University of Southern California Dissertations and Theses

The purification and study of inulin

(USC Thesis Other)

The purification and study of inulin

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content TH E PURIFICATION AND STUDY
O F INULIN
A . Thesigs
P r e se n te d to th e
D epartm ent o f C hem istry
U n iv e r s ity o£ Southern C a lif o r n ia
I n P a r t i a l F u lfilm e n t
o f th e R equirem ents fo r th e D egree
M aster o f S cien ce.
By
A rthur Y /illia m B a r te l 2nd.
June 19&7
UMI Number: EP41484
Alt rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
Published by ProQueSt LLC (2014). Copyright in the Dissertation held by the Author.
Dtesartaisft Pub!
UMI EP41484
Microform Edition © ProQuest LLC.
All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 48106-1346
This thesis, written by
^ ....
under the direction of h± s.- Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements for the degree of
M ASTER OF SCIENCE
Dean
Secretary
Faculty C om m ittee
T able of C o n te n ts
In tr o d u c to r y
I O b ject o f w o r k .................................................... 1
I I G en eral In fo rm a tio n C onaerning I n u lin . . . . 1
A. H i s t o r y .............................................................................1
B . O ccurence . . . . . . . . . . . . . . . 1
C. P r o p e r tie s . . . . * . * .................................. 2
D .> S tr u c tu r e .................................. 6
E. M olecu lar W eight . . . ., . . 1
E xp erim ental. Work
I Method o f P re p a r in g I n u l i n ............................. 9
I I P u r if ic a t io n . . . . ............... . . . . . . . 11
III. M icroscop ic Study • • • • • * * ■ • - * * * * 16
C o n c lu sio n s
I G e n e ra l C o n c lu sio n s . .
21 .
FIGURE PAGE
1 S p h e r o -c r y s ta ls o f I n u lin in D a h lia tu b e r s . . %
2 I n u l i n S p h e rO -c r y s ta ls w i t h i n th e C e lls . . . . 4
3 A G lu c o s a z o n e .......................................................... . . 5,
4a P u r if ie d I n u lin , R e fle c te d L ig h t ................................... 12
4b P u r if ie d I n u lin , T ran sm itted L ig h t .............................. 13
5 Graph on Ashas of I n u l i n ....................................... . . . 1£
6 M u cilagin ou s Background . . . . .......................... . . .lb
7 A g g reg a tes o f I n u l i n .................................................................19
8. Large I n u l i n S p h e ro s . .............................. 20
The P u r i f ic a t Ion. and Study
o f
I n u lin
I n u lin has b een p rep ared in th e C hem ical L ab oratory o f th e
U n iv e r s ity of S o u th e rn C a l i f o r n i a to be used i n th e p r e p a r a t i o n
1 2 " 5
o f le v u lo s e by s e v e r a l w o rk ers. ’ 9 3 The o b je c t o f t h i s r e
se a r c h i s to prep are an in u lin f r e e from o th e r a s s o c ia t e d m atter
and to d eterm in e i f p o s s ib le * th e d if f e r e n c e b etw een in u lin as>
co m m ercia lly produced and th a t p u r if ie d a f t e r many c r y s t a l l i z a
t io n s .
@ Q
I n u lin was d is c o v e r e d in 1804 by Rose,-. - I n u lin l i k e sta r c h *
s e r v e s a s th e r e se r v e m a te r ia l in underground s to r a g e o r g a n s.
However,, i t i s p r e s e n t in c o n s id e r a b le amounts in ab ove-grou nd
~ i
organs and i s s a id to be p r e s e n t even in sm a ll amounts in th e
4
se e d s o f c h ic o r y .
I n u lin fin d s i t s w id e s t d i s t r i b u t io n in th e C om positae
and r e la t e d g ro u p s, where i t w h o lly r e p la c e s s ta r c h and a c
c u m u la te s, d u rin g th e dormant season * in th e rhizom es and
n o d u le s . I n u lin i s found in th e d a h lia to as h ig h a s 42 p er
c e n t o f th e dry m a te r ia l, and to 44 p e r c e n t in In u la h e liu m .^
6
"The d a h l i a i s a h a rd y , tu b e ro u s r o o te d p l a n t in d ig e n o u s to
th e h ig h e r p a r t s of M exico. I t was named in h o n o r of P r o f e s -
V lv ia n , L e v u lo s e . A t h e s i s P r e s e n te d to th e U n iv e r s ity of
S o u th e rn C a l i f o r n i a in June 1 9 2 2 .
B lack* Commercial Value of L e v u lo s e , A T h e sis P r e s e n te d to th e
U n iv e r s ity of S o u th e rn C a l i f o r n i a in June 1927 *
R ie g e r, Com mercial P r e p a r a t io n o f L e v u lo s e , A T h e sis P r e s e n te d
. to th e U n iv e rs ity o f S o u th e rn C a l i f o r n i a in June 1934-•
^ G-rafe and Vauk, B iochem . Z . , 4.3, 4 2 4 , (1912)
5
D ra g e n d o rf, M a t e r i a l i e n zu. e l n e r M onograph!e d es I n u l i n s
( S t . P e t e r s b e r g , 1870]
6 T. H,. B a ile y * The S ta n d a rd C y c lo p e d ia of H o r t i c u l t u r e , v o l . I , 951
2
so r Andreas D ah l, a Sw ed ish p u p il o f L in n a e u s.w I t has a lso ,
b een ob served In o th e r c la s s e s and has been found even in
A lg a e . 1 I t may be p rep ared from th e tu b er o f d a h lia and c h ic o r y
r o o ts and from Jeru salem a r t ic h o k e s .
By r e d is s o lv in g in u lin in w ater a t 6 0 ° - 10° and r e c r y s t a l -
l i z i n g , w h ite , d ou bly r e f r a c t in g s p h e r o c r y s ta ls are o b ta in e d . 2
W inton s t a t e s ,^ th a t a f t e r d r y in g th e d a h lia tu b e r s , th e in u lin
s e p a r a te s in am orphus, g la s s y m asses; ( f i g . 1) i f , however*
th e f r e s h m a te r ia l i s p la c e d in str o n g a lc o h o l, th e in u li n ,
w hich i s in s o lu b le in t h i s l iq u i d , se p a r a te s a s b e a u t if u l a g
g r e g a te s o f s p h e r o c r y s ta ls w h ich g iv e p o la r iz a t io n d r o ss e s
w ith c r o s s e d N i c o ls . A ccord in g to A. V o g l,^ io d in e g reen c o lo r s
th e membrane o f th e parenchyma c e l l s b lu e and th e w a lls o f th e
v e s s e l s g r ee n ; c h lo r z in c io d in e c o lo r s a l l th e membranes b lu e
. 4
and th e w a lls o f th e v e s s e l s g r e e n is h y e llo w , ( f i g . 2) Hoche
s t a t e s th a t t h i s typ e o f in u lin can o n ly be g o t te n when i t i s
h e a te d t o 94° to 95°C , o th e r w ise th a t th e r e i s o b ta in e d a c ic u la r
1
c r y s t a l s . The r e p o r ts o f th e s p e c i f i c r o t a t io n have been g iv e n
a t v a r io u s v a lu e s from -3 1 ° to -4 0 ° .^ T h is l a s t v a lu e was
o b ta in e d a f t e r a l l a s s o c ia t e d m atter h a v in g a low er r o t a t io n was
rem oved.
1 Gzapek* B iochem ie d er P fla n z e n (J e n a , 1 9 1 3 ), v o l . 1 , p . 45b
2 Bechamp, B u l l , s o c . ch im . , ( 3 ) 9 > * 213 (1893)
• z
Winton,, M icroscopy o f T ec h . P ro d u c ts
^ Hoche, Z. V e r. D eu t. Z u c k e rin d ., 1926, 621
^ P rin g sh eim and J . R e i l l y , B e r . , 6 2 , 23Tb (1929)
T a n ret, B u l l , s o c . ch im . , T S T 9 , 2 0 0 * 2 2 7 , 625 (1693)
(a ) S p h e r o -c r y s ta ls o f I n u lin in D a h lia tu h er s a f t e r
tre a tm en t w ith h y d r o c h lo r ic a c id . iR eess).
C D j)) I n d i v id u a l c r y s t a l s more s t r o n g l y m a g n ifie d t h a n (a)
f ig . 3 L
4
I n u lin spherQ:-<2 ;rR atals
f i g . 2
Dean1 and P rin g sh e im 2 v/ere u n a b le to se c u re th e i n u l i n
w ith c o n s t a n t s d e s c r ib e d by T a n r e t. So u n c e r t a i n i s i t s
o c c u rre n c e in i t s v a rio u s c o n d it i o n s , t h a t i t must be s t r o n g
l y d e p e n d e n t on th e se a so n and o t h e r u n c o n tr o l la b l e c irc u m
s t a n c e s . N orm ally i n u l i n i s s a id to show a l e v o - r o t a t i o n o f
a b o u t -3 6 ° and i s n o t red u c ed by F e h lin g s s o l u t i o n . A f t e r
b o i l i n g for tw e n ty - f iv e m in u tes i n w a te r i t w i l l form a g l u -
co sazo n e ( f i g . 3) w ith p h e n y l h y d r a z in e . There i s th u s a
fiS- 3
1 Dean,, Am. Chem. J . , 32, 69 (1904)
^ P rin g sh e im and Aronowsky, B e r . , 54, 1261 (1921)
re sem blance to cane su gar w hich i s known to have in common
th e p a r t ic u la r ly l a b i l e f r u c t o s e s t r u c t u r e . I n u lin i s g en
e r a l l y c o n sid e r e d to be c o n str u c te d s o l e l y o f f r u c to s e u n it s ,^
a lth o u g h i t i s p o s s i b e l th a t th e r e may be a ls o g lu c o s e rem ain
d e r s p r e s e n t.^
I n u lin i s v ery h y g r o sc o p ic and g iv e s up th e w ater w ith
d i f f i c u l t y . When a i r d r ie d i t c o n ta in s 10 p e r c e n t w a te r * w hich
3
co rresp o n d s to one mole o f w a te r . There i s no c o lo r a t io n p r o
duced by io d in e s o l u t i o n . I n u lin i s s o lu b le o n ly to th e e x t e n t
o f 0 .0 1 gram i n 100 m l. b f w a ter a t 0 °G, w h ile th e r e i s s o lu b le
- o 4
36*5 grams in 100 m l. o f w a ter a t 100 C and p r e c i p i t a t e s slo w
l y when a llo w ed to c o o l. In some s o lu tio n s * c r y s t a l l i z a t i o n
d o es n o t tak e p la c e , ev en when "seeded" w ith i n u l i n - s p h e r i t e s .
"As to th e c o n s t i t u t i o n o f i n u li n , c o n c lu s io n s deduced
from s o d iu m -in u lin , are n o t j u s t i f i e d by th a t compound whose
5
c o m p o sitio n has b een s t a t e d to be G^E^O^ *haOH, *HaOH,
or ev en a s C c^H^qO^) ^ *HaOE, ^ d ep en d in g on th e c o n c e n tr a tio n
o f th e a lc o h o l used in th e p r e c ip it a t io n " .. G^E^0^*Ha and a
co rr esp o n d in g p o ta ssiu m d e r iv a t iv e can be form ed in liq u id
8
ammonia.
* * * Haworth and L earn er, J . Chem. S o c . 1928, 619
^ Sch lu b ack and E is n e r , H er.* 6 2 , 149-3 (1929)
3 P rin g sh e im , The Chem. o f th e M onosaccharides and o f th e P o ly s a c c h .
^ B e i l s t e i n » I , 1095
^ K arrer, Staub* and W & lti, He It?. Chim. A c ta * 5* ^30 (1922)
^ P f e i f f e r and T o lle n s , A nn. , 2 1 0 , 285 (1881)
^ P rin g sh eim and Aronaw sky, B e r . , 5 5 , 1414 (1922)
^ L . Schmid and B eak er, B e r ., 1966 (1925)
7
I r v in e 1 was one o f th e f i r s t to d e s c r ib e th e m e th y la tio n
o f in u lin * M eth y la tio n was o b ta in ed up to ab ou t 40 p e r c e n t
m eth oxyl by u s in g d im e th y l su lp h a te and by r e p e a te d su b s e
qu en t m etjsy la tio n w ith m eth yl io d id e and s i l v e r o x id e . I r v i n e 1 s
ex p erim en ts showed th a t th e fr u c to s e in in u lin i s p r e s e n t in
p
th e same u n sta b le -form as in s u c r o s e . Haworth a ls o m eth y l
a te d in u lin in 1 9 2 8 .
By u s in g a c e t i c anh ydride in th e p r e se n c e o f p y r id in e ,
in u lin t r i a c e t a t e i s form ed w hich may be s a p o n ifie d w ith th e
p r o d u c tio n o f I n u lin . T h is in u lin shows th e same X -ray spectra-^
a s th a t o f th e o r ig in a l i n u l i n . A lso t h i s was s u b s ta n tia te d
by th e f a c t th a t th e two a g reed a s to th e k i n e t i c s o f f e r -
4
m en ta tio n by th e same fun gus enzym e.
A number o f d i f f e r e n t r e s u l t s have been o b ta in ed o f th e
m ic e lla r m agnitude o f in u lin in w a te r . T a n ret, D d ll and
L in tn e r , and Brown and M orris,^' a l l o b ta in h ig h v a lu e s w h ile
K ilia n i ob serv ed a m olar w e ig h t o f w hich a g ree
w ith e b u llio s c o p ic and c r y o sc o p ic m easurem ents th a t conform to
l0 6 H100i V 7 ’S
1 I r v in e and S t e e l e , J . Chem. S o c ., 1 1 7 > 1474 (1920)
2
Haworth and L earn er, J . Chem. S o c ., 1928, 619
^ P rin g sh eim and Aronowsky, B e r . . 5 4 , 1281 (1920)
^ P rin g sh eim and Perew osky* Z. p h y s i a l . chem . , 153 > 13& (1926)
Karrer, Elnfuhrung, in d ie Chemie aer polymeren K ohlenhydrate.
( Le i p z i g , 1 9 2 5 ), P2W
^ P rin g sh eim , The Chem. o f th e S a c c h a r id e s (1932)
^ P rin g sh eim and F e lln e r , Ann. , 4 6 2 , 331 (1928)
^ P rin g sh eim and J . R e i l l y , B e r ., 6 1 ,s 2018 (1928)
However, the c o m p le x ity o f in u lin in w ater may be in flu e n c e d
in v a r io u s ways in d e p e n d e n tly o f h y d r o ly tic a c t io n and c o n se
q u en tly th e se v a lu e s can n ot be taken a s a b s o lu t e . Even in b o i l
in g w ater the in u lin p a r t i c l e s sto p t h e ir d is a g g r e g a tio n a t th e
d is a c c h a r o se stag.**' A lso in acetam ide^ and liq u id ammonia^ i t
i s in th e d is a c c h a r o se s t a g e .
The m o lecu la r w e ig h t o f in u lin a s th e t r i a c e t a t e has
been d eterm in ed c r y o s c o p ic a lly in g l a c i a l a c e t i c a c id , p h e n o l,
and n a p h th a le n e . The r e s u l t s le a d one to b e lie v e th a t i t
h
c o n s i s t s o f 9 f r u c to s e rem a in d ers. However, Bergmann has
shown th a t i t i s d im o le c u la r in a c e t i c a c id . In t h i s c o n n e e t-
5
io n I t was p o s s i b l e to g e t th e 9 - f o l d m olecule in a 3 p e r
c e n t s o l u t i o n and th e 2 - f o l d m olecule in a 0 .4 - 1 .6 p e r c e n t
s o l u t i o n .
• * * P rin g sh eim , C hem istry o f th e S a c c h a r id e s (1932)
^ L. Schmid and B e ak e r, B e r ., 58* 1966 (1925)
^ H. R e ih le n and R. T. N e s t l e , B e r ., 59, 1159 (1926)
^ P rin g sh e im and F e l i n e r . Ann. , 46 2 , 231 (1926)
^ H ess and S tah n , Ann. „ 455* 106 (1927)
EXPERMENTAL
S e v e r a l m ethods have been su g g e s te d f o r th e p r e p a r a tio n
1
o f i n u l i n . Thorpe s t a t e s th a t a mash i s made o f th e r ip e tu b e r s ,
a l i t t l e ca lciu m ca rb o n a te i s added, and th e m ixtu re e x tr a c te d
w ith b o i l i n g w a te r . The f i l t r a t e i s fr o z e n and th e sed im en t
w hich se p a r a te s d is s o lv e d in h o t w a ter and a g a in fr o z e n . T anret
a d v is e s p r e c i p i t a t io n w ith Ba(0H )2 » d e c o m p o sitio n o f t h i s p r e
c i p i t a t e w ith C O 2 and su b seq u en t p r e c i p i t a t io n w ith 95$ a lc o h o l.
2
A common method em ployed by K ilia n i to remove i n u li n ,
i s to add powdered c a lc iu m carb on ate to th e f i n e l y ground mat
e r i a l and to b o i l th e m ix tu r e . The albumen i s removed w ith
le a d a c e t a t e and th e in u lin i s se p a r a te d by f r e e z in g . A s s o c ia t
ed r ed u c in g m a tter may be removed by means o f th e barium, compound.
4
A ccord in g to S t e in , th e raw m a te r ia l i s c u t f i n e , th en
steam ed w ith th e a d d it io n o f m ilk o f lim e , p r e s s e d in a h y
d r a u lic p r e s s , and th e j u ic e c l a r i f i e d . The l a t t e r i s n e x t
poured in to a v e v o lv in g b a sk e t where i t i s fr o z e n in t o f l a k e s .
The f la k e s are tr a n s fe r e d to a t o s s in g a p p aratu s and to s s e d
up,, a f t e r w hich th e j u ic e i s c e n tr ifu g e d in th e in u lin c e n t r i
fu g e m ach ine, The i n u li n so produced i s washed and c le a n e d .
B e ils t e in ^ g iv e s th e fo llo w in g d ir e c t io n s f o r p r e p a r in g
in u lin : 1 1 The ground up d a h lia b u lb s are h e a te d to b o i l i n g w ith
* Thorpe, D ic tio n a r y o f A p p lied C h em istry, v o l . I I 53
2 K i l i a n i , A n n ., 2 0 5 , 145 (1880)
^ O ppenheim er, D ie M ethodik d e r Ferm ente ( L e ip z ig , 1 9 2 7 ), P 2 8 8 .
^ S t e in , Chem. A b s. , v o l . I I , p 2463 (1908)
B e i l s t e i n , Handbuoh d e r O rganise he Chem ie, v o l . I , p 1095
10
an e q u a l volume o f w a te r , w ith the a d d itio n o f a l i t t l e CaCQ,,
3
a f t e r w h ich a lc o h o l i s added, w hich rem oves th e in u lin a s a
c lo u d y p r e c i p i t a t e . The p r e c ip it a t e i s f i l t e r e d o f f and brou gh t
in t o a f r e e z in g m ix tu r e . A fte r th aw in g, th e p r e c i p i t a t e i s d i s
s o lv e d in h o t w ater and th e f i l t e r e d s o lu t io n a g a in f r o z e n .
The same p u r i f i c a t i o n i s r e p e a te d a few tim es and th e in u lin
i s th en w ashed, f i r s t w ith v e ry d i l u t e , l a t e r w ith 93$ a lc o h o l.
S tron g m u cila g in o u s p la n t p o r tio n s are cooked up w ith w a ter;
th e d is s o lv e d m u cilage p r e c ip it a t e d w ith le a d a c e t a t e ; th e le a d
removed by H^S; ev a p o ra ted u n t i l a scum form s, and p r e c ip it a t e d
w ith a l c o h o l . ”
R ie g e r 's ^ m ethodsoof p r e p a r in g in u lin in th e la b o r a to r y
a r e : ( 1 ) d i f f u s i o n from th e s l i c e d tu b er s in a d i f f u s i o n b a t
te r y ,, 12) (tr e a tin g th e tu b e r s w ith su p e r -h e a te d steam , and e x
p r e s s io n by h y d r a u lic p r e s s u r e .
The in u lin u sed in t h i s in v e s t ig a t io n was p rep ared by
a c o -w o r k e r . 2 I t had been o b ta in e d by e x tr a c t io n from th e ground
tu b e r s by h o t w ater fo llo w e d by e x p r e s s io n w ith p r e s s u r e , f i l
t r a t i o n , and c l a r i f i c a t i o n w ith c h a r c o a l and d iatom aceou s e a r t h .
The sam ple o b ta in ed had undergone one r e e r y s t a l l i z a t i o n . The
c o lo r was g ra y and had a a sh o f 0 .2 p e r c e n t .
I t was th e pu rpose o f t h i s i n v e s t ig a t io n to d eterm in e to
what e x t e n t fu r th e r p u r i f i c a t i o n m ight be brought a b o u t, and
to stu d y th e v a r io u s p r o d u cts o b ta in e d .
R ie g e r , Com mercial P r e p a r a tio n o f L e v u lo s e , A T h e sis P r e se n te d
to th e U n iv e r s ity o f S ou th ern C a lif o r n ia in June 1 9 3 4 .
o
Holzman, Commercial P r e p a r a tio n o f I n u lin , A T h esis P r e se n te d
to th e U n iv e r s ity o f Sou th ern C a lif o r n ia .
11
The in u lin was r e c r y s t a l l i z e d s i x tim es from d i s t i l l e d
w a te r . The p roced u re was t o ta k e 500 grams o f in u lin and d i s
s o lv e in 2000 m l. o f w a ter a t 95°0* T h is s o lu t io n was a llo w ed
to sta n d f o r tw e n ty -fo u r hours and was th en s t i r r e d . T his s o l
u t io n s o l i d i f i e d to a c o n s is t e n c y o f a s t i f f p a s t e . On s t i r -
f in g i t "became more liq u id in n atu re and th e in u lin s e t t l e d
our le a v in g a su p e rn a ta n t la y e r o f w a te r . I t was th en f i l t e r e d
and sucked dry on a Buchner f u n n e l. T h is in u lin was r e d is s o lv e d
in an e q u a l w e ig h t o f w a ter a t 95°C and a llo w e d to sta n d and
was f i l t e r e d as "before. T h is p roced u re was fo llo w e d f o r fo u r
more tim es w ith a t o t a l o f s i x c r y s t a l l i z a t i o n s .
A fte r th e f i n a l c r y s t a l l i z a t i o n th e in u lin was f i l t e r e d and
th e sem i-d ry cake was broken in t o sm a ll p ie c e s and was a llo w e d to
sta n d in a two l i t e r b eaker to in su r e u n iform h y d ra tio n ." 1 " In
t h i s c o n d itio n , th e m a te r ia l c o n ta in s ab ou t 50 p e r c e n t w a te r .
I t was th en m ixed w ith a 55 p e r c e n t s o lu t io n o f a lc o h o l. A fte r
s e t t l i n g , th e d i l u t e a lc o h o l was f i l t e r e d o f f and 66 p e r c e n t a l
c o h o l s u b s t it u t e d . T h is, in tu r n , was s u c c e s s iv e ly r e p la c e d
by 95 p e r c e n t and a b s o lu te a lc o h o l. I t was fo llo w e d by w ashing
w ith a s o lu t io n c o n ta in in g e q u a l p a r ts o f e th e r and a lc o h o l, and
f i n a l l y w ith d ry e t h e r . T h is r a th e r e la b o r a te method o f d e
h y d r a tio n ap p ears to be n e c e s s a r y in o rd er to o b ta in in u lin a s
a f i n e , w h ite mass o f un iform m icr o sc o p ic a p p ea ra n ce. ( f i g . 4 a ,b )
^ I r v in e and S t e e l e , J . Chem. Soc I* 117# 1474 (1920),
Purified Inulin 60X
R e fle c te d l i g h t . N o tic e
th e sm a ll a g g r e g a te s w ith
no f o r e ig n m a tter p r e s e n t .
f i g . 4h
P u r i f i e d I n u l i n 60X.
T ra n s m itte d l i g h t
14
£ sam ple o f s e v e r a l grams o f t h i s p u r if ie d in u lin was
p la c e d in a P e t r i d is h and c a r e f u l ly w eigh ed in o rd er to d e
term in e th e amount o f w a ter on d e h y d r a tio n . The in u lin was
p la c e d o v e r su lp h u r ic a c id and th e d e s s ic a t o r was e v a c u a te d . The
d e s s ic a t o r was h e a ted in an in c u b a to r a t 50°0 f o r tw e lv e h o u r s.
At th e end o f t h i s p e r io d th e in u lin had tu rn ed to a l i g h t o l i v e -
g r e e n c o lo r . S in c e t h i s u n u su al c o n d itio n had a r is e n i t was
n e c e s s a r y to r e p e a t t h i s d r y in g p r o c e s s , b u t w ith a sam ple o f th e
o r ig i n a l in u lin w h ich had n o t had any o f th e '‘p u r i f i c a t i o n * 1
tr e a tm e n t. I t to o d ev elo p ed t h i s g r e e n c o lo r on sta n d in g o v er
o
n ig h t in th e d e s s ic a t o r a t 50 C.
T hinking th a t p erh ap s th e su lp h u r ic a c id fumes m ight ca u se
th e d evelop em en t o f th e c o lo r , phosphorous p e n to x id e was s u b s t i
tu te d a s a d e s s ic a n t . The d e s s ic a t o r was a g a in e v a cu a te d and
p la c e d in th e in c u b a to r a t 50°C f o r tw e lv e h o u r s . B oth th e sam ple
o f o r ig in a l in u lin and th e p u r if ie d in u lin tu rn ed g r e e n .
Now, two new sam ples o f in u lin were p la c e d in th e d e s s ic a t o r
w h ich c o n ta in e d no d e s s ic a n t and was t r e a te d a s b e f o r e . A gain
b o th sam ples tu rn ed th e same g reen c o lo r .
W ith th e b e l i e f tlxat t h i s b a tc h o f in u lin m ight be contam
in a te d in su ch a way th a t th e im p u r itie s c o u ld n o t be removed
e v en a f t e r r e p e a te d r e c r y s t a l l i z a t i o n s ,, in u lin p rep ared by o th e r
w orkers in p r e v io u s y e a r s was o b ta in ed and was t r e a te d in a l l th e
manners d e s c r ib e d a b o v e . These sam ples a ls o d ev elo p ed th e g r e e n
c o l o r .
A fte r th e g r ee n in u lin had been exp osed to th e a i r f o r
s e v e r a l h o u rs, th e c o lo r changed from g r ee n to v a r io u s sh ad es
15
o f l i g h t to dark brown. The dark brown sam ples th en become a
s t i c k y , syrupy,, m ass, w h ile th e l i g h t e r brown sam ples rem aine
d r y . B oth th e g r ee n and brown v a r i e t i e s o f in u lin have a b i t t e r
t a s t e .
S in ce th e in u lin d e v e lo p ed th e g r ee n c o lo r in th e ab sen ce
o f a i r and th e brown in th e p r e sc e n c e o f a i r , and had a b i t t e r
t a s t e , and s in c e i t d id come in c o n ta c t w ith ir o n in th e f i l t e r
p r e s s , i t appeared th a t th e g r ee n c o lo r m ight be a fe r r o u s ir o n
compound and th e brown a f e r r i c ir o n compound. H owever, on t e s t i n g
f o r fe r r o and f e r r i c ir o n w ith f e r r i - and fe r r o c y a n id e and th e
f e r r i c io n w ith ammonium th io c y a n a te , no ir o n was found to be
p r e s e n t . T h is ir o n was t e s t e d f o r in b o th a s o lu t io n o f th e
in u lin and a ls o in th e a sh o f th e burned i n u l i n .
A h b t s o lu t io n o f in u lin comes in c o n ta c t w ith cop p er d u rin g
i t s e x t r a c t io n from th e tu b er and c o u ld p o s s ib ly p ic k up some
cop p er whose s a l t c o u ld be g r e e n . However,, a l l t e s t s f o r copper
were n e g a t i v e .
To make su re th a t t h i s c o lo r c o u ld n o t be cau sed from a
h eavy m e ta l, a sam ple o f i n u li n was p rep ared d i r e c t l y from th e
w ashed d a h lia tu b e r s . The o n ly m etal to be used th rou gh ou t th e
w hole e x tr a c t io n was a s t a i n l e s s s t e e l s p a tu la u sed to s l i c e th e
t u b e r s . The in u lin o b ta in e d was d r ie d in th e manner p r e v io u s ly
d e s c r ib e d . I t to o d e v e lo p ed th e g r ee n c o lo r .
Tannins c o u ld ca u se th e in u lin to d e v e lo p e i t s b i t t e r t a s t e
and w ith t h i s in mind pure in u lin was prep ared by many r e c r y s
t a l l i z a t i o n s as f i r s t d e s c r ib e d , b u t w ith e v e r y c r y s t a l l i z a t i o n ,
Darko a com m ercial a b s o r b tio n medium, was u s e d . I f any ta n n in s
w ere p r e s e n t th e y sh o u ld have been removed by t h i s p u r i f i c a t i o n
p r o c e s s . When the in u lin was d r ie d a s th e p r e v io u s sam ples had
16
d r ie d , th e in u lin tu rn ed g r e e n .
The a sh o f th e u n p u r ifie d in u lin was 0 .2 p e r c e n t and
droped to l e s s than 0 .1 p e r c e n t a f t e r th e secon d r e c r y s t a l
l i z a t i o n from th e o r ig i n a l in u lin o b ta in e d . The a sh o f th e
p u r if ie d p ro d u ct was 0 .0 4 p e r c e n t .
The s p e c i f i c r o t a t io n o f th e o r ig i n a l in u lin w a s-4 4 * 5 °
and o f th e p u r if ie d p rod u ct,, - 3 8 . 5°
@ ® ®
A m ic r o sc o p ic ex a m in a tio n o f in u lin under v a r io u s c o n d it
io n s and v a r io u s d e g r e e s o f p u r it y in d ic a te d t h a t th e in u lin
s p h e r o c r y s ta l have an average d ia m eter o f a p p ro x im a te ly
QhOOl c e n tim e te r . T h is v a lu e was d e r iv e d by m easuring th e
d ia m e ter s o f many c r y s t a l s th a t had been p h otograp hed through
th e m ic r o sc o p e . The av era g e d ia m e ter as p h otograp hed i s d iv id e d
by th e t o t a l m a g n ific a tio n . 0 .0 0 0 6 cm. was found to be th e
deam eter o f in u lin sp h ero s a s ph otographed by an Argus cam era,
and 0 .0 0 0 8 cm. was th e v a lu e tak en from p h otograp h s tak en w ith
th e C hem istry D ep artm en t's eq u ip m en t.
In o rd er to p h otograp h th e i n u li n , a sm a ll amount o f sam p le,
th e amount c a r r ie d on th e t ip o f a d i s s e c t i n g n e e d le , i s tr a n s -
fe r e d to a g l a s s s l i d e and mixdd in a drop o f a b s o lu te a lc o h o l.
A fte r th e drop has e v a p o r a te d , i t le a v e s an e v e n ly d e p o s ite d
f ilm w h ich may be e a s i l y ob serv ed under th e m ic r o sc o p e .
Pure I n u lin app ears a s s in g le g r a in s or a s a g g r e g a te s o f
two to e ig h t g r a in s to a bunch, ( f i g . 4) On th e o th e r hand,
in u lin th a t has been c r y s t a l l i z e d o n ly once or tw ic e a f t e r e x
t r a c t io n from th e d a h lia tu b e r s ap p ears to have a n o th er su b sta n c e
m ixed w ith th e g r a in s . At t h i s s ta g e i t a ls o has a ten d an cy
0.20
0 1
4 2
3 € 5 8
Humber o f K e e r y s t a lliz a t io n s
Four p a r t s o f w a te r t o
one p a r t o f I n u lin .
B efo re any r e c r y s t a l l i z a t i o n , i n u li n
bad an a sh o f 2 .0 0 p e r e e n t .
t is * 5
to clump I n to much l a r g e r a g g r e g a t e s . F ig u re 6 . shows
c l e a r l y th e g e la ti n o u s m a t e r i a l a s s o c i a t e d w ith th e i n u l i n
s p h e r o s , w h ile f i g u r e 7* shows th e la r g e a g g re g a te s d e v e lo p e d .
M a g n ific a tio n 430X
f i g . 6
I f I n u l i n i s a llo w e d to s ta n d f o r a p e rio d of tim e in a
c o ld s o l u t io n o f a p p ro x im a te ly 0 . 1 N h y d r o c h lo r ic a c id ,, th e
p a r t i c l e s of i n u l i n a p p e a r to s w e ll i n to sp h e ro s many tim e th e
d ia m e te r of th e o r i g i n a l p a r t i c l e s . F ig u re shows th e p a r t -
19
l c l e s w ith th e same m a g n if ic a tio n a s f i g u r e s 5* and. 6 .
M a g n ific a tio n 250X
f i g . 7
20
M a g n ific a tio n 250X
f i g . 8
conclusion;
Commercial in u lin can be p u r if ie d to a fu r th e r d eg ree
sim p ly by r e p e a te d r e c r y s t a l l i z a t i o n from d i s t i l l e d w a te r .
I n u lin h as a d e c id e d f l a t w h ite c o lo r a f t e r th e th ir d
t o fo u r th c r y s t a l l i z a t i o n . At t h i s p e r io d i t no lo n g e r d rys
in th e h ard , se m i-tr a n s p a r e n t, h o r n -lik e m a te r ia l, but d rys
in th e p o w d e r -lik e form . A stu d y o f th e graph on page 17
shows th a t a f t e r th e fo u r th r e c r y s t a l l i z a t i o n th e r e i s no
p e r c e p t ib le change in th e a s h . A lso th e r e i s no r e d u c tio n o f
F e h lin g s s o l u t i o n .
Thus a stan d ard o f p u r it y may be ad op ted : (1) th e compound .
sh o u ld be w h ite , ( 2 ) sh o u ld g iv e l e s s than 0 . 1 p e r c e n t ash*
and (3 ) be f r e e from any a c t io n w ith F e h lin g s s o l u t i o n . An
o th e r stan d ard m ight be added and th a t i s , t h a t th e in u lin
sh o u ld d is p la y a c o n s ta n t s p e c i f i c r o t a t io n o f ab ou t — 38°
on s u c c e s s iv e c r y s t a l l i z a t i o n s from w a te r .
In o rd er to remove a l l t r a c e s o f m ucilagen ou s m a te r ia l
w h ich i s a s s o c ia t e d w ith th e in u lin a t th e b e g in n in g , i t I s
a p p a r e n tly n e c e s s a r y to wash th e in u lin w ith a lc o h o l and f i n a l l y
w ith e t h e r . I t i s o n ly a f t e r th o se f i n a l w ash in gs th a t th e
in u lin e x h ib it s f i n e , n o n -a g g reg a ted c r y s t a l s when o b served
th rough th e m icr o sc o p e .
I t was found th a t a g r ee n c o lo r d ev elo p ed on str o n g d e
h y d r a tio n and su b seq u en t h e a t in g . S in ce no h eavy m eta l c o u ld be
found p r e s e n t in th e i n u l i n , and a s f i n a l p r o o f, in u lin p r e
p ared c o m p le tly in g la ss; tu rn ed g reen on s im ila r trea tm en t*
22
a n o th e r e x p la n a tio n a s to th e cau se o f th e c o lo r had to be
d e v e lo p e d . I n u lin may be h e a te d a t v a r io u s tem p era tu res up
to 70gC and no change in c o lo r i s n o t ic e d . S in c e a d r a s t ic
d e h y d r a tio n must take p la c e b e fo r e h e a tin g to g e t th e c o lo r ,
th e l o s o f w ater from th e in u lin m o lecu le i t s e l f may be th e
ca u se f o r c o lo r d e v e lo p em e n t. A long w ith t h i s l o s s o f w a ter
from th e m o lecu le th e r e w ould be p erh ap s a c h a r r in g w hich
m a n ife s ts i t s e l f in th e g reen c o lo r r a th e r than th e u s u a l brown
o r b la c k c o lo r e x h ib ite d in th e g e n e r a l c h a r r in g o f o rg a n ic
compounds su ch a s su g a rs o r s t a r c h e s . H owever, fu r th e r work
whould be done to d eterm in e th e e x a c t c a u se o f t h i s p e c u lia r
c o lo r phenom ena.
S in ce th e r e i s a m u cila g in o u s m a te r ia l a s s o c ia t e d w ith
in u lin when i t i s in an impure s t a t e w hich can be ob serv ed
th rou gh the m icro sco p e, a m icr o sc o p ic ex a m in a tio n may be used
to d eterm in e th e p u r it y o f i n u l i n . T h is extran u ou s m atter
ap p ears a s a se m i-tr a n s p a r e n t f ilm w ith th e in u lin sp h ero -
c r y s t a l s d is p e r s e d th r o u g h to u t the f i e l d a s a g g r e g a te s , I f ig * 5 )
w h ile pure in u lin ap p ears o n ly as in d iv id u a l s p h e r o -c r y s ta ls
s c a t t e r e d th rou gh ou t th e f i e l d . I f ig * 4b)
BIBLIOGRAPHY
B ioch em iseh e Z e i t s c h r i f t .
M a te r ia lie n zu e in e r M onographie d es I n u l i n s .
The Standard C y clo p ed ia o f H o r t ic u lt u r e .
T h eses P r e se n te d to th e U n iv e r s ity o f Sou th ern C a lif o r n ia .
B ioch em ie d er P fla n z e n , Czopek (J en a , 1913)
B u ll e t i n de l a s o c i e t e chem ique de F ra n ce.
M icroscopy o f T e c h n ic a l P r o d u c ts, W inton.
Z e i t s c h r i f t d e r W ir tsc h sftsg r u p p e Z u c k e r In d u s tr ie .
B e r ic h te d e r d e u tsch en ch em isch en G e s e l ls c h a f t .
Am erican C hem ical J o u r n a l.
J o u rn a l o f the C hem ical S o c ie t y (L on d on ).
The C hem istry o f th e M onosaccharides and o f th e P o ly s a c c h a r id e s
B e i l s t e i n , Handbuch d e r Q rganische Chem ie.
A nnalen d er Chemie,, J u stu s L i e b i g ' s .
Z e i t s c h r i f t fu r p h y s io lo g is c h e Chem ie.
D ic tio n a r y o f A p p lied C hem istry* Thorpe.
C hem ical A b s tr a c ts .

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The purification and conductance of sodium tetradecyl sulfate

PDF

Synthesis and reactivity studies of cyclopentadienylmanganese diphosphine complexes

PDF

Studies of 2-nitrobenzenesulfenyl bromide and 2,4-dinitrobenzenesulfenyl bromide

PDF

Second-order nucleophilic substitution reactions of cyclobutane derivatives: A study of the stability of pentacoordinate carbon

PDF

The acid catalyzed isomerization and dimerization of styrene oxide

PDF

Kinetics of the reductive elimination reaction of trans-bromohydroxytetracyanoplatinate ion with thiosulfate and tetrathionate ions

PDF

Kinetics and mechanism of the substitution of water in aquapentacyanocobaltate (III) by pyridine

PDF

Studies on the determination of thorium in the presence of rare earths

PDF

Studies on the ignition of Lanthanum oxide

PDF

Studies on alpha-naphthylglyoxylyl derivatives

PDF

The stereochemical course of the nucleophilic displacement of cyanide from alpha-ferrocenyl-alpha-aminonitriles, and model experiments for the synthesis of optically active alpha-ferrocenyl alkyl...

PDF

The rate of aquation of the sulfito ligands in disulfitotetracyanocobaltate (III) ion

PDF

The reaction of phenylmagnesium bromide with methyl B-benzoylpropionate and with diethyl succinate

PDF

Tests and properties of crude petroleum emulsions

PDF

Studies in the production of levulose from inulin.

PDF

A kinetic study of the base hydrolysis of chloroamminebis (dimethylglyoximato) cobalt (III)

PDF

Some properties of the tetrakis (ethylenediamine)-u-dioldichromium (III,III) and - Dicobalt (III,III) complex ions

PDF

The chlorinolysis of carbon-sulfur bonds

PDF

The catalytic conversion of parahydrogen by paramagnetic ions in aqueous solution

PDF

The chemistry of 3-methyl-3-nitro-1, 2-butanedicarboxylic acid

Asset Metadata

Creator Bartel, A. W (author)

Core Title The purification and study of inulin

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Degree Program Chemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, organic,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-787075

Unique identifier UC11348125

Identifier EP41484.pdf (filename),usctheses-c17-787075 (legacy record id)

Legacy Identifier EP41484.pdf

Dmrecord 787075

Document Type Thesis

Rights Bartel, A. W.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA