t , th e l e v e l i n th e f l a s k rem ained e s s e n t i a l l y
c o n s ta n t o v e r a p e r io d o f s e v e r a l d a y s .

S l i g h t a d ju s tm e n ts o f th e s o lv e n t

l e v e l w ere made p e r i o d i c a l l y w ith th e ja c k , r e f e r r i n g t h e l e v e l t o a
f i d u c i a l mark w ith t h e a i d o f an e y e - p ie c e a tta c h e d t o t h e o u t- g a s s in g
tu b e .

25

The o u t-g a s d e v ic e i s shown i n F ig u re i ; 0 O u t- g a s s in g i s ev en more
e s s e n t i a l w ith c a p i l l a r y colum ns th a n w ith l a r g e r colum ns, b e c a u s e even
v e r y s m a ll b u b b le s ca u se s o lv e n t flo w i n t e r r u p t i o n i n s m a ll- d ia m e te r
co lu m n s*>
D e t e c t o r s e n s i t i v i t y was found t o in c r e a s e a lm o st l i n e a r l y a s t h e
flo w r a t e was d e c re a s e d down t o 3 - 5 m i l l i l i t e r s a day*,

The flo w r a t e

s e l e c t e d f o r m ost o f t h e l a t e r w ork was 5 m i l l i l i t e r s a day*,

A flo w r a t e

o f 2o5 m il l .11,i t e r s p e r day in c r e a s e d th e s e n s i t i v i t y by a f a c t o r o f 1*7>
b u t t h e e l u t i o n tim e became p r o h i b i t i v e f o r t h e s i z e column employed*
More n o is e was a ls o o b serv ed a t t h i s flo w r a t e , b e c a u s e t h e w ick seemed
t o d ry p e r io d ic a lly * ,
5« Sam ple I n j e c t i o n
One o f t h e m ajo r p ro b lem s e n c o u n te re d i n t h i s s tu d y i s th e re p ro d u c ­
i b l e i n j e c t i o n o f a l i q u i d sam ple i n t o a moving l i q u i d e lu e n t*

I f th e

s o lv e n t flo w i s i n t e r r u p t e d , th e e q u ilib r iu m i n t h e d e t e c t o r cham ber i s
d istu rb e d * .

I f , on t h e o t h e r h a n d , t h e sam ple i s in tr o d u c e d d i r e c t l y ,

c o n s id e r a b le s p re a d in g o f t h e s o lu t e band may r e s u l t b e c a u s e o f tu r b u le n c e
and d i f f u s io n *
The i n j e c t i o n a rra n g e m e n t u sed i s shown i n F ig u re 5*

I f th e u s u a l

l i q u i d ch ro m ato g rap h y te c h n iq u e i s u sed f o r sam ple i n j e c t i o n , t h e t h r e e way s to p c o c k i s opened so a s t o c u t t h e flo w from t h e s o lv e n t r e s e r v o i r
and a llo w a lo n g hypoderm ic n e e d le t o be i n s e r t e d i n t o t h e sp ace j u s t
abxjve t h e column*

The e x c e ss s o lv e n t i s removed from t h e column and th e

sam p le i n j e c t e d i n t o t h e to p o f th e c a p i l l a r y *

The sam ple m ust be pushed

26

FIGURE h °

OUT-GAS DEVICE, LOCATED IN SOLVENT LINE BETWEEN RESERVOIR AND
SAMPLE INJECTION FQRT. HEAT OF LAMP DISPLACES DISSOLVED AIR,
WHICH IS VENTED THROUGH STOPCOCK AND DRYING TUBE.

FIGURE 5 .

ARRANGEMENT FOR SAMPLE INJECTION.

28

i n t o t h e c a p i l l a r y so a s t o a v o id w e ttin g t h e o u ts id e o f th e n e e d le
w ith s a m p le .

The n e e d le i s w ith d raw n as t h e sam ple f i l l s th e c a p i l l a r y ,

b u t i t m ust b e w ith d ra w n s lo w ly t o av o id tr a p p in g a i r b u b b le s w hich would
s to p s o lv e n t flow o

O r d i n a r i l y i n column chrom atography th e sam ple i s

allo w e d t o flo w down i n t o th e to p o f th e column u n t i l t h e to p m eniscus o f
t h e sam ple j u s t s t a r t s o n to t h e colum n, b u t w ith a c a p i l l a r y column th e
s o lv e n t l e v e l may b e r e s t o r e d im m e d ia te ly , s in c e th e s o l u t e band i s n o t
s p re a d b y m ixing i n t h e c a p i l l a r y *

I t i s b e s t t o r e s t o r e th e norm al

s o lv e n t p r e s s u r e a s soon a s p o s s i b l e , s in c e th e d e t e c t o r i s d is tu r b e d b y
th e sam ple i n j e c t i o n *
S u rp ris in g ly l i t t l e

s p re a d in g r e s u l t s from i n j e c t i o n o f th e sam ple

d i r e c t l y i n t o th e s o lv e n t s tre a m , p r o v id in g th e n e e d le used i s v e r y s m a ll
and th e sam ple i s Q o Q l - m i l l i l i t e r o r le s s *

T h is s i z e sam ple can b e i n j e c t e d

i n t o t h e s tre a m r e p r o d u c ib ly b y means o f a hypoderm ic m ic ro - s y rin g e w ith
a Chaney a d a p to r*
6* T h e rm o s ta tin g and T em p erature C o n tro l
T em p eratu re c o n t r o l i s one o f th e m ajor l i m i t a t i o n s t o s e n s i t i v i t y *
The th e r m o s ta t f o r a l i q u i d chrom atography d e t e c t o r sh o u ld b e c o n s tr u c te d
c a r e f u l l y w ith th e b e s t a v a i l a b l e equipm ent! how ever, a b a th w hich r e g u l a t e s
t o ± G«01°G* i s a d e q u a te f o r d e t e c t i o n o f sam ples of l a r g e r c o n c e n tr a tio n
Q
—3
th a n lC f m oles i n 0*01 m i l l i l i t e r s (10
M) e The r e g u l a t i n g e le m e n ts f o r
t h e b a t h m ust b e i s o l a t e d from th e b r id g e c i r c u i t t o p r e v e n t any e l e c t r i c a l
p ic k - u p from t h e r e l a y s o r h e a t e r s *

29

The o n - o f f h e a t e r u sed was a 1 5 0 -w a tt I n f r a - r e d bulb* o p e ra te d b y a
m ercu ry r e l a y s w itc h *

The r e l a y was a c t i v a t e d w ith a F i s h e r - S e r f a s s

E l e c t r o n i c R e la y , c o n t r o l l e d b y a m ercu ry m ic r o - s e t th e r m o r e g u la to r .

The

s te a d y h e a t e r and c o o le r ( 125- w a tt k n i f e h e a te r 9 and copper c o i l co o led
w ith t a p w a t e r , r e s p e c t i v e l y ) w ere a d ju s te d so t h a t th e o n - o f f tim e was
35 seco n d s*

The d e t e c t o r sy stem was i n s u la te d so t h a t i t r e q u ir e d 195

s e co n d s t o re sp o n d t o b a t h te m p e r a tu re c h a n g e s.
The b a th i t s e l f was i n s u l a t e d and c o v e re d , w ith th e e x c e p tio n o f a
s m a ll o p en in g f o r th e i n f r a - r e d lam p, w hich was
w a te r le v e l *

set 5 in c h e s from th e

The w a te r was c i r c u l a t e d w ith a s m a ll c e n t r i f u g a l pump,

w hich was shock-m ounted t o re d u c e v i b r a t i o n .

The w a te r l e v e l i n t h e b a th

was m a in ta in e d w ith a sip h o n *
The d e t e c t o r cham ber was p la c e d i n th e c e n te r o f th e b a th i n s i d e a
l a r g e g l a s s c y lin d e r *

The chamber and th e r m o s ta tin g j a c k e t f o r th e columns

w ere i n s u l a t e d w ith g l a s s wool*

The g la s s - w o o l i n s u l a t i o n a l s o a c te d a s

a c u s h io n t o i s o l a t e t h e d e t e c t o r cham ber from v i b r a t i o n and sh o ck , s in c e
t h e s u s p e n s io n f o r th e c y lin d e r c o n ta in in g th e chamber i s s u p p o rte d b y
t h e wool*
7 * W h e atsto n e B rid g e
The W h ea tsto n e b r id g e w hich i s u sed t o d e t e c t t h e change i n r e s i s t a n c e
r e s u l t i n g from th e d i f f e r e n c e i n te m p e ra tu re o f th e t h e r m is t o r s need n o t
b e d e s ig n e d t o m easure t h e a b s o lu te r e s i s t a n c e .

T h is a s p e c t o f th e

re q u ire m e n t s i m p l i f i e s t h e d e s ig n o f th e b r id g e c o n s id e ra b ly ^ how ever,
t h e s m a ll s i g n a l s w hich m ust be m easured do r e q u i r e c a r e f u l s h i e l d i n g

30

and g ro u n d in g .

Mich o f th e random n o is e i n th e c i r c u i t can be e lim in a te d

b y dam ping, s in c e th e s i g n a l s of i n t e r e s t a r e lo n g i n d u r a t io n , a llo w in g
u s e o f tim e c o n s ta n ts as lo n g as 30 seco n d s t o f i v e m in u tes *
A s c h e m a tic d ia g ra m o f th e d e t e c t o r i s shown i n F ig u re 6 .

The

t h e r m i s t o r s ( r s and r r ) w ere s e le c te d as d e s c r ib e d i n th e t h e r m i s t o r
s e c t i o n o The v a r i a b l e e x t e r n a l r e s i s t a n c e s ( r ° and r ° ) w ere m atched
«L

d e cad e r e s i s t a n c e b o x es *

S

The p o t e n t i a l s o u rc e (B) was a 6 - v o lt s to r a g e

b a t t e r y w hich had b e e n ch arg ed i n a s te p w is e m anner, sh ak en t o re d u c e
p o l a r i z a t i o n , and a llo w e d t o s ta n d two o r t h r e e days b e f o r e i n c o r p o r a tio n
i n t o t h e b r id g e „

The s to r a g e b a t t e r y , when t r e a t e d i n t h i s m anner, i s a

v e r y s t a b l e p o t e n t i a l so u rc e f o r p e r io d s o f tim e as lo n g a s a m onth,
p ro v id e d t h a t th e to p o f t h e b a t t e r y i s c a r e f u l l y c le a n e d o f a l l a c id
and k e p t d r y « The u n b a la n c e o f th e b r id g e was d e t e c t e d w ith a 1 .0 m i l l i v o l t f u l l - s c a l e e l e c t r o n i c r e c o r d e r (V )j th e r e c o r d e r u sed was a
Brcwn E l e c tr o n l k r e c o r d e r w ith a d j u s ta b le z e ro and v a r i a b l e sp a n from 0 -1
rav <►t o Q -£ l mv. f u l l - s c a l e ) .

A change o f one ohm on th e b r id g e caused a

d e f l e c t i o n c o rre s p o n d in g t o 60 p e r c e n t o f f u l l s c a l e on th e maximum
s e n s i t i v i t y s c a l e ( 0 -1 mv») »

Thus, w ith 2000-ohm th e r m is to r s h a v in g

te m p e r a tu re c o e f f i c i e n t s o f ab o u t f i v e p e r c e n t p e r d e g re e , a te m p e ra tu re
d ifT e r e n c e o f 0o001°G o b etw een th e th e r m is to r s c o u ld e a s i l y b e d is c e rn e d
on t h e r e c o rd e r o
T h is s e n s i t i v i t y i s b y no means th e l i m i t o f t h e s e n s i t i v i t y con­
c e iv a b le f o r t h i s ty p e o f d e t e c t o r . I t i s , how ever, s u f f i c i e n t f o r
_,a
-9
d e t e c t i o n o f 10 -1 0
m oles o f sam ple i n 0*01 m i l l i l i t e r s o f s o l u t i o n .

FIGURE

6.

BRIDGE

CIRCUIT

32

8 . T re a tm e n t o f S o lv e n t and P r e p a r a tio n o f Columns
One o f t h e co n v e n ie n c e s o f gas chrom atography i s t h e f a c t t h a t t h e
same colum n may b e u sed f o r r e p e a te d s e p a r a tio n s w ith o u t s i g n i f i c a n t
changes i n i t s r e t e n t i o n tim e s f o r p a r t i c u l a r com ponents *

T h is a llo w s

t h e o p e r a to r t o make r e p e a te d runs^ u s in g th e tim e r e q u ir e d f o r e l u t i o n
o f a p a r t i c u l a r com ponent band f o r i d e n t i f i c a t i o n o f th e com ponent.
I n l i q u i d ch ro m ato g rap h y a g iv e n column i s u s u a ll y packed f o r a s e p a r a t i o n
and th e n d is c a rd e d * s in c e i t may become p a r t i a l l y d e a c tiv a te d * T h is
p r o c e s s i s v e r y in c o n v e n ie n t* ^nd i t i s p a r t i c u l a r l y so f o r th e new
d e te c to r * s in c e s t a b i l i z a t i o n

o f te n r e q u i r e s s e v e r a l h o u rs o r lo n g e r

f o r a new colum n.
The tim e r e q u ir e d f o r s t a b i l i z a t i o n o f new ly-packed columns was
re d u c e d c o n s id e r a b ly by e q u i l i b r a t i n g th e s o lv e n t u sed w ith an e x c e s s o f
a d s o r b e n t.

T h is tr e a tm e n t o f th e s o lv e n t was found t o be more c o n v e n ie n t

and e f f e c t i v e th a n d i s t i l l a t i o n and o th e r means o f p u r i f i c a t i o n .

When th e

s o lv e n t was p r e p a re d i n t h i s manner* th e columns d id n o t become d e a c tiv a te d
o v e r i n d e f i n i t e p e r io d s o f tim e .

One s e t o f a lu m in a columns was u sed f o r

s e p a r a t i o n s o f e r g o s t e r o l and c a l c i f e r o l f o r a p e r io d o f f i v e m onths w ith
no a p p a r e n t d e a c t i v a t i o n .

D u rin g t h a t tim e th e columns w ere exposed t o

s e v e r a l d i f f e r e n t k in d s o f sam ples o f q u e s tio n a b le p u r i t y and became
d is c o l o r e d t o a d u l l y e llo w brown* b u t th e r e t e n t i o n tim e rem ain ed a p p ro x i­
m a te ly t h e sam e.
T w o -m illim e te r i . d . c a p i l l a r y columns w ere packed w ith an alu m in a
s l u r r y i n th e s o lv e n t u sed and s e a le d a t th e to p and b o tto m w ith c o tt o n
p lu g s .

The c o t t o n p lu g s s e rv e d two e s s e n t i a l f u n c t io n s ;

to p re v e n t

33

t h e column from b e in g d is tu r b e d by sam ple i n j e c t i o n , and t o s to p s o lv e n t
flo w i f a i r o r d is s o lv e d gas became tra p p e d in t h e c a p i l l a r y .

These

t i g h t c o t t o n p lu g s p r o te c t e d th e columns from c e r t a i n d e a c t i v a t i o n on two
s e p a r a t e o c c a s io n s when th e s o lv e n t l e v e l r a n down i n t o t h e c a p i l l a r y .
9 » P u r i f i c a t i o n o f R eag en ts and P r e p a r a tio n o f S o lu tio n s

The cy clo h ex a n e (95 p e r c e n t , v o / v .) - a c e t o n e (5 p e r c e n t, v . / v . )
s o lv e n t u sed f o r th e s o l u t i o n s employed f o r m o le c u la r w e ig h t and concen­
t r a t i o n - r e s p o n s e d e te r m in a tio n s was p re p a re d by tr e a tm e n t w ith an e x c e ss
o f a d s o r b e n t.

Sam ples o f th e s o lv e n t w ere chrom atographed t o d e te rm in e

w h e th e r th e b ack g ro u n d was a l t e r e d b y th e s o l v e n t .
Eastm an ¥ h i t e - L a b e l Grade s o l u t e s w ere u sed d i r e c t l y w ith , no f u r t h e r
p u rific a tio n .

The s o l u t i o n s w ere p re p a re d b y d i r e c t w e ig h in g o f t h e s o l u t e

and a d d i t i o n o f th e s o lv e n t from a g ra d u a te d hypoderm ic s y r in g e .

3h

I I I . QUALITATIVE APPLICATIONS OF THE DETECTOR
Ao G e n e ra l
Most o f t h e r e s e a r c h i n th e developm ent o f t h i s d e t e c t o r h a s b e e n
d i r e c t e d to w a rd s d e v is in g a t o o l s u i t a b l e f o r g e n e r a l u se i n l i q u i d
ch ro m ato g rap h y * The i n i t i a l g o a l was t o d e m o n s tra te i t s a p p l i c a b i l i t y t o
v a r i o u s s o lv e n t sy stem s and t o a l a r g e v a r i e t y o f s o l u t e s i n t h e s e system s..
The g e n e r a l e x p l o r a t o r y r e s u l t s w i l l b e p r e s e n te d i n th e f i r s t t h r e e
p a r ts o f th e q u a li t a ti v e a p p lic a tio n s s e c tio n .
Once t h e p o t e n t i a l was c l e a r l y d e m o n s tra te d , th e i n t e r e s t s h i f t e d t o
two s p e c i f i c a p p l i c a t i o n s o f th e d e t e c t o r .

B ecause o f i t s im p o rtan ce t o

o th e r s t u d i e s b e in g c a r r i e d o ut i n t h e s e l a b o r a t o r i e s , th e a n a ly s is o f
t h e e r g o s t e r o l i r r a d i a t i o n m ix tu re re c e iv e d s p e c i a l a t t e n t i o n .

F o r work

w ith f a i r l y d i l u t e s o l u t i o n s o f th e s e h ig h m o le c u la r w e ig h t com ponents,
i t was fo u n d n e c e s s a r y t o in c r e a s e t h e s e n s i t i v i t y o f t h e d e t e c t o r h y a
f a c t o r o f 100 above i t s l e v e l a t th e tim e o f i n i t i a t i o n o f t h i s s p e c i f i c
s tu d y .

T h is new g o a l sp a rk e d f u r t h e r in s tr u m e n ta tio n t o o b ta in th e

d e s ir e d s e n s i t i v i t y .
The second o f t h e s p e c i f i c a p p li c a t i o n s e x p lo re d h a s b e e n th e p o s s i b l e
u s e o f th e d e t e c t o r f o r m o le c u la r w e ig h t d e te r m in a tio n s .

T h is le d t o th e

m a th e m a tic a l and q u a n t i t a t i v e s tu d i e s p r e s e n te d i n S e c tio n s IV and V.

35

Bo W ater ( S o lv e n t ) - C e l lu l o s e (A d so rb e n t) System
T h is s o l v e n t - s o r b e n t sy stem was employed i n th e e a r l y s ta g e s o f t h e
d ev elo p m en t o f t h i s d e t e c t o r .

S in c e i t i s n o t v e r y u s e f u l c h ro m a to g ra p h ic -

j Up s e p a r a t i o n s w ere observed,} how ever, t h e g e n e r a l n a tu r e o f t h e t l e t e c t o r re s p o n s e w h ich was c l e a r l y e s t a b li s h e d h e lp e d i n mapping o u t th e s u b se ­
q u e n t d e v e lo p m e n ts.

The p a t t e r n o f p o s i t i v e d e f l e c t i o n ( h e a tin g o f t h e

^sample t h e r m i s t o r ) f o r s o l u t e s g iv in g s o lu t i o n s l e s s v o l a t i l e th a n t h e
s o l v e n t , and n e g a tiv e d e f l e c t i o n ( c o o lin g o f t h e sam ple t h e r m is to r ) f o r
s o l u t e s g iv in g s o l u t i o n s more v o l a t i l e th a n th e s o lv e n t was o b s e rv e d .
T a b le I I g iv e s a l i s t o f th e s o lu t e s used i n t h i s sy ste m , t o g e t h e r w ith
th e ir d ire c tio n s of d e f le c tio n .
TABLE I I
SOLUTES OBSERVED WITH WATER-CELLULOSE SISTEM

S o lu te

B o ilin g P o in t
°C.

D ire c tio n of
D e f le c ti o n

G ly c e ro l

290

p o s itiv e

E th y le n e G ly c o l

197

p o s itiv e

B u ta n o l- 1

117

n e g a tiv e

P r o p a n o l- 2
E th a n o l
S u g a rs

82. k
78 *h'
n o n -v o la tile

n e g a tiv e
n e g a tiv e
pos i t i v e

P a r k e r B lu e - b la c k In k

p o s itiv e

S a l t s (N a c l, NaBr, N a l, KOI, KBr, L iC l, CuCl2)

p o s itiv e

36

The p a t t e r n o f p o s i t i v e and n e g a tiv e d e f l e c t i o n i s c l e a r l y e s t a b li s h e d
i n T a b le 11^ e x c e p t p o s s i b l y f o r b u ta n o l - 1 , w hich h as a b o i l i n g p o in t
h ig h e r t h a n t h a t o f w a t e r .

The v a p o r p r e s s u r e o f a b u ta n o 1 -w a te r m ix tu r e ,

h o w ev er, i s g r e a t e r th a n t h a t of p u re w a te r a t 25°£«

I t i s n o t p o s s ib le

t o make q u a n t i t a t i v e e s tim a tio n s o f t h e v a p o r p r e s s u r e , b e c a u s e t h e concen­
t r a t i o n o f th e s o l u t i o n a s i t p a s s e s th e t h e r m i s t o r i s n o t known,
A s t r i n g colum n was u sed i n t h i s p r e lim in a r y work*

T h is f i r s t d e t e c t o r

was s e n s i t i v e t o m o d e ra te ly s m a ll sa m p le s , b u t , u n f o r t u n a t e l y , t h e r e a s o n
f o r t h i s s e n s i t i v i t y was n o t re c o g n iz e d u n t i l much l a t e r .

The s t r i n g

colum n b y i t s n a t u r e r e q u ir e d v e r y slew flo w r a t e s , so th e q u a s i- e q u i lib r i u m
r e q u i r e d f o r a te m p e r a tu re r i s e co u ld b e a p p ro a c h e d .

A f te r th e s t r i n g

colum n was r e p la c e d w ith packed colum ns, s e v e r a l months o f f r u i t l e s s i n v e s t i ­
g a t i o n w ere r e q u i r e d t o r e g a i n t h i s s e n s i t i v i t y , b e c a u s e o f th e h ig h e r
flo w r a t e s w hich r e s u l t e d .
C. B u ta n o l S a t u r a te d w ith W ater— C e llu lo s e System
When packed colum ns w ere f i r s t used w ith t h i s s y ste m , i t was p o s s i b l e
t o o b se rv e c h ro m a to g ra p h ic s e p a r a t io n s f o r th e f i r s t tim e w ith t h e new
d e te c to r.

The s e n s i t i v i t y o f th e d e t e c t o r was red u ced b e c a u s e o f l a r g e

flo w r a t e s , so t h a t i t was n o t p o s s ib le t o o b se rv e s m a ll sam p les; how ever,
i t was p o s s i b l e t o o b t a i n some s e m i - q u a n ti ta t iv e r e s u l t s t o d e m o n s tra te
p o s s i b l e u s e o f t h e d e t e c t o r a s an a n a l y t i c a l t o o l .
The m ost i n t e r e s t i n g , and p e rh a p s th e m ost im p o r ta n t, o f th e o b s e rv a ­
t i o n s made w ith t h e b u ta n o l- w a te r sy stem i s th e n e g a tiv e - p e a k e f f e c t due
t o t h e ch an g es i n t h e s o lv e n t c o n c e n tr a tio n upon th e i n t r o d u c t i o n o f a

37

s o l u t e - c o n t a i n i n g sam ple on a c h ro m a to g ra p h ic colum n«> B ecause o f i t s
im p o rta n c e , t h i s e f f e c t w i l l be d is c u s s e d i n d e t a i l i n a l a t e r s e c t io n
( S e c t io n VT-A)o
T a b le I I I g iv e s a l i s t o f t h e s o lu t e s u sed w ith t h i s s o lv e n t- s o r b e n t
sy s te m , t o g e t h e r w ith t h e i r d e f l e c t i o n d i r e c t i o n s „
TABLE I I I
SOLUTES OBSERVED IN BUTANOL-CELLULOSE SI5TEM

S o lu te

B o ilin g P o in t
° C o

D ire c tio n of
D e f l e c tio n

G ly c e r o l

290

p o s itiv e

E th y le n e G ly c o l

197

p o s itiv e

M in e ra l O i l

n o n - v o la tlie

p o s itiv e

A cetone
Benzene
A c e tic A cid

5 6 .5
80 e l

118 e

l

n e g a tiv e
n e g a tiv e
n e g a tiv e

P a r t i a l s e p a r a t i o n o f a m ix tu re o f e th y le n e g ly c o l and g l y c e r o l was
o b s e rv e d .

The r e l a t i v e peak h e i g h ts could b e c a l i b r a t e d t o g iv e t h e i r

r e l a t i v e c o n c e n tr a tio n s w i t h i n f i v e p e r cen to

Com plete s e p a r a tio n s w ould

h av e b e e n p o s s i b l e u s in g l e s s c o n c e n tr a te d sam ples o r lo n g e r columns *
S in c e t h e sy ste m d id n o t a f f o r d co m p lete s e p a r a t i o n , th e i n d i v i d u a l p eak s
co u ld be d i s t i n g u i s h e d o n ly when an y one s o l u t e c o n s t i t u t e d no more th a n
70 p e r c e n t o f t h e t o t a l s o l u t e i n th e sam p le.

38

Bo C yclo h ex an e (95 p e r c e n t, v . / v , .) and A cetone (5* p e r c e n t , v . / v . )
( S o lv e n t) — Alum ina (S o rb e n t)
( C a p i l l a r y Column— 2 mm. iodo x 23 cm*)
T h is s o lv e n t ” s o r b e n t sy stem was s e le c t e d b e c a u s e i t was s u i t a b l e f o r
t h e s e p a r a t i o n o f t h e e r g o s t e r o l i r r a d i a t i o n m ix tu re .

When t h i s s o lv e n t-

s o r b e n t sy stem was f i r s t em ployed, t h e d e t e c t o r was n o t s u f f i c i e n t l y
s e n s i t i v e f o r d i r e c t a n a l y s is of th e i r r a d i a t i o n m ix tu re .

A la r g e v a r i e t y

o f d i f f e r e n t s o l u t e s , in c lu d in g e r g o s t e r o l and c a l c i f e r o l , w ere chrom ato­
g rap h ed d u r in g t h e p e rio d o f developm ent o f th e d e t e c t o r s e n s i t i v i t y .
T a b le IV i s a l i s t o f th e n o n - v o l a ti l e s o lu t e s ru n w ith t h i s s o lv e n ts o r b e n t s y s te m .
TABLE IV
SOLUTES OBSERVED IN CICLOHEZANE-ALUMINA SYSTEM

M in e ra l o i l

A ro c lo r

P a ra ffin

P o ly s ty r e n e

p-D ic h l o r ob enz ene

A n th racen e

N a p h th alen e

A crid en e

Camphor

B enaoic a c id

T r ip h e n y l m ethane

H exam ethyl b en zen e

'Sudan re d

E r g o s te r o l

S udan y e llo w

C a lc ife ro l

The s a t u r a t e d h y d ro c a rb o n sam ples ( p a r a f f i n , m in e ra l o i l , and
p e tro le u m e t h e r ) w h ich w ere chrom atographed showed s p re a d in g due to t h e i r
ra o le c u la r-w e ig h t d i s t r i b u t i o n , b u t no c l e a r s p re a d in g o f com ponents,

39

b e c a u s e t h e a lu m in a column employed was to o s h o r t t o r e s o lv e t h e s e s o l u t e s .
W ith lo n g e r co lu m n s, h o w ev er, t h i s sy stem sh o u ld be u s e f u l i n h y d ro c a rb o n
a n a l y s i s f o r th e n o n - v o l a t i l e s .
A ro m a tic -h y d ro c a rb o n c o a l - t a r d e r i v a t i v e s , such as a n th r a c e n e , a r e
c l e a r l y s e p a r a te d from c o a l - t a r h e t e r o c y c l i c s on th e s h o r t alu m in a column
used.

A s p h a lt sam p les w ere s e p a r a te d i n t o two m ajo r com ponents, each o f

w hich i t s e l f showed some r e s o l u t i o n .

Longer columns sh o u ld r e s o lv e th e s e

sam ples i n t o s e v e r a l p eak s w hich would b e s u i t a b l e f o r c h a r a c t e r i z a t i o n
o f a s p h a l t s a m p le s .
Some s o l u t e s h av in g s o l u t i o n s more v o l a t i l e th a n th e s o lv e n t w ere
a l s o ch ro m ato g rap h ed f o r co m parison o f th e s e n s i t i v i t y o f t h e d e t e c t o r
f o r v o l a t i l e s and n o n - v o l a t i l e s .

The s e n s i t i v i t y o f th e d e t e c t o r f o r

v o l a t i l e com ponents in c r e a s e s as th e v a p o r p r e s s u r e i n c r e a s e s , u n t i l th e
v a p o r p r e s s u r e o f t h e s o l u t e i s so la r g e t h a t th e s o lu t e e v a p o r a te s from
t h e w ick b e f o r e t h e sam ple re a c h e s th e t h e r m i s t o r .

The maximum d e f l e c t i o n

o b se rv e d showed t h a t t h e d e t e c t o r was as much as 100 tim e s more s e n s i t i v e
t o v o l a t i l e sam p les th a n t o n o n - v o l a t i l e sam ples.

The maximum o b serv ed

s e n s i t i v i t y o c c u rre d w ith p e tro le u m e t h e r as a s o l u t e i n c y c lo h e x a n e .
T h is s o lv e n t - s o r b e n t sy stem was employed i n th e l a t e r q u a n t i t a t i v e
s t u d i e s o f d e t e c t o r re s p o n s e as f u n c tio n s o f c o n c e n tr a tio n and sam ple
v o lu m e.

T hese r e s u l t s a r e t a b u la t e d in t h e A ppendix and d is c u s s e d i n

S e c t i o n V.

Uo

E . The E r g o s te r o l I r r a d i a t i o n M ixture
T h is p a r t i c u l a r a p p l i c a t i o n was ch o sen b e c a u s e o f i t s im p o rtan ce
and b e c a u s e i t r e p r e s e n te d a t y p i c a l c a s e o f a b io c h e m ic a l o r n a t u r a l p ro d u c t a n a l y s i s a p p ro a c h in g th e l i m i t s o f d e t e c t o r s e n s i t i v i t y .

The

c h ro m a to g ra p h ic r e s u l t s co u ld be compared w ith r e s u l t s o b ta in e d b y
s p e c tr o p h o to m e tr ic te c h n iq u e s r e c e n t l y d ev elo p ed i n t h i s l a b o r a t o r y (1 5 ) >
i f t h e d e t e c t o r co u ld b e s u f f i c i e n t l y d ev elo p ed t o b e a b le t o d e t e c t v e r y
s m a ll sam p les o f d i l u t e s o l u t i o n s .

I n t e r e s t i n t h i s problem sp a rk e d new

e f f o r t s t o i n c r e a s e th e s e n s i t i v i t y o f t h e d e t e c t o r .

In o rd e r f o r a

c h ro ra a t© g ra p h ic a lly o b s e rv a b le change i n e r g o s t e r o l t o b e in d u ced b y
r a d i a t i o n , w ith th e l i g h t s o u rc e and m onochrom ator a v a i l a b l e , t h e i r r a d i -3
-4
a t io n s o l u t i o n m ust be 10 -1 0
m o la r. I f th e sam ple s i z e t o b e a n a ly z e d
i s t o b e 0 .0 2 m l ., and i f d i r e c t s a m p lin g , w ith o u t p r e v io u s ly c o n c e n tr a tin g
_s
_9
t h e s o l u t i o n i s t o b e p o s s i b l e ) , th e d e t e c t o r m ust b e s e n s i t i v e t o 10 -1 0
m oles o f s o l u t e i n a sa m p le .
The r e f i n e d in s tr u m e n ta tio n r e q u ir e d t o g e t t h e d e s ir e d s e n s i t i v i t y
was co m p leted o n ly s h o r t l y b e f o r e th e t h e s i s was w r i t t e n .

U n f o r tu n a te ly ,

q u a n t i t a t i v e r e s u l t s hav e n o t y e t b e e n o b ta in e d f o r t h i s a n a ly s is .; how ever,
q u a l i t a t i v e s e p a r a t i o n s o f p r e p a re d m ix tu re s o f c a l c i f e r o l and e r g o s t e r o l
h av e b e e n r u n , and changes i n e r g o s t e r o l s o lu t i o n s s u b je c te d t o s u n li g h t
h av e b e e n o b s e rv e d .
The a lu m in a c h ro m a to g ra p h ic columns u sed w ere checked f o r a c t i v i t y
w ith a m ix tu re o f Sudan re d and Sudan y e llo w dyes ( 1 6 ) .

The Sudan y e llo w

was e l u t e d fro m t h e column and d e te c te d as a s y m m e tric a l band w ith a peak

hi

a t 215 m in u te s o

The Sudan re d was o b serv ed as a b ro a d d iff h .s e b a n d ,

f i r s t d e t e c t e d a t 260 m in u te s .
U20 m in u te s o

I t was f i n a l l y e lu te d c o m p le te ly a f t e r

T h is s e p a r a t i o n in d ic a te d a d e q u a te a c t i v i t y o f th e alu m in a

f o r s e p a r a t i o n o f t h e e r g o s t e r o l i r r a d i a t i o n m ix tu re .
P u re sam ples o f e r g o s t e r o l and c a l c i f e r o l s o lu t i o n s w ere chrom ato­
g rap h ed t o e s t a b l i s h th e r e t e n t i o n tim e s and p u r i t i e s .

C a l c i f e r o l had

one p eak a t 2Q5 m in*, w h ile e r g o s t e r o l had two p e a k s , one m ajor p eak a t
280 rain * , and a m in o r one c o rre s p o n d in g t o th e c a l c i f e r o l *
A 0pQ2-mlo sam ple o f a 1 :5 d i l u t i o n o f a s a tu r a t e d e r g o s t e r o l s o lu ­
t i o n i r r a d i a t e d w ith i n d i r e c t s u n li g h t f o r f i v e days was ch ro m ato g rap h ed ,
show ing a s u b s t a n t i a l d e c r e a s e i n th e e r g o s t e r o l peak and an in c r e a s e i n
t h e c a l c i f e r o l peak*

S u b se q u en t i r r a d i a t i o n red u ced th e e r g o s t e r o l u n t i l

i t was i n d i s t i n g u i s h a b l e from th e background t r a c e .

Two o th e r s m a ll

p ea k s a p p e a re d b etw een th e e r g o s t e r o l and c a l c i f e r o l d u r in g t h e p r o g r e s s
of th e i r r a d ia tio n .

T hese p eaks w ere n o t i d e n t i f i e d , b u t th e y may b e

t a c h y s t e r o l , p r e c a l c i f e r o l , o r o x id a tio n p r o d u c ts , s in c e i n t h i s p r e l im i ­
n a r y e x p e rim e n t no e f f o r t was made t o e x clu d e oxygen.

F ig u re 7 shows

a r e p r e s e n t a t i v e d e t e c t i o n o f th e s e p a r a ti o n o f an i r r a d i a t i o n m ix tu re .
W hile o n ly v e r y p r e lim in a r y r e s u l t s have b e e n o b ta in e d w ith t h i s
s y ste m , t h e r e seems t o b e l i t t l e re a s o n t o d oubt t h a t th e d e t e c t o r can
b e a p p lie d t o t h i s and many o th e r sy stem s of b i o l o g i c a l i n t e r e s t .

IRRADIATION

h2

\X 2 s
c

Ct:

LU

O
I— >UJ <£

<c 2
O

^X

LU

LOJ

X

^
° o<
^
GO

o

¥

LU

ERGOSTEROL

3 .®
I

<c
\! £ S

LU

rvj —»
05 3

LU

oc>

(—

z:

o

O

*
<C ( 3

Ml.

Co

Co

OF

>

00 ^
cs ^

C
rx

—

ERGOSTEROL

co

CL

^
o

— NC

«o
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or

LU

m

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£
o
a s 'N O d s a a

FIGURE

7.

(N

1

CHROMATOGRAM
M IXTURE.

LU

REPRESENTATIVE

LU

IV . MATHEMATICAL ANALYSIS OF THE DETECTOR RESPONSE

A . G e n e ra l
I t i s p o s s i b l e a t th e p r e s e n t tim e t o p ro p o se a q u a n t i t a t i v e
m a th e m a tic a l d e s c r i p t i o n o f th e d e t e c t o r r e s p o n s e .

S e v e r a l s im p lif y in g

a s su m p tio n s a r e made t o c a s t th e e q u a tio n s i n t o u s e f u l form j m ost o f
t h e s e a s su m p tio n s can b e j u s t i f i e d b e c a u s e o f th e v e r y s m a ll te m p e r a tu re
u n b a la n c e in v o lv e d i n a c t u a l m easu rem en ts.

I t i s n e c e s s a r y , how ever, t o

r e s t r i c t p r e s e n t c o n s id e r a t i o n s t o th e s im p le s t c a s e o f a s in g le -c o m p o n e n t
s o lv e n t .
I n t h e a n a l y s i s w i l l b e c o n s id e r e d , i n t h a t o r d e r , th e e l e c t r i c a l
b a la n c e c o n d i t i o n , t h e t h e r m i s t o r t e m p e r a t u r e - r e s i s t a n c e r e l a t i o n s h i p s ,
t h e th e r m a l b a la n c e c o n d itio n f o r c e r t a i n s p e c i a l c a s e s , and t h e shape
and s i z e o f t h e component p eak s i n t h e re c o rd e d chrom atogram .
B . E l e c t r i c a l B a la n c e C o n d itio n
The b r id g e c i r c u i t em ployed i s shown i n F ig u re 5>.
s u p p li e s a f ix e d v o l t a g e , E , t o t h e b r i d g e .

The b a t t e r y , B,

The t o t a l c u r r e n t , i ,

is

d iv id e d I n to t h e tw o p a th s i s and i r th ro u g h t h e sam ple and r e f e r e n c e
th e rm is to rs , re s p e c tiv e ly .

T hese th e r m i s t o r s have r e s i s t a n c e s r g and r ,

and a r e in s e r i e s w ith e x t e r n a l r e s i s t a n c e s r ° and r ° , r e s p e c t i v e l y .
Then

w ith

(3 )

i_s =

E o
r3 * r5

and
i

(10

E
r

o
rr * rr

S in c e t h e r e f e r e n c e arm i s n o rm a lly u n a lte r e d i n a d e te r m in a tio n , i t i s
c o n v e n ie n t t o d e f in e
(v5 )'

Rr = r r * r °r

The r e c o r d in g p o te n tio m e te r , V, m easures t h e p o t e n t i a l d i f f e r e n c e ,
e , b etw een p o i n t s a and b , u n d e r such c o n d itio n s t h a t c u r r e n t d oes n o t
flo w from a t o b th ro u g h th e m easu rin g c i r c u i t .
(6}
v J

e * i rr r - i sr s
or

(7 )

e = s

Rl
r

-

k

' 5o
r + r
s
s

and
e
E

(8 )

S o lv in g f o r r

s

r

r
R
r

r
r

s
s

+ r

i n te rm s o f e g iv e s

o
s

Then

"When e as 0 9
( 10)

If

r

r *

» r ° , and e = 0 , th e n r g ® r * T h is i s th e c o n d itio n w hich would
r

i d e a l l y o b t a i n f o r m atched t h e r m i s t o r s w ith p u re s o lv e n t on e a c h w ic k ,
e q u a l flo w r a t e s , and i d e n t i c a l w icks*
C * T h e rm is to r T em p eratu r e*t|te$is t ance R e la tio n s h ip s
The r e s i s t a n c e o f t h e th e r m is to r i s , o f c o u r s e , s tr o n g l y d ep en d en t
upon te m p e ra tu re *

The n o rm al e x p r e s s io n f o r t h e r m is to r r e s i s t a n c e as a

f u n c t i o n o f te m p e r a tu re o v e r a ra n g e o f te m p e r a tu re i s
(1 1 )

r = rQ e

-b(T ~T Q)
v

O ver a v e r y s m a ll te m p e r a tu re ra n g e t h i s may b e ap p ro x im a te d by th e l i n e a r
e x p r e s s io n
(12)

r - r 0 —a (T -T 0 ) ,

w h ere r 0 i s t h e r e s i s t a n c e a t T0 and a i s th e te m p e r a tu re c o e f f i c i e n t o f
r e s is ta n c e *

The r e s i s t a n c e o f th e sam ple th e r m is to r may th e n b e e x p re s s e d

i n te rm s o f th e r e s i s t a n c e o f t h e r e f e r e n c e t h e r m is to r a s
(1 3 )

- a (T - T ) .
' s
r'

I t i s c o n v e n ie n t t o r e a r r a n g e t h i s e x p r e s s io n t o o b ta in T i n te rra s o f r .
s
s

U6

Do T herm al B alan c e C o n d itio n s
At each t h e r m i s t o r i n th e d e t e c t o r c i r c u i t y th e c o n d itio n o f th e r m a l
b a la n c e can b e e x p re s s e d i n term s of t h e h e a t in p u t and h e a t rem o v al
f a c t o r s w hich h av e b e e n c o n s id e re d i n S e c tio n I I - A .

H eat in p u t r e s u l t s

fro m e l e c t r i c a l h e a tin g o f th e t h e r m is to r and from s o lv e n t c o n d e n s a tio n .
S in c e t h e t h e r m i s t o r te m p e r a tu re i s alw ays h ig h e r th a n t h a t o f i t s s u rro u n d ­
i n g s , a l l o t h e r f a c t o r s le a d t o h e a t re m o v a l.

Most im p o rta n t o f th e s e

i s v a p o r i z a t i o n o f th e s o lv e n t ; n e x t m ost Im p o rta n t i s p ro b a b ly c o n d u c tio n
o f h e a t away b y t h e flo w in g l i q u i d .

L ess im p o r ta n t, and p ro b a b ly n e g l i g i b l e ,

h e a t rem o v al o c c u rs th ro u g h r a d i a t i o n l o s s e s from th e th e r m i s t o r t o th e
s u r r o u n d in g s , and b y th e r m a l c o n d u c tio n and c o n v e c tio n l o s s e s t o t h e a i r ,
N o n - v o la tile s o l u t e s have b e e n assum ed.
1 . R a te s o f H e a t I n p u t.
a . E l e c t r i c a l h e a tin g o f th e th e r m is to r ;

(i? )

qE “

R e fe re n c e t h e r m i s t o r ,
(1 6 )

r

qgy -

Sam ple th e r m is to r .
(u )

qEs -

b . C o n d e n sa tio n o f v a p o r ;
(1 8 )

A

AHvap

r .

kl

w h ere k ^ » r a t e c o n s ta n t f o r c o n d e n s a tio n , i n m oles p e r u n i t a r e a p e r u n i t
o
p r e s s u r e p e r se c o n d ; P x « v a p o r p r e s s u r e o f p u re s o lv e n t a t th e th erm o ­
s t a t e d te m p e ra tu re * T0; A » e f f e c t i v e a r e a of w ick i n c o n ta c t w ith
t h e r m i s t o r ; and

AHv

= h e a t o f v a p o r i z a t i o n o f s o lv e n t a t T0 (assum ed

te m p e r a tu r e in d e p e n d e n t) .

The h e a tin g due t o c o n d e n s a tio n o f v a p o r

i s assum ed t o b e i d e n t i c a l f o r b o th sam ple and r e f e r e n c e th e r m is to r s *
p r o v id e d t h a t A i s th e same f o r b o th w icks* s in c e b o th a r e in c o n ta c t
w ith th e same v a p o r r e s e r v o i r *
2 . R a te s o f H eat Rem oval.
a . V a p o r iz a tio n cf s o lv e n t ;
(1 9 )
w h ere

=

Bv e_AHv a p /RT

A A H ^ -X ^

* p re * -e x p o n e n tia l f a c t o r f o r r a t e c o n s ta n t f o r v a p o r iz a tio n *

i n m oles o f s o lv e n t v a p o r iz in g p e r u n i t a r e a o f w ick p e r second* and
AHv a p i s assum ed t o be t h e a c t i v a t i o n e n e rg y f o r th e v a p o r i z a t i o n p r o c e s s .
i s t h e mole f r a c t i o n o f s o lv e n t a t t h e t h e r m i s t o r .

I t h a s b e e n assum ed

h e r e t h a t t h e s o l u t i o n i s i d e a l ; o th e rw is e i t i s n e c e s s a r y t o r e p l a c e
b y a x = Xx y; .

S in c e sam ple and

te m p e ra tu re s * th e te m p e r a tu re T

r e f e r e n c e th e r m is to r s mayb e a t d i f f e r e n t
may b e e i t h e r Tg o r T .

b . C o n d u ctio n o f h e a t b y flo w in g l i q u i d :
(2 0 )
w h ere k

qF = kpF (T - T0 ) ,

i s th e r a t e c o n s ta n t f o r t h e h e a t rem oval, i n c a l o r i e s p e r second
F
p e r u n i t flo w r a t e p e r u n i t te m p e r a tu re d i f f e r e n t i a l b etw een s o lv e n t* a t

U8

T0 , and w ic k , a t T .

F i s t h e volume flo w r a t e o f t h e s o lv e n t down t h e

w ick (h e n c e t h e flo w r a t e o f th e column) 0 F and T may d i f f e r f o r sam ple
and r e f e r e n c e t h e r m i s t o r s , b u t k^ and T0 a r e assumed i n v a r i a n t .
Co R a d ia tio n o f h e a t t o s u rro u n d in g s^
(2 1 )

=

k ^ (T -

Tq)

a

T h is te r m " is c o n s id e re d n e g l i g i b l e a t th e s m a ll te m p e ra tu re d i f f e r e n c e s
em p lo y ed .
d . C o n d u c tio n and c o n v e c tio n o f h e a t t o t h e s u rro u n d in g s th ro u g h th e
gas p h a s e :
(2 2 )
w h ere k „ i s
Cjr

qQ « k Q A(T - T0 ) ,

t h e r a t e c o n s ta n t f o r th e h e a t rem o v al, i n c a l o r i e s p e r

p e r u n i t a r e a p e r u n i t te m p e r a tu re d i f f e r e n t i a l .

second

T h is term w i l l p ro b a b ly

b e n e g l i g i b l e , b u t i s in c lu d e d f o r c o m p le te n e s s.
3 . The B a la n c e C o n d itio n s

A l l te rm s e x c e p t r a d i a t i o n l o s s from th e t h e r m is to r a r e in c lu d e d ,
a . R e fe re n c e t h e r m i s t o r :
R ate o f h e a t in p u t » r a t e of h e a t re m o v a l.

(2 2 )

( 2 3 )----'

qgr +

!c “

%jrT *

+

*

^G r’

—----- on r . k P i 0 A„ A H
“ Be"
° 2, r
c
r
vap
v
(\ r r «■ r r 7)

AHv a p /RTr A AH
+
r
van
^

kFFr (Tr - T° ) + k GAr ^ r ' To)S in c e p u re s o lv e n t i s u se d as th e r e f e r e n c e , Xi r =* 1 .

D u rin g a ru n , t h e

c o n d itio n s a t t h e r e f e r e n c e t h e r m i s t o r a r e assumed c o n s ta n t.

b9

bo Sam ple t h e r m i s t o r ;

(21*)

4Es ♦ 40 -

4^3 ♦

qFs ♦ 4Rs * qGs-

2

(2 ^

TZ~\

s

V U'P
s

r s + k 0P l °

* W
S in c e t h e r e s i s t a n c e r

s

V

As

AHvap -

T<>> * W

AIW

RTs

Ag A H v ap . Xx

Ts - To)

and te m p e ra tu re T a r e i n t e r s r e l a t e d b y e q u a tio n
s

(1 3 )., e q u a tio n (2 5 ) may b e c o n s id e re d t o b e th e r e l a t i o n s h i p b etw een T
s
( o r r ) and c o n c e n t r a t i o n ( e x p re s s e d a s X-jJ a t f ix e d b r id g e v o l t a g e ,
t h e r m o s t a t te m p e r a tu r e , and colum n flo w r a t e .

C om bination o f t h i s

e x p r e s s io n w ith e q u a tio n (9 ) f u r n is h e s th e d e s ir e d r e l a t i o n s h i p o f o b serv ed
r e c o r d e r v o l t a g e , e , t o c o n c e n tr a tio n ,,

Thus th e problem h as b e e n s o lv e d

i n p r i n c i p l e , b u t th e e q u a tio n s o b ta in e d a r e n o t i n r e a d i l y u s e f u l fo rm .
C o n s id e r a b le s i m p l i f i c a t i o n c a n be e f f e c t e d by c o n s id e r in g th e e q u a tio n s
s u b j e c t t o t h e r e s t r i c t i o n o f v e r y s m a ll te m p e ra tu re d i f f e r e n c e b etw een
sam p le and r e f e r e n c e t h e r m i s t o r s , and b y making o th e r h i g h ly p l a u s i b l e
a s s u m p tio n s .
E„ D e r i v a t i o n o f R esp o n se-C o n cen t r a t io n R e la tio n s h ip s
f o r I d e a l S o l u t i o n s o f N o n -V o la tile S o lu te s
The s t a r t i n g e q u a tio n s i n t h i s tr e a tm e n t a r e th e th e r m a l b a la n c e
e q u a tio n s (2 3 ) and (2 5 )*
g iv e s

S u b t r a c tio n o f e q u a tio n (23) from e q u a tio n (25)

50

(2 6 )

E 2 \ --------- 2— --------L<r B * - # a

-

—[ * k P i0
Rr

A H -an (A -A ) =

j

S

By . A Hyap [As Xi e ' AHvap/RTs _

AHyap/RTr j +

♦ k p [F s (Ts -T 0 ) - Fr (Tr -T 0 ) ] ♦ kGU s (Ts - T 0) - V l j - I , ) ] .
I t w i l l b e assum ed t h a t t h e w ick s a r e e q u i v a l e n t , so t h a t As » Ar ■ A.
Then
(27)

E2 [ --------------—

-

- ( r S * r s 3*

- H

] =

Bv - OJlvar) -A[X1e ~ * HvaP/i,T s -

V

e '^ a j / ^ r ]

♦ kF (Fs (Ts -T 0 ) - Fr (Tr -T 0 ) ] +
k cA (Ts -T r ).

I t i s c o n v e n ie n t n e x t t o s im p l i f y e q u a tio n (2 7 ) te rra by te r m .
The te rm on t h e l e f t - h a n d s id e o f t h e e q u a tio n i s th e d i f f e r e n c e i n
e l e c t r i c a l h e a t i n g o f sam ple and r e f e r e n c e t h e r m i s t o r s .

I t may be

s i m p l i f i e d b y t a k i n g a d v a n ta g e o f th e r e l a t i v e l y s m a ll d i f f e r e n c e i n
r e s i s t a n c e o f t h e two arms o f t h e b r id g e .
(2 8 )

r ♦ r °
— ------ —
r ♦ r G
r
r

*

( r ♦ r °) - ( r
— §---------r ♦ r °
r
r

U

♦ r G)
- -—

T h is i s o f t h e form 1 * x w ith s m a ll x , and
(2 9 )

(X ♦ x )

2

= X ♦ 2x ^ x

X ^ 2x f op sjuclUl. x •

Hence
(

(30)

/ r ♦ r 0^
[
s
s \
\
o j
\ r ♦ r
/
' r
r /

aj

n
1 ♦

2 rs
a_____
o
r + r
r
r

+

2 r ° - 2 ( r + r °)
s_______v r
r J
o
r + r
r
r

and* t o t h i s a p p ro x im a tio n ,
(3 1 )

(r

* r G) 2 » 2 ( r * r ° ) r ♦ ( r + r ° ) ( 2 r ° - r - r ° )
s
r
r
s
r
r
s
r
r

s

Thus
r
(3 2 )

r
- ---a-----------------( 2 r ♦ 2 r ° - H )R
v s
s
r r

§----(r + r °)2
s
s

From e q u a tio n ( 1 3 ) ,
(3 3 )

r

« ( r - oT ) ♦ aT
v r
r'
s

s

C om bining e q u a tio n s (3 2 ) and (33)>
r

r

(3U )
(r + r °)2
x s
s '

r

* a «a T

[2a® AT + 2 ( r * r ° ) - R JR
v r
s '
r r

The e l e c t r i c a l h e a t i n g d i f f e r e n c e term th e n becomes

(33)

E2 C
n

r

,

- r l
n

( [2 (r ♦ r ° ) - R ] ♦ 2a°A T
v- v r
s
r

,

B_ I

e2
R 2
r

« 2r ^ r r ° ~ r s G) * ( r r °~r r ) ° ' A T
R - 2 (r
r
r

r °) + 2« A T
s '

I t i s r e a d i l y s e e n t h a t t h i s term i s s m a ll and can be made e s s e n t i a l l y
z e r o i f r ° » r ° and r ° » r i b o th o f t h e s e c o n d itio n s can b e met
r
s
r
r
e x p e r i m e n t a l ly .

Two c a s e s w i l l be c o n s id e re d s e p a r a t e l y .

In th e f i r s t

c a s e , r ° « r ° and r ° « r , so t h e e l e c t r i c a l h e a tin g te rm d ro p s o u t.
r
s
x*
I n t h e second c a s e , r r 0 « r s ° , and th e term becomes

52

w h ere t h e a p p ro x im a tio n i s b a s e d on t h e f a c t t h a t 2 a
(F o r 0 . 1 M .b ip h e n y l, 2 a . ^ T ^

1*0 ohms, Rr

^

< < Rp.

1*000 ohms,

- ■

CzL OoOlj ev en a 0 . 1 M s o l u t i o n i s c o n s id e re d t o b e a h ig h c o n c e n tr a tio n ,

p r o b a b ly h ig h e r t h a n w ould n o rm a lly b e e n c o u n te re d i n c h ro m a to g ra p h ic w o rk ).
The f i r s t te rm on th e r ig h t- h a n d s id e o f e q u a tio n (2 7 ) r e p r e s e n t s
t h e h e a t a b s o rb e d b y v a p o riz a tio n * .

I t can be c o n s id e r a b ly s im p l i f i e d by

t h e f o llo w in g a s s u m p tio n .

( 3 7 ) - i »^ A.EHvao/RT-r,
T ‘
iv ap/

R

e

s

r
A RVap

The e x p o n e n t,

^ rp

-A ^vap
■■■■■ ■1 . Z^T, can b e e v a lu a te d f o r a c a s e s tu d i e d .
R±r

b ip h e n y l i n c y c lo h e x a n e (95
w ith Tr ^

r s

F o r 0 . 1 M,

c e n t , v . / v . ) — a c e to n e (5 p©** c e n t, v . / v . ^

3 0 0 °K a d e f l e c t i o n o f a p p ro x im a te ly 20 ohms was o b ta in e d $ s in c e

t h e t h e r m i s t o r r e s i s t a n c e ch an g es ab o u t 100 ohms p e r d e g r e e , / \ T — 0 . 2 ° .
F o r c y c lo h e x a n e , Z^Hv ap » 6620
a t 25°C . (1 7 )*
Hence
• AT
3
— 7 x 10” , f o r a c o n c e n tr a tio n c o n s id e re d t o b e i n th e h ig h r a n g e . The
e x p o n e n tia l te rm i n e q u a tio n (3 7 ) may b e c o n s id e r e d , t h e r e f o r e , t o b e o f
t h e fo rm eX w ith s m a ll x , so t h a t
- AHyap/RTs
(38)

^*V ap o

A t ^

^vap

e - R ^ -

^ AT
T r 2.

Thus t h e v a p o r i z a t i o n te rm i n e q u a tio n (2 7 ) becomes
(3 9 )

By . Z i H __ . A

vap

[ x x e ~ ^ Hv a p /RTs _ e -A H vap/RTr j

By •A H v a p A e " ^ HvaP/RTr [xx ♦ x j£ 5 z aE_ * 2 - l j
R
T
1r 2

53

I n tr o d u c i n g t h e m ole f r a c t i o n o f s o l u t e , x 2 ■ 1 - x 2,
(1*0)

By

Hyap ■>Atx1e“^ a p /R T s- e ' 4 H v a P/,RTl']

- Bv ^ H v a p -A .e " ^

a P/R T r [ ^ k a p . 0

_ x a( l ♦

I t i s a l s o c o n v e n ie n t t o r e w r i t e th e second te rm on t h e r ig h t- h a n d s id e
o f e q u a tio n (2 7 ) t o in c lu d e s p e c i f i c a l l y th e q u a n t i t y
(1*1)
For equ al
(1*2)

kF (FS(T S-T 0 ) -

® Tg - Tr .

Fr (Tr -T 0 )J - kF [Fs »AT +(F s -F r )(T r -T 0 ) J .

flo w r a t e s , F g= Fp « F , and
kj. [FS(T S-T 0 > -

Fr (Tr -T 0 ) ] - kF - F « ^ T -

The e x p r e s s io n s i n e q u a tio n s ( 3 6 ) , (UO), and (U l)

can now be

in tro d u c e d i n t o e q u a tio n ( 27 ) t o o b ta in

(1*3) — ( r r ° - r r ) a - ^ T - By-/*Hv a p *A e " ^ V RTr [^ % a p .
Rj.3 - .
H
ir
^^vap
* 2 (1 ♦

— R—

* fE
T
!r 5J )J

* kf [Fs 'A T ♦ (F s -F r )(T r -T 0) J + k(jA-xiT.
R ecom bining te r m s , g ro u p in g th o s e l i n e a r i n / A T, and s o lv in g f o r x 2
g iv e s

(UU)

X2

“

-

(Tr -T 0 )

;___________
B y ^ H y ap A e - ^ v a p / R T r (1 * ^

L

.

Jfc )

5h

F o r f i x e d flo w r a t e s , th e r m o s ta t te m p e r a tu r e , and b r id g e v o l t a g e , t h i s i s
©f t h e form
a oAT ♦ b

0*5)

(1 + c o A T )
w here

By-

(1*8)

b -

A H ^p-A e"

AHvap/EiTr

.

kp (F -F _ ) (Tr -T )
.S .r ------- — 2----------------

By- *4H

A ^vap
RT 2
r

9

-A-e” AHvap /HTr

and

I t w i l l g e n e r a l l y b e t r u e t h a t c* A T < < 1 , a s was shown i n th e d i s c u s s io n
_3

b e tw e e n e q u a tio n s (3 7 ) and ( 3 8 ) , w here c* A

t

— 7 x 10

f o r 0 . 1 M.

b ip h e n y l , w h ich i s c o n s id e re d t o b e i n th e h ig h ra n g e o f c o n c e n tr a tio n *
Hence a n e r r o r ©f l e s s th a n one p e r c e n t w i l l g e n e r a lly r e s u l t from n e g l e c t
o f t h e c *A T te r m , and
(^ 0 )

x3 m

a* A T * b

E q u a tio n (5 0 ) can be e x p re s s e d in te rm s o f t h e o b se rv e d v o l t a g e , e ,
b y u s e ©f e q u a tio n s (5?) and ( li t) *

Then

55

S e t t i n g t h e e x t e r n a l r e s i s t a n c e s e q u a l ( r r °» r s ° ) ,

(5 2 )

x2 -

a ( r ♦ r °)
-1 ^ — ^

©
1 -----

(£e_ ♦
'‘Rr

♦ b

1)
E'

S o lv in g f o r t h e m easured v o l t a g e , e ,
rr°

(5 3 )

e -

E

-IT —
■ ®P

(x 2 - b)

a ( r r ♦ r q° )

_ ( Xs _b )

a

When x 2 » 0 ( p u re s o lv e n t on sam ple t h e r m i s t o r ) , e * e 0 , and

(5U )

©o 88

- r r°
Rr
a ( r r + r g °)

E q u a tio n (5 3 ) shows a n e a r l y l i n e a r v a r i a t i o n o f o b serv ed v o lta g e
w ith t h e m ole f r a c t i o n , x 2, o f s o l u t e .

D e v ia tio n s from l i n e a r i t y m ig h t

b e e x p e c te d a t h ig h e r c o n c e n t r a tio n s , b e c a u s e o f th e x 2 term i n th e
d e n o m in a to r ,of e q u a tio n (53 )s t h i s sh o u ld ca u se a m o re-rap i d - t h a n - l i n e a r
i n c r e a s e ©f e w ith x 2 a t h ig h e r c o n c e n tr a tio n s ♦ The o r i g i n o f t h i s te rm
can b e t r a c e d back t o t h e rs te rm i n th e d en o m in ato r o f e q u a tio n ( 5 2 ) •
S in c e r r ° , Rp, e , and E have a l l b een m easu red , th e im p o rta n c e o f t h i s
rr
te rm can b e e s ti m a t e d . The v a lu e o f g — i s ab o u t 0 . 5 . The b r id g e
v o lta g e , E, i s 6 v o l t s .

O bserved v a lu e s o f e ra n g e from l e s s th a n one

m i l l i v o l t t o p e rh a p s 2 0 m i l l i v o l t s a t t h e h i g h e s t c o n c e n tr a tio n s employed
-3
§ i s i n g e n e r a l l e s s th a n 3 x 10 , w hich i s l e s s th a n one p e r
r °
cent of —
Thus , e x c e p t f o r c o n c e n tr a tio n s h ig h e r th a n 0 . 1 M.,
"r

Hence

56

e q u a tio n ( 5 2 ) becom es
a(r
(5 5 )

x2 •

♦ r °) R
s ' r

' v

and e q u a tio n (5 3 ) becom es
a rr° E
(5 6 )

e -

(x , - b ).

a ( r r + r s °)E r
When x 2 = 0 , e . e0 , g iv e n b y
b , so t h a t

(5 7 )

a ( r r ♦ r s ° )Hr
(5 8 )

e ~ e0 ■

a (r r *

E q u a tio n (5 6 ) ©r (5 8 ) may b e ta k e n as t h e fu n d a m e n ta l e q u a tio n f o r
d e s c rib in g th e d e te c to r re sp o n se .

F u r th e r j u s t i f i c a t i o n f o r th e

s i m p l i f i c a t i o n o f e q u a tio n (5 3 ) t o t h e form o f e q u a tio n (5 6 ) can b e
i n f e r r e d from th e a v a i l a b l e d a t a .

I n th e q u a n t i t a t i v e s tu d y o f b ip h e n y l

i n c y c lo h e x a n e -a c e to n e s o l v e n t , a re s p o n s e o f U*l5 mv. was o b ta in e d f o r
a 0 .0 2 2 M. s o l u t i o n , i n a ra n g e w here t h e r e s p o n s e - c o n c e n tr a tio n d ep endence
was fo u n d t o b e l i n e a r .

U sing

i s fou n d t o b e 5*25 i n t h i s s o l u t i o n w ith x 2 o n ly 2.U x 10

.

Thus th e

x 2 term i n t h e den om in ator o f e q u a tio n (5 3 ) sh o u ld b e n e g l i g i b l e up t o
p erh a p s *1 M. s o l u t i o n s , so t h a t u s e o f th e l i n e a r e q u a tio n (5 6 ) i s
c le a r ly j u s t if ie d

i n m ost o f t h e c a s e s o f i n t e r e s t .

I t h a s , t h e n , b e e n shown t h a t , w i t h i n t h e fram ew ork o f th e assu m p tio n s
made, t h e r e s p o n s e o f t h e d e t e c t o r sh o u ld be a c o l l i g a t i v e p r o p e r t y ,

57

d e p e n d e n t o n ly upon th e m ole f r a c t i o n o f s o l u t e i n th e s o l v e n t .

S in c e

d i l u t e s o l u t i o n s a r e u s e d , th e re s p o n s e i s a l s o a l i n e a r f u n c t io n o f
t h e c o n c e n t r a t i o n e x p re s s e d i n m o l a l i t y o r m o l a r i t y .
(5 9 )

m2 ^

(6 0 )

c 2—

Thus

-x *

and
m2 d x

w h ere m2 = m o l a l i t y o f s o l u t e , c 2 » m o lar c o n c e n tr a tio n o f s o l u t e ,
Mx « s o lv e n t m o le c u la r w e ig h t, and d^ « s o lv e n t d e n s i t y .
The a s su m p tio n s w hich h av e b e e n made a re s
1) th e r m a l b a la n c e a t each t h e r m i s t o r ,
2} e q u iv a le n t w ic k s ,
3 ) c o n s ta n t r a t e o f c o n d e n s a tio n , d e te rm in e d b y th e s o lv e n t v a p o r
p r e s s u r e a t th e r m o s ta t te m p e r a tu r e ,
U) n e g l i g i b l e r a d i a t i o n h e a t l o s s from th e t h e r m i s t o r s ,
5 ) no change i n c o m p o sitio n o r te m p e ra tu re o f t h e s o l u t i o n as i t
d e sce n d s t h e w ick p r i o r t o r e a c h in g t h e t h e r m i s t o r ,
6) no h e a t c a p a c i t y f o r t h e w ick o r th e t h e r m i s t o r ,
7 ) s m a ll p e r c e n ta g e ch an g es i n a b s o lu t e te m p e ra tu re and r e s i s t a n c e
a t th e sam ple t h e r m i s t o r i n com parison t o th e r e f e r e n c e
t h e r m i s t o r , and
8) i d e a l s o l u t i o n s o f n o n - v o l a t i l e s o l u t e s .
E ach o f t h e a ssu m p tio n s sh o u ld a lm o st c e r t a i n l y be v a l i d e x c e p t f o r th e
n e g l e c t o f te m p e r a tu re changes i n th e s o l u t i o n a s i t d e sc e n d s t h e w ic k ,
qriH t h e n e g l e c t o f t h e h e a t c a p a c i t i e s o f t h e w ick and t h e t h e r m i s t o r .

58

The h e a t c a p a c i t i e s o f th e w ick and th e r m is to r a r e p r o b a b ly e f f e c t i v e l y
in c o r p o r a te d in t o t h e term kQA° £^T, b u t th e s o l u t i o n tem p era tu re
a ssu m p tio n i s p r o b a b ly l e s s v a l i d .

However, a s shown b e lo w , th e form o f

e q u a tio n s ( 5 6 ) and ( 5 8 ) i s u n a lte r e d b y t h i s assu iq p tio n j o n ly t h e
d e f i n i t i o n s o f some o f th e p a ra m eters a r e ch an ged .
P erh ap s t h e m ost p l a u s i b l e a ssu m p tio n f o r t h e d e s c e n d in g s o l u t i o n
i s t h a t i t e q u i l i b r a t e s th e r m a lly w ith i t s su rro u n d in g s th rou gh th e
c ondens a t io n -v a p o r iz a t io n e q u ilib r iu m .

Then, e q u a tin g c o n d e n s a tio n and

v a p o r iz a t io n r a t e s ,
(6 1 )

k ^ 0 « By e~ A H vap/RT ^

w here T i s th e te m p er a tu r e on th e sam ple w ick j u s t b e f o r e th e t h e r m is t o r .
For t h e r e f e r e n c e w ic k , x x ** 1 and T = T0 , s o
(6 2 )

kcPx0 - Bv e" ^ Hvap/H T0

( 63)
(63J

x

Hence

=

^

1

- ^ H vap/RT

-1

R

(T - I p )
T0 2

e
or

(6U)

x3 -

^ Hvap

(T - T nl

R

T° 2

and
(6 5 )

T -

x s + To

^^vap
The te m p er a tu r e T0 i n e q u a tio n ( 2 5 ) , i n th e secon d term on th e r ig h thand s i d e sh o u ld b e r e p la c e d by T .

Hence

59

2

(6 6 )

k j F s (Ts -T 0 ) ------- >

kf F s (Ts -T ) . kFFs (Ts -T 0 ) - kFFs -

“^ ^ v a p

Xs

The o n ly change i n th e d e r iv a t i o n i s , t h e n , t h e a d d it io n o f t h e term

HT 2
k-F^s 0 __ —

° x 2 t o th e r ig h t-h a n d s i d e o f e q u a tio n s ( 2 5 ) , ( 2 6 ) ,

and (U 3)«

( 27) ,

I n t r o d u c t io n o f t h i s terra would m o d ify e q u a tio n (UU) t o

r ea d

[kpF

r

(6 7 )

JJs

U

J *

( r - r ° ) |
a +
r r
ly a

GF

Bv ~.A

*2 =

A e~AHv a p /R T r ]& T

♦ kF (F s -F r ) (Tr -T 0 )
2

Bv -A H vap A e - * IW

Hrr ( i *

k p F s. _ | | n
* ~ “ iAvap

r
E q u a tio n (U5) would th e n become
(6 8 )

a - &T # b

x2

1 * C«^T + W
RT0.
- Bv AHyap A e “ ^ Hv a p ^ r
S in c e c .
Aq^
(6 9 )
{

<

< 1 , a s h as b een d is c u s s e d ,

-v — ^
T ■» b
2 “
2
1 + h p F g . RTq

. Bv * A j4

p

=
______

A e ^ W

KTr

w here
2

kpFq » BTq
(7 0 )

P -

1 ♦

(7 1 )

x 2 “ a** ^ T ♦ b»

Thus

w here

. Ae - * Hvap/RTr .

a«

d

^ b

>

60

( ? 2 ) a* m -p

and b* = ^ *

The form o f e q u a tio n (7 1 ) i s i d e n t i c a l t o t h a t o f e q u a tio n ( 5 0 ) ,
and e q u a t io n s p a r a l l e l t o ( 5 1 ) th rou gh ( 5 8 ) can now b e w r i t t e n b y
r e p la c in g a and b b y a 1 and b* r e s p e c t iv e l y .,
I t h as b e e n e s t a b lis h e d t h a t th e r e s p o n se o f th e d e t e c t o r sh o u ld be
a c o l l i g a t i v e p r o p e r ty , v a r y in g l i n e a r l y w ith m ole f r a c t i o n (o r concen­
t r a t i o n ) o f s o l u t e in th e s o lv e n t f o r i d e a l s o l u t i o n s .

The c o l l i g a t i v e

n a tu r e o f th e r e s p o n se le a d s t o a h ig h g e n e r a li t y o f a p p l i c a b i l i t y o f th e
d e t e c t o r f o r chrom atograp h ic p u r p o se s , and s u g g e s t s i t s u s e f u ln e s s f o r
t h e d e te r m in a tio n o f m o le c u la r w e ig h ts and thermodynamic p r o p e r t i e s .
I t i s p o s s i b l e th rou gh u se o f known c o n c e n tr a tio n s o f sam p les o f
known m o le c u la r w e ig h t t o d eterm in e t h e c o n s ta n t ~ ( * Y + r s ) , and i t may
th e n b e assumed t h a t t h i s param eter i s t h e same f o r d i f f e r e n t s o l u t e s ,
p r o v id e d th e th e r m o s ta t te m p e r a tu r e , s o l v e n t , column flo w r a t e s , and
b r id g e v o lt a g e a r e un ch an ged .

As w i l l b e shown in t h e n e x t s e c t i o n , th e

l i n e a r r e l a t i o n s h i p b etw een r e sp o n se and c o n c e n tr a tio n makes p o s s i b l e
q u a n t i t a t i v e chrom atograp h ic a n a ly s is u t i l i z i n g in t e g r a t e d a r e a s o f th e
component p e a k s .
The m a th e m a tic a l d evelop m en t h a s b een b a sed on a r e a s o n a b le s e t o f
a s s u m p tio n s , and i t le a d s t o c o n c lu s io n s e n t i r e l y c o n s i s t e n t w ith
o b s e r v a t io n s th u s f a r made.
The r e s p o n s e p er u n i t c o n c e n tr a tio n can be s e e n from e q u a tio n (5 6 )
t o I n c r e a s e w ith b r id g e v o l t a g e , E, and w ith th e tem p era tu re c o e f f i c i e n t
o f r e s i s t a n c e , a , o f t h e t h e r m is to r s u s e d .

The e f f e c t o f f lo w r a t e can

61

b e s e e n i n t h e c o n s ta n t b ! , w h ich i s p r o p o r t io n a l t o ( F

-

and p rod u ces

an u n b a la n c e s i g n a l w ith p u re s o lv e n t on th e sam ple t h e r m is t o r .

The flo w

r a t e a l s o a i i e c t s a 1, w here i t s prim ary c o n t r ib u t io n e n te r s in t o th e

n u m e ra to r

i n a way w h ich le a d s t o an e s s e n t i a l l y in v e r s e dependence o f

r e s p o n s e on f lo w r a t e , a s was s o s t r i k i n g l y ap p aren t d u rin g t h e e v o lu t i o n
o f t h e d e t e c t o r i n t o i t s p r e s e n t form ( s e e S e c t io n I I - C ) .

The e f f e c t o f

t h e h e a t o f v a p o r iz a t io n o f t h e s o l v e n t i s q u it e com p lex, s i n c e a h ig h
v a lu e r e d u c e s t h e r a t e o f v a p o r iz a t io n , b u t in c r e a s e s th e h e a t e f f e c t p er
m ole v a p o r iz e d ; a v e r y h ig h h e a t o f v a p o r iz a t io n w ould ten d t o d im in is h
resp o n se.

The term kp», w h ich in c lu d e s h e a t c a p a c it y and th er m a l c o n d u c t iv it y

o f t h e l i q u i d , p r o b a b ly d o e s n o t v a r y a p p r e c ia b ly from one s o l v e n t t o
a n o th er.

The e f f e c t i v e a r e a , A, i s im p o rta n t in in c r e a s in g th e r e s p o n s e ,

s o w ic k d e s ig n i s o f o b v io u s im p o rta n c e.
F . C o n c e n tr a tio n P r o f i l e o f S o lu t e Bands
"When a sam ple i s in j e c t e d o n to a ch rom atograp h ic colum n, th e sam ple
i n i t i a l l y may be c o n s id e r e d t o h a v e a p p r o x im a te ly a r e c ta n g u la r co n cen ­
t r a t i o n p r o f i l e , w it h a w id th p r o p o r tio n a l t o th e v o lu m e, V0, o f t h e
sa m p le , and a h e ig h t e q u a l t o t h e i n i t i a l c o n c e n t r a t io n , CQ.

When t h e

band p a s s e s th rou gh th e colum n, i t s s p r e a d in g w i l l ten d t o g iv e i t a
G a u ssia n s h a p e , p r o v id e d no c h e m i-s o r p tio n o c c u r s .

D epending upon t h e

r e l a t i o n s h i p b etw een V0 and th e s p r e a d in g , t h r e e d i s t i n c t s i t u a t i o n s can
a r i s e , a s r e p r e s e n te d i n F ig u r e 8 .

F ig u r e 8-A shows a c a s e i n w hich th e

s p r e a d in g le a d s t o a c o n c e n t r a t io n a t t h e c e n te r o f t h e band a p p r e c ia b ly
lo w e r th a n th e c o n c e n t r a t io n , CQ, i n th e i n i t i a l sa m p le.

F ig u r e 8-B

CONCENTRATION

TIM E

FI6URE
8 . G A U S S IA N S P R E A D OF CONCENTRATION
FOR S A M P L E I N J E C T I O N S O F V A R Y I N G S I Z E * .
A, S M A L L S A M P L E ,
< CQ J B, L A R G E R S A M P L E ,
CMA= CQ , NO F L A T T E N I N G ; C , S T I L L L A R G E R
S A M P L E , CMAX= CQ , W I T H F L A T T E N I N G AT P E A K .

63

^ rep resen ts t h e s p e c i a l c a s e i n w hich th e s p r e a d in g and V0 a r e so r e la t e d
t h a t t h e peak h e ig h t i s j u s t Go, b u t t h e shape i s G a u ssia n .

I n F ig u r e 8-C

I s shown i h e c a s e , f o r s u f f i c i e n t l y la r g e sam ple v o lu m e s, where th e
le a d in g and t a i l i n g ed g es o f t h e band a r e G a u ssia n , b u t where t h e c e n t r a l
p o r t io n h a s l e v e l l e d o f f t o g iv e a f l a t t e n e d peak w ith h e ig h t C0 .
F ig u r e 8-A p r o b a b ly r e p r e s e n t s th e s i t u a t i o n m ost f r e q u e n t ly en co u n tered
i n ch rom atograp h ic w ork, w here s m a ll sam ple volum es a re employed t o p r o v id e
maximum r e s o l u t i o n o f component p e a k s .

F ig u r e 8-C r e p r e s e n ts th e c a s e o f

g r e a t e s t i n t e r e s t f o r m o le c u la r w e ig h t and therm odynam ic s t u d i e s , where
i t i s d e s i r a b l e t h a t th e d e t e c t o r r e s p o n se b e d i r e c t l y a s s o c ia t e d w ith a
m easured c o n c e n tr a tio n ^ t h i s c a s e i s a l s o m ost u s e f u l in a s s e s s i n g th e
r e s p o n s e c h a r a c t e r i s t i c s o f th e d e t e c t o r i t s e l f .

I t i s w o r th -w h ile t o

lo o k i n t o t h e m ath em atics o f th e c o n c e n tr a tio n p r o f i l e t o s e e w hat can b e
in f e r r e d from m easu rab le p r o p e r t ie s o f t h e component p e a k s .
S in c e th e rec o r d e d chromatogram i s p r e s e n te d in term s o f m i l l i v o l t s
o f r e s p o n s e v s . tim e in m in u te s , th e m a th em a tica l tr e a tm e n t o f th e co n cen ­
t r a t i o n p r o f i l e w i l l be s e t up on t h i s b a s i s .

Assuming a G aussian sh ap e

f o r th e c o n c e n tr a tio n p r o f i l e ,
(7 3 )
w here

Ct -

V

W

2

« m olar c o n c e n t r a t io n o f s o l u t e a t tim e t ,

r\

C «= m olar con cen ­

t r a t i o n o f s o l u t e a t t h e band p e a k , ? « tim e t o r e a c h peak (from sam ple
i n j e c t i o n t i m e ) , and p i s th e b ro a d en in g f a c t o r , su ch t h a t a h ig h p
in d i c a t e s much s p r e a d in g , w h ile a low p i n d ic a t e s a sh a r p , narrow p e a k .
I f an i n i t i a l volu m e, V0 , o f sam ple o f c o n c e n tr a tio n G0 i s o r i g i n a l l y

6k

I n j e c t e d , th e t o t a l number o f m oles o f sa m p le, n0 , i s g iv e n by
(7U )

n 0 « C0V0 o

T h is t o t a l number o f m oles must a l s o be o b ta in e d by a d d in g up t h e numbers
o f m oles a c r o s s th e s o l u t e band— i . e . , b y i n t e g r a t io n o f th e ban d.

For

i n t e g r a t i o n , th e c o n c e n tr a tio n e x p r e s s io n i n e q u a tio n ( 7 3 ) m ust b e
m u lt ip l ie d by th e volum e flo w r a t e , F , in l i t e r s p er m in u te , and th e n
in t e g r a t e d o v e r a tim e i n t e r v a l w hich in c lu d e s th e e n t i r e b an d.

Then t h e peak c o n c e n tr a tio n i s g iv e n b y
(76)

G -

C0

CSO

F
t« o
The d e f i n i t e i n t e g r a l can b e e v a lu a te d from a t a b le o f i n t e g r a l s , r e w r it in g
i t i n th e form

(7 7 )

H ence

The p a r a m e te r P may b e r e l a t e d t o t h e band w id th , e x p re s s e d a s t h e h a l f —
w id t h ,

A t a , a t th e h a l f - h e i g h t , Ct =
2

'c ,

from e q u a tio n ( 7 3 ) .

65

Thus

(80)

p =

Vln~2
and
(8 1 )

V CL

'G = G0 .
F *

2

\Zff / i n 2

or
C F • ^ t , V t f / In 2
(8 2 )

0o -

2l

V0
s o t h a t t h e i n i t i a l sam ple c o n c e n tr a tio n can b e d eterm in ed from t h e peak
h e i g h t , C, th e h a lf - w id t h a t h a l f - h e i g h t ,

^ ie volum e flo w r a t e o f
2

t h e colum n, F , and th e sam ple volum e i n j e c t e d , V0 .
S in c e i t h as h e e n shewn i n th e p r e c e d in g s e c t i o n t h a t t h e r e s p o n s e ,
e - e Q, i s p r o p o r tio n a l t o c o n c e n tr a tio n (e q u a tio n ( 5 8 ) ) , c o n c e n tr a tio n
can b e c a lc u la t e d from th e ob served r e s p o n s e , i f th e peak i s G a u ssia n .
F u rth erm ore, even f o r n o n -G a u ssia n p e a k s , th e in t e g r a t e d a r e a w i l l b e a
m easure o f number o f m oles in tr o d u c e d , and t h i s i n c o m b in a tio n w ith t h e
i n j e c t e d sam ple v o lu m e, V0 , i s s u f f i c i e n t t o d e te r m in e c o n c e n t r a t io n .
Thus

(8 3 )

or

e _e =
e eo

« r r° B
-----------------------a * ( r r + r g 0 )! ^

x2 -

S -S t-5
•
n
m nn
a l ( r r * r s )^ r

ca

h

1

66

(81*)

e -e 0 -

a • C2 -

<r . "c e ' ( t “ ~ )2 /fi2

(85)

a r r ° B_______
<r « a t ( r + ^
'

w here

x1

M,
1000 d-L

m illiv o lts
m o le /lite r

*

S

a c o n s ta n t c h a r a c t e r i s t i c o f t h e s o l v e n t sy ste m and d e t e c t o r under p a r t i c u ­

(86)

(e - e0 ) =

The maximum r esp o n se* ( e - e 0 )* i s th e n

<?

C »

O' •

—Q
0o
F . A tj, V f f / l n 2

-

l a r o p e r a tin g c o n d i t i o n s .

or
( 87)

Gq =

~~ e o ) ^

2

cr . V
Q
The a r e a under a G au ssian r e s p o n se curve i s g iv e n b y
( 88)

f

CL=

^00
( e - e 0 )dt

= cr S'

t= o
w

o

VnGn
F

^

f

00
e " (t

S3

_

nn
F

w

J mL m *

Thus th e number o f m oles in tr o d u c e d i s

(89)

no - f . - CL,

and t h e i n i t i a l c o n c e n tr a tio n i s
(90)

C0 «

Yo

• C l*

The c o n c lu s io n s r e g a r d in g a r ea a r e n o t r e s t r i c t e d t o G a u ssia n curve
sh ap es* b u t a r e a co n seq u en ce o f t h e l i n e a r i t y o f r e s p o n se o f t h e d e t e c t o r
A g e n e r a l r e s p o n s e cu rve can b e fo r m u la te d as

67

(91)

e-e0 =

Cr

'g * s ( t )

w here S ( t ) i s th e c u rv e -s h a p e f u n c t io n , w hich was e “ ( t ” t ) 2/ P 2 f o r th e
G a u ssia n shape*

Then
ca

(92)

= fr

no =

/nQ°

J ( e - e 0)d t = F C
t= o

J

S ( t ) d t = C0V0

t= o

The r e l a t i o n s h i p b etw ee n C and C0 i s , o f c o u r s e , d ep en d en t upon th e cu rv e
sh ap e.

(93)

'O = C0

2 aCO
F

J

S (t)d t

I t i s o f i n t e r e s t t o d e te rm in e th e sam ple v o lu m es, V0, w hich w i l l
g iv e each o f t h e t h r e e cu rv e sh a p e s shown i n F ig u re 8 .
v e n i e n t l y found b y s e t t i n g ^ * C0 i n e q u a tio n ( 8 1 ) .
(9U)

V0 =

F*

T h is i s con­

Then

V tf/ln 2
2
<»s

i s t h e volum e w hich w i l l j u s t g iv e th e maximum, C, e q u a l t o th e i n i t i a l
c o n c e n t r a t i o n , C0 .
t o Vn <

T h is i s c a s e B in F ig u re 8*

Case A c o rre s p o n d s

F* A't’j . V ff / i n ~ 2 , and Case C c o rre s p o n d s t o VG '> F* A t / i n
2

2.

"2

G. F a c to r s C au sin g N on-L inear R esponse
I n th e m a th e m a tic a l dev elopm ent o f th e d e t e c t o r re s p o n s e r e l a t i o n s h i p s ,
i t was fo u n d t h a t l i n e a r re s p o n s e i s t o b e e x p e c te d , a t l e a s t up t o concen­
t r a t i o n s o f p e rh a p s 0 . 1 - 1 M ., i n s o f a r as th e th e r m a l and e l e c t r i c a l
c h a r a c t e r i s t i c s o f th e d e t e c t o r a r e c o n c e rn e d .

I t i s p o s s i b l e , how ever,

68

f o r a p p r e c i a b l e d e v i a t i o n s from l i n e a r i t y , o r a p p a re n t d e v i a t i o n s , t o
o c c u r a t much lo w e r c o n c e n tr a tio n s th ro u g h f a i l u r e o f t h e s o lu t i o n s t o
obey i d e a l s o l u t i o n la w s , o r th ro u g h s p e c i f i c s o lu t e - s o l v e n t or s o l u t e s o l u t e i n t e r a c t i o n s w hich can be c la s s e d a s m o le c u la r-c o m p le x f o r m a tio n .
I n a p p l i c a t i o n o f t h e d e t e c t o r t o system s of o rg a n ic s o l u t e s and
s o l v e n t s , i n th e c o n c e n tr a tio n ra n g e below 0 .1 M, a c t i v i t y e f f e c t s w ould
n o t b e e x p e c te d t o be p ronounced * However, i t m ust b e re c o g n iz e d t h a t
n o n - l i n e a r i t y can r e s u l t from t h e s e e f f e c t s , and i t sh o u ld b e p o in te d
o u t t h a t t h e d e t e c t o r d o es in d eed o f f e r a p o s s i b l e t o o l f o r s tu d y o f
th e se e f fe c ts *
Of more a p p a r e n t c o n c e rn t o th e p r e s e n t s tu d y i s th e p o s s i b i l i t y o f
m o lecu lar-co m p o u n d ( o r com plex) f o r m a tio n , p a r t i c u l a r l y th ro u g h s o l u t e s o lu te in te ra c tio n ..

I t i s p o s s i b l e f o r com plexes to form from i n t e r 'V

a c t i o n b etw een d i f f e r e n t s o l u t e s p e c i e s , o r from s o l u t e - s o l v e n t i n t e r ­
a c t i o n , b u t t h e c a s e o f d i r e c t i n t e r e s t t o th e p r e s e n t s tu d y i s
d i m e r i z a ti o n o f a p a r t i c u l a r s o lu t e s p e c i e s , and o n ly t h i s c a s e w i l l be
t r e a t e d i n any d e t a i l .
I f a s o l u t e s p e c i e s , A, u n d erg o es d im e r iz a tio n

" (9 5)

C
2A
C-2x

iz z z r

A2

,

X

an o r i g i n a l c o n c e n t r a t i o n , C, w i l l a p p e a r a s a t o t a l m o lar c o n c e n tr a tio n
o f G - 2x ♦ x « G - x a t e q u ilib riu m .,

The c o n c e n tr a tio n , G, and h en ce

t h e d im e r c o n c e n t r a t i o n , x , w i l l v a r y a c r o s s a band w ith th e n o m in al sh ap e
shown i n F ig u r e 8 A o r B , and t h e r e s u l t a n t shape w i l l b e c o m p lic a te d by
th e d i m e r i z a t i o n .

I n t h e c a s e shown in F ig u re 8 C, how ever, i n th e

69

f l a t t e n e d peak C =2 c o, and th e peak h e ig h t w i l l c o rre s p o n d t o C0 t h i s s i t u a t i o n , t h e n , i s th e d e s i r a b l e ‘one f o r s tu d y o f th e d i m e r iz a tio n
e q u ilib r iu m c o n s t a n t , K^, w hich i s g iv e n b y

(96)

Kd -

=
(A )2

2--(C0 - 2 x ) 2

T h is e q u ilib r iu m c o n s ta n t can b e o b ta in e d r e a d i l y from a re s p o n s e v s .
c o n c e n t r a t i o n c u r v e , w hich sh o u ld ap p ro ach l i n e a r i t y a t lo w er concen­
tra tio n s o

I f t h e l i n e a r p o r ti o n i s e x tr a p o la te d t o a c o n c e n tr a tio n , C0,

w here d i m e r i z a ti o n o c c u r s , th e v e r t i c a l d e p r e s s io n o f th e a c t u a l re s p o n s e
compared t o t h e e x t r a p o l a t e d re s p o n s e i s a d i r e c t m easure of c r x , and a
h o r i z o n t a l l i n e drawn from th e p o i n t o b ta in e d a t C0 t o t h e e x tr a p o la te d
l i n e a r r e s p o n s e l i n e w i l l i n t e r s e c t th e re s p o n s e l i n e a t a c o n c e n tr a tio n
o f C0 - x .

T h is m ethod i s i l l u s t r a t e d In th e n e x t s e c t i o n , w here i t i s

a p p lie d t o an e x p e r im e n ta l c a s e .

I t must be r e c o g n iz e d , how ever, t h a t

t h e v a l i d i t y o f t h e m ethod r e s t s upon th e a ssu m p tio n t h a t th e d im e r iz a ti o n
i s w h o lly r e s p o n s i b le f o r th e o b serv ed n o n - l i n e a r i t y .
r e q u i r e s more e x p e r im e n ta l d a t a t o become j u s t i f i e d .

T h is a ssu m p tio n

70

V o QUANTITATIVE OBSERVATIONS— CORRELATION WITH THEORY
A* G e n e ra l

Q u a n t i t a t i v e d a t a became o b ta in a b le o n ly a t a v e r y l a t e s ta g e o f
t h e I n v e s t i g a t i o n , a f t e r e v o lu tio n o f th e d e t e c t o r t o i t s p r e s e n t fo rm .
E a r l i e r q u a l i t a t i v e o r c r u d e ly s e m i - q u a n tita tiv e s t u d i e s had in d ic a te d
a ro u g h p r o p o r t i o n a l i t y b etw een c o n c e n tr a tio n and r e s p o n s e , and a
q u a n t i t a t i v e s tu d y was u n d e rta k e n t o b e t t e r d e f in e t h i s r e l a t i o n s h i p f o r
v a r i o u s sam ple s u b s t a n c e s ,
The q u a n t i t a t i v e d a t a o b ta in e d a r e ta b u la t e d i n co m p lete form i n
A p p en d ices I and I I j a l l d a t a w ere o b ta in e d w ith th e cy clo h ex an e (95>
p e r c e n t v » /v o ) and a c e to n e (5 p e r c e n t, v „ /v „ ) s o lv e n t- a lu m in a s o r b e n t
sy ste m o

The d a t a o b ta in e d f a l l i n t o two d i s t i n c t gro u p s z

d a ta o b ta in e d

f o r s m a ll sam ple volum es w ith C < C0 (c o rre s p o n d in g t o F ig u re 8 -A ), and
d a t a o b ta in e d f o r l a r g e r sam ple v o lu m es, g iv in g f l a t t e n e d peaks w ith
'd « c 0 (c o rr e s p o n d in g t o F ig u re 8-C )»

The second group o f d a t a i s more

c o n v e n ie n tly t r e a t e d fro m th e s ta n d p o in t o f d e t e c t o r r e s p o n s e th e o r y
and f a c t o r s c a u s in g n o n - l i n e a r i t y of r e s p o n s e , w h ile t h e f i r s t group o f
d a t a r e q u i r e s i n a d d i t i o n th e a p p l i c a t i o n o f th e c o n c e n tr a tio n p r o f i l e
a n a ly s is .

I t i s m ore c o n v e n ie n t t o t r e a t t h e f l a t t e n e d p eak s f i r s t , and

th e n t o exam ine t h e sm a ll-sa m p le -v o lu m e d a ta i n l i g h t o f th e r e s u l t s
o b ta in e d w ith l a r g e r s a m p le s ,

71

Bo L a rg e Sam ple Volumes* F la tte n e d P eaks (2 => C0 )
O nly one s o lu te * b ip h e n y l* h a s b e e n s tu d ie d e x te n s iv e ly * w ith
s u f f i c i e n t l y l a r g e volum es o f sam ple (V0 — 0o05 m lc) a t d i f f e r e n t s o l u t e
c o n c e n t r a ti o n s t o make p o s s i b l e t h e m ap p in g -o u t o f t h e c o n c e n tr a tio n r e s p o n s e cu rv eo

A few s c a t t e r e d o b s e r v a tio n s w ith h ex am eth y lb en zen e and

w ith trip h e n y lm e th a n e s o l u t i o n s have a ls o b een o b ta in e d « The d a t a o b ta in e d
a r e p r e s e n te d i n A ppendix I* and th e re s p o n s e i s p l o t t e d as a f u n c t io n o f
c o n c e n t r a ti o n f o r b ip h e n y l i n F ig u re 9 c
The d a t a p l o t t e d i n F ig u re 9 w ere n o t o b ta in e d u n d e r th e c o n d itio n s
o f t h e m a th e m a tic a l d e r iv a tio n * w ith r s ° = r r ° c However* e s s e n t i a l l y no
change i n re s p o n s e t o sam ple was found t o r e s u l t when i d e n t i c a l sam ples
w ere i n j e c t e d w ith r ° sa r ° » 2000 ohms* e 0 = -2 8 d mv0* as compared
jy

w ith Ty,
1

O

*3 2000 ohms* r u

O

19U9 ohms* e 0

0 nw«

L a te r d a t a w ere o b ta in e d a f t e r t h e d e t e c t o r was opened and th e
p o s i t i o n s o f t h e w ick s w ere a lte r e d * and th e s e l a t e r r e s u l t s show a lo w ered
re s p o n s e a t h ig h e r c o n c e n tr a tio n s * a lth o u g h n e a r ly th e same re s p o n s e
was o b ta in e d i n th e lo w e r c o n c e n tr a tio n ra n g e (GoOl-O.OU H . ) .
S in c e t h e d e v i a t i o n s from l i n e a r i t y a t h ig h e r c o n c e n tr a tio n s d i f f e r
from s o l u t e t o s o lu te * i t a p p e a rs l i k e l y t h a t th e s e a r e a f u n c t io n o f th e
sam p les th e m se lv e s* r a t h e r th a n o f th e d e t e c t o r .

In p a r tic u la r*

i t would

a p p e a r t h a t d im e r iz a tio n o r h ig h e r a g g r e g a tio n o f th e s o lu t e s p e c ie s
s t u d i e s co u ld o c c u r th ro u g h fr™comp l e x in g o f th e ty p e rev iew ed by
Andrews ( l8 ) «

The n o n - l i n e a r i t y i s th e n a s c r ib e d t o th e r e s u l t a n t h ig h e r

a v e ra g e m o le c u la r w e ig h t and low ered r e l a t i v e m o lar c o n c e n tr a tio n a t

72

RESPONSE

(MILLIVOLTS)

15

10

5

0

0 .0 2

0 .0 4
0 .0 6
0 .0 8
0 .1 0
C O N C E N T R A T I O N ( M O L E S / LI TER )

FIGURE
9.
D E P E N D E N C E OF R E S P O N S E ON
C O N C E N T R A T I O N FOR S O L U T I O N S OF P I P H E N V L
IN C Y C L O H E X A N E ( 9 5 % , V / V ) - A C E T O N E
(5 % , V /V ) SOLVENT.

73

h i g h e r sam ple c o n c e n t r a t i o n s .

A ttem pted q u a n t i t a t i v e tr e a tm e n t o f th e

a s s o c i a t i o n would be p re m a tu re on such l i m ite d d a t a .
The t h r e e m ost im p o rta n t c o n c lu s io n s w hich can be drawn from th e s e
p r e l im i n a r y q u a n t i t a t i v e s t u d i e s a r e as f o llo w s .
1) The r e s p o n s e a p p e a rs t o be v e r y n e a r ly l i n e a r w ith c o n c e n tr a tio n
f o r c o n c e n tr a tio n s below 0 .0 1 M* and re s p o n s e t o d i f f e r e n t s o l u t e s
a p p e a rs e q u iv a le n t i n t h i s r a n g e e

T his s u g g e s ts t h e p r e d ic te d

a p p l i c a b i l i t y o f t h e d e t e c t o r t o o b ta in q u a n t i t a t i v e a n a l y t i c a l *
m o le c u la r w eig h t* and therm odynam ic d a t a .
2 ) D e v ia tio n s a t h ig h e r c o n c e n tr a tio n s a p p e a r t o b e d ep en d en t on
th e n a tu r e o f th e s o lu te * and p ro b a b ly a r e a m easure o f com plexin g o r a s s o c i a t i o n phenom ena.
3 ) From t h e re s p o n s e t o low c o n c e n tr a tio n s * of b ip h e n y l* th e in h e r e n t
s e n s i t i v i t y o f th e d e t e c t o r i s e s tim a te d t o be 1 8 8 .5 mv./M.* w ith
a p e a k - to -p e a k n o is e l e v e l in th e p r e s e n t d e t e c t o r of a b o u t 0 .1 mv.
Thus t h e p r e s e n t s e n s i t i v i t y i s such t h a t a 0 .0 0 1 M. s o l u t i o n i s
d e te c ta b le a t s ig n a l- to - n o is e of 2 s i .

T h is h as b e e n v e r i f i e d f o r

an e r g o s t e r o l s o l u t i o n .
Co S m all Sample Volumes (C < C0 )
S o lu t i o n s of v a r i o u s m o l a r i t i e s o f b ip h e n y l* t r i p h e n y l methane*
h ex am eth y lb en zen e* and a c r id e n e have b e e n i n j e c t e d w ith V0 e q u a l t o 0 .0 1
and 0 .0 2 m l.

Peak h e ig h ts * h a lf - w id th s * and a r e a s have b e e n e v a lu a te d

f o r t h e s e sa m p le s£ t h e d a t a a r e p r e s e n te d in A ppendix I I .

71*

Ttijo a s p e c ts o f t h e s e d a t a a r e p a r t i c u l a r l y s t r i k i n g .

F i r s t , th e

r e p r o d u c i b i l i t y o f i n t e g r a te d a r e a s i s e x c e l l e n t , even when sam ples w ere
in tr o d u c e d w ith o u t f i r s t w ith d ra w in g th e s o l v e n t - - i . e . , w ith sim p le
i n j e c t i o n below t h e s u r f a c e o f th e s o lv e n t o n to t h e column w ith o u t d i s t u r b ­
in g t h e s o lv e n t f lo w .

A reas a r e r e p r o d u c ib le to ± 2 p e r c e n t .

The

i n t e g r a t e d a r e a sh o u ld be a d i r e c t m easure o f th e number o f m oles o f
s o l u t e i n t h e l i n e a r ra n g e o f r e s p o n s e .

U n f o rtu n a te ly , i t was found l a t e r

t h a t t h e s e sam ples w ere ru n i n t h e n o n - li n e a r ra n g e o f th e re s p o n s e c o n c e n t r a ti o n c u rv e ; t h i s cau sed a n o n - l i n e a r i t y o f a r e a a s a f u n c tio n o f
t h e sam ple volum e, V0 , a l s o , s in c e th e peak c o n c e n tr a tio n depends upon V0 .
F u rth e rm o re , t h e n o n - l i n e a r i t y le d t o n o n - e q u iv a le n t a r e a s f o r e q u iv a le n t
c o n c e n tr a tio n s and sam ple s i z e s o f d i f f e r e n t s o l u t e s , so t h a t m o le c u la r
w e ig h ts w ere n o t o b ta in a b le from th e s e d a t a .
S ec o n d , t h e d ependence o f G/G0 on V0 f o r b ip h e n y l s o lu t i o n s c o r r e l a t e s
w e l l w ith t h e p r e d i c t i o n s o f th e G au ssian c o n c e n t r a t i o n - p r o f i l e tr e a tm e n t
and makes p o s s i b l e t h e c a l c u l a t i o n of th e b ro a d e n in g f a c t o r , p, in
e q u a tio n ( 7 3 ) ; v a lu e s of ^ /G 0 w ere o b ta in e d from F ig u re 9 .

W ith sam ple

i n j e c t i o n b e n e a th th e s o l v e n t , G/C0 i s a p p ro x im a te ly 0 .3 f o r 0 .0 1 m l.
sam p le s and 0.1*5 f o r 0 . 0 2 m l. sam p les; th e s e g iv e v a lu e s o f p o f 6.1*
and 8 .5 m in ., r e s p e c t i v e l y .

A gain th e n o n - l i n e a r i t y o f re s p o n s e i s

r e f l e c t e d i n th e v a lu e s of p , w here th e h a l f - h e i g h t i s lo w er th a n i t
s h o u ld b e , g iv in g h ig h e r v a lu e s o f P , f o r th e 0 .0 2 m l. sam ples a s compared
t o t h e 0 .0 1 m l. s a m p le s .

F o r 0 .0 2 m l. sam ples in tro d u c e d o n to th e column

a f t e r w ith d ra w in g t h e s o l v e n t , (?/C0 i s ab o u t 0 .5 7 , i n d i c a t i n g t h a t th e
s p r e a d in g f a c t o r , P , i s re d u c e d t o ab o u t 7 °k m in. b y t h i s te c h n iq u e o f

75

sam p le i n j e c t i o n »

These r e s u l t s p r e d i c t a f l a t t e n i n g o f peaks t o o ccu r

f ° r V0 g r e a t e r th a n 0 oQU5 m l ., and t h i s was v e r i f i e d e x p e r im e n ta lly in
o b ta in in g t h e d a t a d is c u s s e d i n S e c tio n V-B and t a b u l a t e d in A ppendix I .
The flo w r a t e , F , was 5
J
day

.

76

V I „ DISCUSSION AND CONCLUSIONS
Ac The P re -P e a k E f f e c t
The m ost i n t e r e s t i n g and u s e f u l a s p e c t o f th e r e s u l t s o b ta in e d w ith
t h e b u ta n o l and w a te r s o l v e n t - c e l l u l o s e s o rb e n t system is* perh ap s* t h e
o b se rv e d p r e -p e a k e f f e c t »

The p re -p e a k e f f e c t a p p e a rs a t th e flo w ­

th ro u g h tim e o f t h e s o lv e n t* and h as b e e n o b serv ed o n ly i n th o s e s o lv e n ts o r b e n t sy stem s t h a t have a s t a t i o n a r y p h ase w hich c o n s t i t u t e s one o f th e
s o l v e n t com ponents o

T h is o c c u rs w ith a c e l l u l o s e a d s o rb e n t and a w a te r -

c o n ta in in g s o lv e n t* s in c e a l a y e r o f w a te r i s h e ld on t h e c e l l u l o s e .
T h is la y e r* w hich may b e many m o lecu les deep* a c t s as th e a d s o rb in g sub­
s ta n c e b in d in g a th in * p e rh a p s m ono-m olecular* l a y e r of b u ta n o l* t h e
o t h e r corapbnent o f th e s o l v e n t .
The p r e - p e a k h a s two p a r t s s

a n e g a tiv e d e f l e c t i o n o c c u r r in g f i r s t

a t t h e flo w -th r o u g h tim e c o rre s p o n d in g t o th e tim e r e q u ir e d f o r a s o lv e n t
sam ple r i c h i n b u ta n o l t o b e d e t e c t e d , and a p o s i t i v e peak a t t h e tim e
c o r re s p o n d in g t o t h e flo w -th ro u g h tim e of a s o lv e n t sam ple r i c h in w a te r .
F ig u r e 10 shows a r e p r e s e n t a t i v e p r e - p e a k .
R a t i o n a l i z a t i o n o f th e p re -p e a k may f o llo w two c o u rs e s w hich a t f i r s t
a p p e a r t o be e n t i r e l y d i f f e r e n t b u t a r e a c t u a l l y t h e same e x p la n a tio n
fro m two d i f f e r e n t v ie w p o in ts .
The f i r s t *

and p e rh a p s t h e m ost s tr a ig h t- f o r w a r d * ap p ro ach i s t o

c o n s id e r t h e le n g th o f t h e a d s o rb e n t w hich i s o ccu p ied by th e s o lu t e
sam ple when i t h a s j u s t c o m p le te ly ru n i n t o t h e colum n.

The s o lu t e sam ple

77

o
cc
LU

o

S O L V E N T — N - BUTAN 0 L-W ATER
F L O W R A T E — 10 M L / H O U R
S O R B E N T — CELLULOSE
— I ML
3 0 % GLYCEROL SOLUTION

LU

CO

LU

QC

Q_
LU

CC
Cl .

l—

INJECTION

FI6URE

10 .

REPRESENTATIVE CHROM ATOGRA M
SHOWING
P RE - P E A K
EFFECT.

78

w i l l i n i t i a l l y d i s p l a c e th e a lc o h o l w hich i s h e ld on th e w a te r l a y e r *
so t h a t an a l c o h o l - r i c h band w i l l flo w down t h e column a t i t s n a t u r a l
flo w r a t e j s in c e th e s o l u t e m ust now be i n e q u ilib r iu m w ith th e w a te r
l a y e r a d so rb e d on th e s t a t i o n a r y phase* some o f th e w a te r w i l l be d is p la c e d
and flo w down th e column a t i t s n a t u r a l flo w r a te *

The w a te r peak

p a r t i a l l y o v e r la p s th e a lc o h o l peak* so t h a t i t i s c a n c e lle d t o a c e r t a i n
e x te n t o
The a l t e r n a t i v e e x p la n a tio n i s by a n a lo g y t o gas ch ro m ato g rap h y .
The phenomenon h a s b e e n o b serv ed in th e d e te r m in a tio n o f im p u r itie s in
c e r t a i n o f t h e g a s e s u s u a l l y u sed a s c a r r i e r s i n gas ch ro m ato g rap h y .

The

phenom enon h a s b e e n o b serv ed by J . P . M ickel (1 9 ) and E . E rb (20)
in d e p e n d e n tly i n t h e d e te r m in a tio n o f oxygen and n itr o g e n in hydrogen o r
helium * u s in g Golay -”ty p e c a p i l l a r y colum ns.
The n e g a tiv e - p e a k e f f e c t * as i t i s named i n gas chrom atography*
a p p e a rs when a p u re h y d ro g en sam ple i s i n j e c t e d i n t o a stre a m o f im pure
hydrogen* w hich s e r v e s a s th e e lu e n t s tre a m f o r a c a p i l l a r y colum n.
N e g a tiv e oxygen and n i t r o g e n p eak s a p p e a r a t t h e tim e s r e q u ir e d f o r e l u t i o n
o f oxygen and n itro g e n * r e s p e c tiv e ly * i f th e s e g a se s a r e ru n i n th e same
colum n w ith a p u re h y d ro g e n e l u e n t .

T h is e f f e c t can be r a t i o n a l i s e d * if*

one p i c t u r e s b a n d s o f oxygen o r n itr o g e n d e f ic i e n c y moving th ro u g h th e
colum n w ith t h e same r e t e n t i o n tim e s as th e s e g a s e s would have a s sam ples
i n a s tre a m o f p u re h y d ro g e n .
I n l i q u i d chrom atography* one may c o n s id e r t h e e l u e n t as a nonc o m p re s s ib le g a s .

The p o r t i o n o f th e s o lv e n t w hich c o n ta in s t h e s o l u t e

c o u ld t h e n b e c o n s id e re d as a band w hich i s red u ced i n s o lv e n t

79

c o n c e n tra tio n .

T h is s o lv e n t band sh o u ld move th ro u g h t h e column a t th e

r a t e o f flo w o f th e com ponents o f th e s o lv e n t .
T h is phenomenon h a s a l s o b e e n o b serv ed b y Bo F« M eyers (2 1 ) i n th e
io n -e x c h a n g e ch ro m ato g rap h y o f amino a c i d s , u s in g a c o n tin u o u s ly - r e c o r d ­
in g r e f r a c t o m e t e r f o r a d e te c to r ,,

The p r e - p e a k , o r co m p en satio n peak

(a s M eyers named i t ) , was n o t r a t i o n a l i z e d t o a g r e e w ith th e e x p e rim e n ta l
d a t a h e o b ta in e d , and th e 'i s i g n i f i c a n c e o f i t s p re s e n c e was n o t re c o g n iz e d .
The e x p la n a tio n he p o s tu l a t e d r e q u i r e s th e m o r e - ti g h tl y - h e ld component of
t h e s o lv e n t t o a p p e a r a t t h e b o tto m o f th e colum n, j u s t b e f o r e th e s o l u t e
band a p p e a r s .

H is r e c o r d e r t r a c e s show, how ever, t h a t b o th th e com ponents

o f th e p r e - p e a k a p p e a r a t th e r e t e n t i o n tim e of t h e s o l v e n t .

M oreover,

h e d id n o t d e t e c t t h e amino a c id s a t t h e i r r e t e n t i o n tim e s w ith th e
r e f r a c t ©meter | h e a t t r i b u t e d t h i s f a i l u r e t o t h e com pensation o f th e
r e f r a c t i v e in d e x by th e changes i n th e s o lv e n t c o m p o s itio n .

The amino a c id s

w e re , h o w ev er, d e t e c t e d by n in h y d rin r e a g e n t a s much as s i x h o u rs a f t e r
th e a p p e a ra n c e o f th e p r e -p e a k ; t h u s , th e p re -p e a k co m p en satio n o f th e
r e f r a c t i v e in d e x b y th e changes i n th e s o lv e n t c o n c e n tr a tio n co u ld n o t
h a v e b e e n due t o t h e p re s e n c e o f th e amino a c i d , b u t m ust h av e b e e n caused
by a s o l v e n t - d e f i c i e n t band moving th ro u g h th e column w ith t h e r e t e n t i o n
tim e o f t h e s o l v e n t .

H is r e f r a c t i v e - i n d e x d e t e c t o r a p p a r e n tly was n o t

s u f f i c i e n t l y s e n s i t i v e t o d e t e c t th e r e f r a c t i v e - i n d e x ch an g es due t o th e
am ino a c i d , a t t h e c o n c e n tr a tio n s p r e s e n t i n th e s o lu t e b an d s, sp re a d by
p a s s a g e th ro u g h th e colum n.
The n e g a tiv e p eak e f f e c t , co m p en satio n e f f e c t , and p r e —p eak e f f e c t
a p p e a r t o be t h e same phenom enon, even th o u g h th e y o c c u r i n d i f f e r e n t

80

b r a n c h e s o f c h ro m a to g ra p h y 0 The p re -p e a k may p o s s i b l y b e c a l c u l a t e d f o r
l i q u i d ch ro m ato g rap h y t o g iv e q u a n t i t a t i v e d a t a as t o t h e s i z e o f th e
sam p le i n j e c t e d i n t o th e colum n, and i t can b e used t o c a l c u l a t e th e R„
f
v a l u e , w h ich i s u s e f u l i n i d e n t i f i c a t i o n o f component b a n d s .
Bo The U ltim a te S e n s i t i v i t y o f th e D e te c to r
The d e t e c t o r , i n i t s p r e s e n t s t a t e o f d ev elo p m en t, i s c a p a b le o f
„9
d e t e c t i n g above t h e b ackground U x 10
m oles o f n o n - v o la ti l e s o l u t e .
T h is i s by no means th e u l t i m a t e i n s e n s i t i v i t y w hich t h i s d e t e c t o r can
c o n c e iv a b ly r e a c h .

The minimum r e s i s t a n c e change o r u n b a la n c e w hich ca n

b e o b se rv e d w ith a W h eatsto n e b r id g e i s l im i t e d by th e fo llo w in g s
th e r m a l n o is e s i n th e t h e r m i s t o r s , l e a d s , c o n n e c tio n s , and r e s i s t a n c e s ;
c o n ta c t p o t e n t i a l s i n t h e c o n n e c tio n s ; r e s i s t a n c e f l u c t u a t i o n s due t o
e x t e r n a l te m p e r a tu r e ch an g es o f t h e b r id g e e le m e n ts n o t th e r m o s ta te d ;
n o i s e s w h ich a r i s e i n t h e p rim a ry s ta g e s o f a m p lif ic a tio n ; v o lta g e f l u c t u ­
a t i o n s i n t h e p o t e n t i a l s o u rc e ; and th e th e r m o s ta tin g o f th e d e t e c t o r .
I f a W h ea tsto n e b r id g e w ere employed u s in g th e rm a lly -c o m p e n s a tin g
e le m e n ts , and i f t h e w hole b r id g e w ere th e r m o s ta te d , n o is e s due t o changes
i n t h e r e s i s t a n c e o f th e b r id g e e le m e n ts could be red u c e d below th e d e t e c t ­
a b le ra n g e .
S in c e i n th e d ev elo p m en t o f a chrom atogram s e v e r a l m in u te s a r e
r e q u ir e d f o r one s o l u t e band t o p a s s a t h e r m i s t o r , th e r m a l n o is e s can be
e l i m i n a t e d , b e c a u s e th e y a r e r e l a t i v e l y o f h ig h f re q u e n c y .

I f a tim e

c o n s ta n t o f 30 sec o n d s i s em ployed, th e h ig h e s t fre q u e n c y p a s se d w i l l b e
l / 3 0 c y c le s p e r s e c o n d .
10“

9

The v o l t a g e from th e r m a l n o is e th e n would b e

v o l t s , w hich may b e d i s r e g a r d e d , even f o r v e r y h ig h a m p l i f i c a t i o n .

81

N o ise s due t o c o n ta c t p o t e n t i a l s a r i s i n g .f r o m b i - m e t a l l i c j u n c ti o n s
i n t h e c i r c u i t may be red u ced b y u se of s p e c i a l s o ld e r and le a d s -

The

re m a in in g j u n c t i o n p o t e n t i a l s * i f p re s e n t* can b e m a in ta in e d c o n s ta n t
w ith t h e r m o s ta tin g o f th e c o n t a c t s .
C a r e f u l l y s e l e c t e d c i r c u i t com ponents may b e u sed in t h e f i r s t s ta g e s
o f a m p lif ic a tio n o . S in c e m ost o f th e n o is e a r i s i n g i n t h e a m p l i f i e r i s o f
h ig h fre q u en c y * a s i s t h a t a r i s i n g from th e rm a l n o ise* t h e s e may a l s o b e
re d u c e d by damping..

W ith p r o p e r dam ping and s h ie ld in g * a d .c # a m p lif i e r

may b e d e s ig n e d w hich w i l l h av e a g a in o f 10

3

w ith l e s s th a n 3 p e r c e n t

n o is e o

Thus* a o n e - m i l l i v o l t f u l l - s c a l e re c o rd e r* co u p led w ith a d . c .
s
a m p lif ie r * co u ld d e t e c t a s i g n a l o f 10
v o l t s w ith a n o is e l e v e l o f l e s s
-7
t h a n 5 x 10
v o lts .
—

N o ise a r i s i n g from p o t e n t i a l changes i n th e v o lta g e s o u rc e may be
e lim in a te d b y p a r a l l e l i n g s e v e r a l s to r a g e b a t t e r i e s w hich have b een
d e s ig n e d f o r n o i s e - f r e e o p e r a tio n and w hich have b e e n ch arg ed s lo w ly and
c a re fu lly .
F in a lly * b a th te m p e r a tu re f l u c t u a t i o n s co u ld b e re d u c e d t o ± Q.Q01°C
s© t h a t w ith t h e r m i s t o r s m atched w i t h i n ± 2 p e r cent* b a th te m p e ra tu re
„

f l u c t u a t i o n s w i l l r e p r e s e n t ± 1 .2 x 10

6

v o lts .

T h is n o is e can b e re d u c e d

s t i l l f u r t h e r by dam ping th e d e t e c t o r chamber w ith i n s u l a t i o n and r e d u c in g
t h e o n - o f f tim e o f t h e r e g u l a t o r u n t i l th e re s p o n s e tim e f o r th e d e t e c t o r
t o te m p e r a tu r e changes i s lo n g e r th a n th e on—o f f r e g u l a t i o n .
I f th e c i r c u i t now i n o p e r a tio n w ere m o d ifie d a s s u g g e s te d i n th e
-12

p r e c e d in g p a ra g ra p h s * t h e d e t e c t o r co u ld s e n s e 10
m oles o f sam ple and
_ ii
d e te rm in e q u a n t i t a t i v e l y 10
m o le s. T h is i s not* however* th e

82

i n - p r i n c i p l e lim it, o f t h i s d e t e c t o r , s in c e s m a lle r t h e r m i s t o r s , m o d ifi­
c a t i o n s o f t h e w ic k , and re d u c e d flo w r a t e s co u ld c o n c e iv a b ly in c r e a s e
t h e s e n s i t i v i t y t o d e t e c t a s l i t t l e as 10

w13

o r 10

14

m oles o f sam ple*

I f su c h an e x te n s io n i s p o s s ib le ^ th e n t h i s t o o l w ould become com parable
w i t h t h e m ost s e n s i t i v e d e t e c t o r s now a v a i l a b l e i n an y a r e a o f c h e m is tr y .
I f s e n s i t i v i t y o f t h e d e t e c t o r i s th e o b j e c t , th e n t h e p r e s e n t
d e t e c t o r c an b e e x ten d e d s t i l l f u r t h e r , s in c e i t i s more s e n s i t i v e t o
v o l a t i l e s u b s ta n c e s th a n t o n o n - v o l a t i l e s u b s ta n c e s .

F o r s o lu t e s h a v in g

v a p o r p r e s s u r e s tw ic e t h e v a p o r p r e s s u r e o f t h e s o lv e n t a t th e d e t e c t o r
te m p e r a tu r e , th e n t h e d e t e c t o r i s more s e n s i t i v e by a f a c t o r o f 100.

T h is

w ould p la c e t h e l i q u i d ch ro m atography th e r m i s t o r d e t e c t o r a lr e a d y n e a r ly
on a p a r w ith th e m ost s e n s i t i v e gas chrom atography d e t e c t o r s .
C o S u g g e s tio n s f o r F u r th e r Work
S e p a r a t i o n s i n l i q u i d c h ro m ato g rap h y , in p a r t i c u l a r l i q u i d - p a r t i t i o n
c h ro m a to g ra p h y , a r e o f te n b e s t co n d u cted u s in g s o lv e n ts w hich change i n
C o m p o sitio n d u r in g th e d evelopm ent o f a chrom atogram .

The new d e t e c t o r

can b e e x te n d e d t© work i n th e s e sy stem s i f th e fo llo w in g c o n d itio n s a r e
raets

t h e t h e r m i s t o r s m ust b e c a r e f u l l y m atched, so t h a t s o lv e n t changes

w i l l n o t a f f e c t t h e u n b a la n c e ; t h e flo w r a t e s o f th e two colum ns m ust b e
n e a r l y i d e n t i c a l ; and t h e two t h e r m is t o r s sh o u ld b e a tta c h e d i n i d e n t i c a l
f a s h i o n t o t h e i r r e s p e c t i v e w ic k s .

A d a p ta tio n o f t h e d e t e c t o r t o th e s e

sy ste m s w ould g r e a t l y i n c r e a s e th e p o t e n t i a l u s e f u ln e s s o f th e d e t e c t o r .
P a p e r ch ro m ato g rap h y i s s e le c t e d i n p r e f e r e n c e t o column ch ro m ato g rap h y
f o r many b io c h e m ic a l a p p l i c a t i o n s , b e c a u s e i t i s p o s s i b l e t o u s e v e iy s m a ll,

83

d i l u t e sam ple s o l u t i o n s , and t o d e v e lo p th e chrom atogram i n more th a n one
d im en sio n *

The u s e o f c a p i l l a r y columns t o some e x te n t o f f s e t s th e

f i r s t a d v a n ta g e , b u t p a p e r ch rom atography may s t i l l b e more c o n v e n ie n t
f o r many a p p l i c a t i o n s o

T h is d e t e c t o r can and sh o u ld be a d a p te d f o r th e

d e t e c t i o n and l o c a t i o n o f s o l u t e bands in p a p e r chrom atography*

No m ajo r

m o d i f i c a t i o n s need b e made f o r p a p e r s t r i p s » I t sh o u ld b e m e c h a n ic a lly
p o s s i b l e t o d e v is e a tw o -d im e n s io n a l scaii mechanism f o r t h e l o c a t i o n o f
s p o ts on p a p e r * W ith o u t d i s t u r b i n g th e p r o g r e s s o f t h e chrom atogram ,
su ch a d e v ic e w ould a llo w th e o p e r a to r to d e te rm in e th e l o c a t i o n , s h a p e ,
and s i z e o f t h e s p o ts on paper*
The c o n t r i b u t i o n made b y t h i s d e t e c t o r w ould b e s e v e r e ly lim ite d i f
i

i t w e re n o t p o s s i b l e t o a d a p t i t t o m o n ito r th e p r o g r e s s o f a p r e p a r a t i v e s c a l e chrom atogram *

The a d a p ta tio n need o n ly c o n s i s t o f a s tre a m d i v i d e r ,

w h ich w ould m o n ito r o n ly a s m a ll p o r ti o n o f th e e f f l u e n t s tre a m from a
l a r g e column*

T h is sh o u ld b e u s e f u l n o t o n ly f o r la b o r a t o r y - s c a l e

p r e p a r a t i o n s , b u t a l s o f o r m o n ito rin g s tre a m c o n c e n tr a tio n s i n l a r g e - s c a l e
i n d u s t r i a l p ro c e s s e s *

T h is d e t e c t o r sh o u ld e a s i l y be a d a p ta b le t o a u to ­

m a tio n c o n t r o l , j u s t as h a s b een done w ith gas chrom atography*
T h is d e t e c t o r i s p a r t i c u l a r l y w e ll s u it e d t o e x p lo r a to r y r e s e a r c h
i n new c h ro m a to g ra p h ic s y s te m s , b e c a u se o f i t s s i m p l i c i t y and a d a p t a b i l i t y *
p r e -p e a k e f f e c t a l s o le n d s i t s e l f t o i n t e r e s t i n g i n v e s t i g a t i o n s i n t o
t h e s tu d y o f t h e n a tu r e o f a d s o r p tio n and d is p la c e m e n t*

I t sh o u ld b e

p o s s i b l e t o d e te rm in e from t h e s iz e s o f t h e p re -p e a k s t h e r e l a t i v e amounts
o f d i s p l a c e d com ponents o f t h e s o lv e n t*

I t sh o u ld be p o s s i b l e t o u s e th e

tim e o f a p p e a ra n c e o f t h e p r e -p e a k f o r d e te r m in a tio n o f th e Rf f a c t o r s ,
s in c e i t em bodies t h e s o lv e n t f r o n t*

8U

A n o th er im p o rta n t a d a p t a tio n o f th e d e t e c t o r would b e t o t h e f i e l d
o f io n -e x c h a n g e c h ro m a to g ra p h y .

T h is a d a p ta tio n i s p e rh a p s th e l e a s t

p r o b a b le o f t h e s u g g e ste d e x te n s io n s , s i n c e i t r e q u i r e s d e t e c t i o n o f
s u b t l e chan g es i n e f f l u e n t a c t i v i t y a t a c o n s ta n t io n i c s t r e n g t h .
S in c e t h e d e t e c t o r re s p o n s e h a s b e e n shown t o b e a l i n e a r f u n c t i o n
o f ishe mole f r a c t i o n o f th e s o l u t i o n p a s s in g th e sam ple t h e r m i s t o r , i t
may b e u se d f o r d e te r m in a tio n o f a c t i v i t y i n d i l u t e s o l u t i o n s .

O nly th e

p r e l im i n a r y s t u d i e s h av e b e e n com pleted to w ard t h i s en d , b u t th e te c h n iq u e
shows c o n s id e r a b le p ro m ise f o r m o le c u la r-w e ig h t d e te r m in a tio n , a s s o c i a t i o n c o n s ta n t d e t e r m in a ti o n s , and d e te r m in a tio n s o f a c t i v i t y .
E s s e n t i a l l y t h e same d e t e c t o r m ight a l s o b e u sed f o r s tu d y o f th e
h e a t o f r e a c t i o n , i f t h e sam ple th e r m i s t o r w ere p la c e d a t t h e c o n flu e n c e
o f tw o w ic k s , one o f w h ich c a r r i e s one r e a c t a n t w h ile th e o th e r c a r r i e s
th e o th e r r e a c t a n t .

The te m p e ra tu re - o f th e chamber could b e v a r ie d

g r a d u a l l y , and a b re a k i n th e cu rv e would i n d i c a t e r e a c t i o n .

85

VII „ SUMMARY
A new, g e n e r a l method h a s b een d ev elo p e d f o r d e te c t i n g and r e c o r d in g
t h e p r e s e n c e o f s o l u t e b an d s in th e e f f l u e n t s tre a m o f a l i q u i d - e l u e n t
c h ro m a to g ra p h ic column*

S in c e t h i s method i s b a se d upon changes i n v a p o r

p r e s s u r e ■which o c c u r i n t h e s o lv e n t due t o t h e p re s e n c e o f a s o l u t e b a n d ,
i t s a p p l i c a b i l i t y i s n o t l i m i t e d t o s p e c i f i c c l a s s e s o f s u b s ta n c e s .
The d e t e c t o r d ev elo p ed u t i l i z e s th e te m p e ra tu re d if f e r e n c e w hich i s
e s t a b l i s h e d b etw een a w ick b a th e d w ith th e e f f l u e n t o f a c h ro m ato g rap h ic
colum n and a r e f e r e n c e w ick b a th e d w ith p u re s o lv e n t> t h e w icks a r e
e n c lo s e d i n a th e r m o s ta te d chamber t h a t i s s a tu r a te d w ith s o lv e n t vapor*
The te m p e r a tu re change i s d e te c te d th ro u g h th e d i f f e r e n c e i n r e s i s t a n c e
o f a p a i r o f t h e r m i s t o r s , one o f w hich i s i n c o n ta c t w ith th e w ick b a th e d
w ith p u r e s o l v e n t , w h ile t h e o th e r i s i n c o n ta c t w ith th e w ick b a th e d
w ith t h e e f f l u e n t s o lu tio n *

The t h e r m is t o r s c o n s t i t u t e two arms o f a

W h ea tsto n e b r i d g e , w hich can b e b a la n c e d when b o th o f th e th e r m is to r s a r e
i n c o n ta c t w ith w ick s b a th e d w ith p u re s o lv e n t*

Any u n b a la n c e i n t h e

b r id g e w hich o c c u rs a s a r e s u l t o f s o lu t e bands p a s s in g i s c o n tin u o u s ly
r e c o rd e d on a r e c o r d in g p o te n tio m e te r*
D e t e c t o r r e s p o n s e i s a c o l l i g a t i v e p r o p e r ty o f th e s o l u t i o n p a s s in g
o v e r th e t h e r m i s t o r s *

A- m a th e m a tic a l tr e a tm e n t h a s b e e n d ev elo p e d and

p r e d i c t s a l i n e a r d ep en d en ce of d e t e c t o r re s p o n s e on mole f r a c t i o n o f
s o lu te *

The m a th e m a tic a l a n a ly s is i s b ased on a c o n d itio n o f th e rm a l

b a la n c e , and r e q u i r e s s u i t a b l e a p p ro x im a tio n s , b ase d on t h e s m a ll m agnitude

86

o f t h e o b se rv e d changes* f o r d e r i v a t i o n of t h e l i n e a r r e l a t i o n s h i p *

The

k i n e t i c s o f t h e s o lv e n t v a p o r i z a t i o n and c o n d e n s a tio n p ro c e s s e s * as
in f lu e n c e d b y th e p r e s e n c e o f s o lu te * a r e p o s tu l a t e d t o p l a y t h e m ajo r
r o l e i n d e t e c t o r re sp o n s e *
F u r th e r m a th e m a tic a l a n a l y s is o f th e c o n c e n tr a tio n p r o f i l e f u r n is h e s
a r e l a t i o n s h i p b etw een t h e c o n c e n tr a tio n g iv in g th e o b serv ed re s p o n s e and
t h e i n i t i a l sam ple c o n c e n tr a tio n *
The e v o lu tio n o f t h e d e t e c t o r and i t s r e l a t i o n s h i p t o o th e r means o f
c h ro m a to g ra p h ic d e te c tio n * and t o r e l a t e d m easu rin g te c h n iq u e s * a r e
d e s c rib e d *

Q u a l i t a t i v e r e s u l t s o b ta in e d in d i f f e r e n t s o lv e n t- s o r b e n t

sy stem s f o r a v a r i e t y o f s o l u t e s a r e p r e s e n te d ! th e re s p o n s e t o v o l a t i l e
and n o n - v o l a t i l e s o lv e n t s i s d is c u s s e d *

P a r tic u la r a tte n tio n has been

d i r e c t e d to w ard s th e a n a ly s is o f th e e r g o s t e r o l i r r a d i a t i o n m ix tu r e .
Some q u a n t i t a t i v e r e s u l t s a r e p r e s e n te d and i n d i c a t e th e n a tu r e and
o
s e n s i t i v i t y o f t h e re sp o n se * A t 33 o
w ith a sam ple column flo w r a t e o f
5 m lo/day* a r e f e r e n c e column flo w r a t e o f 2 m l*/day* c o tto n th r e a d w icks*
n o m in a l 2000-ohm t h e r m i s t o r s w ith a te m p e r a tu re c o e f f i c i e n t o f U*5 p e r
c e n t p e r d eg re e * f ix e d b r id g e r e s i s t a n c e s of 2000 ohms* and an a p p lie d
b r id g e v o l t a g e o f 6 v o l t s * t h e d e t e c t o r h a s a s e n s i t i v i t y o f 188*5 rav./M**
o r t h e c a p a c i t y t o d e t e c t s o l u t e in 0*01 ml* o f 10 M* s o lu t io n s ! t h i s
_8
c o rre s p o n d s t o d e t e c t i o n o f 10 —10
m oles of m a te r ia l*
The new m ethod sh o u ld complement gas chrom atography as an a n a l y t i c a l
method* w ith i t s m ost im p o rta n t a p p l i c a t i o n s i n a r e a s w here gas ch rom atography
i s w e a k e s t— a n a l y s i s o f h i g h - b o i l i n g and th e r m a lly - u n s ta b le com ponents*

87

I t sh o u ld b e o f p a r t i c u l a r v a lu e i n b io c h e m ic a l, n a t u r a l p r o d u c ts , and
p h a r m a c e u tic a l worko

P h y s ic o -c h e m ic a l a p p l i c a t i o n s o f th e d e t e c t o r

in c lu d e i t s u s e i n d e te r m in in g m o le c u la r w e ig h ts , e v a lu a tin g a c t i v i t y
e f f e c t s , s tu d y in g m o le c u la r com plexing and d i s s o c i a t i o n , and o b ta in in g
fu n d a m e n ta l in f o r m a tio n on th e n a tu r e o f th e c h ro m a to g ra p h ic p r o c e s s ®

88

BIBLIOGRAPHY
( 1 ) W. C . K enyon, J* ,E. C a r le y , E . G. B o u ch er, A. E . R o b inson and
A . K . ¥ h i t e , A n a l. Chem., 2 £ , 1888 ( 1 9 $ $ ) .
( 2 ) F . Baumann and B . J . B la e d e l, A n a l. Chem., 2 8 , 2 (1 9 5 6 ).
( 3 ) G. C la x to n , J . o f Chrom atog., 2 , 139 (1 9 5 9 ).
(U) D . Spaokm an, ¥ . H. S t e i n , and S . M oore, A n a l. Chem., ^ 0 , 90 ( 1 9 5 8 ).
( 5 ) G* V". S c h u l t z , 0 . Bodmann and H. J . Cantow, Z . N a tu r f o r s c h ., 7 a ,
760 (1 9 5 7 ) •
( 6 ) F h o e n ix F r e c i s i o n I n s tr u m e n t Company, B u l l e t i n R1000.
( 7 ) E . J . B a id e s , B io d y n am ica, U6. 8 (1 9 3 9 ).
( 8 ) G. B . T a y lo r and M. B . H a l l , A n a l. Chem., 2 £ , 9U7 (1 9 5 1 ).
(9 ) A . F .

B ra d y , H . H u ff and J . W. McBain, J .

F h y s. Chem., £ £ , 305 (1951)

(1 0 ) R . H. M il l e r and H. J . S t o l t e n ,, A n a l. Chem., 2 £ , 1103 (1 9 5 3 ).
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(1 2 ) S . B . K u l a r n i , N a tu re , 1 7 1 . 219 (1953)*
(1 3 ) W. I . H ig u c h i, M. A . S c h w a rtz , E . G. R ip p ie and T . H ig u c h i,
J . F h y s . Chem ., 6£ , 996 (1 9 5 9 ).
(1 h ) H . H . S t r a i n , A n a l. Chem., 3 0 . 621 (1 9 5 8 ).
(1 5 ) H . S . S t i l l o , P h . D. T h e s is , M ichigan S t a t e U n iv e r s ity , 1959*
(1 6 ) H . Brockmann and H. S c h o d d e r, B e r . ,

73 (19U 1).

(1 7 ) N. B . S . , A .P . 1 . , R e se a rc h P r o je c t UU(1 8 ) L . J . A ndrew s, Chem. R ev. j>l±, 713 (195H ).
(1 9 ) J . P . M ic k e l, P e r s o n a l C om m unication.
(2 0 ) E . E rb , 2nd B ia n n u a l I n t e r n a t i o n a l Symposium on Gas C hrom atography
Ju n e 1 0 , 1959*
(2 1 ) B . F . M eyers, J r . , P h . D. T h e s is , N o rth w e ste rn U n iv e r s ity , 195U.

APPENDICES

89
APPENDIX I
QUANTITATIVE CONCENTRATION-RESPONSE DATA FOR LARGE-VOLUME SAMPLES
(FLATTENED PEAKS)

Compound
B ip h e n y l
B ip h e n y l
B ip h e n y l
B ip h e n y l
B ip h e n y l
B ip h e n y l
B ip h e n y l
T r iphenylm e th a n e
T rip h e n y lm e th a n e

Co n c e n tr a t io n
(m o le s /lite r)

Peak
H e ig h t
(mv. )

0.0 2 1 5 6
0.01+312
0 .06U68
0.08621;
0 .1 0 7 8
0.1078
0 .1 0 7 8
0.101+2
0.10U2

U*0 9
9 .2 0
10.01;
13.22
11;.26
l l ;.9 6
1U *95
1 2 .9 1
H .8 9

D e te c to r removed and a l t e r e d — changed re s p o n s e below t h i s lin e *
B ip h e n y l
T rip h e n y lm e th a n e
T r ip h enylm e th a n e
T rip h e n y lm e th a n e
T rip h e n y lm e th a n e
T rip h e n y lm e th a n e
T rip h e n y lm e th a n e
T rip h e n y lm e th a n e
T rip h e n y lm e th a n e
H exam ethylhenzene
H ex am ethylbenzene

O.OU312
0.02081;
0 . Ol;l68
0.0 6 2 ^2
0 .08336
0 .0 8 3 3 6
0 .08336
0.1Gl;2
G.10l;2
0.0613 8
0.1023

6 .5U
U *09
6.21;
7 *78
7 *27
7*98
7*83
9*32
8 .7 9
5*52
8.1;8

A l l d a t a i n t h i s t a b l e w ere o b ta in e d a t 33*5°C*> u s in g cy clo h ex an e (95
p e r c e n t , v . / v . ) and a c e to n e (5 p e r c e n t , v . / y . ) s o lv e n t- a lu m in a s o r b e n t,
w ith an a p p lie d b r id g e v o l t a g e o f 6 v o l t s .

D ata w ere o b ta in e d i n p a r t

w i t h r °» r °« 2000 ohms, e 0 « - 2 8 .6 rav., i n p a r t w ith r s °« 1929 ohms,
s
r
r r °» 1999 ohms, e G « 0 , and i n p a r t w ith r s °= 19U9 ohms, r r °=* 2000 ohms,
ec » 0 .

T he^flow r a t e , F , was 5 m l./d a y i n th e sam ple column and ab o u t

2 ml . / d a y , i n t h e r e f e r e n c e colum n.

90

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