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Some degradation products from humic acid
Item Type text; Thesis-Reproduction (electronic)
Authors Nordby, Harold Edwin, 1931-
Publisher The University of Arizona.
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Download date 28/07/2021 00:47:33
Link to Item http://hdl.handle.net/10150/551382
SOM E D EG R A D A TIO N PR Q D U C T S FROM HUMIC ACID :
' , A T h e s is S u b m itted to th e F a c u lty o f th e
,' i : . “P E P ^ ^ ^ M E N T V O F CHEI^lSTR.Y:
In P a r t ia l F u lf i l lm e n t o f th e R e q u ir e m e n ts ■
; ' : y t
M A ST E R O F SC IE N C E
In th e G raduat e C p ile ge
:: ::;^ ^ ^ r U N W m S I T Y :Q F AR1 e o n a :
195?
STATEM ENT BY AUTHOR
T h is t h e s is has b een su b m itte d in p a r t ia l fu lf il lm e n t o f r e q u ir e m e n ts fo r an a d v a n ced d e g r e e at th e U n iv e r s ity of A r iz o n a and is d e p o s ite d in the U n iv e r s ity L ib r a r y to be m a d e a v a ila b le to b o r r o w e r s u n der r u le s o f the L ib r a r y .
B r ie f q u ota tion s fro m th is th e s is a r e a llo w a b le w ith ou t s p e c ia l p e r m is s io n , p r o v id e d that a c c u r a te a ck n o w led g m en t o f s o u r c e is m a d e . R e q u e sts fo r p e r m is s io n fo r ex ten d ed q u ota tion fro m o r r e p ro d u ctio n of th is m a n u sc r ip t in w h o le o r in p a rt m a y be gra n ted by the h ead of the m a jo r d ep a r tm en t o r th e D ean o f the G raduate C o lle g e w hen in th e ir ju d g m en t th e p r o p o se d u s e o f th e m a te r ia l is in the in te r e s t s of s c h o la r s h ip . In a l l o th er in s ta n c e s , h o w e v e r , p e r m is s io n m u st be o b ta in ed fro m th e a u th or .
SIGNED:
A P P R O V A L BY THESIS D IR EC TO R
T h is th e s is h as b een a p p ro v ed on th e date sh ow n b e lo w :
^ ' S TC o r n e liu s S tee lin k
A s s is ta n t P r o f e s s o r of C h e m is tr yD ate
ACKNOWLEDGMENT
T he au th or w is h e s to e x p r e s s h is s in c e r e g ra titu d e to
D r . C o r n e liu s S te e lin k fo r h is c o n s tr u c t iv e gu id an ce and e n c o u r a g e
m e n t th rou gh ou t th e c o u r s e o f th is r e s e a r c h .
T A B L E O F C O N T E N T S
C H A P T E R : ^ ' ' / . 'P a g e
: 1 I N T R O D U C T I O K / ^ L L / , . ; . V ^ o ^ . % . . o \ o a L . 1
D e fin it io n and o r ig in o f laum ic a c id , , Q , , 1S tr u c tu r e o f h u m ic a c id , , , , , , , , , , , , , » , , , , ,, 4P r o b le m s o f s e p a r a t io n and a n a ly s i s , , , , 8P u r p o s e o f th is in v e s t ig a t io n , ............... . 1 0
II DISCUSSION O P R E SU L T S, \ . . . . . . . 11
P r o to c a te c h u ic a c id and o th er p h e n o lic c o m pounds o- o o :o e* » o » e e o o- o o o e o e e o o o e » o » o e o o o o o o 11
C hrdm atograpfaic and sp ec .tro p h o to m e tr ic ■tecliniiciu.es, , , , ® &, , , * « @ , *, , , , , @ * o ® © , © , , ®« , 1,2
III £/R.Uvi T* -A -X j.o 0 0 0 0 0 0 0 0 , 0, q o o o o o o o o o o o o o o o © o o lb
S o u r c e o f s o il s arnp 1 e o o o , © o d o 0 0 , 0 0 0 0 o o , , , , , © , lbE x tr a c t io n o f h u m ic acid* , * ».» ,» , » , * * * , » , ,» , * , lbA cid h y d r o ly s is , , 18P o ta s s iu m h y d ro x id e fusidri, , . * * & , *»». , ... . 19S o lv en t fr a c t io n a t io n . * * » , . , . . . . , . . . , , . , . . . . ' 20C h r o m a to g ra p h ic a n a ly s i s ............ ' 22S p ec tr o p h o to m e tr ic a n a ly s is . . . . 24Id e n tif ic a t io n o f p r o to c a te c h u ic a c id . . . . . . . . . . . 27Id e n tif ic a t io n o f v a n il l in . . . . . . . . . . . * . , . . . . . . . . , ,30I so la t io n o f a p h lo r o g lu e in o l d e r iv a t iv e . . 31
T V S U M M A R Y . . . . . . . . . . . . . . . . . . . 33
L IT E R A T U R E C I T E D . . . . . . ______ . . . . . 35
A P P E N D I X .. ........ ................. .............................. ..........37
: : i v : ■
I. INTR O D U C TIO N
D e fin it io n and o r ig in o f h u m ic a c id .
H uraic a c id h a s no tr u e defin ition^ T h is su b s ta n c e a lo n g w ith
h u m ic fr a c t io n , s o i l o r g a n ic m a t te r , etc* s h as b een a su b je c t o f s c ie n - .
t i f ic in v e s t ig a t io n fo r o v e r tw o c e n tu r ie s* A lth ou gh r e s e a r c h in th is
f ie ld h a s b een go in g on fo r th is le n g th o f t im e , c o n fu s io n c o n c e r n in g the
n a tu re o f th is 1’com pound*1 s t i l l e x is ts* ‘ One d e fin it io n o f the com pound
i s : th e b la ck a lk a l i - s o lu b le a c id - in s o lu b le m a te r ia l found in th e o r g a n ic
m a tte r o f th e so il* T h is d e f in it io n d o e s n o t h o ld fo r a l l th e s u b s ta n c e s
r e c o r d e d a s h u m ic a c id in the l i t e r a t u r e * U n c e r ta in t ie s in id e n tity
a r i s e out o f th e fa c t th at th e n a tu re o f h u m ic a c id m ay. v a r y w ith th e ty p e
and lo c a t io n o f th e s o i l * S in c e a tr u e d e f in it io n of a su b s ta n c e d ep en d s
on in h e r e n t p r o p e r t ie s and n ot on m eth o d s o f ob ta in in g th e su b s ta n c e ,
w e can n ot a c c e p t th e ab ove d efin ition* A , G ille t (1) h a s d efin ed h u m ic
a c id a s ’’T h at p a r t o f th e m a te r ia l in th e s u r fa c e la y e r w h ic h h a s p r o p
e r t ie s o f f r e e o r g a n ic a c id and w h o s e a lk a li s a lt s a r e w a te r so lu b le* ”
FV S c h e ffe r and E:» W elte (2) h a v e a d v a n ced th e fo llo w in g s c h e m e a s a
s y s te m of c h a r a c te r is in g h u m ic acid*
1
nondark, e o lo r e d (n o n h u m ic} d ark c o lo r e d (hum ic)
H u m ie a c id Humins c o a lF u lv ic a c id s
J„ M . B r e m n e r (3) in a rev iew ' o f s o i l s ta te s th at
th e m o d e r n d e f in it io n o f th e te r m is b ro a d en ed to in c lu d e th e
m a tte r th at i s s o lu b le in a lk a li o r n e u tr a l r e a g e n ts and in s o lu b le in a c id
;©r a lc o h o l. T he te r m " so il" is r a th e r
e v e n b e e n in c lu d ed a s
s o u r c e s o f .h u m ic a c id . U ntil r e c e n t ly the d e f in it io n o f h u m ic a c id w a s
co n fin ed to " so il" h u m ic a c id . H« R au d n itz (4) in a r e c e n t a r t ic le 'g iy e s
o c c u r r e n c e o f h u m ic in j le a v e s o f R hod ed en d ron
p o n tic u m . T h is h u m ic to th e d e fin it io n o f the c o m
is le f t out
a c id is d e fin ed a s th e a lk a li s o lu b le , a c id
organic m atte r 'fdund in
T he fo r m a tio n o f th e h u m ic a c id h a s b een th e su b je c t o f d is c u s s io n
fo r s o m e t im e 0 T h r e e m a in th e o r ie s h a v e b een p o s tu la te d to ex p la in th e
m o d e o f it s fo r m a tio n , ; - „
a„ S y n th e s is by m ic r o o r g a n is m s fr o m lig n in S o u r c e s 0
-b. O x id ation o f c o a l o r l ig n i t e ,
: ' Co S y n th e s is by s o i l m ic r o o r g a n ism s; fr o m ca rb o h y d ra te
s o u r c e s .
T h e o r ie s (a) and (c) h a v e b een a c c e p te d by m a n y in v e s t ig a to r s *
w h ile th e o r y (b) h a s b een p o s tu la te d r e c e n t ly by G ille t ( l ) v L ign in is a
s u b s ta n c e o f h ig h m o le c u la r w e ig h t and unknow n s tr u c tu r e found in th e
w ood o f t r e e s and s o m e p la n ts . T he c a rb o h y d ra te s o u r c e s m a y in c lu d e
c e l lu lo s e and o th e r p o ly s a c c h a r id e s found in p la n ts . S o m e of th e s o i l .
m ic r o o r g a n is m s th at m a y s y n th e s iz e th e h u m ic a c id fr o m th e d e c a y e d
le a v e s and s t e m s o f p la n ts in c lu d e M y c o b a c te r ia c e a e and A c t in o m y c e -
f a e e a e , B r e m n e r (3) h a s re v ie w e d th e o r ie s (a) and (c) an d r e c e n t in v e s -
tig a tio n S h a v e y ie ld e d m o r e e v id e n c e to su b s ta n tia te th e s e t h e o r ie s ,
F la ig and c o -w o r k e r s (5) h a v e sh o w n a c t in o m y c e te s (a s o i l b a c te r iu m )
in c o r p o r a te n itr o g e n in to th e p o ly c y c l ic c e n te r o f h u m ic a c id . He c i t e s
a r e c e n t a r t ic le by B r e m n e r (6) in w h ic h an a p p r e c ia b le p e r c e n ta g e o f
the n itr o g e n in h u m ic a c id w a s n o n a c id h y d r o liz a b le , th u s in fe r r in g th a t
it w a s bound in h e t e r o c y c l ic fo r m , M o r r is o n (7) in h is s tu d ie s on c o m
p a r is o n o f p h e n o lic a ld e h y d e s in p la n ts and o rg a n ic s o i l m a t te r g iv e s .
e v id e n c e o f l ig n in -d e r iv e d a ld eh y d es fr o m h u m ic a c id . R oy (8) h a s
ob ta in ed h u m ic a c id b y th e o x id a tio n o f A s s a m c o a l w ith n itr ic a c id .
T h is la te x e v id e n c e s e e m s to su p p o rt G il le t 's p o s tu la te . B r e m n e r (3)
h as r e v ie w e d a fou rth th e o r y by C .E n d erS in w h ic h the fo r m a tio n o f
h u m ic a c id i s lik e n e d un to the fo r m a tio n o f rn e la n o id in s d u rin g the
m a n u fa c tu r e o f b e e r .
S tr u c tu r e o f h u m ic a c id . ■
A lth ou gh th e n a m e and o r ig in o r fo r m a tio n o f h u m ic a c id is in
d isp u te , th e r e s e e m s to be no. g r e a t d if fe r e n c e o f o p in io n as to th e g e n
e r a l c o n s t itu e n ts o f h u m ic a c id . T he com p ou n d c o n s is t s o f a m in o a c id s
and c a r b o h y d r a te s c o n n e c te d by e s t e r o r e th e r l in g a g e s to an " inner
c o r e " w h ic h is p o ly e y .c lic in n a tu r e . T h is , " in n er c o r e " i s p o ly e a r -
b o x y lic and p o ly h y d r o x y lic , thus a c c o u n tin g fo r i t s c h e la t in g p r o p e r t ie s
w ith c a lc iu m , ir o n , and o th e r m e t a ls . T h e a m in o a c id s and c a r b o h y
d r a te s a r e r e a d i ly h y d r o ly z e d by d ilu te a q u eou s a c id . A r e c e n t a n a ly s is
o f h u m ic a c id fo r i t s ca r b o h y d r a te c o n s t itu e n ts h as b een c a r r ie d out by
D , Li, L yn ch (9), and fo r am in o a c id s b y R . I . D a v is (10) and B r e m n e r
( H i . ■ . ; .
T he " in n er c o r e " w h ich is c o n s id e r e d to be th a t p o r t io n of h u m ic
a c id not h y d r o ly z a b le by d ilu te a q u eou s a c id i s th e s u b je c t o f th is S tudy.
E sh and G u h a -.S ir ca r (12) s tu d ie d th e d eg ra d a tio n p r o d u c ts o f p ea t h u m ic
a c id . T he a c id w a s s u b je c te d to p e r o x id e o x id a tio n , fo llo w e d by p o ta s s iu m
h y d ro x id e fusion* m e th y la t io n ,. and p o ta s s iu m p e r m a n g a n a te o x id a tio n .
T he com p ou n d s id e n t if ie d w e r e V e r a tr ic ( I ) , , V e r a tr o y lfo r m ic (II), and
D ra g u n o y (13) and B p tte r i (14) h a v e a ls o , sh ow n th at a lk a li fu s io n o f
h u m ic a c id p ro d u ces , lig n in d eg ra d a tio n p ro d u cts in the; fo rm of p y r o -
catB ch p l (IV ) and. p fd te c a te e h u ic a c id ('Vf)a : ;: :
m w
'M, K,oy (8)yw orking w ith h u m ic a c id oh ta in ed fr o m n i t f i c a c id o x i - -
d ation o f A s s a m c o a l , s u b je c te d th e a c id to a lk a lin e p o ta s s iu m p e r m a n
gan ate o x id a tio n , fo llo w e d by s u c c e s s iv e e x tr a c tio n s w ith e th y l e th e r ,
a c e to n e y and e th a n o l. T h e w a te r s o lu h le p o r t io n p f th e . r e s id u e w as . ■:
su b je c te d to paper. c h r o m a to g r a p h y , and th e com p ou n d s p y r d th e llit ic
a c id (V I), t r im e l l i t i c (VII), and b e n z e n e p en fa ca rb O x y lic a c id (VIII) 1
: w e r e i^ e h t i f ie h h th e ir B f y a l^ ; ' - ' 'ly. V: )
6
u q c , v; < : ' v : r: : - . 1 4 0 . C.Q.H
■':: -yi: , \ . : - : - VIII , ;■ ■■
G ottlieB-and H e n d r ick s (15) a ttem p ted a d eg ra d a tio n p r o c e d u r e u s e d
p r e v io u s ly fo r lig n in d e g r a d a t io n : a lk a li n itr o b e n z e n e o x id a tio n . In
th is m eth o d ca rb o n s id e 'c h a in s on th e b e n z e n e r in g a r e o x id iz e d to an
a ld eh y d e group . T h ey r e p o r te d fin d in g a t r a c e o f v a n il l in (IX) w hen
th is m e th o d w a s a p p lied to a m u ck s o i l . . '.
cm a-.
:'K./T^'-Mo#'iisopfc';,(o7)V in th e w ork r e p o r te d e a r l ie r , im p r o v e d on. th e '
n itr o b e n z e n e p r o c e d u r e o f G o ttlieb and. H en d r ick s and w a s a b le , th ro u g h
q u a n tita tiv e e x tr a c t io n s o f t h e r e s id u e fr o m th e o x id a tio n , to sh ow th e
p r e se n .e e p f S y r in g y l a ld eh y d e • (X), g u a ia c y l a ld eh yd e (X I), and p a r a - ;
h y d ro x y b en za ld eh y d e (X II). Kukharenkp (16) h a s d e s c r ib e d a m eth o d
of d eg ra d a tio n u s in g so d iu m in liq u id a m m o n ia . N o m e n tio n w a s g iv en
of th e is o la t io n o f any s p e c if ic com p ou n d . . ' '
xx • x n ' ,
T h e p r e s e n c e o f th ese , com p ou n d s su g g e .s ts a p a r e n t m o le c u le s im i la r v
to l ig n in in g e n e r a l s t r u c tu r e . - ; ' ;r.[: i ' S
B ro o k s (17) d e s c r ib e s tw o p r o c e d u r e s fo r the d e g r a d a tio n o f c o a l ■
. h u m ic a c id . W ith h y d ro g eh p e r o x id e th e h u m ic - a c id i s oxidirzed t o
" su b h um ic a c id s '1. N o d ata ; i s ! g iv e n c o n c e r n in g any p ro d u cts i s o la t e d . ' ■'
W ith o z o n e th e " su b h u m ic a c id s H a r e fo r m e d w ith ou t th e n e e d o f o th er . . :
r e a g e n ts . F la ig (5) in h is r e c e n t p a p e r c i t e s r e f e r e n c e (18) in w h ich >
- a lk a li fu s id n o f b itu m in u s c o a l h u m ic : a c id g a v e th e fo llo w in g c o m - > , ’
p o u n d s : is o p h th a lic a c id (XII) 3 m e ta -h y d r o x y b e n z o ic a c id (XIV), and -
2 , 6 -tn ih y d r o x y iS 0 p id h a lic a o id (XV).^^ v- v-V;'’'
.XIII . ' XIV . X V
A lthough th e m a jo r ity o f in v e s t ig a to r s h a v e e v id e n c e fo r the o a r b o -
c y c l ic n a tu re o f th e " in n er core'* , o n e in v e s t ig a to r g iv e s e v id e n c e su p
p o r tin g a h e te r o c y c l ic " in n er c o r e " , F la ig , in v e s t ig a t in g b la ck e a r th
h u m lc acid# o b ta in ed a fte r a c id h y d r o ly s is a r e s id u e co n ta in in g n itr o g e n .
W hen th is r e s id u e w a s fu se d w ith p o ta s s iu m h y d r o x id e , th e d is s o lv e d
p ro d u ct w a s a d ju sted to pH 5 and th en e x tr a c te d w ith d ie th y l e th e r . T h e
com p ou n d s in th e e th e r e x tr a c ts had id e n t ic a l R f v a lu e s and c o lo r r e a c t io n s
w ith s p r a y r e a g e n ts to th oS e e x h ib ite d by in d o le co m p o u n d s . A lth ou gh
no d e fin ite p r o o f w a s g iv e n fo r s p e c if ic co m p o u n d s , s p o ts on th e c h r o
m a to g r a m s ex h ib ite d th e p r o p e r t ie s o f (XVI) and X V II),
X V I X VII
P r o b le m s o f S ep a ra tio n and a n a ly s i s . .
I ln til r e c e n t ly is o la t io n o f d e g r a d a tio n p r o d u c ts o f h u m ic a c id
h a s b een c a r r ie d out u s in g s ta n d a r d s o lv e n t e x tr a c t io n and c r y s t a l l i s a
t io n m e th o d s . T h e s e m e th o d s h a v e n o t b een v e r y .s u c c e s s f u l fo r s u c h
c o m p le x m ix tu r e s and a tten tio n w a s tu rn ed to p a p e r ch r o m a to g r a p h y a s
a m o r e fr u itfu l m e th o d , M o r r iso n (7) a s w e l l a s B r e m n e r (19) h a v e
u s e d th e m e th o d o f S tone and B lu n d e ll (20) w h e r e b y a ld e h y d e s w e r e
s a t is f a c t o r i ly is o la t e d . H a i g (5), u s in g a m e th o d by S en and L eo p o ld
(21) fo r in d o le com p ou n d s in p la n ts , o b ta in ed th e r e s u lt s c ite d a b o v e .
A s s ta te d e a r l ie r M . M . R oy (8.) a ls o o b ta in ed h is r e s u lt s by p a p er •
ch ro m a to g ra p h y .
A lthough m a n y in v e s t ig a to r s h a v e e m p lo y e d p a p e r c h r o m a to g r a p h ic
p r o c e d u r e s fo r p h e n o ls , p o ly p h e n o ls ,a n d c a r b o x c y lia y a c id s , no p r o c e
d u re h a s b een d e v is e d e s p e c ia l ly fo r h u m ic a c id d e g r a d a tio n p r o d u c ts .
A good g e n e r a l b ack grou n d on p a p er c h r o m a to g r a p h y o f p h e n o lic c o m
pounds i s g iv en by B lo c k (22) and L e d e r e r and L e d e r e r (23)= M ore
r e c e n t r e v ie w s on p a p e r c h r o m a to g r a p h y o f p h en o ls a r e by C p u l s o n and
E v a n s (24), R e io (25), and P r id h a m (26).
In a sm u c h a s th e m eth o d o f p a p e r c h r o m a to g r a p h y i s r e la t iv e ly
n ew in th e f ie ld o f h u m ic a c id , th e m e th o d h a s b een l im it e d to a g r e a t
ex ten t to d e te r m in in g th e R f v a lu es' and c o lo r r e a c t io n s o f h u m ic p r o d u c ts
M o r r iso n (7) q u a n tita tiv e ly e s t im a te s th e a ld e h y d e s in h is h u m ic a c id
fr a c t io n s by c o m p a r in g th e o p t ic a l d e n s it ie s of th e ir e lu a te s fro m th e
c h r o m a to g r a m w ith .s ta n d a r d s o lu t io n s . B r a d fie ld and F lo o d (27) h a v e ,
u s e d a m e th o d w h e r e b y th e u lt r a v io le t a b so r p tio n s p e c tr u m o f a c o m
pound ca n be ta k en d ir e c t ly fro m th e p a p e r c h r o m a to g r a m . T h e s p e c
tru m s o o b ta in ed can n ot be u s e d fo r an y q u a n tita tiv e m e a s u r e m e n t n o r
do th e m a x im a o f the p a p er a b so r p tio n S p ectru m c o r r e s p o n d e x a c t ly
w ith m a x im a ob ta in ed fro m a s d lu tiom s p e c tr u m .
10
P u r p o s e o f th is m v estig a tib n o ,
F o llo w in g .acid h y d r o ly s is and a lk a li fu s io n o f th e h u m ic a c id , th e ;
res id u e , i s su b je c te d to so lv e n t fraction a tion ^ T he e x tr a c ts thus o b ta in ed
a r e s u b je c te d to p a p er ch ro m a to g ra p h y w h e r e b y p h e n o lic com p ou n ds a r e
d e te c te d by p h e n o lic sprays: and by u lt r a v io le t f lu o r e s c e n c e . T h e c h o ic e
of a su ita b le so lv e n t s y s t e m , w h ich e f f e c t s a r e a s o n a b le d e g r e e o f s e p a r
a tio n o f th e s e p h e n o lic c o m p o n e n ts , i s a c r i t i c a l p a r t o f th is p r o c e d u r e .
T he s e c o n d s ta g e o f p r o c e d u r e i s to e lu te th e s e p h e n o lic a r e a s , su b je c t
th em to fu r th e r s e p a r a t io n by p a p er c h ro m a to g ra p h y , and f in a lly to
id e n tify th e p h e n o lic c o n s t itu e n ts by sp e c tr o p h o to r a e tr ic a n a ly s is . T o
th is end au th en tic s a m p le s o f know n p h e n o ls a r e ch r o m a to g r a p h e d and
th e ir c o lo r r e a c t io n s w ith s p r a y r e a g e n t s , R f v a lu e s w ith d iffe r e n t
s o lv e n t s y s t e m s , and th e ir u lt r a v io le t a b so r p tio n s p e c t r a a re d e t e r
m in e d . The; is o la t e d p h e n o lic com p ou n d , w h o se R f v a lu e s and c o lo r
r e a c t io n s c o r r e s p o n d to an a u th en tic com p ou n d , is th en ex a m in e d
sp e c tr ophot ora e t r Ic a l ly . Its u lt r a v io le t a b so r p tio n sp e c tr u m is d e t e r
m in e d both in s o lu t io n and d ir e c t ly fr o m th e p a p er c h r o m a to g r a m as
ob ta in ed by B r a d fie id and F lo o d (27)* T h is in v e s t ig a t io n d if fe r s fro m
p r e v io u s in v e s t ig a t io n s o f H um ic a c id in th at th is i s th e f i r s t t im e th at
'a p h e n o lic su b s ta n c e On a c h r o m a to g r a m i s fu r th e r p u r if ie d by e lu tio n
and r e -c h r o m a to g r a p h in g . A s e c o n d u n iq u e m eth o d i s th e u s e of s o lu
tio n and p a p er u ltr a v io le t .a b so rp tio n s p e c tr a in s u b s ta n t ia t in g the
s tr u c tu r e o f th e is o la t e d p h e n o lic com p ou n d .
II. DISCUSSION O F R E SU L T S
P r o to c a te ch u ic a c id and othe r ph e n o lic c6m p ou n d s 0
P r o to c a te c liu ic a c id (3 5 4 » d ihy dr ox yben z o ic ac id ) h a s b een id e n t if ie d
as a d eg ra d a tio n p ro d u ct o f th e h u m ic a c id e x tr a c te d fr o m a C a lifo r n ia
p o d zoL T he p r e s e n c e o f th is a c id le n d s su p p o r t to th e th e o r y th at h u m ic
a c id a r i s e s fr o m lig n in s o u r c e s . A lth ou gh p r e v io u s in v e s t ig a to r s h a v e
d e te c te d th is com p ou n d in S o il o r g a n ic m a t te r , th is i s th e f i r s t t im e th at
ch r o m a to g r a p h ic e v id e n c e fo r i t s p r e s e n c e h a s b een su p p o r te d w ith s p e c -
tr o p h o to m e tr ic e v id e n c e . One m a y p o s tu la te th at p r o to c a te q h u ic a c id is
o n ly one o f a n u m b er o f c a te c h o l d eg ra d a tio r i p r o d u c ts . ;-E vid.ence fo r t h e s e
m in o r c o n s t itu e n ts is' g iv en by th e p r e s e n c e o f b la ck and lig h t v io le t c o lo r s
p ro d u ced -b y th e F e C lg sp r a y in g o f th e p r o to c a te c h u ic a r e a o f the c h r o m a
t o g r a m s . One m a y p o s tu la te th at th e s e co n stitu en ts' a r e g a ll ic a c id and
s y r in g ic a c id . It i s know n th at s y r in g ic a c id i s d e m e th y la te d at 25Q0 C» to
g a ll ic a c id . T he b la ck band p ro d u ced by th e F e C lj s p r a y c o r r e s p o n d s to - -
th e R f v a lu e and c o lo r p ro d u ced by a c o -c h r o m a to g r a p h e d sa m p le o f g a l l ic
a c id . L a ck o f e v id e n c e fo r th e p r e s e n c e o f p r o to c a te c h u ic acid; in. English'*
h u m ic a c id m a y be due to in s u f f ic ie n t in v e s t ig a t io n o f th e E n g lish s o i l- ' 1
p r io r to th e in v e s t ig a t io n o f th e C a lifo r n ia s o i l .
V e r y r e c e n t w o rk in th is la b o r a to r y h a s sh o w n th e p r e s e n c e o f p r o to c a te ch u ic a c id in th e a lk a lin e d e g r a d a tio n o f E n g lish h u m ic a c id .
T he p r e s e n c e o f v a n ill in (IX)5 a lth ou gh not u n e q u iv o c a lly e s ta b lis h e d
in th is la b o r a to r y , m a y a cco u n t fo r th e o c c u r r e n c e o f p r o to c a te c h u ic a c id ,
s in c e it i s w e ll-k n o w n th at p h e n o lic e th e r s a r e d e m e th y la te d u nder the
co n d itio n s o f a lk a lin e fu s io n 0 A ld eh y d es are- v e r y e a s i ly o x id iz e d to a c id s
and thus in the p r o c e s s o f fu s io n , v a n ill in m a y be o x id iz e d and d e m e th y la te d
to p r o to c a te c h u ic acid»
T he p r e s e n c e o f a p h lo r o g lu c in o l- l ik e com p ou n d w a s d e te c te d by
c h ro m a to g ra p h y o f th e a lk a lin e fu s io n e x t r a c t « H o w e v e r , th e c o lo r r e a c t io n s
of th is com p ou n d w e r e not id e n t ic a l to th o s e g iven by an au th en tic s a m p le
o f p h lo r o g lu c in o l •(X .yil)vunder id e n t ic a l c o n d itio n s ,. : . ‘
It i s qu ite r e a s o n a b le to a s su m e th at th is d if fe r e n c e in c o lo r s is n o t due
to e x p e r im e n ta l e r r o r s and th at th e com p ou n d m a y n ot be p h lo r o g lu c in o i.
S in ce th e R f v a lu e s fo r th e unknow n and p h i or o glue in o l a r e a lm o s t id e n t ic a l
in th r e e s o lv e n t s y s t e m s , one m a y a s s u m e th at the unknow n is p h lo r o -
g lu c in o l- l i lc e , i . : e , , a m e th o x y , m e th y l, or e th y l s u b s t itu te d p h lo r o g lu -
cinolo D ue to w ea k s p e c tr a l data and la c k o f o th e r m o d e l cqm pounds in
la te r d e g r a d a tio n s , no fu r th e r a n a ly s e s w e r e c a r r ie d out on th is com p ou n d .
12
C h ro ra a to g ra p h ic and sp e c tr o p h o to m e tr ic t e c lm iq u e s «,
One m a y a g r e e w ith Dr* B r e m n e r {3) th at ch r o m a to g r a p h y is one o f
the few m eans, w h e r e b y th e c e n tr a l c o r e o f h u m ic a c id m a y be elucidated^
.A s can be s e e n by r e v ie w in g th e e x p e r im e n ta l data* c o n s id e r a b le im p r o v e
m e n t o f th is p r o c e d u r e is requ ired^ T he p r e s e n c e o f in s o lu b le b lack
m a t e r ia l in th e e x tr a c t s a m p le s I n te r fe r e d w ith th e c h r o m a to g r a p h in g o f
th e s a m p le s as w e l l a s co n ta m in a ted the s a m p le s u se d fo r s p e c tr a l
a n a ly s is * It w a s n e c e s s a r y to ob ta in e x tr a c ts that w e r e not s tr o n g ly
a c id ic , s in c e u n d er su ch c o n d itio n s th e f i l t e r p a p er w a s d e s tr o y e d upon
d ry in g th e sa m p le . A ch a n g e in d e g r a d a tio n p r o c e d u r e th a t m a y im p r o v e
th e fu se d p ro d u ct w ou ld be s u b s t itu t io n o f s o m e a lk a l i - in e r t c o n ta in e r fo r
th e g la s s flask* A lth ou gh th e fu s io n m ix tu r e w a s c e n tr ifu g e d fo llo w in g
th e fu s io n to r e m o v e a l l s i l i c a t e s , l o s s o f s o m e p h e n o la te s m a y occur*
It w a s n o ted th at in p r e l im in a r y d e g r a d a tio n s th at d ry in g a g en ts ten d ed to
a d so r b th e c o lo r e d m a te r ia l w hen the e th e r e x tr a c ts w e r e dried* R e a l i s - . i'
in g that h u m ic a c id i s a p o w e r fu l lig a n d and th u s c o u ld c o m p le x w ith e v e n
so d iu m and p o ta s s iu m io n s , th is in v e s t ig a to r d isc o n tin u e d the u s e o f d r y
in g a g e n ts «, A lth ou gh no s tu d ie s w e r e c a r r ie d out on th e p h e n o lic s u b
s ta n c e s oh th e ‘e th y l a lc o h o l, e th y l a c e ta te , and w a te r e x t r a c t s , u n d ou b ted ly
th e s e s u b s ta n c e s e x i s t i n th e s e so lv e n ts* Upon sc a n n in g c h r o m a to g r a m s
o f th e s e e x t r a c t s y o n ly lig h t c o lo r e d s t r e a k s w e r e d e te c te d fo r F eC lg
sp r a y e d p a p ers* F u r th e r e x p e r im e h ta t io n is s u g g e s te d in th is area*
■ It w ag n o ted th at upon s ta n d in g ev e n in t ig h t ly s e a le d v ia l s , th e ; ; ; •
v:;:;; e x tr a c ts in c r e a s e d in s o l id in e r t m a t e r ia l con ten t- T h is i s p ro b a b ly due . , \ '
to o x id a tio n o r o x id a tiv e p o ly m e r iz a t io n . S to ra g e of t h e s e sa m p le s u n d er : '
■ , an in e r t gas w o u ld .re d u ce , th e rate., o f th is , reaction.'^:;)' :. ' u y
', , ■ : . R e v ie w in g th e r e s u l t s ob ta in ed on th e p h lo r o g lu c in o l- l ik e com p p u nd , .
, one pan s e e the great n eed for sp e c tr a l data to com plem ent ch rom ato -
t grap h ic data fo r d e te r m in a t io n o f s tr u c tu r e ; S in c e a d e f in ite u lt r a v io le t X
■ ' sp ec tru m .:w;as not o b ta in a b le fo r th is s tr u c tu r e , one ca n c o n c lu d e that th e t / ' .
i, com p ou n d w a s n o t p h lo r o g lu e in o l . F o r th e p u r p o se o f id e n tify in g co rn - ; t
pounds in th e v a r io u s h h in ic a c id fr a c t io n s , th e u lt r a v io le t a b so r p tio n ■
s p e c tr a o f a n u m b er o f p h e n o ls w e r e c o l le c t e d . T h e s e w e r e r e c o r d e d in
V T a b le 5. S in c e a u th o rs a r e n o t in a g r e e m e n t .as to th e p r e fe r r e d s o l v e n t ' . , ;
• . o r m e a n s o f r e c o r d in g s p e c tr a l v a lu e s and u n it s , a l l v a lu e s o b ta in ed fr o m -
th e l i t e r a tu r e w e r e ch a n g ed to mp! (m il l im ic r o n s ) and lo g f u n its . , '
. ' ii W 'U itravlolet. spectr'a obtained d ir e c t ly from a ' - i t ; ;:
pap er ch rom atogram w ith that obtained fro m a so lu tion , one can con clu d e / ■
; ’ that so lu tion s p e c tr a are far Superior to sp e c tra obtained d if e c t ly from
y. ':/.; p a p e r .. : F ir s t , the p eak s .of the sp e c tr a l cu rves, from so lu tio n are m u ch ’ r . i . :;;
' s ha r pe r than th p s e o b t a i n e d fro m d ir e c t ab sorp tion , on p a p e r . S eco n d , th e , '
i v ir : h e ig h ts o f th e ,m a x im a p ea k s fro m s o lu t io n s p e c tr a a r e .t r u e m e a s u r e s o f ; ' i : i /. :..h
th e c o n c e n tr a t io n o f the d is s o lv e d s p e c i e s . One can n ot m a k e su ch quan
t ita t iv e m e a s u r e m e n ts w ith s p e c tr a fr o m p a p e r . M a x im a v a lu e s ab ove 300
14
t n M a r e d isp la ce ,d to lo n g e r w a v e le n g th s than the r e s p e c t iv e v a lu e s
o b ta in ed fr o m so lu t io n s p e c tr a . T he n e g a t iv e a b so r b a n c e r e c o r d e d fr o m
th e d r ie d p a p er a b so r p tio n sp e c tr u m o f p r o to c a te c h u ic a c id ( s e e C hart 4)
i s in e x p lic a b le at th e p r e s e n t t im e . F u r th e r in v e s t ig a t io n o f th is p h e n o m
enon w o u ld p r o v e fr u itfu l in lig h t o f th e fa c t th at o th er com p ou n d s m a y
r e a c t in a s im i la r m a n n e r .
D e s p ite th e s e d isad v a n ta g es^ th e m e th o d o f d ir e c t p a p e r a b so r p tio n
ca n be u s e d to g r e a t ad van tage fo r r a p id d e te r m in a t io n s w h en o n ly s m a ll
am ou n ts o f s a m p le a r e a v a ila b le . T h is ra p id p r e l im in a r y m eth o d o f
id e n t if ic a t io n can th en be c o n fir m e d w ith th e m o r e e x a c t so lu tio n m eth o d .
In the c a s e o f the d e te r m in a t io n o f v a n il l in in h u m ic a c id th e p a p er m ethod^
a lth ou gh not e x a c t , w a s e x tr e m e ly u sefu lo In A ppend ix C a few d ir e c t
p a p er u ltr a v io le t s p e c tr a h a v e b een in c lu d e d to i l lu s t r a t e th is m eth o d , A
c o m p le t e .ta b le o f th e s p e c tr a of p h e n o lic com p ou n ds w o u ld be v e r y u s e fu l
to in v e s t ig a to r s in th e f ie ld o f n a tu ra l p r o d u c ts . C o m p a r iso n o f u lt r a v io le t
s p e c tr a o f s ta n d a rd s w ith the s p e c tr a o f d eg ra d a tio n com p ou n d s su ch a s
p r o to c a te c h u ic a c id sh o w th at u n d er p r o p e r c o n d itio n s r e p r o d u c ib le r e s u l t s
c a n be o b ta in ed . T h e s e c o n d itio n s in c lu d e p r o p e r c o n c e n tr a t io n and a c id ity .
To ob ta in r e p r o d u c ib le s p e c tr a fr o m d ir e c t a b so r p tio n on p a p e r , it w a s
f ir s t n e c e s s a r y to dip the c h r o m a to g r a m in d ilu te a c id and d ry i t . N on u n i
fo r m ity o f th e s e s p e c tr a is. p ro b a b ly due to tw o fa c to r s : (1) the v a r ia b i l i ty
o f pH o f th e c h r o m a to g r a p h ic p a p e r , and (2) th e v a r ia b i l i t y in the pH o f th e
15
,sp |^e^;.;gystem :. B oth fa c to r s w ou ld a ffe c t the. e t iu iiib r iu m c o n c e n tr a tio n
o f th e io n iz e d a.nd u n io n iz e d fo r m s , o f th e phen .olic com p ou n d u n d er in v e s t i -
'V-
III. E X P E R IM E N T A L ■
S o u rce o f s o i l s a m p le . - : A /
T he h u m ic a c id s th at w e r e u s e d for. t h e s e e x p e r im e n t s w e re o b ta in ed
fro m p o d z o l-B h o r iz o n s from; tw o d ifferen t' s o u r c e s . T h e m a jo r ity o f th e '
d eg ra d a tio n s tu d ie s w e r e c a r r ie d out on a sa m p le o f h u m ic a c id e x tr a c te d
ahd p u r if ie d in th e U n iv e r s ity o f L iv e r p o o l la b o r a to r ie s in L iv e r p o o l, . .
E n g lan d . T he E n g lish h u m ic a c id w a s e x tr a c te d w ith la c t ic a c id fro m a .
p o d zo l-'B h o r iz o n in th e D e la m e r e f o r e s t .o f C h e sh ir e , E n g lah d . It w a s
p r e c ip ita te d fro m th e la c t ic e x tr a c t w ith d ie th y l e th e r « T he sa m p le as ' . h-
ob ta in ed fr e m E h glan d w a s d ark b row n in c o lo r , qu ite s o f t in te x tu r e , w ith
no detectable odor . : ; ';:i', : . . - ■, ; ‘ ■ C;
T he C a lifo r n ia s o i l s a m p le .w a s o b ta in ed fr o m M en d o cin o C ounty, . ■
■ C aliforn ia , fr o m a podkO lrB h o r iz o n . At the t im e o f e x tr a c t io n , w h ich w a s
approximately 60 d ays a fte r th e s a m p le w a s ob ta in ed , th e s o i l w as q u ite
E x tr a c t io n o f h u m ic a c id . vv-; • J ' i
T he e x tr a c tio n o f th e C a lifo r n ia s a m p le o f h u m ic a c id w as c a r r ie d
: o a t as' f o l lo w s : One k ilo g r a m o f s o i l w a s p la c e d in an. e x tr a c t io n f la s k o f
th e ty p e sh ow n b e lo w . ' , .
17
T he bottom of the f la s k w a s c o v e r e d w ith a o n e - in c h m a t o f g la s s w o o l.
T he s y s te m w a s f lu sh e d w ith n itr o g e n . F ro m a s e p a r a to r y funnel a c o o le d
so lu tio n o f 0. 5 N . so d iu m h y d ro x id e w a s a llo w e d to flo w th rou gh the s o i l .
A c o n sta n t h ead o f liq u id w as m a in ta in e d ab ove the s o i l . One hundred m l.
o f c o n c e n tr a te d h y d r o c h lo r ic a c id w a s p la c e d in the r e c e iv in g f la sk . F iv e
h undred m l. o f the 0. 5 N . so d iu m h y d ro x id e so lu t io n w a s added , and th e
so lu tio n w as a llo w e d to p e r c o la te th ro u g h th e s o i l . T he e x tr a c to r w as
r e p le n ish e d w ith fr e s h so lu tio n o f a lk a li w hen n eed ed and th e e x tr a c tio n
w a s con tin u ed fo r s e v e n d a y s . At the end o f th is t im e tw o l i t e r s of e lu a te
had b een c o l le c t e d . T he h u m ic a c id p r e c ip ita te d as a b la c k c r e a m y s o l id
fro m the e lu a te upon a c id if ic a t io n . T he c o lo r o f the a lk a li e lu a te , as
o b ta in ed fro m the U tube o f the e x tr a c t in g f la s k , had ch a n g ed fro m an in it ia l
dark b la ck to a dark o ra n g e c o lo r at the end o f s e v e n d a y s .
18
T he e x tr a c t w a s a d ju ste d to pH 1 w ith c o n c e n tr a te d h y d r o c h lo r ic , .
a c id . T he p r e c ip ita te w a s c e n tr ifu g e d and th e cru d e h u m ic a c id w as w a sh e d
w ith ic e w a te r u n til f r e e o f .m in e r a l a c id . The: p r e c ip ita te w a s p la c e d in ': :
ah eixapdrating: d ish ahd/w ah::a llow ed t o d r y th r ee d ays at rciom tem p era-/';;;'\- '' v
t u r e . : T he. prpduct. w a s ground tq a f in e p o w d er arid w a s d r ie d s t i l l fu r th e r / ;
/ / in an overi for' fou r h o u r s 'a t 4 5 °C . T he p ro d u ct c o n s is t e d of. 18. 5 g. o f .
v e r y fin e c o a l-b la c k p o w d er w ith a. n o t ic e ab le l i g n i t e c o a l o d o r . T h is p r o
duct w a s fu r th e r p u r if ie d by d is s o lv in g it in 450 m l. o f 0. 5 N . so d iu m
h y d r o x id e . T h is a lk a lin e m ix tu r e w a s . c e n tr ifu g e d . A la r g e quantity o f
' ■ g ra y s i l t r e m a in e d in the bottom o f th e c e n tr ifu g e b o tt le . T he c e n tr ifu g a te
w a s d eca n ted and a d ju ste d to pH 1 w ith c o t ic e h tr a te d h y d r o c h lo r ic ac id ., i . ■ :
T h e p r e c ip ita te d h u m ic acid w a s washed w ith i c e w aterX and th en w a s d r ie d ' ■ :
as a b o v e . . T he p u r if ie d p ro d u ct c o n s is t e d o f 1 2 . 8 g. o f a v e r y -b la c k p p w d er
.with no a p p r e c ia b le o d o r . • • .
. v:\.Acid:h y d r o l^ i s l : i x . j -
. . A s a m p le o f 3. 2 9 4 g. o f h u m ic a c id w a s p la c e d in a 2 5 0 m l., ro u n d -
■ b ottom f la s k . A c id -w a sh ed b o ilin g ch ip s w e r e added and th e .s a m p le w a s
m ix e d w ith 100. m l . o f ‘2% s u lfu r ic a c id . ; D u r in g th e f i r s t tw o h o u rs o f : X : .
.. r e f lu x , c o n s id e r a b le fr o th in g o c c u r r e d . T h e s a m p le w a s r e f lu x e d fo r fo u r • ' ,■
h o u rs and th en w a s a llo w e d to c o o l . T he m ix tu r e w a s c o o le d s t i l l fu r th e r
in an ic e bath and th en w a s c e n tr ifu g e d . T he h y d r o ly sa te , c o n s is t e d o f a - .
' dark hroWn,clear solution. T he residue; was washed once with ice water
19
, to r e m o v e m o s t Of ttie lig h t brow n c o l lo id a l m a t e r ia l , and th en w as w a s h e d
f r e e o f f e r r ic io n w ith th r e e m o r e w a sh in g s . The s a m p le w a s p r e s u m e d
to be f r e e o f f e r r ic io n w hen the w a sh s o lu t io n w ould not fo r m a b lu e - to -
g r e e n c o lo r w ith K4 Fe(CN)^V A ll w ash in gs, w e r e c o m b in e d w ith the
' h y d r o ly s a te . T he r e s id u e , d r ie d in an o y en fo r fou r h o u rs at 6 0 °C . , " . ;
w e ig h e d G. 978 g. T h is r e s id u e w a s a p o w d er , a l i t t le l ig h te r in sh a d e
than th e . sa rh p le th a t w a s h y d r o ly z e d . N o a p p r e c ia b le o d o r w a s d e te c te d .
T h is r e s id u e c o n s t itu te s 30% of th e o r ig in a l h u m ic acid .. T h is p e r c e n ta g e
(p lus hr m in u s 10 %) w a s c h a r a c t e r is t ic o f s e p a r a te a c id d e g r a d a tio n s o f
th e E n g lish h u m ic a c id . T he r e s id u e ob ta in ed frona th e .C a lifo rn ia h u m ic
■ a c id w a s 71 % o f the h u m ic a c id . , V ■; ■ ■f.. - . ' .
P o ta s s iu m h y d ro x id e fu s io n . i i . d i
A ty p ic a l fu s io n e x p e r im e n t w a s c a r r ie d ou t a s fo llo w s : A n ew 125 .
m l . . p y r e x .E io ra n ce f la s k w a s s e le c t e d . T h is s e l.e c f io n w a s n e c e s s a r y in: ;
o r d e r to ob ta in a f la s k th at w a s f la w le s s and n o t S u b ject to ra p id c o r r o s io n
by KOH. T he f la s k w a s f it te d w ith a tw o -h o le ru b b er s to p p e r c o n ta in in g
a g la s s tube c o n d e n se r and a s h o r te r tu b e , th e la t te r c o n n e c te d b y r u b b e f
tu b in g to a n itr o g e n t a n k . . Upon in s e r t io n o f th e ru b b er s to p p e r , th e f la s k
. w a s fLushed w ith n itr o g e n . T he f la s k w a s p la c e d in a m o lte n m e ta l bath
and twO g ra n ts o f r e a g e n t grad e p o ta s s iu m h y d ro x id e p e l le t s was. ground
in a m o .r ter and p la c e d in the f la s k . T h r e e d r o p s ;o f w a te r w as added a n d .
th e bath w a s h e a te d s lo w ly to 150 C. u n d er n itr o g e n at w h ich p o in t th e
20
a lk a li had m e lte d to a s lu r r y . T he p o w d er ed sa m p le (Q„ 978 g . ) w as
added and. w a s m ix e d w ith th e s lu r r y by g en tly sh a k in g th e f la s k . T he bath
■ V ■ :■ '' . 0 ;; : . .t e m p e r a tu r e w a s in c r e a s e d to 210 C. and w a s h e ld th e r e u n til th e fr o th in g
h ad su b s id e d and th e m o is tu r e had b een d r iv e n o ff (ap p rox . 15 m in . )•. T h e
te m p e r a tu r e th en w a s r a i s e d to 250 C. and w a s kep t at th a t te m p e r a tu r e
fo r an a d d itio n a l th r e e and o n e -h a lf h o u r s . T h is t e m p e r a tu r e w a s m a in -
' ta in e d w ith in 'a 10° C . r a n g e . T he s y s t e m w a s m a in ta in e d u n d er n itr o g e n
d u rin g th e e n t ir e r e a c t io n t im e . T h e r e a c t io n f la s k w a s a llo w e d to c o o l
to n e a r ro o m te m p e r a tu r e and, s u b se q u e n t ly , w a s c o o le d s t i l l fu r th e r in
an i c e bath .
T he fu se d s a lt s w e r e d ilu ted to 50 m l . w ith ic e w a te r and th en w e r e
c e n tr ifu g e d . T h e su p e rn a ta n t liq u id w a s a c id if ie d to pH T w ith c o n c e n
tr a te d s u lfu r ic a c id fo r m in g a la r g e q u antity o f f lo c c u le n t p r e c ip ita te ,
v T h is p r e c ip ita te w a s p ro b a b ly S iC ^ .nH ^ G d is s o lv e d fro m th e r e a c t io n
f la s k b y th e KOH.
S o lv e n t fr a c t io n a t io n .
T h e aq u eou s m ix tu r e o f d e g r a d a tio n p ro d u cts w a s .tr a n s fe r r e d to a
l iq u id - l iq u id e x tr a c to r and th en w a s d ilu ted w ith w a te r to 2 0 0 m l. One h u n
d red f if ty m l . o f d ie th y l e th e r w a s p la c e d in th e b o i le r . . T h e e x tr a c to r w a s
f it te d w ith a c o n d e n se r 'co n n ec ted to a w a te r p um p. Ice w a te r w a s c i r
c u la te d th rou gh th e c o n d e n se r d u rin g th e e x tr a c t io n . T h e r a te o f co n d e n
sa t io n w a s s e t at a p p r o x im a te ly one drop p e r se c o n d by c o n tr o lle d h e a tin g .
A fte r 2 4 h o u rs th e e th e r re tu r n in g to the b o ile r w a s c le a r . T h e e x tr a c t
w a s dark r e d . An a p p r e c ia b le am ount o f r e s id u e had c o l le c t e d on th e ' .
s id e s and b ottom o f th e b o ile r . T he e th e r w a s r e m o v e d b y red u ced
p r e s s u r e : . .■ T h e r e s id u e on th e s id e s , t o g e t h e r vdth th e r e d u c e d e th e r
s a m p le , w a s d is s o ly e d in a b so lu te e th a n o l and th en w a s r e d u c e d to d r y
n e s s at ro o m te m p e r a tu r e . : T h e r e s id u e fro m th e e x tr a c t w a s 0 . 4 3 4 g.
D ie th y l e th e r w a s r e m o y e d fr o m th e a q u eou s so lu t io n . T h e la t te r w a s '
a d ju ste d t o p H 7 w it h c o n c e n tr a t ed KOH so lu tio n and w a s r e due ed to n e a r .
d r y n e s s on a hot p la t e , T h e c b io r o f th e s a lt s at th is p o in t w a s lig h t
brown.: T he s a lt s w e r e tr itu r a te d w ith fo u r d rop s o f d o n e . H^SO^. and
w e r e p la c e d in a S o x h le t e x tr a c t io n th ir n b le . , T he tr itu r a te d s a lt s w e r e . . ;
e x tr a c te d w ith 150 m l. o f ethyL a c e ta te fo r 10 h o u r s , a f te r w h ich the e
e x tr a c tin g so lv e n t r e tu r n in g to th e b o ile r Was c le a r . T h e e th y l a c e ta te
so lu tio n w a s r e d u c e d to a p p r o x im a te ly tw o m l. by d is t i l la t io n . The s o lu - .
t io n a lo n g w ith th e r e s id u e w a s d is s o lv e d in e th y l a lcohol.: T he r e s id u e ■
fro m th e e th y l a c e ta te e x tr a c t io n th en w a s e x tr a c te d w ith 150 m l. o f 95%
e th a n o l in th e s a m e m a n n e r . A ga in , s o m e s a lt s and o th e r r e s id u e w e r e
e x tr a c te d in to the a lc o h o l d u rin g th e e x tr a c t io n . T h e s e r e s id u e s a p p e a r e d '
d u rin g th e r e m o v a l o f the a le o h o l b y d is t i l la t io n . T he sa m p le Was: r e d u c e d ,
to a yblume of two ml; r ;v • y yd,:; ■ ; : f ; V -:-- : - . ; . ' ' ' . ■ : ■ ■ p.d
: ' . T h e r e s id u e le f t in th e e x t r a c t io n , cup Was l i g h t b row n in c o lo r . T h is:
c o n tr a s te d w ith th e b la ck c o lo r o f th e s a m p le b e fo r e th e tw o e x tr a c tio n s . •
T h is r e s id u e w a s r e m d y e d , w a sh e d w ith 50 m l. o f d is t i l l e d w a te r , and
f i l t e r e d . T he c le a r f i l t r a te w a s r e d u c e d in v o lu m e by d is t i l la t io n . F r o m
t im e to t im e d u rin g th e d is t i l la t io n , the in o r g a n ic s a lt s w e re r e m o v e d by
' f iltra tio n ,. T he sa m p le w a s r e d u ced to a v o lu m e o f tw o m l .
T h e h y d r d ly s a te fro m th e a c id h y d r o ly s is w a s su b jected ; to so lv en t,
e x tr a c tio n in a m a n n e r s im i la r to th e p r o c e d u r e ab ove fo r the a lk a li fu se d .
s a m p le . T h e s o lv e n ts w e r e a g a in r e d u c e d in v o lu m e to tw o m l. The c o m
p le te s c h e m e o f h u m ic a c id d e g r a d a tio n and e x tr a c tio n is , g iv e n In F ig u r e i
• / T he s a m p le s p r e p a r e d fro m the d eg ra d a tio n and e x tr a c t io n p ro ced u res ,
;a b o v e w e r e su b je c te d to p a p er ch rp m a tp g ra p h y . T he gen .era l p r o c e d u r e w a s
as f o l lo w s : D e sc e n d in g p a r t it io n ch r o m a to g r a p h y w ith W hatm an #1 p a p e r
w a s u se d fo r a ll c h r o m a to g r a m s . T he p a p er w as, cu t in to s tr ip s 23: c m . by
50 c m . A l in e w a s draw n s e v e n and o n e -h a lf c m . f r o m th e le a d in g ed g e o f
th e p a p e r w ith a le a d p e n c i l . F ro m f iv e to s e v e n s a m p le s w e r e sp o tte d .
a lo n g th is l in e . T he s a m p le s w e r e a p p lie d , u s in g c a p i l la r y tu b es d raw h ;
out at on e end and a r e s tr ic t io n , in the m id d le o f the tu b e . T he so lv e n t w a s
e y a p P fa te d w ith the a id . o f a h a ir d r y e r . F r o m f iv e to tdnzzdrops w e r e p la c e d
at: each/,areaY': . T h e 'n u p ib er pT d rop s/.w c o u c e n tr a tio h y b f. /:-,
the. s a m p le and th e am ount o f f e s id u e in th e s a m p le . F o r c o m p a r iso n p u r
p o s e s a u th en tic s a m p le s o f p h en o ls w e r e c o -c h ip m a to g r a p h e d a lon g w ith •
23
th e h u m ic a c id s a m p le s » T h e s e s a m p le s w e r e d ilu te ' a lc o h o lic so lu tio n s
of th e p h en o ls p r e p a r e d to g iy e w e ll -d e f in e d sp o ts w hen th e ch ro m a to g ra m s;
w e r e scannedo
. T h e .ch ro m a to g ra p h ch a m b er c o n s is t e d o f a c o m m e r c ia l c h r o m a - V
to gra p h ic tank (R e s e a r c h S p e c ia lt ie s # 1 0 1 ), S ta in le s s s t e e l r a c k s h e ld
fr o m one to fo u r g la s s tro u g h s in w h ich th e m o b ile s o lv e n t w a s p laced * A
d ish o f the m o b ile s o lv e n t w a s p la c e d in the b ottom o f th e tank in o r d e r to
sa tu r a te th e c h a m b e r » W hen a tw o -p h a s e s o lv e n t s y s t e m w a s u se d , one '
o f th e p h a se s w a s p la c e d in th e g la s s tr o u g h and the, o th er in the dish* T he
tan k s w e r e a llo w e d to e q u ilib r a te fo r 24 h o u rs b e fo r e th e c h r o m a to g r a m s
w e r e . in s e r t e d , ■ 'v'1 ■ " y ' : .v:';.
T he le a d in g ed g e o f the c h r o m a to g r a m w as p la c e d in th e tro u g h and ;
w a s h e ld in p lace, by a g la s s r o d ,/ T h e c h r o m a to g r a m c u r v e d o v e r an a n t i-
s ip h o n rod and d e s c e n d e d to w ith in 10 c m , o f the b ottom o f th e tanks A
g la s s p la te w a s f it te d on th e top o f th e tank by s e a lin g th e e d g e s w ith s t o p
c o ck g r e a s e . T he c h r o m a to g r a m w a s le f t in th e c h a m b er u n til the s o lv e n t
le a d in g ed g e c a m e w ith in 10 cm , o f th e end o f the p a p e r . T he c h r o m a to
gram w as r e m o v e d fro m th e ch a m b er and th e n w a s d r ie d fo r tw o h o u r s ,
T he p a p er w a s sc a n n e d u n d er u lt r a v io le t l ig h t . A ll f lu o resce n t; a r e a s
w e r e m a r k e d w ith a le a d p e n c il , A c o n c e n tr a te d s o lu t io n o f a m m o n ia w a s
p la c e d u n d er the p a p er and any ch an ge in c o lo r f lu o r e s c e n c e w as n o ted and
r e c o r d e d , R f v a lu b s fo r th e s e f lu o r e s c e n t sp d ts w e r e c a lc u la te d . T he
24
c h r o m a to g r a m w a s sp r a y e d w ith one o f a n u m b er o f p h e n o lic sp r a y s
(T ab le 1)» A fter a tw o -h o u r d r y in g period^ the c o lo r e d a r e a s w e r e
m a r k e d and th e ir r e s p e c t iv e R f v a lu e s w e r e ca lcu la ted ^ T ab le 2 l i s t s
th e s o lv e n t s y s t e m s u s e d .
S p e c tr o p h o to m e tr ic a n a ly s i s .
To ob ta in an is o la t e d com p ou n d in s u ff ic ie n t q u antity fo r s p e c t r o -
p h o to m e tr ic a n a ly s is^ a sa m p le w a s a p p lied as a lo n g s tr fp e or s tr e a k .
T h is s t r ip e c o n s is t e d o f a 20 c m . band o f sa m p le s o lu t io n p la c e d 7 1 /2 c m .
fro m th e le a d in g ed g e o f th e p a p e r . T he sa m p le w a s c o n c e n tr a te d in th is
s tr ip e by f iv e to e ig h t a p p lic a tio n s and th en w a s d r ie d . T he c h r o m a to
g ram w a s d e v e lo p e d in o n e o f th e s o lv e n ts s d r ie d , sc a n n e d u n der u lt r a
v io le t l ig h t , and th e f lu o r e s c e n t b ands w e r e m a r k e d . A tw o c m . s tr ip e
on one s id e o f th e p a p e r (le n g th w ise ) w a s r e m o v e d and w a s sp r a y e d w ith
one o f the; sp ra y s.. T h e c o lo r s w e r e m a r k e d o ff and th e bands c o r r e s p o n d
in g to th e s e c o lo r e d a r e a s or f lu o r e s c e n t a r e a s w e r e cu t out.
T he bands w e r e p la c e d in an e lu tib h tank* T he tank c o n s is t e d o f a
d e s ic c a to r co n ta in in g a d ish o f 70% eth a n o l p la c e d on an in v e r te d b e a k e r .
One end o f th e band w a s d ipped in to th e s o lv e n t d ish , fo ld e d o v er th e ed g e
of th e d ish , and w a s p la c e d in to a 50 m l . b e a k e r . T he p a p e r band w a s
h e ld in p la c e by s m a ll g la s s h o o k s p la c e d on the ed g e o f d ish . The e lu a n t ;
w as a llo w e d to f lo w u n til 15 m l. had c o l le c t e d in th e b eak er* The e lu tio n
tank w a s s a tu r a te d w ith th e e lu tan t at a l l t im e s .
T he e lu te d sa m p le w a s c o n c e n tr a te d and r e -c h r o m a to g r a p h e d
u s in g a n o th er s o lv e n t s y s t e m . M m o s t c a s e s th is p r o c e d u r e e f fe c te d
fu r th e r p u r ifica tio n o A u th en tic s a m p le s w e r e a g a in c o -c h r o m a to g r a p h e d
w ith th e s a m p le . T he r e -c h r o m a to g r a p h ic p r o c e d u r e w a s .rep ea ted i f
n e c e s s a r y u n til i s o la t e d bands or a r e a s w e r e o b ta in ed . A fter th e s e sp o ts
o r bands w e r e p u r if ie d b y-the e lu tio n and r e -c h r o m a to g r a p h ic te c h n iq u e s *
th e y w e r e a n a ly z e d fo r th e ir c h a r a c t e r is t ic u lt r a v io le t sp ectra^ T h e s e
s p e c tr a w e r e ob ta in ed by tw o m e a n s : d ir e c t ly fr o m th e p a p er c h r o m a
to g r a m and fro m so lu t io n s p e c tr a o f th e band e lu ted fr o m th e p a p e r .
T he c h r o m a to g r a m i s sc a n n e d u n d er u lt r a v io le t lig h t and any
f lu o r e s c e n t a r e a s w e r e m a r k e d o ff w ith a le a d p e n c il . If th e com p ou n d
to be is o la t e d d id not h a v e a c h a r a c t e r is t ic f lu o r e s c e n c e u n der u l t r a
v io le t l ig h t 9 th e fo llo w in g p r o c e d u r e w a s c a r r ie d ou t.
In p la c e o f a s in g le sp o t o f th e s a m p le , a s t r e a k o f sa m p le w a s
p la c e d o n th e s ta r t in g l in e . T h is s t r e a k m a y be fou r to f iv e c m . w id e .
A fter th e c h r o m a to g r a m w a s d e v e lo p e d and d r ie d , o n e -h a lf o f the band
ch ro m a to g ra p h ed by th e s a m p le w a s cu t out in th e fo r m o f a s tr ip . T h e
p h e n o lic a r e a s w e r e d e te c te d by sp r a y in g th e s tr ip , and th e c o r r e s p o n d
in g bands on th e c h r o m a to g r a m w e r e m a r k e d w ith a le a d p e n c il . T he
c h r o m a to g r a m w a s d ip p ed in a 5% a c e t ic a c id so lu t io n and th en w a s
d r ie d . , T h is acid , tr e a tm e n t w a s to in s u r e a s l ig h t ly a c id ic m ed iu m fo r
th e u lt r a v io le t s p e c tr u m . The c h r o m a to g r a m w a s d r ie d fo r one h o u r .
26
T he e n c ir c le d band, p r e s u m e d to co n ta in the com p ou n d to be a n a ly sed ,'
: w a s cu t out and an a r e a a lo n g s id e o f th e e x tr a c te d a r e a w as cu t out to
s e r v e as. a b lank . T he c h r o m a to g r a m is i l lu s tr a te d in F ig u r e 1. R ec-.
ta n g le s on e by th r e e -cm . a r e cu t fr o m th e c e n te r s o f th e s e b an ds. '
T h e se s a m p le s (com p ou nd p lu s b lank) w e r e p la c e d in a B eck m a n D . U.
. sp e c tr o p h o to m e te r . T he h o ld e r , th e s a m e s i z e a s a r e g u la r B eck m a n ' . .
D . U. c e l l h o ld e r , is d e s c r ib e d by S te e lin k (29) ( s e e F ig u r e 2 ). '
T he a b so r p tio n s p e c tr u m w a s ta k e n in th e u s u a l m a n n e r b e tw e e n
2.00 and 400 m / f . The s l i t w id th u s e d fo r p a p er S p e c tr a w a s v a r ie d . ... ■
fro m two: m m . to m a x im u m w id th . In c a s e the :sam ple w a s too c o n c e n - '
tr a te d , a n o th e r r e c ta n g le w a s cu t n e a r e r th e ed g e o f th e sa m p le band
and th e sp e c tr u m w a s r e p e a te d . I f th e sa m p le w a s tod d ilu te , a s e c o n d i
c h r o m a to g r a m w a s p r e p a r e d w ith a g r e a te r am ou n t o f sa m p le b e in g ■
a p p lie d . F o r s p e c tr a in 'b a s ic m ed iu m a m o d if ic a t io n o f a s y s te m o f
Jurd (28) w a s u s e d . In th is m eth o d th e r e c ta n g le s p r e v io u s ly u se d in th e
a c id p a p e r sp e c tr u m a n a ly s is , w e r e p la c e d in a s a tu r a te d s o lu t io n o f
. so d iu m " aceta te , w e r e d r ie d for" on e h o u r , and th e a b so r p tio n m e a s u r e -
m e h t Was 'r e p e a te d . N e u tr a l p a p er s a m p le s w e r e a ls o a n a ly z e d and th e
m a x im u m p ea k s h if t s w e r e o b s e r v e d .
S o lu tio n S p e c tr a w e r e ob ta in ed on s a h ip le s e lu te d fr om th e c h r o m a -
; . to g ra m S w ith 70% eth a n o l. In p r e p a r a t io n o f the c h r o m a to g r a m fro m :
■ w h ic h th e e lu tio n s w e r e to be m a d e , o n ly on e -h a lf o f th e w id th o f th e : . '
27
p a p er w a s s tr e a k e d w ith th e sa m p le (a llo w in g tw o c m . on both s id e s fo r
a border)'. T he o th er h a lf o f th e p a p e r w a s le f t b lank to a llo w fo r the
p r e p a r a t io n o f an e lu tio n b lan k . Upon d e v e lo p in g th e p a p e r in a s u ita b le
so lv e n t and d ry in g fo r one h o u r , th e a r e a w a s d e te c te d b y u ltr a v io le t
l ig h t so r by a p o r tio n o f th e a r e a b e in g sp r a y e d . T h e band w a s cut out
and an a r e a o f th e s a m e S ine w a s cu t out fr o m th e b lank s id e o f th e
p a p e r . Upon e lu tio n o f th e sa m p le and th e b lank bands., th e sa m p le and
blank w e r e d ilu ted w ith 95% eth a n o l u n til a q u a lita t iv e s p e c tr u m 'o f th e
s a m p le co u ld be o b ta in ed . F o r c o m p a r is o n p u r p o se s th e so lu t io n
s p e c tr a w e r e run on s a m p le s in s l ig h t ly a c id ic and b a s ic s o lu t io n s . T h e
a c id ic so lu t io n s w e r e p r e p a r e d by ad d in g one drop o f v e r y d ilu te (a p p ro x .
0 . 01 N» H CL) a c id to th e sa m p le in th e c e l l . T he b a s ic so lu tio n s w e r e
ob ta in ed by sa tu r a t in g th e sa m p le in th e s a m p le b lo ck w ith so d iu m
a c e ta te (28).
Id e n tif ic a tio n o f P rO to ca tech u ic a c id .
P r o to c a te c h u ic a c id w a s id e n t if ie d and is o la t e d in th e fo llo w in g
m a n n er: A sa m p le (5. 634 g. ) o f th e C a lifo r n ia h u m ic a c id w a s s u b je c te d
to a lk a lin e d e g r a d a tio n a s o u tlin ed . U pon r e m o v a l o f th e S o lv en t fo l lo w
in g th e e x tr a c t io n o f th e h y d r o ly s a te , a s e m is o l id r e s id u e r e m a in e d
(0. 237 g. ). T h is r e s id u e (6A) w a s d is s o lv e d in 10 m l . o f a b so lu te e th a n o l.
T he s o lu t io n w a s t e s t e d fo r p h e n o lic co n ten t by the a d d itio n o f f iv e d ro p s
o f a v e r y d ilu te s o lu t io n o f a q u eou s F e C l. to five- d ro p s o f sa m p le in
one m l . o f w a te r , A h e a v y b la ck p r e c ip ita te d e v e lo p e d in d ic a tin g the
p re s e n c e o f p h e n o lic c o h s t itu e n ts . ; , ■ ;■.
The so lu tio n w a s su b je c te d to p a p er c h r o m a to g r a p h ic a n a ly s is . .
T he sa m p le w a s p la c e d on th e c h r o m a to g r a m in th r e e d is t in c t sp o ts
c o n s is t in g o f '6, 14 , and 20 d r o p s , r e s p e c t i v e ly . T he c h r o m a to g r a m
was. p la c e d in a b u ta n o l-p y r id in e -w a te r (B . P . W, , s e e T a b le 2) tank fo r
1.2 hpura> - ;jiffer d e v e lo p in g andV dyyihg/ t o an
a r e a w h ic h : f lu o r e s c e d p u r p le u n d er th e u lt r a v io le t la m p . T h is a r e a
(Rf . 58) , w hen subj e c te d to the v a p o r o f a m m on ia ,/ sh o w e d b lu e - g r e e n
f lu o r e s c e n c e . W h en :th e c h r o m a to g r a m w a s sp r a y e d w ith 1 % F e C lo , a
• g r e e n -b la c k a r e a d e v e lo p e d at R f . 63 . A se c o n d c h r o m a to g r a m w a s
run c o n s is t in g o f th r e e sp o ts o f 6A , to g e th e r w ith tw o sp o ts ea ch o f
g a llic a c id and p r o tb c a te c h u ic a c id , .- T h e r e s u lt s o b ta in ed u pon sc a n n in g
u n d e r u ltr a v io l et l i ght w e r e not v e r y: de f in lt e i W hen th e c h r o m a to g r a m - :
w a s s p r a y e d , . w e l l - d e fin ed sp o ts w e r e o b s e r v e d . R f v a lu e s and c o lo r s
o b ta in ed a r e l i s t e d in T a b le s 3 and 4 . C h r o m a to g ra m P r 3 co n ta in ed a
c o n c e n tr a te d band of: 6A on the fu ll w id th o f a c h r o m a to g r a m , f (T h is con-,
r p e h ty a te d d ep b a it 'Vras- ob ta in ed b y a p p ly ih g th e s o lu t io n 1 5 t im e s ov er . th e
s a m e a r e a ») T he p a p er was' d e y e lo p e d in B , P . W. A tw o c m . s tr ip w a s
sp r a y e d w ith F e C F and a b la ck a r e a .e ig h t c m . in w id th w a s d e te c te d . .
T h is a r e a w a s d iv id ed in to fou r s t r ip s and th e p h e n o lic s u b s ta n c e s w e r e .
e lu ted fr o m th e s e fo u r s t r ip s w ith 70% eth an b l. C o n s id e r a b le b lack
r e s id u e w as in c lu d ed in th is e lu tion ,: T h e e lu te d s a m p le w a s r e d u ced to .
n e a r d r y n e s s by h e a tin g in a c a s s e r o le o y e r a v e r y lo w m ic r o b u rn er
f la m e . T he sa m p le w a s w a sh e d w ith tw o m l. o f a b so lu te eth an o l and. th e
b la ck r e s id u e w a s r e m o v e d by c e n tr ifu g a t io n giving- 53a m p le h&A. :
A p a p er c h r o m a to g r a n i P r 4, c o n s is t in g o f 20 d ro p s o f 6AA, w as.
c o -c h r o m a to g r a p h e d w ith 20 d ro p s o f au th en tic p r o to c a te c h u ic .acid in '
a b u ta n o l-a c e t ic a c id -w a te r (B , A . W. ) so lv e n t s y s t e m . P a p e r u lt r a v io le t
s p e c tr a w e r e ta k en on th e s e tw o s a m p le s . A n e e d fo r fu r th e r is o la t io n
o f t h e p h e n o lic com p ou n d w a s o b s e r v e d . P a p e r s P r 5 and P r 6 c o n ta in e d
C drieen tfated bands, o f 6A in B. P . W , and B . A . W. , r e s p e c t iy e ly . T he
p h en o lic a r e a s w e r e e lu te d w ith 70% e th a n o l. . T h e se e lu t io n s w e r e 6A A 2
and 6A A 3, r e s p e c t iy e ly . P a p e r P r 7 c o n s is t e d o f 6A A s tr ip e d the fu ll
w id th and d e v e lo p e d in 5% .H A c,. T h e p h e n o lic a r e a w a s g r e e n w hen '
sp r a y e d w ith F e C lj and had an R f o f > 59. T he u n sp r a y e d a r e a w a s e lu te d
W ith 70% ethanol: g iv in g sa m p le 6A AA , f r e e fr o m r e s id u e and p a le ' yeiloyy-
in colon* T h e sa .m ple w a s r e d u ced in v o lu m e to tw o m l. P a p e r s P r 8
and P r 9 w e r e ,deveiO ped in B. A . W. and. B . P , W,. r e s p e c t iy e ly .. T h e s e
p a p e r s co m p a r e d c o lo r and R f v a lu e s o f 6 A AA w ith th e staridards g a l l ic ,
p r o to c a te c h u ic , and v a n il l ic a c id s . In' ad d ition to sp r a y in g w ith F e C lg ,
th e d r ie d c h r o m a to g r a m w a s S p ra y ed w ith a d ilu te aq u eou s so lu tion , o f
NH-j w h e r e u p o n , th e F e -p r o to c a te c h u ic and F e -h u m ic a c id sp o ts tu rn ed
p u r p le . S a m p le 6A A A w a s fu r th e r t e s t e d by a t e s t tu b e r e a c t io n s im i la r
to th at co n d u cted on 6A. T h is t im e a g r e e n c o lo r w a s o b s e r v e d . Sariip le
6A A A w a s p la c e d oh p a p er F’r 10 a lo n g w ith p y r o c a te c h o l and p r o to c a -
u te c h u ic a c id and w a s d e v e lo p e d in' 20% K C h P a p e r P r 11 , co n ta in in g a
c o n e e n tr a te d sp o t o f bAAA, w d s d e v e lo p e d in B. A. W . U nder, u lt r a v io le t
..light a b lu e a r e a (R f . 88) w hs detected:^ At R f . 85 th e g r e e n p h e n o lic - I - -
a r e a w a s d e te c te d w hen sp r a y e d w ith F eC l^ . A r e c ta n g le w a s cut fr o m ■
the p h e n o lic a r e a and a p a p e r s p e c tr u m w a s ta k en in th e 'm an n er ■
;';d ea cr lb ed p r e v ix ^ T h is s p e c tr u m is sh ow n - in C hart. 3. " A s a fu r th e r ,
c h eck on R f v a lu e s and c o lo r r e a c t io n s ,, s a m p le s '.6A, 6A A , and 6A A A
w e r e sp o tte d on p a p er P r 12, to g e th e r w ith an a u th en tic s a m p le -o f p r o to -
' c a te c h u ic a c id and d e v e lo p e d in a r n - c r e s o l - a c e t i c a c id -w a te r s o lv e n t
s y s t e m u s in g th e w a te r p h a se d f the s y s t e m . - T he p a p e r s w e r e a llo w e d •
to d ry fo r th r e e h o u rs and th en w e r e -sprayed with, a N H j-A gN O ^ sd lu tio n .
T he .a r e a s w e r e d e te c te d by the' l ig h t ta n c o lo r on a d ark purple, b a c k
ground . A so lu t io n sp e c tr u m o f th e su b s ta n c e 6A A A w a s ob ta in ed by
e lu t in g .a band w h ich had b een p u r if ie d by r e c h r o m a to g r a p h y in v a r io u s
s o lv e n t byStem s ( s e e l i a b l e n and Chppti 1 ). ' d'-iy :
Id e n tif ic a t io n o f V a n illin . . . .
.; ■ A sa m p le (1 . 668 g. ) o f th e E n g lish himriic a c id w a s su b je c te d to a
pot as S m m ' h y d ro x id e fu s io n w ith ou t p r e v io u s a c id h y d r o ly s is and th e
g e n e r a l e x tr a c tio n p r o c e d u r e w a s c a r r ie d out as d e s c r ib e d ab o v e .
W hen the e th e r so lu b le p o r tio n (3A) w a s su b je c te d to p a p e r c h r o m a to -
graphy, tw o or p o s s ib ly th r e e p h e n o lic sp o ts w e r e d e te c te d . The r e s u lt s
.'0.i:this a n a ly s is (p ap er V 1) a r e r e c o r d e d in T a b le 7. . ; , . y
. / W hen th e f i r s t 'd eg fad ation ' p r o c e d u r e s w e r e p a r r ie d put, a ld e h y d e s
w e r e not cph S ide r e d as. p o s s ib le d e g r a d a tio n p r o d u c ts , s in c e o x id a tio n
to th e a c id w a s a s s u m e d in th e fu s io n m ix tu r e . L a te r , th e s e com p ou n d s
v /e r e c o n s id e r e d a s ,p o s s ib le r e a c t io n p r o d u c ts and th e c h r o m a to g r a m s ■
rv ere .sc a n n e d fo r th e m . U ltr a v io le t s p e c tr a w e r e d e te r m in e d fo r a
n u m b er o f a ld e h y d e s , in c lu d in g v a n ill in . A sa m p le w a s is o la te d and
p u r if ie d by r e c h r o m a to g r a p h y and u ltr a v io le t a b so r p tio n S p ec tra w e r e - .
tak en d ir e c t ly o ff th e c h r o m a to g r a m o f th is com p ou n d and au th en tic
y a n ill in ( s e e F ig u r e 5). :: ; n ; i v ':;i" ;:V 1 :•
I so la t io n o f a p h lo r o g lu c in o l d e r iv a t iv e . ,
W hen .S'ample 4A w a s su b je c te d to c h r o m a to g r a p h y in 2% BOH :
• (p ap ers P 1 and 2 ), an o r a n g e -r e d sp o t w a s d e te c te d at R f . 70 w ith - -
2 , 4 -d N P H . S u b seq u en tly , p a p e r s P 3, 4 , and 5 w e r e p r e p a r e d c o n ta in
in g 18 au th en tic p h en o l s a m p le s c o -c h r o m a to g r a p h e d w ith 4 A. T he R f
v a lu e s a r e ta b u la ted in T a b le 4. T he o n ly a u th en tic p h en o l R f th at c o r
r e s p o n d e d to th e o .ran ge- r e d band o f 4A w a s p h lo r o g lu c in o l. In s u b s e
quent e x p e r im e n ts this: e lu te d sa m p le w h en su b je c te d to s p e c tr a l a n a ly s is
32
w a s to o d ilu te to y ie ld a w e l l -d e f in e d s p e c tr u m . T h is band (4AA) w a s
is o la t e d . R e s u lts o f c o -c h r o m a to g r a p h y w ith p h lo r o g lu c in o l in v a r io u s
so lv e n ts a r e g iv en in T a b le 8. ,
' - r ■■ suM M AJiY . ;; ; ' \
: /. .F rom th e e v id e n c e p r e s e n te d in th is p a p e r , th e fo llo w in g c o n c lu -
.; ; sihn-s -can' he d raw n . -
•1. P r o to c a te c h u ic a c id has b een id e n t if ie d a s b e in g a d eg ra d a tio n
p rd d n ct o f th e " in n er co r e " o f h u m ic a c id , o b ta in ed fr o m C a lifo r n ia s o i l . .
;, ThiS; ac id w as id en tified by .m eans of ch ro m a to g f aphy and w as \Verified.; 'v. , v:
through p ap er and so lu tio n u ltr a v io le t s p e c tr a l a n a ly se s . : y:-''"".
2 . V a n illin w a s d e te c te d a s a d eg ra d a tio n p ro d u ct o f E n g lish h u m ic
. a c id by c b -c h r o m a to g r a p h y w ith a n a u th en tic v a n ill in s a m p !e . Further- ^ ^ ^ ;, . : .
" ev id en ce fo r th e e x is t e n c e o f th is com p ou n d in E n g lis h h u m ic a c id w a s
: a ■:' in f e r r e d fr o m th e s im i la r i t y o f th e p a p er u ltr a v io le t s p e c tr a of th e / .
unknow n an d th e a u th en tic y a n ill in sa m p les:. ; ■ ; . » ;
■ t:;:- ■ ■ " ; , ' ... 3. A. p h lo r o g lu e in o l- l ik e com p ou n d w a s d e te c te d a s a degradati,Qn;e?yv V’ ••
V p rd d u ct o f E n g lish h u m ic a c id . H o w e v e r , th is com p ou n d w a s sh ow n by
r e a c t io n s w ith s p r a y s and by paper- u lt r a v io le t a n a ly s is to be n o n id e n tic a l <
w ith p h lo r g lu c in p h - ' v\. : - ;" ' ■■ v '
4 . C h r o m a to g ra p h ic a n a ly s e s o f th e e x tr a c ts Show th e m a jo r ity
; o f th e d e te c ta b le p h e n o lic s u b s ta n c e s o f th e fu se d h u m ic a c id to be e th e r .
; 5. It h a s b een sh ow n th a t.p a p e r u lt r a v io le t s p e c t r a l a n a ly is i s .a • ‘
v e r y u s e fu l to o l to c o m p lim e n t p a p er ch ro m a to g ra p h y in q u a lita tiv e
a n a ly s is o f d eg ra d a tio n p rod u ctg of.-htunlc a c id . ’ 1
, ' 6.„ F u r th er j it h a s b een sh ow n th at in sQm e c a s e s ( s e e s e c t io n on j
p h io rog lu ein o l-i-lik e :com pound) sp e c tr o p h o to m e tr ic a n a ly s is i s e s s e n t ia l
for. v e r i f lc a t io n o f h -hfigt^dation p r o d u c t. ,1 • ' / ■ ■ ' ■ i,;
■ 7. S e p a r a tio n by d e te c t io n of th e c o lo r - s p r a y e d c h r o m a to g r a m as.
o p p o se d to s e p a r a t io n by f lu o r e s c e n c e u n d er u ltr a v io le t l ig h t is m o r e
8. T he d e te r m in a t io n o f v a n il l in and p r o to c a te c h u ic a c id in h u m ic
a c id g iv e s fu r th e r •support fo r th e th e o r y th a t h u m ic a c id is. d e r iv e d fr o m
L IT E R A T U R E C IT E D
1. A. G ille t , B r e n n s to ff -C h e m , , 38 , 1 5 1 -6 (1957).
2„ vE. S c lie f fe r and. E„ W elte , M a tu rw issen sc lia ften ., 37 , 3 2 1 -9 (1 9 5 0 ).
3. : J /M V B r e m n e r , j . S o il S c i . , 5, 2 1 4 (1954 ).
4 . H . R au d n itz , C h em . and Ind. , 5 1 , 1 6 5 0 -1 (1 9 5 7 ). .
5. W. E la ig and T h. B rey h a n , Z. P f la n z e n e r n a h r . D ung. B od en k . ,75 , 1 3 2 - 5 (1956); C . A . , 51, 6061 (1957 ).
6. J. M . B r e m n e r , j . A g r . S c i. , 46 , 2 4 7 -5 6 (1955 ).
7. R. I. M o r r iso n , J. S o il S c i . , _9, 1 3 0 -4 0 (1958).
8. M . M . R oy , R e s e a r c h (L ondon), 1 0 , 78 (1957).
9. D . L . L yn ch , L . M . W righ t, and H., O . O lney , S o il S c i . , 8 4 , 4 0 5 -1 1(1 957). ... V :: - 4 , . ' . V : .
10 . R . I. D a v is , C . B . C o u lso n , and C a r lo s L una, C h em . and Ind. ,51, 1 9 4 4 -4 (1 9 5 7 ).
11. : J. M . B r o m n e r , AT. A g r . S c i . , 4 8 , 352 (1 9 5 7 ).
12. G. C . E s h and S. S . G u h a -S ir c a r , J. Indian C h em . S o c . , 17 , 3 2 6 -3 1 (1 9 4 0 ),
13. S . S . DragunoVj N , N . Z h e lo k h o y tse v a , and E . I . S tr e lk o v a , P o c h v o v e d e n ie , 1 5 1 -7 (1950); C , A . , 4 4 , 6995 (19 5 0 ).
14 . M auro B o tte r i , A nn. t r ie s t in i c u r a u n iv . T r ie s t e s e z , , 2 , 20 , 1 5 3 -7 0 (1951); C . A . , 4 6 , 7131 (1952).
15. S id n ey G o ttlieb and S . B., H e n d r ic k s , S o il S c i, S o c . A m . P r o c . ,10, 1 1 7 -2 5 (1 9 4 5 ). y
, : . : : : 35 ■ ; ' ■ ... ' ' '
36
16. T . A . K ukharenko and T . E„ Y n ed erisk oya , K him i T ek h n o l. T o p liv a . N o . 6, 2 5 - 3 4 (1956); C . A . , 50, 16005 (1 9 5 6 ).
17. V„ T . B r o o k s5 G. J . L a w so n , and S , G. W ard, F u e l, 3 8 5 -9 7 (1956) G. A . , ^ 0 , 17378 (1956 ).
18. H . T r o p s c h and A. S c h e lle n b e r g , G e s , Abh.. K en n tn . K dh le, b,191=5 (1921); c . A. 18 2872 (1924). • ' '
19. J . M . B r e m n e r , - Z= P f la n z e n e r n a b r . D ung. B od en k . , 6 9 , 3 2 -8(1955). . T "
20 . J , E.- S ton e and M . J. B lu n d e ll , A n a l. Chezru , 2 3 , 7 7 1 -4 (1951).
21.' S . P . S en and A . C. L e o p o ld , Phyis.lol. P la n ta r u m , 7, 9 8 -1 0 8 (1954)
2 2 . R . J , B lo c k , " P a p er C h r o m a to g ra p h y and P aper. E le c t r o p h o r e s is " , • 2nd ed . , A c a d e m ic P reSB In c. , N ew Y ork , N . Y. , 1 958 .
2 3 . E . L e d e r e r and M» L e d e r e r , 1'C h r o m a to g r a p h y : A R e v ie w o f P r i n c ip le s and A p p lic a tio n s^ , 2nd ed i , Y an N p stra n d , -P r in c e to n , N ew
; J e r s e y , 1957 . a "
24 . C . B . C o u lso n and W., C . E v a n s , J. C h r o m a to g ., 1, 3 7 4 -9 (1 9 5 8 ).
25 . L . R e io , J . C h ro m a to g , , 4 , 3 3 8 -7 3 (1958).
26 . John B . P r id h a m , A nal. C h em . , 29 , 1 1 6 7 -9 (1 9 5 7 ).
2 7 . A . E . B r a d fie ld and A , E . F lo o d , J . C h em . S o c . , 4 7 4 0 -4 (19 5 2 ).
28 . L eo n a rd Jurd and R o b e r t M . H o r o w itz , J . O rg. C h e m ., 22 , 1618 (1957 ). _ ~
2 9 . C o r n e liu s S te e lin k , P h . D . T h e s is , U niv. o f C a lif , at L o s A n g e le s( 1 9 5 6 ). a a; ..-' . a a ' v ' Y # . : . ■ :' ; ' ; ; ■ ■: a a '" /;
30. H . W. L em o n , J . A m . C h em . S o c . , 6% 2998 (19 4 7 ).
31 . R.a A . M orto n and A . L . S tu b b s, J . C h em . S o c . , 1347 (1940).
32 . T od W. C a m p b e ll, J. A m . C h em . S o c . , 73 , 2 7 0 8 (1951 ).
33 . R . F= P a t te r s o n and H, H ib b er t, J. A m C h em . S o c . , 65, 1862v; (1 943). /_ ; -
.A K ^ E N E IX :: „
' ; , ‘ : i W r P ^ . T A B i .b s :
S p ra y R e a g e n ts . ,.:; .-yV ' '
:;V ; ■ y - ;. >:;.
C o lo r R e a c tio n s w ith P h e n o lic S p r a y s ,
R f V a lu e s fo r F o u r S o lv en t S y s te m s .
M a x im a fo r
I so la t io n o f a P h lo r o g lu c in o l- l ik e C om pound.
38
T a b le 1,' S p ra y R e a g e n ts » , -
S p ra y A bbr e v ia t io n ’ R eta ren c e'
ly ' l % f e C l ^ ' .■‘ . J' ■ :F e : A,-1r I'r. .
2 2 . :
2«- D ia z o t iz e d S u lfa n ilic a c id - :::V y ' DSA.;-'^;:■:> 1 '
3. ; D ia z o t iz e d p - N i t r o a n i l in e : '■ v x ^ D P N A ^ -: \ ,r.'\
t .. ; 22 ’. :;R;v>: i;.
•4. ■ 2, 4 - D in itr o p h e n y lh y d r a z in e■ ' ; ■■■' •: , 2 s 4dN P H ■ ■ ' T ■, ' - * 22 'V
5 . :¥ a n in in in HC1 . ; V H C 1 '■>. I..;. :’
6 i A m m on ia ca l. S i lv e r N itr a te ; ■ AgOH; : y t ; ,
1 22 " A ,;-:
-7. N y d r a z ln e R ' ' ' . v / ' ' v H r '
T a b le 2„ S o lv e n t S y s te m s , • ' , •;
• ■■■ . '< ; ^Solvent y Y " -
>ompOs.iiib:
imlBHxe „
n' v '- T v Aiati.r e *” , 1V v ia t io n i •
R e fe r -en ce
T y \:5,% .& q eilc:ac ld ' 'r# 5%HAc V - i r •
2v B u ta n o l, A c e t ic a c id , HoG r (3:5:2) ' BAW :r ' 1 '
22 '/A y'
3, B u tan o l, P y r id in e ,. HgO . ; ' 1 (6 :4:3) . ■'''b p W '-v : ' i ;■■■ I,
4 , B u tan d l, s a t , w ith 2% NH^.OH 1 . (o rg a n ic p h a se ) " ; . 1 '
1 a%BG)H 4 -r - . ; : V;
■ * : . ; !5, m - C r e s o l , A c e t ic a c id , H2 O '- I ; ' (or gan ic p h a s e ) i:1 .
(50:S:48,);
.1 ' I- -
22 ■ :
6. 20% KG 1 ■ ; ' ' •■V, ' KC1 • 1. r' 1 ' i'.:
f M
7, B u tan o l, B e n z e n e , s a t . N aC l 1(o rg a n ic p h a s e ) '
(1 :1 :2 ) , .. ’ BBKfaC 1 :i■' ■iV;/:
Gv
22
40
T a b le 3. C o lo r * R e a c t io n s w ith P h e n o lic S p r a y s .
SPR A Y SC om pound !______Re 1 P S A 1 PNA.
' O bs. L it .* ^ O bs. 'L it . ** ' O b s. 'L it.*** ' V1. P h e n o l i. P i Y i Y f Y -G ' ! .2 . P y r o c a te c h o l ' P G -B k ! T i P k ' Y ' R -P '3. R e s o r c in o l ' . - t B * y * Y -O ' Y -O > : V B l' '4 . H yd roq u in on e ' W - ! T i W ' W 1 !5. P y r p g a l lo l 1 P - V ‘ B i B * B ^ Y -T I 16. P h lo ro g lu e in o l t *=* f B i Y ! R V Y -O ' B l-G /N H g7, p -H y d r o x y b e n z o ic t t i i t . *
: a c id • R -V Y .1 Y ( y ( Y * R8. m -H y d r o x y b e n z o ic i r i I ! !
a c id • • ' B l- P - ■ T Y 1 y 1 Y ' R '9. G a llic a c id 1 P -V Gy ? T ' Gy ' Y -T f ' ! ; - ;
10. S a l ic y c l ic a c id ' P - V ' R -V .1 Y i y 1 ; Y . R . i ' B i ;■11. - R e s o r c y l i c a c id f - V R -V i Go 1 y 1 O -R12, - R e s o r c y l i c a c id 1 .. B r Go r i Y 1 Y 113 . % - R e s o r c y l ic a c id I V t O ' Y ' Y -G o 1 Y !
14 . G en tisic* a c id ' B l B l i T ' W ' W ' Y ' B l .15 . P r o to c a te c h u ic ! . i i i f ■ -v. •■
a c id ' G G t Y -G o ' P k ' Y -O ' R '1 6 . V a n ill ic a c id r Y i Y- ' Y ' R -P ! i
17 . S y r in g ic a c id ' T B t O -P k 1 R ' Y T f
1 8 . P h lo r o g lu c in o l ' j t I T 1
c a r b o x y lic a c id :r j Y 1 . 1 G o I t -vl
19. V a n illin ' B l - 1 Y 1 1 P • t * 1 ,, ,
2 0 . P r o to c a te c h u a l- T i ' ■; ' I I
d ehyde It
G !I
T i i i r
f ! f . 1
^ D efin itio n o f c o lo r t e r m s r ;-■ B l—■ B lu e Go ~ G old ' P k - P in k Y - Y e llo w
Bk - B la c k G y - G r ey R « R ed W - W hiteB - B row n O - G range T - T an - no c o lo r
. G - G reen . P - . P u r p le ; V - V io le t UV yitrftY|oli.et: l ig h t
>' 5‘> V alueB ta k en fr o m R e io (R ef. 2 5 ).?{> |c
V a lu e s ta k en fr o m C o u lso n and E v a n s (R en 24)*
41
T a b le 4. V a lu e s in F o u r S o lv en t S y s te m s (R f X 1 00 ).
' C om ppund ' 7 " ' \ .;'.:.vS,bl.venta :'
; '* :5 S iA c :"j; RAW . : B P W :4 ? : '2 :%BGH'4
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4.44:844:J;:9''
4 . H y d r 0 quinoxie i: ;-74' 1 ' .■ svv ' 89 .: i 82 ;y4'
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■ vi:; i■j
6 . P h lo ro g lu G in o l} ■ ' . r
' i . 75 '•'4 . 88 v '4- .1 ■
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! ; 1.8 . :
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1GV S a lic y c l ic a o ld ' ‘ ; • ' .V:, p - 1 '::92:4I" »
i: 63 • 4?
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1 RV $ -B .e s o r c y l ic a c idr •
■1
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44.,4.'i424l'4 4'44V
1 2 , ' R e s o r e y l ic a c id • . . ! 611r ■ -I 9 6 / 4
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1 5-y P r o to c a te c h u ic a c id • 57 :,:v : ; 4 y
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4 :67';44^:,:
V a n ill ic a c id V ,, '.;:62 ;■ ■ ■■.■I-:-./ : # 4 4y BR ' 4; •444'
■4%v S y r in g ic a c id ' •
1 : " ■
57;i ‘I
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y .i : : . :
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'1
t. ' 56 I :
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f
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V a n illin? . .
i ■
1 ■ i : ■
' 14/
/ i71
T a b le 5. M a x im a fo r U ltr a v io le t A b so r p tio n S p ectra , in 95% E th a n o l.
C om pound' ! xn.pi. j i xnM R e f.
1 -P h en ol2 P y r o c a te c h 13 R e s o r c in o l
'* 215 . 0 4 . 00't ' I.':'/' A I,/." .
6;:E i i lo r : o g l n c i n o l :-7 • p>- Hyd r oxyb e n z o i c .. •
a c id :' 8 in H yd roxyb en z ode.
9 C a llic : a c id ;10 S a l ic y c l i c . a c id1 1 - R e s o r c y l i c a c id12 - R e s o f c y l i c a c id '
;13 I " R e s o r c y l ic a c id ,14 G ent is tc,; a c id .>' ■15 P r o to c a te c h u ic a c id
■ : y;... ; .(p a p er l;: • 1 :16 ;Y h n illic acld^ ;
17 S y r in g ic . a c id18 P h lo r o g lu c in o i a c id19 V a n illin ; . . : ■ ;v
; (pape r ) '20 2122 '2 -
;23;'3-2 4 4 -2 5 2 -M eth o x y b en za ld eh y d e 2 6 V e r a tr a ld e h y d e
.27 .B en zo ic acid:28 P h th a llc a c id ^ '29 Iso p h th a lic a c id30 T e r e p h th a lic a c id
i . i ■
: T '; tip
I . I
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; :h :yi,;.'i;' ’:. ; if;i;
■ tA :
1..'
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;: V :a;'- !
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2 7 1 . 0
2 3 3 . 0
2 5 9 . 0 305 . 5 2 9 6 . 0 308 . 0 308 303 . 0 3 3 4 . 0 2 9 5 .0 . 293
260 :2 9 7 . 5 310 . 0
e1c'.I:
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310:313
: 3 2 5
y 3i.6.;;2 2 :1
319 . 5 308 :308
' 2.27 .: a 2 8 1
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'4 .3 2 3. 68 3. 34
'31,39;.
'2 .1126 4.22y;:; 3. 70
3. 44 3 ,9 6
3 .4 8 ;3 v 4 6 4 l l 4 .3 . 633 .4 9
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:272v5 ' '; 2 3 6 . 0 2 5 5 . 0 2 5 0 . 0 :25:1; :• 252 . 0 236 . 0 .259. 0 2 6 0
:;235: 2 8 6 . 0
'290 2 7 3 ,0 262 . 5
1279. O 2 3 0 ' : 281 280 308 .
.255 ■' ': 2 5 4 I .3 8 4 2 5 3 . 0 278 276
;267v: y 2 7 4 280 2 8 4
2 ,4 8
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^ ;-'.;3 l- .Cbs;! '
:P-; ■ - .•'••;' . 32;
3. 95 314 . 6 8: O b s.3 ^ 7 4 ' Obs ,:
* (rvL-j, ’
2 .4 2: r'P "S>-
- Cb:s:.';. . ObS:. •4 . 39
3 , 9 6 O b s,. ' O b s.
""'Obsr"2 ,:6 2 ::yy
' O b 33; ; Obs.:,"4 .0 1 ' 334 ,1 2 ; : 324 ,6 3 y 3 3
■ 6yJoba.'y l'':Obaiy
"’’ OKq-14 .1 1 ;
O S &v , 3',y;82
4 , 000 Cfi 0 ' ’ 631i . ■ 3-6,. qf’t,.
4 / 2 4 .■ V " 6 '3 1 .4 ,0 7 :: 1: 314 . 05 6 3 0
6; o d e . ,■■■*;• :a '■ - ;3. ZD
3 . 0 1 y• ,3d
313 . 1 1 !. 31
(C ary ) I(C ary)
(Cary)-,Itianylv::(C ary l
|B U )
3 .2 4 3.1
R ite r a tu r e : v a lu e s ta k en fr o m ..R e fe r e n o e s ''C ite d .
Table 6, Is olation of P rotocatech u ic .Acid,
Ghro.N um ber
SolventSpray
i H.: A r'Sam p le t P r p t . A, j C ol or R f.i ' Rf,
G allic A,■ : 'R f. %
:Pr: 1
P r 2- -Pr: 4 A ; :P r 7:P x [ 8 • -P P : ;'9: '
P r 10 -
III
v- r 1:1 ’
1
' f
6A ':
6A ; 6AA
6 A A A 6 AAA
6AAA
BPW BP W . 5%HAc.BA% <::
,20%KC1
.v:i.
iii'i
- i : f
F e
_ P e - . P e -
F eF e
; Fe . and, NHo
F e -
b b :-g - uV' P: ' (Bl ' B l- G 1 B l-G
":G ' G ■1 P ' G
:583> ' 56 '
' ' *
59 ' 85 80 1:
streak .
56
Ri- SO
: y p strea k
51
; 79
Pr- 11 v-/;V ; 6AAA ' P 1 BAW V. : - 9, : F e -: ' 9G .;.9; 9 : :. ** ' r .P r ■1 2 p -J:yP ' 6A , '' r 'G r c a p ,I: --- . . I" -AgOH - , 1 ta n /P ' 37 ' ;56': f a h /p
6A A ' ; ■f - CAH -: 9 "I. A g© B 1 ta n /P - 35 ' v 36 tan/P; , f •;' : . .! ■6AAA ; > CAHVr ' ' v AgCDH ' ■ 9 ta n /P 36 ' 36 ta h /P 1 ' :
P r 1 4 -a , b 1 ■ 6AA2 ’ ' BAW . 1 . ■ F e ' Bk ' : 69 '- ; , : ", , 99 ; . ";99- 975 ' .‘P;
P r , 1 5 -a , b ; M A 2 ' :. ; P f; • 5%PAcg 'G'i: ::9 - ,,FeG g --P.G9G --• 9 5:9P r - ' 16 ■■';G < iA 1 v,'P% h P c? . 1 9 ' F e - ' • ' : 71 '
.. - - F ' - , G 58 9 Gr :; 'P r 17 : n 6A , 5%HAc , ' F e 9 1 G . -streak ,' 57 : ?:
„ . i 6AA2 , 5%HAc . , F e 9.G 54:, 57 p -V.; ;6 AAA : 5%HAc : : Fe9 , G ' 55 9 57
P r i s ; , 6AA3. ‘ ; t • , 5 %H Ac . V - F e f : ,t G.9;:, 9;, 9' 56 :,9 ;\:p^GPP r- -19 .A:, : 6AA2 - - ' ,5%HAc - . : FA : " ; - G-G ' G,/ "57-v: -t ' ■ - ' : . GG 9 : ipP r 20 . '6AA4 ; ' BPW 9 ' , F e - ■’ G ■" 9 91P : p . ; AP r ■2l' v : 6AA2A 1 BPW A F e - ' G ##** : . 86 1 1 <P r .22 ; ; 6AA4 I BPW ; 1 . F e - ' ! P *** 78 G 1 >[<
y (C hart 3):* Solution sp ectru m
S ol, s p e c « (Ch. 1)ion
i A 6AA, ;• iA A A r
onE lution of 6A fro m BPW pap er
6AA26AA3.6AA4
E lution of 6A : fr om E lution of 6A on
- E lution of 6A on d 70. E lution of 6AA2
paper
r.'fr'Orh er , : 43
Table 7. Iso lation and. Identification of V anillin . '
Chro,; , S a m p l e• '■A: - . - '
, Solvent ■ 1 r. t H„ A. '... . .. .|; Sample- , ; Vanillin A h . A„ A ! S a m p l e
' P a p e r , . N u m h e r ■ z, .'Sygteiri .i Spray:;;:' , Color Rf.:, Color Rfc 1 ColorB Y: V Rf.
3A; ■ ' ■ ■ :
':BPir :v .:; v ; : -
:' B 1
T ;;V V
v' ' si -76 1
v :> ' : ' ' W 62 ' t . ' .
.i : V- - v h 1
' '' 'I-" : v\ $
3A A h : : '
. A h
1 . ■•*' I . .
: i,; , - h " A Y '
- '
- ' h''-A ■ ■ ' ^
' B 1' p . h"'V. -P - A P ;
'A.V 9 0 A:- A, A 7 7 A ,■ A.A'- v 721
A 4 8 . ... ■•:,>: . . 1; ; ■ .1 . 1 " 'A - , - ' 131 A: 4 2 5 -- - '
: Y 3 , ;v - v ' v , a ;-f;-,.
/T 7 1
■ •• S A A ■-
: : A ;
v : < j B A W -
, V ' B P W' J I 1 ' ’ ; i ■" i
' R ' ' Go^./I : V-hr 1 - - '
A 9 1 A:: ■... 8 4 ,
A : A - Ar
r : W'A:AP,
AS,;A 9 6 Av - h 7 :
A l'A;' - 9 6 -A -83 .
: : v s # ; , i a a : B A W A , ■ • P N A - - , G o; : ' 1. . G o 1 : :
' . ' - , f - - ■ V 6. .. .. 1A-A ? h % B Q H - 1 : T-;yA
170 V.'? Y" 71A ■A iW '. ;A a A - ^ - a ’.
- E lu ted g iv in g 3AA„
; | vE luted.:giving 1 AA.„; iy ' l A . '1 . .;N: / :l ' ;■ ' .Papof:":d p e o t n ^ tak en on 1AA (C hart 5) and v a n ill in (C hart 6)1 . .■
t a b l e 8. Iso lation of a P h io ro g lu c in o l- l ik e Compound.
. • C hro,. P a p e r
t
' tS a m p le
NiimbeT. . ■
r ' " p a :;"- : 'i ■Solvent. :i:. S y s te m ''.7 S p ra y ;
' : .■' ,H . A ,::
C olorS am p le
A Rf.
'■ : ; - , P lil o r . . ,C b ior
7: - u v x(b a 7 7 '/ R f., tC o lo r • ' .
> . n 1
•! =■ 4A | :Z%BQEv :
' r ■ ■ . ■ - p .2, 4dSPH -
' 'i 0 “R 70
' 83 ■;
I;.f AA- ; ,■i-■: y-v;-:,-, 7 .
. ■ ■ ■ . ■ ■
;:P 2 ; ; 1 a- . -■ \: 4 A ■ v 2 % BC H :
i . \ ' : ■ ' ■' . ; : ‘
- ' 2 S 4dN PH: ■ r:: ■ ; / '
i ' g - p77 T-! . . " '
7 708.9
' : 7;p ;7 : :::,7i.;:7'Ar -
■ 7-7, 7 l:P ' •: ' ; 7 93 '■" ' B1 7 78
' : f ■ . t s
4a . ; Z % B Q U ^ \: , , 2,, 4 d E P E ■C lC '-Kn : A
63 A 85
V ;v # ,- '7 :' .
‘ 1 4A - : 1 '2% B0H ' -1 n - - Af ' "
' 2 , 4 d |fP H ' O -R ' Yf
7 63 88
AA.A'
.P 5 •- 1 '
■;4A : ' i ' l l .1.- ■ 2%B©H 7 V ; r :- 2 9.4dbrPH
p:l QAEA , Y .
- 65■:p 88
- -f :;^ .4A A r m A : ' '2 %BQH
I ■ -/
1 ' . ' - ' ‘ DSA •
, - : f . ; ' ,i .
'r y -Y ':
!
7 64 ' Go .1 •i ■ ■ ’ • ;• .
66 ;■. -« - A ' ' 7 7 (B i-G /N H g)'"
P 7 "/«■
4A A ; - i: ' b e w : V A V H G l; ' Y-C: ■: 85 7:r - o^Ap:--p;'
86 1 .u: ; ; . , (B1-G /N H 3)
p 8 -"’y'A
f V ■ 4A A . i A ' ' ;^HC1 V 7:r '■.:7::m,7- A
■'Y - © r.1 7r
A 77 R w oi ; 77 -'77^' . : : ; 7 . A (B l-G /b fH 3 )7
: • " U ltr a v io le t c o lo r u n d er NH^ v a p o r .fo r H„ A. sa m p le and p h lo r o g lu c in o l. '
l i s t o f f i g u r e s ■ „ .
lv . j& Typieal c h r o m a to g r a m fo r s p e c tr a l a n a ly s is . , ; ' • ■ ' ,
2 . T he p a p er a b so r p tio n c e l l h o ld e r . . '
: 3. S ch e m e o f h u m ic a c id d eg ra d a tio n . ;'v,■ \
■4. S ch e m e o f p u r if ic a t io n o f sa m p le 6A A 2A fo r sp e c tr o p h o to - :V' ■■ ■ , : metric anaiysis. : ' :i '> ■ ;;
. 5. S ch e m e o f p u r if ic a t io n o f s a m p le s 1A A and 3A A fo r s p e c t r o - ■
46
4 7
cut
a p p lied s a.rnple
cuts a m p le
cut
'r"- b lank
ch ro m a to g ra p h eda r e a
s o lv e n t fron t
cut i
F ig u r e 1. A ty p ic a l c h r o m a to g r a m fo r s p e c tr a l a n a ly s is .
Ift:gd41O3
w .
0. 312 ± 0, 005"
m tn-
Sil
3F
h in g e s -'
2. 91 5 * 0. 005H
F ig h r e 2. T he p a p e r a b so rp tio n c e l l h o ld e r .
b a ll and sp r in g
0. 005" open ing fo r p ap er
Hurn-ic A c id49
(H y d r o ly sa te )
R e flu x ed 4 h r s =:..(2% h 2s o 4 ) ;
E th er e x tr a c te d 24 hrs<
(R es id u e ) '' _ -KOH fu s io n 4 h r s = @ 2 5 0 °C t D is s o lv e d in. 50 m l . H^O
(E th er s o l . )
A : , ^.. D is c a r d
(A queous s o l . ) (E th er s o l . )
(E th y l A cetate./ SOl. ) '
to .p H 7 . . R ed u ced to •. d r y n e s s A c id if ie d ■
. . I V . ; - . . :E ther, ox tracte .d 24 hrS
(Aqueous s o l . )N e u tr a lis e d
to pH 7 - " .R ed u c ed to
A
e x tr a c te d ' '
(R es id u e ) . (E th y l A c e ta te
C ' ■■■ ;—(E th an o l s o l . )
r s o l . )
E th an o l • e x tr a c te d
(R e s id u e | i •
A c id if ie d E th y l A c etati
e x tr a c te d ‘‘
(R esid u e ) ;
E th an o le x tr a c te d
D ,(E th an o l
s o l . )
W a ter : e x tr a c te d
(R esid u e )
E .
1 y : D is c a r d
e r s o l . )
W atere x tr a c te d
' aK■H D is c a r d
F ig u r e 3. S c h e m e o f H u m ic a c id d eg r a d a tio n .
506A
16 -
S o lv en t 5% H Ac % r a y F eC lg
. , S la c k ;B ands R f • 69
B lack: R f . 71
"G reen R f . 58
E lu ted E lu ted
Spectruna Spectrum , (too. d ilu te ) (im p u re)
P a p e r P r 13 S o lv e n t B P W
E lu te d :R e s id u e r e m o v e d
6A A2
P a p e r P r 14 S o lv en t SAW
B ands v
G reen R f „ 7 5
P ap e r P r 1 5 .S o lv en t 5%. HAc S p r a y s P N A , F e C l3uv light '
(E eC lg G reen ^ ^ v(P N A )G d ld ;; B lu e . ■ ■ ; '
R f . 60 ■ (iJT) R f . 57:- , ' - ' / -
E lu ted R e s id u e /
/y
Zz :
z
u v
P a p e r P r .21.- S o lv e n t BPW
FeCl>
| E lu ted
B lu e R f . 43
UY lig h t
E lu t e d '
■ '' y ' - . , 'G r een R f .. .86
(too d ilu te ) : (C h a r t 1)
Figure; 4 . S c h e m e o f p u r if ic a t io n o f sa m p le 6A A 2A fo r sp e c tr o p h p tp -a n a ly s is
P a p e r V 4 S o lv e n t BPW Wvlight V ' ; ;
p a p e r Y 2 Solvent BPW
■ v ' f iig iif :S o lv e n t BPW
B an d s B ands
B lu e ' P u rp le R f . 90 : R f . 77 B ands
Slue , / : TUii;;::,White ■ R f ,, 81 R f ; j 6 ■ R f .6 2
S o lv en t BAW
Bahds
. ' , G old R ed
Rf .^lGold 3 m in o r bands
(C hart 3)
1A A and 3A A for
A E P ENptiC :' C 1
• l i s t o f . c h a r t s -
■ , ' 1. S o lu tio n s p e c tr u m o f 6A A 2A (P r o to c a te c h u ic acid)., ' ;
• Z. S o lu tio n spectrurD. o f P ro to ca ,tech u Ic a c id . , ■
; 3 . P a p e r s p e c tr u m o f BAA A (P r o tb c a te c h u ic a c id ) .
. 4 . P a p e r sp e c tr u m o f P r o to c a te c h u ic a c id . ■ ' ■
:■ ' ‘ v , 5. P a p e r s p e c tr a o f 1.AA and 3A A (V 3.n illin ).
• ' . 6. P a p e r sp e c tr u m o f .Y an illin .
: ■ A S o ltition spectirum o f V a n illin . •, ' . • , > ' .
8. P a p e r sp e c tr u m o f m -H y d ro x y -b en zo ic a c id .
9. P a p e r sp e c tr u m o f G e n t is ic a ld eh y d e .
10. S o lu tio n s p e c tr u m o f G en tisic : a ld e h y d e . :
IT. S o lu tio n s p e c tr u m o f G en tisio 'V acid .
. 12. S o lu tio n s p e c tr u m o f P r o to c a te c h u a ld e h y d e .
. 13. S o lu tio n s p e c tr u m o f Z d -D ih y d r o x y b e n z o ic a c id .
■ 1 4 . S o lu tio n s p e c tr u m o f 2, 6 -D ih y d r o x y b en zo ic acid .,
. 15. S o lu tio n S p ectru m o f 3 , 5 -D ih y d r o x y b e n z 6 ic a c id . .
V. 16. P a p e r sp ectru m of 3, 5 -D ih ydroxybenzo ic a c i d . v
52
53
1. 5
1 .4
1. 3
1 . 2 -
X m ax6A A 2A (— .4 2 5 5 2946A A 2A - H (----- ) 260 2956 A A 2A & N aA c 249 288
O. D.
0 . 8 ~
0. 7 u
0 . 6 -
0. 4 - /
0 . 2
X V
240 260 280 300 320 340 360
Chart 1. Solution sp ectru m of 6AA2A (P rotocatech u ic acid).
_L — I 380
54- -
1.
0.
0.
O. D.
0.
m a x .294295289
P r . a . ( ---4 257P r . a . - H ( — ) 260P r . a . - N aA c (— H 249
-
0260240 300220
X r* M
C hart 2. S o lu tio n sp e c tr u m o f I^ rotocatech u ic a c id .
55
. 5m a x .
6 A A A */6 A A A - H ( — )
234231
2 97 295. 4
3
2
1
0
9
8
7
6
5 L_
4
3
2
1
02 0220 24 0 300 320 360340 380
A rn ><
C hart 3. P a p e r s p e c tr u m o f bAAA (P r o to c a te c h u ic a c id ).
A. m a x . - 2 3 5 , 295
O .D .
220 240 280 300 320 340 360 380
Chart 4. P a p er spectru m o fP ro to ca te c h u ic acid .
57
0. 7
0 . 6
0. 5 r
0 . 2 -
0. 1
^ m a x .3AA (----- -) 287 3051AA (-— ) 234 278 310
1 / \ /\ •\ t
\
3 r
\
\\
\
\0 U-, t < i— i 1 . 1 ,220 240 2 0 2 CO 300 320 340 3 >0
A rn m
Ch.irt 5. P a p e r s p e c t r a of 1A A and 3AA (V in i l lm ) .
I380
58
. 5m a x
310Z81232. 4
. 3
. 2
. 1
. 0
. 9
. 8
. 7
. o
. 5
. 4
. 3
0 . 2
0. 1 L
1----------1---------1---------J--------J---------1---------i---------1----------1--------1----------i---------1---------1---------1---------1-------i.220 240 260 280 300 320 340 360
X M M
Chart 6. P ap er sp ectru m of Vanillin.
- /380
59
1 .4
1 .3 L
1 . 2
1. 1
X m ax231 280
1. 0
O . E0. 7
0.
0.
0.
0.
0.
0.
u
5
4
1
0220
i-
x _L i 1x - XX
240 2 0 180 300
m m320 340 0
Chart. 7. S o lu tio n sp e c tr u m of V a n illin .
I I n i t / z v f A r i - r n n o I i K r o n /
309
i------1330
60
. 4A_ m a x .
?.26 289. 3
. 2
. 1
. 0
. 9
0. 7
0. h
0. 5
0. 4
0. 3
20.
0. 1
L ZC320 340 3802o0240 280 300220
A /n m
Chart 8. P a p er sp ectru m of m -H y d ro x y b en zo ic acid.
5
4
3
2
1
0
9
8
7
o
5
4
3
2
1
2
61
m a x40 365
J i i L L0 240 280 300 3Z0
— t------ 1----- 1____i____>310 3u0 380
h a r t 9. P a p e r s p e c t r u m of G vn t i s ic a ldehyde .
62
1 .4
1 . 3 I-
1 . 2
/ "r, ax .2 0 3 , 4
1. 1
1 . 0 -i
0 . 9 j-
O. D.0 . 8 k
0. 7 1
0 . 6 -
0 . 5 r
0 . 4 _
0 . 3 -
0 . 2 _j
0. 1 -
20 :4C 2 0 2 8 0 300 320_i---------L—.—I----------1
3 4 , 3 0 380
X M ^
Ch . rt 10. So lu t ion s p e c t r u m o f C„ it i s ic - I n / a i i.
63
, 9
. 0
. 4
L og c
. 8
m a x .. 2 334
. 6
J JL380360340280 300 320240
A
Chart 11. So lu t ion s p e c t r u n , o f Cu i t i s i c a c id .
64
m a x .?S0233 312
. b
. 5
. 4
. 3
. 1
260220 240 1:80 300 320 340 380
A. m><Chart 12. So lu t ion s p e c t r u m o f i r o t o c a t c c h u a l d e h / d e .
65
4 . 4 0 p______ A. rr a x ._______
1 Z55 296
4. 20
4. 00
3. 30
3.
3. 40
3. 20
3. 00 _i j
340220 240 2 0 280 300 320\ m . K
C h a r t 13. Solution s p e c t r u m of 2, 4 - D ih y d r o x y b e n z o ic icid.
66
4. 00
m ax2 52 303
3. 80
3. 60
3. 40
20
3. 00
. 60
____ i_230220 240 300 320 340
A
C h a r t 14. Solu t ion s p e c t r u m of 2, - D i h / d r o x y b e n z o i c a c id .
67
4. 00 _/ m "i x .
2 50 303
3. 60
3. 40
3. 00 L
2, 60
2 . 602 0 4L ,60 2 60 300 320 340
A rn.^
Caa t 15. Solution sp ectru m of 3, 5 -D ih /d ro x y b en zo ic acid .
1 J 1 X 1 I I t I______ I_____ I i
220 240 260 280 300 320 340% t n M
J____(
360
Chart 16. P a p er spectru m of 3, 5-D ihydroxy ben zo ic acid.
