PA-SPECTROSCOPIC INVESTIGATION ON FLOWER PETALS

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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�

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 t far Physikalische Chemie I der U n i v e r s i t a t Dtisseldorf, U n i v e r s i t t i t s s t r a s s e 26.43, 0-4000 DZEsse Zdorf 1, F.R. G .

RGsume. - Les s p e c t r e s p h o t o a c o u s t i q u e s e n a m p l i t u d e 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 o b t e n u s 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 - m i s s i o n d i f f u s e s e t aux s p e c t r e s d e s pigments e n s o l u t i o n . A b s t r a c t .

-

PA a m p l i t u d e and phase s p e c t r a of i n t a c t f l o w e r p e t a l s a r e compared w i t h t h e c o r r e s p o n d i n g 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 . I n t r o d u c t i o n

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 p h y t o g e n i c 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 o f t h e UV-VIS a b s o r p t i o n s p e c t r a o f 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) s p e c t r o s c o p y .

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 p r o v e s 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 o f 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 o f PAS i s d e m o n s t r a t e d f o r a b i o l o g i c a l and b i o c h e m i c a l a p p l i c a t i o n . 2 . E x p e r i m e n t a l

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 r a n g e o f 30 t o 500 Hz. Phase l a g s p e c t r a were r e - corded 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

C6-138 JOURNAL DE PHYSIQUE

D i f f u s e r e f l e c t a n c e ( D R ) and s c a t t e r e d t r a n s m i t t a n c e (ST) spec- t r a were r e c o r d e d 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 a t t a c h m e n t .

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 w a t e r .

3 . D i s c u s s i o n

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 a n t h o c y a n i n s , g l y c o s i d e s o f 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 o f t h e pe- t a l s d i s c u s s e d i n t h i s p a p e r .

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 a n t h o c y a n i n [ 5 ] , t h e a b s o r p t i o n s p e c t r u m o f 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 o f t h e i o n i c a n t h o c y a n i n s a r e v e r y 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 c o r r e s p o n - d i n g chemical r e a c t i o n s can b e i n t e r e s t i n g . B e s i d e s ,

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 o f 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 o f 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 c o n f i n e d 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 o f 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 f r e q u e n c y and t h e a l t e r n a t i v e o f a m p l i t u d e 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 com- p a r i s o n o f methods. A p a l e b l u e p e t a l o f l a r k s p u r ( d e l p h i n i u m s p p . ) 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 - p a r e n c y 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 o f a l l methods a r e s h i f t e d

bathochromic compared t o t h e Xmax v a l u e s of t h e p u r e components i n MeOH/HCl [5] ( f i g . l a )

.

---

PAS s c a t t e r e d t r a n s m i s s i o n

...

d i f f u s e r e f l e c t a n c e

. _ . _ ^. _ .

t r a n s m i s s i o n o f 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 ( p a p a v e r 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 e x p e c t e d . 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 f r e q u e n c y . Thus c o i n c i d i n g Amax v a l u e s o f t h e p i g m e n t s ' components cyanin- 3 - g l u c o s i d e and cyanin-3,5-glucoside [6] can b e d e t e c t e d a t a modulation f r e q u e n c y o f 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 o f t h e PA-signal f o r s p e c t r a l changes o f 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 o f g r e a t o p t i c a l d e n s i t y , n o t o n l y t h e t h i c k n e s s

us

o f t h e a n a l y z e d l a y e r h a s t o b e 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 genera- t e d n e a r t h e s u r f a c e b r i n g s i n a g r e a t e r c o n t r i b u t i o n t o t h e 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 h a v i n g a d i s t a n c e of

us

from t h e s u r f a c e . T h i s s p a t i a l depen- dency 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 o f 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

C6-140 JOURNAL DE PHYSIQUE

and t h e s c a t t e r i n g power o f t h e sample. I t i n c r e a s e s w i t h modu- l a t i o n f r e q u e n c y 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.

T h i s 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 o f t h e s i g n a l a m p l i t u d e 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 p r o v i d e d 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 o f 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 d e p t h 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 0

COO 5 0 0 600

-

^{h / n m}

-

F i g . 2a) PA-amplitude spectrum F i g . 2b) PA-amplitude s p e c t r u m a t 500 Hz ( a ) and PA-phase lag a t 200 Hz ( a ) and PA-phaselag spectrum a t 105 Hz ( b ) o f a spectrum a t 6 0 Hz ( b ) o f a r e d poppy p e t a l p u r p l e pansy p e t a l

The stem o f a p u r p l e pansy ( v i o l a t r i c o l o r ) was b r o u g h t 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 o f ammonia i n w a t e r . To t h e d e g r e e 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. S i m u l t a n e o u s l y 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 o f which i s e s t i m a - t e d t o be a t 385 nm.

When a d r o p o f ammonia was added t o an e x t r a c t o f 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

of aqueous a m n i a _4: PAS-spectrum 24 h a f t e r the 1 : PAS-spectrum before exposure beginning of exposure

2 : PAS-spectrum 10 min a f t e r the 5.1 : Transmittance spec- of a

beginning of exposure neutral extract

3: PAS-spectrum 33 m i n a f t e r the ^5.2: Transmittance spedrum of an

beginning of expsure 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 PA- spectrum 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 g i v e n i n f i g . 4

r i l .

The pH-dependent composition o f t h e s e chemical e q u i l i b r i a ex- p 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 P A S i n t h e p e t a l s and t h o s e g i v e n i n l i t e r a t u r e (51 measured i n an a c i d s o l u t i o n .

JOURNAL DE PHYSIQUE

dn purple

- H Q

4,. -,,@I

^{+ H I A}

^.,,

^{- 530nm}

Fig.4 R e a c t i o n p a t h s and chemical e q u i l i b r i a o f 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 m o l e c u l a r UV-VIS s p e c t r a . For t h e m a j o r i t y o f 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 p u r p o s e . F u r t h e r improvement o f 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 s t u d y i n g t h e p h o t o a c o u s t i c e f f e c t i t s e l f .

[I]

D. Cahen et al. ; Photochem. Photobiol.

2,

803 (1980) FEBS Letters

91,

131 (1978)

FEBS Letters

91,

339 (1978) [2] G.F. Kirkbright et al.; Analyst

101,

553 (1976)

Analyst

s,

281 (1977)

[3J p. Helander, J. Lundstrom; J. Appl. Phys.

521

1 1 46 (1981)

[q

H.-H. Perkampus; Naturwissensch.

69,

162 (1982)

[5] J.B. Harborne; Comparative biochemistry of flavonoids, Academic Press, London, 1967

[L6] W. Karrer; Konstitution und Vorkomen der organischen Pflanzenstof fe

Zweite Auflage, Birkhauser-Verlag Basel, 1976 [7] J.B. Harborne; The flavonoids, Advances in research,

Chapmann and Hall, London, 1982