IN D U S T R IA L aiENCINEERIMi C H E M IS T R Y
Vol. 30, C onsecutivo N o. 11
ANALYTICAL EDITION
20,300 Copies of This Issue Printed
March 15, 1938
Harrison E. Howe, Editor
Vol. 10, No. 3
T h e U l t r a c e n t r i f u g e a n d I t s F i e l d o p R e s e a r c h . .
...The Svedberg 113
D i s c u s s i o n ...Elmer O. Kraemer 128
D i s c u s s i o n ...HughS. Taylor 129
P o t e n t i o m e t r i c T i t r a t i o n i n N o n a q u e o u s S o l u t i o n s
... A. E. Ruehle 130
An a l y s i s o p Ma p l e Pr o d u c t s...
... Ira E. Puddington and J. F. Snell 132
R a p i d C o l o r i m e t r i c D e t e r m i n a t i o n o f L e a d i n M a p l e S i r u p ...J. L . Perlman 134
C o l o r i m e t r i c D e t e r m i n a t i o n o f I r o n w i t h S a l i c y l i c A c i d . S p e c t r o p h o t o m e t r i c S t u d y . . J. P . Mehlig 136
P l a t i n i z e d S i l i c a G e l a s C a t a l y s t f o r G a s A n a l y s i s .
... Kenneth A . Kobe and Walter I . Barnet 139
De t e r m i n a t i o n o f Un s a t u r a t i o n i n Or g a n i c Co m
p o u n d sb y Me a n s o f Me r c u r y- Ca t a l y z e d Re a c t i o n w i t h St a n d a r d Br o m a t e- Br o m i d e...
... Howard J. Lucas and David Pressman 140
Di r e c t De t e r m i n a t i o n o f Av a i l a b l e P 20 5 Co n t e n t o f Fe r t i l i z e r s...
. . . W. H. M aclntire, W. M. Shaw, and L. J. Hardin 143
Pr o x i m a t e An a l y s i s o f Ga s o l i n e ...
Charles L. Thomas, Herman S. Bloch, and James Hoekstra 153
C h e m i c a l a n d X - RayD i f f r a c t i o n S t u d i e s o f C a l c i u m Ph o s p h a t e s...
Harold C. Hodge, Marian L.LeFevre, and William F. Bale 156
' I \
A n a l y s i s o f C a u s t i c L i q u o r s f o r T r a c e s o f I m p u r i t i e s ... O . S . Duffendack and R . A . Wolfe 161
M e l t i n g P o i n t D e t e r m i n a t i o n s u n d e r M e r c u r y . .
... Myron A . Coler 164
P h o t o e l e c t r i c R e l a y U n i t . . . George W . Josten 165
Me l t i n g Po i n t St u d i e s o f Bi n a r y a n d Tr i n a r y Mi x t u r e s o f Co m m e r c i a l Wa x e s...
. . . J. R. Koch, George J. Hable, and Lewis Wrangell 166
O n e - P i e c e S t a n d a r d P i p e T e e P i e z o m e t e r R i n g . .
... Frank C. Vilbrandt 168
Hi g h- Va c u u m Ga s- An a l y s i s Ap p a r a t u s... ...
... Edward C. Ward 169
Mo d e r n La b o r a t o r i e s:
M i c r o c h e m i c a l L a b o r a t o r y o f A m e r i c a n M e d i c a l A s s o c i a t i o n . . J. B . Peterson and E. W. Schoeffel 172
T h e A m e r ic a n C h e m ic a l S o c ie ty a s s u m e s n o r e s p o n s ib ility fo r th e s ta t e m e n ts a n d o p in io n s a d v a n c e d b y c o n t r ib u to r s to its p u b lic a tio n s .
P u b lic a t io n O ffice* E a s t o n , I*a.
E d it o r ia l O ffic o : R o o m 7 0 6 , M i lls B u i l d i n g , W a s h i n g t o n , D . C . A d v e r t is in g D e p a r t m e n t ! 3 32 W e s t 4 2 n d S t r e e t , N o w Y o r k , N . Y . T e l e p h o n e : N a t i o n a l 0 8 1 0 . C a b le : J ie c h e m ( W a s h in g t o n ) T e l e p h o n e : B r y a n t 9 -4 4 3 0
P u b lis h e d b y t h e A m e r ic a n C h e m ic a l S o c ie ty , P u b lic a tio n O ffice, 2 0 th &
N o r t h a m p t o n S ts ., E a s t o n , P a . E n t e r e d a s s e c o n d -c la s s m a t t e r a t th e P o s t O ffice a t S S a s to n , P a ., u n d e r th e A c t of M a r c h 3 , 1879, as 4 8 tim e s a y e a r.
I n d u s t r i a l E d i t i o n m o n t h ly o n t h e 1 s t; A n a ly tic a l E d i tio n m o n th ly o n th e 1 5 th ; N e w s E d i tio n o n t h e 1 0 th a n d 2 0 th . A c c e p ta n c e fo r m a ilin g a t s p e c ia l r a t e o f p o s ta g e p r o v id e d f o r in S e c tio n 1103, A c t of O c to b e r 3 , 1917, a u t h o r iz e d J u l y 13, 1918.
A n n u a l s u b s c r ip tio n r a t e s : In d u s t r i a la n d En g i n e e r i n g Ch e m i s t r tco m p le te $ 6 .0 0 ; (a ) In d u s t r i a l Ed i t i o n$ 3 .0 0 ; (6) An a l y t i c a l Ed i t i o n S 2 .5 0 ; (c)
Ne w s Ed i t i o n $ 1 .5 0 ; (a ) a n d (6) to g e th e r , $ 5 .0 0 ; F o r e ig n p o s ta g e to c o u n tr ie s n o t in t h e P a n A m e ric a n U n io n , $ 2 .4 0 , (a ) $ 1 .2 0 ; (6) $ 0 .6 0 ; (c) $ 0 .6 0 C a n a d ia n p o s ta g e o n e - th ir d th e s e r a t e s . S in g le co p ie s : (a ) $ 0 .7 5 ; (6) $ 0 .5 0 ; (c) $ 0 .1 0 . S p e c ia l r a t e s t o m e m b e rs .
C la im s fo r co p ies lo s t in m a ils to b e h o n o r e d m u s t b e re c e iv e d w ith in 60 d a y s of d a t e o f is s u e a n d b a s e d o n r e a s o n s o th e r t h a n “ m iss in g f r o m files.”
T e n d a y s ’ a d v a n c e n o tic e o f c h a n g e of a d d r e s s is r e q u ir e d . A d d r e s s C h a r le s L . P a r s o n s , B u s in e s s M a n a g e r , M ills B u ild in g , W a s h in g to n , D . C ., U . S. A.
4 IND U STR IA L AND E N G IN E E R IN G CHEM ISTRY VOL. 10, NO. 3
T H E R M A L R E S I S T A N T L A B O R A T O R Y W A R E S
V i t r e o s i l . . . .
( f u s e d p u r e s i l i c a a n d q u a r t z ) . S u p r e m e i n i t s f i e l d f o r o v e r t w e n t y - f i v e y e a r s . M a x i m u m r e s i s t a n c e to a c i d s a n d t h e r m a l s h o c k . U s e f u l a t t e m p e r a t u r e s u p to 1 0 0 0 to 1 1 0 0 ° C .
T h e r m a l
Alumina Ware
( 9 9 . 9 % p u r e ) . A n e w p r o d u c t o f p u r e c r y s t a l l i z e d a l u m i n a .
H i g h l y r e s i s t a n t to m e t a l s , f l u x e s , e t c .
F o r w o r k i n g t e m p e r a t u r e s u p to 1 9 5 0 ° C .
O b t a i n a b l e f r o m A l l D e a l e r s o r f r o m t h e M a n u f a c t u r e r s
T HE T H E R M A L S Y N D I C A T E L T D.
14 EAST 46th ST... NEW YORK, N. Y.
M ARCH 15, 1938 ANALYTICAL E D IT IO N 5
M o r e A c c u r a t e
—M o r e R a p i d — M o r e R e l i a b l e Than A
C o l o r i m e t e r
* s» » .
C E N C O - S H E A R D
-S A N F O R D
P H O T E L O M E T E R
(Patent N o s. 2 ,0 5 1 ,3 1 7 and 2 ,0 5 1 ,3 2 0 )
P
H O T E L O M E T R I C m e th o d s o f a n a ly sis re p la c e m a n y o f th e u su a l c o lo rim e tric m e th o d s w ith s lig h t m o d ific a tio n s in p ro c e d u re . N o c o m p a riso n s ta n d a r d s a re n e e d e d . A sin g le c a lib r a tio n for a n y p a r tic u la r u n k n o w n h o ld s in d e fin ite ly . S u c n sim p lific a tio n in la b o r a to r y p r a c tic e g r e a tly f a c ilita te s th e w o rk a n d sa v e s m u c h tim e in im p o r ta n t a n a ly se s. F o r ex a m p le , in th e c o lo rim e tric d e te r m in a tio n o f m o ly b d e n u m in ste e l, p r e p a r a tio n o f a n ew refe re n c e sa m p le is r e q u ir e d e v e ry few h o u rs ; w ith th e “ P h o te lo - m e te r ,” a sin g le c a lib r a tio n h o ld s in d e fin ite ly .PRESENT ANALYTICAL APPLICATIONS FOR “ PHOTELOMETER”
Food Analysis L e a d in m ic r o g r a m s C o p p e r
I r o n C a r o te n e F la v in
Steel Analysis M o ly b d e n u m M a n g a n e se C o p p e r C h r o m iu m T ita n iu m V a n a d iu m
W ater Analysis A m m o n ia M a g n e s iu m C a lc iu m S u lp h a te s N itr a te s N itr ite s A lu m in u m C h lo rin e
Miscellaneous C h r o m e P la tin g
S o lu tio n s S u lp h ite L iq u o r s T a n n in L iq u o r s S o d iu m C h r o m a te S o d iu m D ic h r o m a te
For Full In form ation , Ask For B u lletin No. 104F
C H I C A G O 1 7 0 0 Irvins P k . Blvd.
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INSTRUMENTS I P LABORATORY
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B O S T O N 7 9 A m herst St.
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6 IND U STR IA L AND E N G IN E ER IN G CHEM ISTRY VOL. 10, NO. 3
Ju st Published
M O D E R N M E T H O D S OF R E F I N I N G
LUBRI CATI NG OI L S
V lad imir A Kalichevsky
Research and D e v e lo p m e n t L ab o ra to rie s , S o c o n y - V a c u u m C o r p o r a t io n and C o - A u t h o r o f C h em ica l Refining o f Petroleum
A . C . S. M onograph N o . 76
T
I h elast few years witnessed the introduction of new revolutionary methods in petroleum refining which include the deasphalting, solvent dewaxing and selec
tive solvent refining processes, and the use of different inhibitors and additives for raising the standards of the finished products well above the limits which could be obtained by the old conventional refining methods.
This new refinery technique is exposed for the first time in a clear and concise manner showing the logical structure of the modern refinery operations and the applicability of the individual processes to the general scheme of refining. Problems connected with everyday refinery practice as well as those of particular interest to research chemists or plant executives are discussed in detail thus giving an invaluable assistance to all persons directly or remotely connected with the actual refinery operations. The book is an outcome of a sys
tematic study by the author of the new refining meth
ods with which he was connected from the early stages of their development.
The book also contains a large amount of informa
tion which appears for the first time in print and which fills important omissions in the present-day literature in this field.
An extraordinarily complete bibliography and patent references are given, which will facilitate research in the original literature.
This book will be of great value to petroleum refiners, chemists, petroleum technologists, research workers, chemical engineering students interested in the chem
istry and refining of petroleum, manufacturing chem
ists using petroleum derivatives, and patent attorneys requiring a survey of patents on the subjects discussed.
2 4 0 Pages Illustrated
REINHOLD PUBLISHING CORPORATION
C H A T T E R H E A D IN G S Preface.
Properties o f Refined Oils.
Modern Refining M ethods.
Petroleum Waxes.
Dewaxing w ith Filter-aids.
Solvent Dewaxing.
Rem oval o f Oil from Petroleum Waxes.
A sphaltic Substances.
Conventional Deasphalting Methods.
Deasphalting w ith Solvents.
General Principles o f Solvent Refining Processes.
Variables Involved in Refining Oils w ith Selective Solvents.
Single Solvents.
Mixed Solvents.
Double Solvents.
Disposal o f Solvent Extracts.
Pour Point Depressants.
Viscosity Index Im provers.
Oiliness Carriers.
Oxidation Inhibitors.
Fluorescence.
Appendix (Miscellaneous T ables)
Patent Index— A u th or Index—
Su b ject Index
$ 6.00
M ARCH 15, 1938 ANALYTICAL E D IT IO N 7
S E T T I N G T H E P A C E I N C H E M I C A L P U R I T Y S I N C E 1 8 8 2 “ T J . .
X I TW K z a q j e s ñ r
B a k e r & A d a m s o n
Division of G EN ER A L CH EM ICAL COMPANY, 40 Rector St., N ew York
C.VA cú / s
ATIANTA • tAlTlMOU • »OSTON * KtffAlO • CHAJUOTTÏ • CHICAGO • ClfVbANO • 0CNVI» ■ KANSAS CITY • IOS ANCtUS • MWNfAKMJS • PXlA DitmA • MTSłUłOH • riO W KNCI • SAN »AMOSCO • ST. IOWS
J u s t where electricity’s benefits begin, or where they will end, would be hard to say. It is safe to predict, however, that the electrical marvels of today will appear but crude beginnings when compared to the developments of tomorrow.
Few industries can boast of such far reaching achieve
ments as those accomplished by the electrical industry . . . fewer still can point to a comparable rapidity of growth.
In the early days of Edison, Westinghouse, Marconi, some of the great electrical discoveries were necessarily a matter of chance because of the lack of accumulated research ma
terial. But once these men gave the industry its start, prog
ress became increasingly determined by research initiative.
In carrying out its carefully planned research programs, the demand on the electrical research worker for new materials is a constant one. His
laboratories must produce new dielectric materials . . . new metallurgical combinations . . . new equipment for ventures into the realms of ultra high frequencies.
To produce these materials the research worker must have only the most precise testing equipment at his finger
tips. His electrical instruments must be capable of measur
ing values infinitesimally small. His reagent chemicals must be absolutely dependable and be adapted to the most exacting kinds of work.
Where reagent chemicals are called into play in the electrical laboratory, the engineer or chemist who uses them will find that B & A Reagent Chemicals meet all requirements for high testing standards. These reagents are outstanding for their consistent quality and their con
formance to the specifications that appear on the Baker and Adamson Product label.
MARCH 15, 1938 ANALYTICAL E D IT IO N 9
KIMBLE GLASS C O M P A N Y v i n e l a n d , n . j .
n e w
Y O R K •• C H I C A G O •• P H I L A D E L P H I A •• D E T R O I T • • B O S T O N
T h r o u g h t h e p o w e r o f e x p lo s iv e s , im p a s s a b le m o u n ta in r a n g e s b o w t o t h e e n g in e e r ’s c o m m a n d — r a ilro a d s , b r id g e s a n d d a m s f o llo w in t h e w a k e o f t h e s e m a n -m a d e e a r th q u a k e s . C o n s t r u c t i o n r id e s o n a p a c e e a c h y e a r as o v e r 3 0 0 ,0 0 0 ,0 0 0 p o u n d s o f d y n a m ite , n itr o g ly c e r in e , b la s tin g p o w d e r a n d o t h e r p e r m is s ib le e x p lo s iv e s c a r v e t h e w a y . In 1 9 3 5 o v e r $ 4 0 ,6 6 7 ,0 0 0 w o r t h o f t h e s e p r o d u c t s w e r e c o n s u m e d b y t h e q u a r r y , m in in g a n d c o n s t r u c t i o n in d u s tr ie s . T h e e x a c t in g q u a l ity c o n t r o l o f th is v io le n t b u t u s e fu l p o w e r d e p e n d s g r e a tly o n K im b le B lue L in e E x ax . In t h e g r e a t in d u s t r ia l a n d r e s e a r c h c e n te r s — in c h e m ic a l p la n ts , c lin ic a l a n d m e d ic a l i n s t i t u t i o n s — B lue L in e E x a x h a s p la c e d c o n t r o l , t e s t a n d a n a ly s is o n a h ig h e r p la n e o f a c c u r a c y a n d A S S U R A N C E . T h e e a s y - to - r e a d , b r illia n t B L U E f u s e d - in c a lib r a tio n s , a n d t h e h ig h ly a n n e a le d ( s tr a in - f r e e ) c o n s t r u c t i o n o f e s ta b l is h e d s t a n d a r d o f in d u s t r y a n d s c ie n c e — f o r s a f e ty a n d s e rv ic e .
» » » T h e V i s i b l e G u a r a n t e e o f I n v i s i b l e Q u a l i t y • • • Stocked by leading Laboratory Supply Houses throughout the U. S. and Canada
10 IND U STR IA L AND E N G IN E E R IN G CHEM ISTRY VOL. 10, NO. 3
ARTHUR H. T H O M A S COMPANY
R E T A IL — W H O LE SA LE— E X P O R T
LA B O R A TO R Y A PPA RA TU S AND R EA G EN TS
W E ST W ASHINGTON SQ U ARE P H IL A D E LP H IA . U .S.A .
Cable Address, “ Balance,” Philadelphia
T W O N E W M O D E L S
B EC K M A N G LA SS ELEC TR O D E pH M E T E R
LABORATORY MODEL
For precise m easu rem en ts in th e laboratory
Similar to the original model b u t with the following improvements:
Combination pH and millivolt scale, permitting oxidation- reduction potential measurem ents
New type, factory filled and sealed glass electrode and charged calomel electrode
A portable, self-contained, direct reading instrument, suitable for practically all substances, regardless of color, suspended solids, col
loids, and oxidizing or reducing agents, requiring only 2 to 3 ml of solution.
A switch shifts the calibration from pH units to millivolts. Range pH 0 to pH 13, or —1300 to +1300 millivolts. The instrum ent is sensitive to 0.01 pH and, in general, a practical accuracy of ±0.05 pH can be expected over the range to pH 9.5.
The condition of balance or unbalance is continuously indicated, per
mitting rapid measurements. Maximum sensitivity occurs a t the bal
ance point, with automatic sensitivity reduction as unbalance increases.
Controls require only a few seconds for adjustm ent. Built-in tem
perature compensator from 10 to 40 °C and asymmetry potential ad
justor are provided. In shielded, hardwood carrying case 11 X 9 X 8 inches, total weight 19‘A lbs.
4 9 1 9 . G la s s E le c tr o d e p H M e te r , B e c k m a n N e w L a b o r a to r y M o d e l G , a s a b o v e d e s c r ib e d , w ith n e w t y p e s e a le d e le c tr o d e s a n d 6 m l g la s s b e a k e r , c o m
p le te in c a r r y in g c a s e $ 1 9 5 .0 0
C o d e W o r d ...F abob
Descriptive pam phlet sent upon request.
INDUSTRIAL MODEL For fa c to ry , field an d laboratory use
A new, portable, self-contained instrum ent of rugged con
struction, without slide wire potentiometer, for continuous pH readings.
W ith new type, factory filled, heavy wall, internally shielded, glass electrode and companion calomel electrode, both charged ready for use. Operates upon a new vacuum tube voltmeter circuit.
The scale reads from pH 0 to pH 14 in 0.1 pH divisions, with double graduations of pH 0 to pH 7 and pH 7 to pH 14, the acid or alkaline range being selected by a switch. The instrum ent is adjusted for normal operation a t 25 °C and a control is provided for the compensation of asymmetry potential.
The electrodes are 5 inches long and are mounted on a support rod in the com partm ent a t the right of the carrying case and can be raised and lowered by pressing a release button in the side of the holder. A 50 ml Pyrex beaker is used as a container for the test solution and readings can be made with as little as 1 0 ml of sample.
The instrum ent is entirely self-contained, with necessary dry cells for operation, in case 1 3 V j X 9‘/< X 9 inches, weight 2 3 lbs.
4918-R. Glass Electrode pH M eter, Beckman Industrial Model, as above described, with shielded electrodes, 50 ml Pyrex beaker, 1 0 0 ml bottle of saturated KC1 solution and 480 ml of Buffer mixture; complete in carrying case... S150.00 Code W ord... Fabnt
Descriptive pam phlet sent upon request.
INDUSTRIAL «»u ENGINEERING CHEMISTRY
A N A L Y T IC A L E D IT IO N ♦ H a r r is o n E . H o w e , E d ito r
T he U ltracentrifuge and Its F ield o f R esearch
T IIE SVEDBEKG, U niversity o f U psala, Sw eden
C
OLLOIDS and high-m olecular substances are th e building stones of th e cells and th e various tissues of the organism s. Life is n o t possible w ithout these substances, be
cause no o ther m aterial lends itself to th e manifold perform ances here required. N o other m aterial offers such a w ealth of varieties, such pliable forms. Accordingly th e biological and medical sciences which aim a t an elucidation of th e processes taking place in living beings are interested in th e properties of high-m olecular substances. Only by a detailed stu d y of the behavior of these com pounds will it be possible to find proper remedies in th e case of disturbances. I t is obvious, there
fore, th a t our bodily welfare is highly dependent on our knowledge of th e properties of th e macromolecules of high- m olecular com pounds. Illness and d eath cannot be fought successfully if we do n o t know th e chem istry and physics of th e high-m olecular m aterial of our own body.
W hen hum an beings began to im prove th eir conditions by m aking weapons, various dom estic appliances, and clothing th e y had to borrow from anim als and plants to supplem ent w h at t h e i r o w n b o d ie s
l a c k e d . Clubs of wood were used as s u b s t i t u t e s for th e h eavy paw of the lion an d th e bear, th e hide of cow or sheep was sw ept around th e body to pro
te c t th e th in hum an skin.
T h u s was laid the founda
tion for th e creation of th e superbeing which m odern m an together w ith all his t e c h n i c a l facilities repre
sents. T he hum an facul
t i e s w e r e extended until now adays m an is a brain in th e center of a super
body.
T he craftsm an o f o ld tim es h ad to use w h at his fellow creatures, th e anim als and plan ts, m ade. In our d a y s powerful industries are busy producing a rti
ficial su b stitu te s for m any o f t h e n a t u r a l m aterials.
P aper m akes papyrus and vellum superfluous, artificial silk m akes it unnecessary to cultivate th e silkworm, syn
thetic rubber makes us inde
pendent of th e rubber plants, etc. N o t only su b stitu te s b u t en
tirely new products, such as cellulose derivatives, artificial res
ins, and other synth etic polym erization products are finding extensive use in th e service of m odern m an. In all these cases we are dealing w ith high-molecular substances and colloids. I t does n o t take deep thinking to conclude th a t m any indispensable articles of our daily life would be unsuitable and too expensive, if we did n o t know th e rig h t w ay to produce them , and this, in its tu rn , requires knowledge of high-m olecular com pounds.
T hree hundred years ago th e first Swedish settlers tried to find their living on th e shores of th e Delaw are, using very simple utensils and obtaining th e necessary products from th e cultivation of plants and anim als. In our days th e sam e place is th e site of powerful industries producing in a b e tte r and cheaper w ay m any of the necessities of daily life.
Development o f the U ltracentrifuge
T he realization of th e im p o rta n t role played b y high- m olecular com pounds b o th in th e life of th e organism s and in
m any industrial processes has aw akened a lively in
terest in system s of this kind and a num ber of new m ethods are being applied in their stu d y . One of th e new tools is the ultracen
trifuge.
Before d e s c r i b i n g t h e present forms a few his
torical notes m ay be per
m itted. T he u ltra cen tri
f u g e o r i g i n a t e d in some work on colloids done in U psala ab o u t 1920 concern
in g p a r t i c l e 's i z e in gold sols {68). W e had tried to determ ine d i s t r i b u t i o n
Fi g u r e I s Ro t o r s a n d Ce l l s
U p p e r . F o r c e n tr if u g a l fie ld s u p t o 7 1 0 ,0 0 0 tim e s th e f o rc e o f g r a v i ty . L a r g e s t d ia m e t e r , 1 0 .4 c m .; m e a n a c tiv e r a d i u s , 3 .2 5 c m .;
h e i g h t of c o lu m n of 's o lu tio n , 0 . 8
cm . L o w e r. F o r c e n tr if u g a l fie ld s u p to 3 0 0 ,0 0 0 tim e s t h e fo rc e o f g r a v i ty . L a r g e s t d ia m e t e r , 18 c m .; m e a n a c tiv e r a d i u s , 6 .5 c m .;
h e i g h t of c o lu m n o f s o lu tio n , 1 . 8
cm .
113
114 IND U STR IA L AND E N G IN E E R IN G CHEM ISTRY VOL. 10, NO. 3
Fi g u r e 2 . Di a g r a m m a t i c Re p r e s e n t a t i o n o f Oi l- Tü r b i n e Ul t r a c e n t r i f u g e
curves by recording the settling of th e particles under th e influence of g r a v i t y . Only very coarse-grained sols (down to ab o u t 1 0 0 m \x) could, however, be studied in th a t way. I t was n a tu ra l for m e to tu rn m y a t te ntion to th e possibility of increas
ing th e force acting upon th e particles b y using a centrifugal field instead of th e field of gravity. E arlier atte m p ts in this direction had n o t been very successful, however, an d when I spoke to m y research stu d en ts ab o u t this possibility they were n o t v ery en th u siastic ab o u t it. I t was n o t until I cam e to th e U niversity of Wisconsin as a visiting professor in 1923 th a t I f o u n d i n t e r e s t in t h i s problem.
J . B. Nichols, now a t th e du P o n t E xperim ental S tation in W ilm ington, w a s w i l l i n g to cooperate w ith me.
W e b u ilt a sm all m achine allowing optical observations of sedim entation to be carried o u t during ro tatio n (67).
T he field was n o t m ore th a n ab o u t 1000 tim es grav ity and sedim entation could be followed only for a sh o rt period, ow
ing to convection currents. W e felt confident, however, th a t th e problem could be solved.
A fter m y re tu rn to Upsala, D r. R inde and m yself undertook a system atic stu d y of th e conditions of convection-free sedi
m entation, using fine-grained gold sols as te st objects (69).
In the first place, we found th a t th e sam ple of solution studied m u st be sector-shaped in order to perm it th e molecules to travel along radii and n o t strik e ag ain st th e walls of the vessel enclosing th e sample. Secondly,* th e tem p eratu re of the colum n of liquid has to be k e p t co n stan t both in space and tim e; otherw ise convection currents caused by density differ
ences se t in. W e therefore spun our first rotors in hydrogen a t atm ospheric pressure so as to reduce m aterially th e h ea t caused b y friction against th e surrounding gas.
In 1924 we were able to perform faultless sedim entation , in centrifugal fields 5 0 0 0 tim es th e force of g rav ity an d to m e ą s-'' ure th e size distrib u tio n in gold sols down to th e m ost fine
grained ones. T he nam e ultracentrifuge was proposed for this new research tool. U sing th e sam e a p p a ratu s F ahraeus and I (1 9 2 5 ) succeeded in determ ining th e particle w eight of hemoglobin by m eans of sedim entation m easurem ents (64).
Fi g u r e 3 . Se c t i o n o f Ce l l s U p p e r . C e ll f o r o p ti c a l o b s e r v a tio n s
L o w e r. C e ll fo r s e p a r a t io n u n d e r o p tic a l c o n tr o l
C o n tra ry to expectations, we found th a t th e solution of this protein is m onodisperse and defined by th e environm ent. One is therefore justified in speaking a b o u t its particle m ass as its m olecular weight. T his finding stim ulated in te rest an d one of the m edical foundations in Sweden (Therese och Johan Anderssons M inne) g ranted m e a sum of m oney sufficient for building a high-speed ultracentrifuge to stu d y th e behavior of protein molecules in intense centrifugal fields. Collabora
tion w ith L jungstrom and Lysholm enabled m e to obtain convection-free sedim entation in fields 1 0 0 , 0 0 0 tim es g rav ity in 1926 (66). In th e spring of 1931 fu rth e r im provem ents of th e m achinery accom plished by B ocstad an d m e m ade pos
sible sedim entation m easurem ents a t 2 0 0 , 0 0 0 tim es grav ity (mean radius x = 65 m m .; height of colum n of solution = 12 m m .; 5 4 ,0 0 0 r. p. m ., 51). U sing th e sam e radius an d th e sam e height of colum n of solution, we reached 2 6 0 ,0 0 0 tunes grav ity early in 1932 (61), 3 0 0 ,0 0 0 in th e spring of 1932 (56), and 4 0 0 ,0 0 0 in the spring of 1933 (49, 52, 57).
E ssentially higher fields cannot be utilized w ith rotors of this size because of failure of th e m aterial. I t seem ed of in te re st to try a sm aller ro to r ty p e capable of giving consider
ably higher intensities although a t th e sacrifice of height of column of solution and hom ogeneity of th e centrifugal field.
Reducing the m ean radius from 65 to 36 m m . and th e height of sam ple from 18 to 8 m m ., sedim entation m easurem ents iu fields u p to 6 0 0 ,0 0 0 tim es g rav ity were m ade in th e fall of 1933 (55) and u p to 9 0 0 ,0 0 0 tim es g rav ity in th e sum m er of 1934 (48, 60). T he rotors used in these experim ents exploded, however, after a few runs. A fu rth e r reduction of th e m ean radius to 3 2 .5 m m . and im provem ents in th e construction have m ade it possible to do regular m easurem ents in fields u p to 71 0 ,0 0 0 tim es grav ity (32). T he com parison of m easurem ents m ade in very intense centrifugal fields, using a low column of solution and a sm all m ean radius, w ith m easurem ents m ade in som ew hat less intense fields using a higher sam ple situ ated farth e r from th e center of ro ta tio n has shown th a t th e ac
curacy is m uch b e tte r in th e la tte r case, a t least as far as sedim entation velocity m easurem ents are concerned.
T heoretical considerations (O. Quensel and K . O. Pedersen) and experim ental te sts have show n th a t th e power of the ultracentrifuge to resolve a m ixture of m olecular species is proportional to oi-xh, w here co is th e angular velocity, x th e distance from th e center of rotation, an d h th e height of column of solution (54). T he largest value for this pro d u ct reached so far is 5 .8 3 X 1 0 8 (7 0 ,0 0 0 r. p. m ., h = 1.65 cm.,
I
0
m%
m M
-vMARCH 15, 1938 ANALYTICAL E D IT IO N
Fi g u r e 4 . Ax i a l Se c t i o n o f Oi l- Tu r b i n e Ul t r a c e n t r i f u g e
tion thermocouple, Th,, near the rotor serve for tem
perature control of the centrifuge. A beam of light from a mercury lamp, L, filtered through Lfu L f2, and Lfi, passes the cell, C, in the rotor on its way to the camera. The exposures are timed by means of the electromagnetic shutters, Si and St. For speed meas
urements and speed control a magnetic generator, M, is used in connection with a reed-frequency meter, an oscillograph, or a frequency bridge.
The pressure oil which feeds the turbines is pro
duced by a special oil compressor, cooled and ther
mostated to a suitable temperature before entering the turbine chambers. A system of channels in the heavy steel casing makes it possible to thermostate the centrifuge by means of oil or water circulation.
The lubrication oil for the bearings passes through an oil filter and is controlled by valve Vs. By chang
ing the speed of the motor which drives the com
pressor and by operating valve V3, the pressure of the oil entering the turbines- may be regulated so as to make possible sedimentation measurements a t any desired speed between 5000 and 140,000 r. p. m. The resistance thermometers, Rh, Rh, and Rh, and the manometers, G\, Go, and Gz, enable the operator to con
trol temperature and pressure in various parts of the machinery.
x — 6.58 cm .). F or sta n d ard equipm ent, therefore, a large ro to r is to be preferred.
F rom th e m any different experim ental m achines bu ilt in Upsala, tw o sta n d ard types have been developed (48). The first is adapted for the region 500 to 15,000, and th e other for th e range 15,000 to 750,000 tim es gravity. T he low-speed m achine is driven directly b y a high-frequency m otor and is provided w ith ball bearings. T he ro tatio n takes place in hydrogen a t atm ospheric pressure and the casing is immersed in a w a t e r t h e r m o s t a t . I t is
used for sedim entation equilib
rium m easurem ents in solutions of high-m olecular substances and for sedim entation velocity m eas
urem ents on heavy particles.
O u r h i g h - s p e e d m achine is driven by oil turbines and has w hite-m etal bearings w ith mov
able, dam ped pistons. T he rotor spins in hydrogen a t r e d u c e d pressure. I t is used for velocity m e a s u r e m e n t s in solutions of high-molecular com pounds and for equilibrium m easurem ents on low-molecular substances.
A few details concerning the oil-turbine ultracentrifuge m ay be of interest. T he sam ple to be studied is enclosed in a sector
shaped cell provided w ith plane- parallel q u artz windows (Figures 1 and 3). R ecently a cell ty p e w ith a dividing m em brane in th e middle has been introduced for analytical determ ination of sedim entation.
A detailed section of th e centrifuge proper through th e axis of rotation is given in Figure 4.
Figure 5 gives a view of th e installation showing the camera, th e centrifuge on its foundation, th e oil coolers, and th e h and rails leading to the p it where the m otor, compressor, and filters are located.
R ecently an air-turbine-driven, self-balancing u ltracen tri
fuge has been developed b y Beam s (5) of th e U niversity of Virginia and im proved and ad a p te d to sedim entation m easure
m ents b y B auer (4) and by W yckoff (7) a t the Rockefeller In stitu te in New York. H ere a light D uralum in rotor hangs
The rotor (Figures 1 and 2) of chromium-nickel steel is supported by horizontal bearings, Bi and Bi, and kept in rotation by means of two small twin-oil turbines, T i and 7j, one on each end of the shaft.
Hydrogen is adm itted a t the periph
ery and constantly pumped off so as to maintain a pressure of a b o u t 20 mm. Thermocouples
Thi in the bearings and a radia Fi g u r e 5 . Oi l- Tu r b i n e Ul t r a c e n t r i f u g e In s t a l l a t i o n
116 IND U STR IA L AND E N G IN E E R IN G CH EM ISTR Y VOL. 10, NO. 3
on a th in steel sh a ft and is supported by an air-film bearing.
T he friction, an d consequently th e energy consum ption, are therefore very low. T h e air turbine is sealed off from the vacuum cham ber in w hich th e ro to r moves b y surrounding th e vertical sh a ft w ith an oil-gland-shaped bearing. Beams 1ms fu rth e r succeeded in spinning electrically a hanging D u r
alum in ro to r supported b y an air-film bearing (6). T hese new types of ultracentrifuge prom ise to be of great service in m any cases, although th e resolving power has n o t y e t been pushed to th e height obtainable w ith th e steel ro to r of the oil-turbine ultracentrifuge.
Sedim enta tion
T he process of sedim entation is in m o st cases followed by optical m eans. Two different properties of th e solute m a y be utilized for th e determ ination of th e concentration distribu
tion in th e ro tatin g solution— nam ely, th e light absorption and th e refraction. I n b o th cases th e thickness of th e layer of liquid studied necessitates long-focus lenses in order to avoid parallactic errors. W hen using th e absorption m ethod, photographic exposures of th e sedim enting column are m ade from tim e to tim e by light of a w ave length absorbed by th e solute. T hese pictures are th e n m easured by means of a m icrophotom eter and give th e relation between concentration c and distance x from center of rotatio n . E ac h m olecular species is bro u g h t o u t as a step on th e c-x curve (Figure 6).
T he change in refractive index can be used in various ways.
T he sim plest w ay is to apply th e Toepler schlieren m ethod (80). T he different m olecular species presen t are then re
corded on th e p la te like th e Unes of a m ass spectrum (Fig
ure 7).
T he m ost accurate procedure for obtaining th e real concen
tra tio n d istribution in th e sam ple studied is to ta k e pictures of a finely ruled scale th rough th e sedim enting colum n of solution by light of a w ave length w hich is n o t absorbed (18, 19). B y m easuring th e displacem ent, z, of th e lines, we get th e concentration gradient, dc/dx, as a function of th e dis
tance from th e center of rotatio n . E ach m olecular species is therefore shown as a m axim um on th e z-x curve (Figure 8).
I n m a n y cases, such as antibodies, enzymes, m ixtures of proteins and carbohydrates, it would be of great value if a m echanical division of th e sam ple studied could be accom
plished after a certain tim e of centrifuging and controlled by optical observations. A nalytical determ ination of sedimen
ta tio n would th e n be possible. E xperim ents of this kind can now be perform ed using th e cell w ith p artitio n m em brane show n in F igure 3 (81). Figure 9 dem onstrates th e com plete
ness of th e separation (pneumococcus antibody).
F i g u r e 6. S e d i m e n t a t i o n P i c t u r e s O b t a i n e d b y L i g h t - A b s o r p t i o n M e t h o d ( L e f t ) a n d C u r v e s o p C o n c e n t r a t i o n D i s t r i b u t i o n f o r L i m u l u s H e m o c y a n i n a t p H 6 . 8 ( R i g h t )
( E r i k s s o n - Q u e n s e l )
S e d im e n ta tio n c o n s ta n ts of c o m p o n e n ts , 5 6 .5 X 1 0 “ 13, 3 4 .6 X 1 0 " 13, 16.1 X 1 0 “ 13, a n d 5 .9 X 1 0 “ 1S, C e n tr if u g a l fo rc e 1 20,000 tim e s g r a v i t y . T im e b e
tw e e n e x p o s u re s , 5 m in u te s
M e n is c u s
P r o te i n c o m p o n e n ts
I n d e x
0 0-2 0 4 0 6 0-8 10 1-2
Distance from Meniscus (x), cm.
In d e x
F i g u r e 7. S e d i m e n t a t i o n P i c t u r e s f o r L i m u l u s H e m o c y a n i n ( E r i k s s o n - Q u e n s e l )
O b ta i n e d b y T o e p le r s c h lifr e n m e th o d a t p H C.8, s h o w in g th e f a s t e s t th r e e s e d im e n tin g c o m p o n e n ts of a =» 5 6 .5 X 10 “ u , 3 4 .6 X 1 0 " 13, a n d 16.1 X 1 0 -13. C e n t r if u g a l f o r c e 1 2 0 ,0 0 0
tim e s g r a v i t y . T im e b e tw e e n e x p o s u r e s , 5 m in u te s
U ltraeenlrifuge M easurem ents
Two kinds of m easurem ents can be m ade by m eans of th e ultracentrifuge. In th e first place, one m ay centrifuge long enough for a sta te of equilibrium to be reached between sedi
m entation an d diffusion. T hen for each m olecular (or p ar
ticle) species th e following form ula is valid (53, 68):
M = 2R T ln m
(1 - F p M ^ - i , » )
where M = m olecular (or particle) weight, R = gas constant, T = absolute tem perature, c = concentration of solute, V = partial specific volum e of solute, p = den sity of solution, x = distance from center of rotation, and to = angular velocity.
I n this w ay one obtains th e m olecular w eight directly, in
dependent of shape or hyd ratio n (22). If several m olecular species are present in th e solution th e m olecular weight values calculated for different distances from th e center of ro tatio n show a m arked drift. Freedom from d rift is a crite
rion of hom ogeneity w ith regard to m olecular weight.
In th e second place one m ay use a centrifugal field strong enough to cause th e molecules or particles to sedim ent w ith m easurable velocity. T his procedure enables us to find how m any different kinds of molecules are present in th e solution.
If th e sedim entation velocity is referred to u n it field an d w ater of 20° C. as solvent, it is called th e sedim entation co nstant:
dx/dt , 1 — Fp0
s
= ^ ^ T ^ V - P sec- (2)
M e n is c u s
P r o te i n c o m p o n e n ts
MARCH 15, 1938 ANALYTICAL E D ITIO N 117
Lifrçulus l}err)ocyaoir}
S -34-6
x, cm.
Fi g u r e 8 . Se d i m e n t a t i o n Di a g r a m f o r Li m u l u s He m o c y a n i n ( Pe d e r s e n) O b ta i n e d b y ( I ( r e f r a c t i v e in d e x m e th o d a t p H 6.8, s h o w in g th e s a m e fo u r m a in c o m p o n e n ts as i n F ig u r e 0 a n d a lso a s m a ll a m o u n t o f a f if th . C e n t r if u g a l fo rc e , 1 2 0 ,0 0 0 tim e s g r a v i ty . T im e
a f t e r r e a c h in g f u ll s p e e d , 3 5 m in u te s Fi g u r e 9 .
m i n a t i o n
An a l y t i c a l De t e r- o f Se d i m e n t a t i o n
where 17 and p are th e viscosity and density of the solution,
770 and po th e sam e q u antities for w ater a t 20° C.
B y combining diffusion and sedim entation d a ta th e w eight of th e different m olecular species is calculated according to th e form ula (53, 69)
1 r R T s ro\
M = 5 ( 1 - V p ) ( 3 )
where s = sedim entation constant, and D = diffusion con
sta n t.
T his equation m ay be deduced in such a w ay th a t the in
dependence of M of shape an d hydration becomes evident.
B oth in E q u atio n 3 an d in E q u atio n 1 th e m olar frictional co n stan t is elim inated, in th e first case because two independ
e n t m easurem ents are carried out, one on sedim entation and one on diffusion, and in th e second case because sedim entation and diffusion are responsible for the sta te of equilibrium reached.
S edim entation m easurem ents in th e ultracentrifuge can also be used for th e determ ination of the w eight distribution or size distribution of molecules or particles in a polydisperse m ixture (3, 21, 29, 41, 45, 69). As th e th eo ry is ra th e r com
plicated, we will n o t go into it here.
M e a s u r e m e n t o f D i f - Horse antib°dy 8erTum againat pneu-
m o c o c c u s T y p e 1 p o ly s a c c h a r id e . FUSION. I n Order t o c a lc u - L e f t. C o n t e n t of u p p e r cell c o m -
late m olecular w eight from saccharide. afAlghtaddContent of lower"
sedim entation velocity de- cel1 oom of po”ysacchariede addition term inations, it is neces
sary to have an independ
en t and accurate m easurem ent of the diffusion constant, D . In m any cases only a small am o u n t of substance is available and a m icrom ethod has therefore been worked o u t (18, 20).
The light from a lamp, b, passes filters, c, and a transparent scale, d, on its way to tne diffusion vessel, /, and the camera, n.
A therm ostat ensures constant temperature. A diffusion cell with plane-parallel windows and requiring only about 1 cc. of solution is used (Figures 10 and 11).
B y m eans of a m ovable slide th e solvent can be placed on to p of th e solution. T he change of concentration w ith tim e a t th e boundary between th e two columns of liquid is then followed optically, by m eans of either th e lig h t absorption or the refraction m ethod.
E l e c t r o p h o r e s i s M e a s u r e m e n t s . A s a supplem ent to the stu d y of th e sedim entation of molecules in centrifugal
2 0 4 - 5 cm.-
Fi q u r e 1 0 . Di f f u s i o n Ap p a r a t u s ( La m m)
118 IND U STR IA L AND E N G IN E E R IN G CHEM ISTRY VOL. 10, NO. 3 fields it is of considerable value to be able to investigate their
m igration in electric fields {71, 76).
T he electrophoretic m obility is m easured using th e m oving boundary m ethod. In a U -tube w ith plane-parallel windows th e solution to be studied is placed undern eath th e solvent and an electric potential gradient is created over reversible electrodes. T he m ovem ent of th e boundary is observed by m eans of th e light absorption or one of th e refraction m ethods.
A plot of th e m obility as a function of p H furnishes two im p o rta n t d ata— viz., th e isoelectric point an d th e m obility per pH u n it in th e isoelectric region.
T he general setup for electrophoresis m easurem ents is sim ilar to th a t used for diffusion determ inations. T he m igra
tion a p p a ratu s has recently been very m uch im proved (77).
T he straig h t lim bs of the tu b e are now m ade rectangular in section, thus offering a larger surface for conducting aw ay th e heat. T he front walls are plane-parallel, so as to allow accu
ra te optical observations to be carried out. T he limbs are divided into tw o p a rts w hich on b o th ends are cem ented to precision ground-glass plates. C orresponding plates are also cem ented to ad jacen t to p and bo tto m p a rts of th e U -tube.
This m akes it possible to divide th e colum n of solution after a suitable m igration tim e. In order to minimize th e danger of therm al convection currents and a t th e sam e tim e allow higher voltages to be applied, th e electrophoresis is conducted a t ab o u t 4° C. where w ater has its density m axim um and where th e change of density w ith tem perature, therefore, is zero. A fu rth e r fea
tu re of considerable im portance f o r t h e analysis of m ixtures consists in giving the w h o le c o l u m n of l i q u i d in which the electrophoretic m i gration takes p l a c e a constant m otion so a s to p r e v e n t t h e b o u n d a r i e s f r o m m oving o u t from the straig h t lim bs of the U -tube in long-tim ed experim ents. To this end a n ebonite cylin
der is slowly l i f t e d o u t o f t h e l i q u i d in one of t h e e l e c t r o d e v e s s e l s b y m e a n s o f c l o c k work.
A p p l i c a t i o n s . T he ultracentrifuge has a wide range of
application. W ith th e aid of this tool m olecular w eight de
term inations have been done from ab o u t 2 0,0 0 0 , 0 0 0 (tobacco mosaic virus) down to ab o u t 40 (lithium chloride). This technic offers th e unique possibility of carrying o u t an analysis of th e various m olecular species or particle sizes presen t in a solution. T he sedim entation co n stan t is a very character
istic m olecular p roperty and, b y m eans of it, it is often possible to follow sensitive aggregation an d dissociation reactions in biological system s. T he com bination of sedim entation equi
librium an d sedim entation velocity m easurem ents allows cer
tain conclusions w ith regard to th e shape of th e molecules or particles. This is often of im portance when investigating high-molecular compounds.
Among th e substances studied so far are proteins, poly
saccharides, polyhydrocarbons, polystyrenes, dyestuffs, and other synthetic organic compounds, as well as inorganic col
loids and inorganic salts.
Results o f Protein Investigations
Some of the m ain results of th e protein investigations car
ried o u t in Upsala m ay be m entioned {48, 50, 52, 54).
A very striking b u t ra th e r unexpected p ro p erty of protein solutions discovered by the ultracentrifugal analysis is th e perfect m olecular hom ogeneity. T his m eans th a t th e solu
tion of a certain protein is either uniform w ith regard to molecu
lar w eight or contains a lim ited num ber of different molecu
lar species, as a rule in equilibrium w ith each other. Change in protein concentration, in pH, or in concentration of o th e r solutes present m ay bring ab o u t dissociation or association.
If th e sedim entation proceeds so quickly th a t no appreci
able diffusion takes place during a run, th e m olecular hom o
geneity can be tested sim ply by studying th e degree of sh arp ness of th e receding boundary (Figures 13 and 14).
In cases where th e sedim entation proceeds more slowly, so th a t noticeable diffusion occurs during a run, th e hom ogeneity can be tested by com paring th e theoretical sedim entation- diffusion curves w ith th e observed ones (Figure 15).
A hom ogeneity te s t m a y also be perform ed b y m eans of sedim entation equilibrium m easurem ents (Figure 16). H ere the m olecular w eight values should be independent of th e distance from center of rotation.
T he dependence of a protein on p H is exemplified by th e sta b ility diagram s of H elix pomatia, H elix arbustorum, H elix nemoralis, and H elix horlensis (Figure 17, 11).
In the case of Helix p o m a t i a a n d Helix nemoralis the protein contains only one com
ponent a t the isoelec
t r i c point, while the hemocyanin of Helix arbustorum and Helix hortensis contains two components in the iso
e l e c t r i c region. On lowering or raising the pH, points are reached w h e r e a very small change in pH causes a great change in t h e molecular state. The o r i g i n a l molecule of Helix pomatia of weight 6.740.000 (s = 98.9 X
1 0 -13) dissociates step
wise into halves (s = 62.0 X 10~13), eighths (s = 16.0 X 10-“ ), and sixteenths (s = 12.1 X 10-” ). The pH-dissociation prod
ucts represent perfectly h o m o g e n e o u s com-
MARCH 15, 1938
M e n is c u s !
ANALYTICAL E D IT IO N 119
P r o te i n
I n d e x
F i g u r e 1 3 . S e d i m e n t a t i o n P i c t u r e s O b t a i n e d b y A b s o r p t i o n M e t h o d ( L e f t ) , a n d C u r v e s o f C o n c e n t r a t i o n D i s t r i
b u t i o n f o r H e l i x H e m o c y a n i n a t pH 5 . 5 ( R i g h t ) M ■» 6 ,7 4 0 ,0 0 0 ; a = 0 8 .9 X 1 0 “ 13. C e n t r if u g a l fo rc e 4 5 ,0 0 0 tim e s g r a v i ty . T im e b e tw e e n e x p o s u re s , 5 m in u te s . S h a r p n e s s of b o u n d a r y a n d s te e p n e s s o f c u r v e s d e m o n s t r a te th e h ig h d e g re e of m o le c u la r h o m o g e n e ity of th is
p r o te in ( E r i k s s o n - Q u e n s e l) . Distance from Meniscus, cm.
ponents. The presence of divalent ions (Ca**, M g * '1) causes a considerable change in the stability diagram of Helix potnalia hemocyanin. Measurements of the Tyndall effect gave the first indication of this interesting phenomenon (9). An analysis by means of the ultracentrifuge (Eriksson-Quensel) has shown th a t upon addition of 0.01 M calcium chloride, the dissociation on the alkaline side of the isoelectric point does not become noticeable until a pH of about 9.5 is reached, where the molecule splits into halves and eighths. W ithout Ca + * the dissociation starts a t pH 7.4.
P r o te i n
I n d e x
F i g u r e 14. S e d i m e n t a t i o n o f H e m o g l o b i n i n C e n t r i f u g a l F i e l d 900,000
T i m e s G r a v i t y ( E r i k s s o n - Q u e n s e l ) T im e b e tw e e n e x p o s u re s , 3 m in u te s
T he reversibility of th e dissociation-association process in
fluenced b y hydrogen-ion concentration is dem onstrated by th e following experim ent (11):
A solution of Helix pomatia hemocyanin a t pH 6 . 8 of sedi
mentation constant 98.9 X 10- 1 3 (molecular weight 6,740,000) was brought to pH 8.0, where it contains three components with the sedimentation constants 98.9 X 10—13, 62.0 X 10”JJ, and 16.0 X 10_13 (molecular weights 6,740,000, 3,370,000, and 842,000). The pH was then changed back to 6 . 8 and a sedi
mentation analysis performed. I t was found th a t all the frag
ments of dissociation had completely united to form the original component of s = 98.9 X 10_15 (molecular weight 6,740,000).
H igh dilution often causes dissociation. T hus, hemoglobin is p a rtly dissociated into half molecules upon dilution (35).
In dilute solutions of thyroglobulin there are present several dissociation products (23). T he addition of an am ino acid or another protein often causes dissociation (33). T hus serum album in m ay be sp lit by adding clupein (Figure 18).
In certain cases even extrem ely sm all am ounts of foreign substances m ay cause dissociation. Thus, th e addition of
0 . 0 0 1 per cent thyroxin gives rise to an appreciable dissocia
tion of thyroglobulin (24).
T he action of a dissociating com pound on a protein is more or less specific. An amino acid w hich acts strongly upon a certain protein m ay have no effect on an o th er protein, and vice versa. T hus arginin plus am m onium chloride dissoci
ates serum album in (Figure 19) b u t n o t Helix hem ocyanin, while lysin plus am m onium chloride splits th e la tte r protein b u t n o t th e form er (10,34). G uanidine chloride affects Helix hem ocyanin very strongly b u t has only a v ery slight effect on serum album in. Clupein splits both, and arginin w ithout am m onium chloride has no effect on either (10, 33, 34).
H igh sa lt concentration m a y cause dissociation or associa
tion. In solutions of thyroglobulin (s = 19.2 X 10-ls , M = 640,000), th e addition of 4 M sodium chloride gives rise to a homogeneous association pro d u ct of s = 196 X 10- l î , corresponding to a m olecular w eight of ab o u t 16,000,000 (23, 24).
M e n is c u s
P r o te i n
I n d e x
Fi g u r e 1 5 .
0 0-2 0 4 0-6 0 8 1 0 1 2
Distance from Meniscus, cm.
Se d i m e n t a t i o n Pi c t u r e s Ob t a i n e d b y Ab s o r p t i o n Me t h o d ( Le f t) a n d Cu r v e s o f Co n c e n t r a t i o n Di s t r i b u t i o n f o r « - La c t a l b u m i n ( Ke k w i c k) ( Ri g h t)
S I : 17 6 0 0 - 8 — 1.9 X 1 0 -13; D = 10.6 X 1 0 “7. O b s e r v e d ( f u ll-d ra w n c u rv e ) a n d th e o r e tic a l (circles) v a lu e s a g re e , s h o w in g t h a t a - l a c ta lb u m i n is h o m o g e n e o u s w ith r e g a r d to m o le c u la r w e ig h t. C e n tr if u g a l fo rc e , 3 1 0 ,0 0 0 tim e s g r a v i ty . T im e b e tw e e n e x p o s u r e s , 4 0 m i n u te s ( P e d e r s e n )
120 IND U STR IA L AND E N G IN E E R IN G CH EM ISTR Y VOL. 10, NO. 3
J _______ l---
8 10 12
H e lix n e m o r a lis H e lix hortensia
Fi g u r e 1 7 . pH — St a b i l i t y Di a g r a m s ( Er i k s s o n- Qu e n s e l)
A detailed stu d y (M cFarlane, Pedersen, an d Tiselius) b rought to light a num ber of new facts. I t was found (25) th a t in con
ce ntrated sera, p a rt of th e globulin molecules dissociated and th a t this effect w as probably due to th e action of serum album in (33, 3/i).
T he effect is different for different species. In F igure 20 sedim entation diagram s for norm al hum an, cow, and horse serum are given.
In diluted condition all three show the m axim a of norm al album in and globulin, th e globulin content decreasing in th e order:
horse, cow, m an. In the undiluted sera th e globulin m axim um is very m uch depressed an d th ere appears in one of th e diagram s (hum an serum ) a new m axim um (the “X - com ponent,” 25) probably corresponding to half or fo u rth molecules of globulin (34).
In th e horse an d cow sera this dissociation pro d u ct is hidden in th e album in maximum.
A com parison of norm al hum an serum w ith pathological sera reveals a num ber of inter
esting differences (Figure 2 1, 26).
R ecently it has been found th a t a protein molecule m ay be split b y the action of ultrasonic waves (8). T hus, Helix hemo- cyanin a t pH 6.2 is p a rtly de
composed into half molecules.
T his process seems to be dif
ferent from th e p H dissocia
tion, in so far as a lowering of p H does n o t cause th e half molecules to unite.
Special Groups of Proteins
T he above survey has aim ed a t giving a general picture of th e physico-chem ical properties of th e protein molecules, espe
cially w ith regard to th e in
fluence of environm ent. In the following, a sh o rt sum m ary of some of th e results obtained in U psala for special groups of pro
teins will be presented.
T he serum proteins are am ong t h o s e which have been m ost fully studied, b u t which still p r e s e n t n o t a b l e difficulties.
E arly sedim entation studies (27) in th e ultracentrifuge showed th a t in dilute norm al serum th ere are two m ain protein con
stitu en ts w ith s = 4.5 an d 7.1 corresponding to th e album in (s = 4.5, D = 6.2, M = 69,000) and globulin (s = 7.1, D = 4.05, M = ab o u t 160,000) frac
tions of th e salting-out process a n d a s m a l l a m o u n t o f a heavier globulin com ponent of s = 18.5. In pathological sera new com ponents often appear side by side w ith th e norm al ones (27).
I I_____
D i s t a n c e jr o m c en ter of rolaHor)
______ I____________ I____________1---L—
5 50 5-60 5-70 5ao 5-90 cm.
F i g u r e 1 6 . R e l a t i o n b e t w e e n M o l e c u l a r W e i g h t a n d D i s t a n c e f r o m C e n t e r o p R o t a t i o n f o r P h y c o e r y t h r i n (M = 290,000) a t pH 6.8 (Er ik s-
s o n- Qu e n s e l)
C o n s ta n c y o f m o le c u la r w e ig h t t h r o u g h o u t t h e w h o le x -re g io n d e m o n s t r a te s h o m o g e n e ity of th i s p r o te in .
7 9
H e lix p o m a tia H e lix a rb u sto ru m
M o l e c u l a r weiqfyt
S e d im e n ta tio n e q u ilib riu m oj P t ) Y C o e r Y i b r m in p h o sphate bujfer o f pH 6 ô w ill) P* Noel a l 2o*c m e a s u r e d a j l e r 10s. I20.I32.I39.15S fjrs .
