PA-SPECTROSCOPIC INVESTIGATION ON
FLOWER PETALS
Xiao Li, K. Brücher, W. Görtz, H.-H. Perkampus
To cite this version:
Xiao Li, K. Brücher, W. Görtz, H.-H. Perkampus. PA-SPECTROSCOPIC INVESTIGATION
ON FLOWER PETALS. Journal de Physique Colloques, 1983, 44 (C6), pp.C6-137-C6-143.
<10.1051/jphyscol:1983621>. <jpa-00223180>
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Submitted on 1 Jan 1983
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JOURNAL DE PHYSIQUE
Colloque C6, suppl6ment au nOIO, Tome 44, octobre 1983
page C6- 137
PA-SPECTROSCOPIC INVESTIGATION ON FLOWER PETALS
Xiao Li, K.H.
~r;cher*,
W. ~ o r t z *and H.-H.
Perkampusrt
Research I n s t i t u t e for Chemical Industry, Shenyang, PR China
* ~ n s t i t u far
t
Physikalische Chemie I der Universitat Dtisseldorf,
Universittitsstrasse 26.43, 0-4000 DZEsse Zdorf 1, F.R. G.
RGsume. - Les s p e c t r e s photoacoustiques en amplitude e t en phase
de p e t a l e s de f l e u r i n t a c t s s o n t compares aux s p e c t r e s c o r r e s pondants obtenus p a r d e s t e c h n i q u e s de r E f l e x i o n e t de t r a n s mission d i f f u s e s e t aux s p e c t r e s d e s pigments en s o l u t i o n .
A b s t r a c t . - PA amplitude and phase s p e c t r a of i n t a c t flower
p e t a l s a r e compared with t h e corresponding s c a t t e r e d t r a n s m i t t a n c e , d i f f u s e r e f l e c t a n c e s p e c t r a and t h e s o l u t i o n s p e c t r a
of pigments.
1. Introduction
PAS h a s s u c c e s s f u l l y been used a s a s p e c t r o s c o p i c method f o r i n
v i v o i n v e s t i g a t i o n s of phytogenic m a t e r i a l [I-37. The o b j e c t o f
t h i s i n q u i r y i s t h e d e t e c t i o n of t h e UV-VIS a b s o r p t i o n s p e c t r a
of n a t i v e f l o w e r pigments by PAS comparing t h e r e s u l t s w i t h
d i f f u s e r e f l e c t a n c e (DR) and s c a t t e r e d t r a n s m i t t a n c e (ST)
spectroscopy.
O p t i c a l p r o p e r t i e s a r e found t o be t h e c r i t e r i a t o d e c i d e
whether PAS proves s u p e r i o r t o t h e o p t i c a l methods c o n s i d e r i n g
t h e s e n s i t i v i t y t o s p e c t r a l a b s o r p t i v i t y of s c a t t e r i n g and
s t r o n g l y a b s o r b i n g b i o l o g i c a l m a t e r i a l . The c a p a b i l i t y of PAS
i s demonstrated f o r a b i o l o g i c a l and biochemical a p p l i c a t i o n .
2 . Experimental
A s i n g l e beam PA-instrument
[4:]
was f i t t e d w i t h a f l a t c y l i n d r i c a l c e l l , t h e f r o n t and r e a r q u a r t z window of which form more
t h a n 70% of t h e c e l l w a l l s ' a r e a . The r e a r window i s used
a s t h e sample h o l d e r . So t h e l i g h t s c a t t e r e d and t r a n s m i t t e d
by t h e p e t a l s can l e a v e t h e c e l l w i t h o u t c a u s i n g an i n t e r f e r i n g
background s i g n a l . A s a t i s f a c t o r y S/N r a t i o was o b t a i n e d i n a
modulation range o f 30 t o 500 Hz. Phase l a g s p e c t r a were recorded u s i n g t h e phase a n a l o g o u t p u t of t h e l o c k - i n a m p l i f i e r .
+address
of correspondence
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1983621
JOURNAL DE PHYSIQUE
C6-138
D i f f u s e r e f l e c t a n c e (DR) and s c a t t e r e d t r a n s m i t t a n c e (ST) spect r a were recorded on a Perkin-Elmer-555-spectrophotometer
equipped w i t h an i n t e g r a t i n g s p h e r e attachment.
I f PA-spectra y i e l d e d i n f o r m a t i o n t h a t c o u l d n o t be o b t a i n e d
from DR o r ST s p e c t r a t h e r e s u l t s of PAS were v e r i f i e d by
t r a n s m i t t a n c e measurements of p e t a l e x t r a c t s . I n o r d e r t o r e duce s o l v e n t induced s h i f t s on t h e o r i g i n a l a b s o r p t i o n i n t h e
p e t a l some o f t h e pigment was e l u t e d from a ground p e t a l w i t h
water.
3 . Discussion
Most f l o w e r pigments can be d i v i d e d i n t o t h e f a t s o l u b l e
c a r o t e n o i d s and t h e w a t e r s o l u b l e f l a v o n o i d s . F r e q u e n t l y
o c c u r i n g f l a v o n o i d s a r e t h e anthocyanins, g l y c o s i d e s of t h e
a n t h o c y a n i d i n s which a r e t h e chromogenic molecules of t h e pet a l s discussed i n t h i s paper.
S i n c e t h e Amax v a l u e s of an UV-VIS a b s o r p t i o n spectrum a r e
d i s t i n c t i v e f o r an anthocyanin [ 5 ] , t h e a b s o r p t i o n spectrum of
a p e t a l s h o u l d i n d i c a t e t h e pigments p r e s e n t . A s t h e UV-VIS
s p e c t r a of t h e i o n i c anthocyanins a r e very s e n s i t i v e t o s o l v e n t
and pH induced s h i f t s , s p e c t r o s c o p i c s t u d i e s of t h e correspond i n g chemical r e a c t i o n s can be i n t e r e s t i n g . Besides,
the 'max
v a l u e s i n l i t e r a t u r e which a r e determined by a s t a n d a r d i z e d
MeOH/HCl e x t r a c t of t h e dye C5] w i l l n o t n e c e s s a r i l y be v a l i d
f o r a pigment i n t h e i n t a c t p e t a l .
The a p p l i c a b i l i t y of DR and ST s p e c t r o s c o p y f o r such s p e c t r o s c o p i c i n v i v o s t u d i e s , however, i s confined t o c e r t a i n o p t i c a l
p r o p e r t i e s of t h e sample, whereas PAS i s a d a p t a b l e t o t h e
problem by means of a v a r i a b l e modulation frequency and t h e
a l t e r n a t i v e o f amplitude o r phase l a g s p e c t r a .
The examples shown i n f i g . 1 a r e t y p i c a l r e s u l t s of t h e comp a r i s o n of methods. A p a l e b l u e p e t a l of l a r k s p u r (delphinium
spp.) shows even w i t h i n t h e a b s o r p t i o n band s u f f i c i e n t t r a n s parency and s c a t t e r i n g power t o d e t e c t t h e v i s i b l e a b s o r p t i o n
maxima (due t o t h e m i x t u r e of p e l a r g o n i d i n , c y a n i d i n , and
d e l p h i n i d i n g l u c o s i d e s ) w i t h ST- and DR-spectroscopy a s w e l l
a s w i t h PAS. The i n s i t u s p e c t r a of a l l methods a r e s h i f t e d
bathochromic compared t o t h e Xmax
i n MeOH/HCl
[5] ( f i g . l a )
.
values of t h e p u r e components
Fig.1
Absorption s p e c t r a of a b l u e l a r k s p u r ( a ) and a
r e d poppy ( b ) p e t a l , measured by
-------
.......
._ ._ ._ .
PAS
s c a t t e r e d transmission
diffuse reflectance
t r a n s m i s s i o n of e x t r a c t
I n c o n t r a s t w i t h t h i s sample an i n t e n s l y c o l o u r e d r e d poppy
(papaver r h o e a s ) p e t a l has an o p t i c a l d e n s i t i y g r e a t enough t o
s a t u r a t e t h e o p t i c a l s i g n a l i n a r e g i o n where t h e Xmax v a l u e
i s expected. PAS, however, a n a l y z e s o n l y a l a y e r n e a r t h e s u r f a c e , t h e t h i c k n e s s o f which depends on modulation frequency.
Thus c o i n c i d i n g Amax v a l u e s of t h e pigments' components cyanin3-glucoside and cyanin-3,5-glucoside
[6]
can be d e t e c t e d a t a
modulation frequency of 500 Hz. The maximum of t h e PA-spectrum
shows a bathochromic s h i f t of 10 nm r e l a t i v e t o t h e Xmax v a l u e
c i t e d i n l i t e r a t u r e r51 ( f i g . l b )
To u n d e r s t a n d t h e s e n s i t i v i t y of t h e PA-signal f o r s p e c t r a l
changes of t h e a b s o r p t i v i t y i n t h e r e g i o n of g r e a t o p t i c a l
.
d e n s i t y , n o t only t h e t h i c k n e s s
us
of t h e analyzed l a y e r h a s t o
be c o n s i d e r e d , b u t a l s o t h e f a c t t h a t an amount of h e a t generated near t h e surface brings i n a greater contribution t o the
PA-signal t h a n t h e same amount d e p o s i t e d i n a l a y e r element
having a d i s t a n c e of us from t h e s u r f a c e . This s p a t i a l dependency of t h e s i g n a l g e n e r a t i o n w i t h i n
us
i s a f u n c t i o n of t h e
l i g h t i n t e n s i t y d i s t r i b u t i o n determined by t h e o p t i c a l d e n s i t y
JOURNAL DE PHYSIQUE
C6-140
and t h e s c a t t e r i n g power of t h e sample. I t i n c r e a s e s w i t h modul a t i o n frequency t h u s improving t h e s e n s i t i v i t y of PAS f o r
s m a l l d i f f e r e n c e s i n t h e a b s o r p t i v i t y of opaque samples.
This wavelength dependency o f t h e o p t i c a l a t t e n u a t i o n f u n c t i o n s
produces a phase l a g of t h e s i g n a l amplitude t h a t a l s o r e p r e s e n t s t h e s p e c t r a l change o f t h e o p t i c a l d e n s i t y provided t h a t
t h e sample h a s no l a y e r s t r u c t u r e .
To have a c o n v e n i e n t comparison of t h e s p e c t r a i n f i g . 2
a
d e c r e a s i n g o p t i c a l p e n e t r a t i o n depth i s i n d i c a t e d by an i n c r e a s i n g phase a n g l e .
I
COO
-
500
h/nm
-
600
0
Fig. 2a) PA-amplitude spectrum
a t 500 Hz ( a ) and PA-phase lag
spectrum a t 105 Hz ( b ) of a
r e d poppy p e t a l
Fig. 2b) PA-amplitude spectrum
a t 200 Hz ( a ) and PA-phaselag
spectrum a t 6 0 Hz ( b ) of a
p u r p l e pansy p e t a l
The stem of a p u r p l e pansy ( v i o l a t r i c o l o r ) was brought i n
c o n t a c t w i t h a d i l u t e s o l u t i o n of ammonia i n w a t e r . To t h e
degree t h e s o l u t i o n p e n e t r a t e d t h e p e t a l , i t s c o l o u r g r a d u a l l y
t u r n e d from p u r p l e t o b l u e and became p a l e brown o v e r n i g h t .
I n t h e PA-spectrum t h e v i s i b l e a b s o r p t i o n maximum m i g r a t e s from
530 t o 565 nm w i t h i n 30 minutes.
Simultaneously an a b s o r p t i o n
band i s r a i s i n g i n t h e n e a r W, t h e maximum of which i s estimat e d t o be a t 385 nm.
When a drop of ammonia was added t o an e x t r a c t of t h e p e t a l i t s
c o l o u r immediately changed t o b l u e , caused by a s h i f t o f t h e
Fig. 3
A pansy petal put in a solution
of aqueous a m n i a
1 : PAS-spectrum before exposure
2 : PAS-spectrum 10 min a f t e r the
beginning of exposure
3: PAS-spectrum 33 min a f t e r the
beginning of expsure
4: PAS-spectrum 24 h a f t e r the
beginning of exposure
5.1 : Transmittance specof a
neutral extract
5.2: Transmittance spedrum of an
alkaline extract
a b s o r p t i o n maximum from 5 4 0 t o 575 nm. The change i n t h e PAspectrum i s c o n s i s t e n t w i t h t h e r e a c t i o n scheme given i n f i g . 4
ril.
The pH-dependent composition o f t h e s e chemical e q u i l i b r i a exp l a i n s t h e d i f f e r e n c e between t h e Xmax v a l u e s d e t e c t e d by PAS
i n t h e p e t a l s and t h o s e given i n l i t e r a t u r e (51 measured i n an
acid solution.
JOURNAL DE PHYSIQUE
dn
-HQ
4,.
Fig.4
purple
-,,@I .,,A
530nm
+HI
Reaction p a t h s and chemical e q u i l i b r i a of a p u r p l e
pansy pigment and t h e r e s u l t i n g c o l o u r s of t h e p e t a l
d e p e n t on pH
5 . Conclusion
Although o p t i c a l l y and t h e r m a l l y inhomogeneous p e t a l s a r e no
c o n v e n i e n t samples f o r q u a n t i t a t i v e c a l c u l a t i o n s , PAS can be
u s e f u l l y a p p l i e d t o them f o r d e t e r m i n i n g q u a l i t a t i v e molecular
UV-VIS s p e c t r a . For t h e m a j o r i t y of t h e p e t a l s i n v e s t i g a t e d
PAS t u r n e d o u t t o b e t h e unique method f o r t h i s purpose.
F u r t h e r improvement of t h e i n s t r u m e n t a t i o n w i t h r e g a r d t o t h e
S/N r a t i o and an c o n v e n i e n t o p e r a t i o n w i l l n e v e r t h e l e s s be
n e c e s s a r y t o make PAS a t t r a c t i v e f o r an u s e r who i s n o t
i n t e r e s t e d i n studying t h e photoacoustic e f f e c t i t s e l f .
6. Literature
[I]D.
Cahen et al. ; Photochem. Photobiol. 2, 803 (1980)
FEBS Letters 91, 131 (1978)
FEBS Letters 91, 339 (1978)
101,553
[
2
]
G.F. Kirkbright et al.; Analyst
Analyst
[3J
p. Helander, J. Lundstrom; J. Appl. Phys.
[q
H.-H. Perkampus; Naturwissensch.
[5]
J.B.
s,
281
69, 162
(1976)
(1977)
521
1 1 46 (1981)
(1982)
Harborne; Comparative biochemistry of flavonoids,
Academic Press, London, 1967
[L6] W. Karrer; Konstitution und Vorkomen der organischen
Pflanzenstoffe
Zweite Auflage, Birkhauser-Verlag Basel, 1976
[7]
J.B.
Harborne; The flavonoids, Advances in research,
Chapmann and Hall, London, 1982