New Applications of Photoluminescence ... - ACS Publications

19 New Applications of Photoluminescence Techniques for Forensic Science

Downloaded by NORTH CAROLINA STATE UNIV on June 22, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0013.ch019

PETER F. JONES T h e Aerospace C o r p . , El Segundo, Calif. 90245

I s h a l l describe the u s e f u l p r o p e r t i e s o f photoluminescence and the current a p p l i c a t i o n o f these p r o p e r t i e s in forensic science. New a p p l i c a t i o n s o f photoluminescence developed or being i n v e s t i g a t e d in our l a b o r a t o r y are a l s o described. We have used photoluminescence techniques t o : (a) l o c a t e and i d e n t i f y seminal s t a i n s , (b) detect l e a d and antimony gunshot r e s i d u e at the nano-gram level, and (c) d i s c r i m i n a t e between d i f f e r e n t g l a s s and human (head) h a i r samples. All of these techniques can be c a r r i e d out r a p i d l y i n the crime l a b o r a t o r y . Luminescence i s a g e n e r a l term and has d i f f e r e n t meanings depending on the field o f a p p l i c a t i o n . I am concerned here with photoluminescence, which can be d e f i n e d as the l i g h t emitted by a chemical species in the ultraviolet-visible wavelength r e g i o n o f the electromagnetic spectrum (300 t o TOO nm) when e x c i t e d w i t h u l t r a v i o l e t r a d i a t i o n (190 t o 380 nm). Absorption o f u l t r a v i o l e t r a d i a t i o n by a luminescent molecule causes it t o undergo an e l e c - t r o n i c t r a n s i t i o n from the ground s t a t e , i . e . , the s t a t e o f lowest energy, t o a higher energy or e x c i t e d s t a t e . When a mole-cule i n the e x c i t e d s t a t e returns t o its ground energy s t a t e , a p o r t i o n o f its excess energy is r e l e a s e d through the emission o f light. Luminescent p r o p e r t i e s o f use are (a) the e x c i t a t i o n and emission s p e c t r a , i.e., i n t e n s i t y versus wavelength (the e x c i t a - t i o n spectrum is a p l o t o f the v a r i a t i o n i n the luminescence i n t e n s i t y as the wavelength o f the e x c i t i n g r a d i a t i o n is v a r i e d ) , (b) the decay time o f the luminescence once the e x c i t a t i o n source i s extinguished, and (c) the quantum yield o f emission, i.e., the r a t i o o f the number of molecules that emit l i g h t t o the number o f molecules that absorb e x c i t a t i o n . The luminescence can c o n s i s t o f both f l u o r e s c e n c e and phosphorescence. The fluorescence o f most molecules appears at shorter wavelengths and has a f a s t decay time (10 to 10 sec), whereas the phosphorescence appears at longer wavelengths and has a longer decay time (10 t o 10 s e c ) . -9

-6

-6

183

In Forensic Science; Davies, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.

184

FORENSIC SCIENCE

Photoluminescence a n a l y s i s has the advantages t h a t (a) i t can be h i g h l y s e l e c t i v e because the absorption, emission, and l i f e t i m e parameters must match; (b) i t i s h i g h l y s e n s i t i v e ; (c) i t i s o f t e n nondestructive ; (d) i t i s inexpensive to perform; and (e) i t o f t e n does not r e q u i r e the separation of complex mixtures.


Current

Uses o f Photoluminescence i n Forensic

Science

The most b e n e f i c i a l advantage o f photoluminescence a n a l y s i s i s i t s high s e n s i t i v i t y , which i s l e s s than a nanogram f o r e f f i cient emitters. Because of t h i s s e n s i t i v i t y , i t has been used e x t e n s i v e l y i n f o r e n s i c science f o r a v a r i e t y of a p p l i c a t i o n s i n v o l v i n g i n s p e c t i o n with u l t r a v i o l e t l i g h t . T y p i c a l l y , a hand h e l d low-pressure mercury lamp i s used with f i l t e r s as an u l t r a v i o l e t e x c i t a t i o n source, and the m a t e r i a l s of i n t e r e s t are v i s u a l l y inspected (sometimes making use o f another f i l t e r to d i s criminate luminescence c o l o r s ) . A p p l i c a t i o n s i n c l u d e the examina t i o n o f documents, e.g., f o r f o r g e r i e s ; the l o c a t i o n of body f l u i d s t a i n s ; the comparison o f o i l s , greases, paint c h i p s , and g l a s s fragments ; and, most f r e q u e n t l y used, the v i s u a l i z a t i o n of spots i n paper or t h i n - l a y e r chromatography. O c c a s i o n a l l y , emission s p e c t r a have been obtained with a r e c o r d i n g spec trophot ο fluorom et er t o compare p a i n t , i n k , g l a s s , minerals, paper f i l l e r s , and p l a s t i c s . More r e c e n t l y , i t has been shown t o be u s e f u l f o r drug analyses such as screening f o r morphine i n body f l u i d s (l_) and f o r the comparison o f motor o i l s (2). Udenfriend (3), G u i l b a u l t (k), Konstantinova-Shlezinger ( 5 ), and K i r k (6) have summarized many of these a p p l i c a t i o n s . New

A p p l i c a t i o n s o f Photoluminescence Techniques

The high s e n s i t i v i t y and s p e c i f i c i t y o f photoluminescence a n a l y s i s should make i t p o s s i b l e to i n d i v i d u a l i z e clue m a t e r i a l s , e.g., h a i r and g l a s s , by the c h a r a c t e r i s t i c luminescence prop e r t i e s o f t r a c e c o n s t i t u e n t s or i m p u r i t i e s . Of p a r t i c u l a r s i g n i f i c a n c e are the newer techniques of a n a l y z i n g the luminescence decay curves. For example, even when the absorption and l u m i nescence s p e c t r a of the i m p u r i t i e s are s i m i l a r , i t i s p o s s i b l e t o determine t h e i r concentrations i f t h e i r luminescence l i f e t i m e s differ. The usefulness o f t h i s technique i s i l l u s t r a t e d i n F i g s . 1 and 2 , where i t i s shown that the fluorescence spectra of naphthalene (N) and 1 , 6 - d i m e t h y l napthalene (DMN) are too s i m i l a r for fluorescence s p e c t r a l a n a l y s i s o f t h e i r mixtures ( F i g . l ) ; yet t h e i r r e l a t i v e concentrations can be r e a d i l y determined from the fluorescence decay curve ( F i g . 2 ) . As i n d i c a t e d by the dashed curve i n F i g . 2 , the observed decay i s the sum of exponential decays from a shorter l i v e d component, i . e . , DMN (lifetime 50 nsec) and a longer l i v e d component, i . e . , Ν ( l i f e t i m e 100 nsec). St. John and Winefordner (j) have discussed t h i s technique i n general and Hoerman and co-workers ( 8 , 9 ) have been


JONES

Photoluminescence


19.

WAVELENGTH, λ

0

20

40

Techniques

185

Figure 1. Representation of the fluores cence spectra of naphthalene (N) and 1,6dimethylnaphthalene (DMN)

Figure 2. Fluorescence decay curve for pulsed excitation of a mixture of naphtha lene (N) and 1,6-dimethylnaphthalene (DMN) with a concentration ratio of 5:95. The fluorescence intensity (arbitrary units) is plotted on a logarithmic scale.



186

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i n v e s t i g a t i n g the p o s s i b i l i t y o f u s i n g i t f o r d i f f e r e n t i a l i d e n t i f i c a t i o n o f micro-organisms and body connective t i s s u e . Luminescence decay curves are a l s o o f t e n used t o v e r i f y that samples do not c o n t a i n i m p u r i t i e s . The absence o f i m p u r i t i e s can be e s t a b l i s h e d i f the luminescence decay curve i s exponential and i f the spectrum does not change with time a f t e r p u l s e d e x c i t a t i o n . However, i n some cases, the luminescence decay curve can be nonexponential even i f a l l o f the luminescing s o l u t e s are c h e m i c a l l y identical. This occurs f o r molecules with luminescence l i f e t i m e s that depend upon the l o c a l environment. In an amorphous matrix, there i s a v a r i a t i o n i n s o l u t e luminescence l i f e t i m e s . Therefore, the luminescence decay curve can be used as a measure o f the i n t e r a c t i o n o f the solute with the solvent and as a probe o f the micro-environment. Nag-Chaudhuri and Augenstein (lO) used t h i s technique i n t h e i r studies o f the phosphorescence o f amino acids and p r o t e i n s , and we have used i t t o study the e f f e c t s o f polymer matrices on the phosphorescence o f aromatic hydrocarbons ( l l ) . This s e n s i t i v i t y o f the luminescence o f a molecule or atom t o i t s micro-environment i s a very important a t t r i b u t e i n the i n d i v i d u a l i z a t i o n o f clue m a t e r i a l . Seminal S t a i n s . As p r e v i o u s l y r e p o r t e d , we have used l u m i nescence decay p r o p e r t i e s t o detect the presence o f semen on strong f l u o r e s c e n t backgrounds (12). We have r e c e n t l y extended the use o f t h i s technique as an a i d i n the i d e n t i f i c a t i o n o f semen (13). In the crime l a b o r a t o r y , absolute proof t h a t a s t a i n i s o f seminal o r i g i n i s o n l y a f f o r d e d by the microscopic o b s e r v a t i o n o f i n t a c t spermatozoa. However, one l a b o r a t o r y r e p o r t e d that spermatozoa were observed i n o n l y approximately 50% of the cases where a s t a i n was suspected t o be o f seminal o r i g i n . In cases where no spermatozoa are found, a l t e r n a t e methods have been developed f o r seminal s t a i n " i d e n t i f i c a t i o n . " Two methods commonly used t o t e s t f o r seminal s t a i n s are the a c i d phosphatase t e s t and the F l o r e n c e t e s t . Both t e s t s were developed on the b a s i s o f the r e a c t i o n o f an i n t r o d u c e d compound with substances that are present i n seminal f l u i d . Positive r e s u l t s f o r these t e s t s are e i t h e r the formation o f a c h a r a c t e r i s t i c c o l o r or the formation o f s p e c i f i c c r y s t a l s . Since the substances t e s t e d are a l s o present i n other body f l u i d s and i n vegetable j u i c e s , the s p e c i f i c i t y of these t e s t s has been questioned ( l U ) . I t has been w e l l - e s t a b l i s h e d that c e r t a i n amino a c i d s , i . e . , phenylalanine, t y r o s i n e , and tryptophan, both f l u o r e s c e and phosphoresce (15). We b e l i e v e t h a t a combination o f these amino a c i d s i s r e s p o n s i b l e f o r the observed luminescence of seminal f l u i d . Furthermore, i t seems reasonable that e i t h e r t h i s combination o f amino acids would not be present or would not occur i n the same proportions i n other body f l u i d s or substances of b i o l o g i c a l origin. Therefore, d i f f e r e n t i a t i o n between seminal f l u i d and



19.

JONES

Photoluminescence

Techniques

187

other substances on the b a s i s o f phosphorescence behavior appears t o be an a t t r a c t i v e technique. The approach i n our study was to use the phosphorescence examination as an adjunct to the a c i d phosphatase t e s t . S t a i n s o f the d i f f e r e n t m a t e r i a l s were prepared, and t h e i r luminescence p r o p e r t i e s were v i s u a l l y noted using hand-held, s h o r t - and l o n g wavelength e x c i t a t i o n lamps. The r e s u l t s o f t h i s simple t e s t are given i n Table 1. Only four of the m a t e r i a l s t e s t e d , i . e . , v a g i n a l f l u i d , almonds, r i c e and r a t t l e s n a k e venom, gave phosphorescence r e s u l t s that were d i f f i c u l t t o separate from those of seminal f l u i d . However, a l l but the v a g i n a l f l u i d were e a s i l y d i s t i n g u i s h e d by other luminescent c h a r a c t e r i s t i c s . Thus, when the phosphorescence and a c i d phosphatase t e s t s are combined with the r e c e n t l y introduced e l e c t r o p h o r e s i s procedures f o r the separ a t i o n of v a g i n a l and seminal a c i d phosphatase ( l 6 , 1 7 , 1 8 ) , a p o s i t i v e i d e n t i f i c a t i o n o f semen i s p o s s i b l e , even i n the absence of spermatozoa. Detection o f Gunshot Residue. When a suspect has been apprehended f o l l o w i n g a shooting, d e t e c t i o n of gunshot residue on h i s hands may provide s i g n i f i c a n t evidence i n the i n v e s t i g a t i o n . Previous methods o f gunshot residue d e t e c t i o n , which are of quest i o n a b l e r e l i a b i l i t y because of t h e i r l a c k of s e n s i t i v i t y or s p e c i f i c i t y , i n c l u d e the c o l o r t e s t f o r n i t r a t e s (19) and the c o l o r t e s t s of Harrison and G i l r o y (20) f o r antimony (Sb), barium (Ba), and l e a d (Fb), the three most c h a r a c t e r i s t i c m e t a l l i c e l e ments found i n gunshot r e s i d u e . U n t i l r e c e n t l y , the method i n general use f o r d e t e c t i n g residue on hands, although the use o f t h i s method i s not n e a r l y as widespread as need would d i c t a t e , was the a p p l i c a t i o n o f neutron a c t i v a t i o n a n a l y s i s to detect antimony and barium ( 2 l ) . This method has serious drawbacks, e.g., the time and inconvenience of sending samples out f o r a n a l y s i s and the i n a b i l i t y to detect l e a d . I describe here the r e s u l t s o f our p r e l i m i n a r y study (22) of the a p p l i c a t i o n o f photoluminescence techniques to gunshot residue d e t e c t i o n . The key o b j e c t i v e i n t h i s study was to develop a r a p i d , r e l i a b l e , and convenient method o f d e t e c t i o n f o r use i n the crime l a b o r a t o r y on the b a s i s of the d e t e c t i o n of l e a d , antimony, and barium. We d i d not attempt to repeat the extensive work already c a r r i e d out with neutron a c t i v a t i o n a n a l y s i s concerning the importance of the d e t e c t i o n of these elements and the i n t e r p r e t a t i o n o f f i n d i n g s . The l i t e r a t u r e concerning photoluminescence was surveyed f o r methods of a n a l y s i s f o r antimony, barium, and l e a d t h a t would be (a) r e l i a b l e , s e n s i t i v e , and q u a n t i t a t i v e ; (b) that would not i n v o l v e a great d e a l o f wet chemistry; and (c) t h a t would be capable of simultaneous determination o f more than one of the three elements. No s a t i s f a c t o r y procedure f o r d e t e c t i o n of barium was found. Low-temperature c h l o r i d e i o n comp l e x i n g with l e a d ( i l ) and antimony ( i l l ) provides the most s e n s i t i v e , convenient, and r a p i d method o f luminescence a n a l y s i s known


In Forensic Science; Davies, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975. None None Weak y e l l o w r i n g None

Weak g r e e n None None

Blue Blue V e r y weak b l u e Blue

Weak b l u e None

Blue

None Blue None Blue

None None None None None

Semen Vaginal Fluid Human M i l k E x p r e s s e d Almonds

Human U r i n e B i n d Weed (Morning G l o r y ) R i c e , Whole G r a i n

Lucerne ( A l f a l f a ) Cow's M i l k Clover R a t t l e s n a k e Venom

Cauliflower B r u s s e l Sprouts Apple Mold Bread Mold Sweet P o t a t o

None None None None None

Weak y e l l o w Blue None None

Long-Wave E x c i t a t i o n

Properties Comments

Yellow fluorescence

Cloth fluorescence quenched

Cloth fluorescence quenched

p r o p e r t i e s o f f r e s h s t a i n s on c l o t h

Phosphorescence

Low-temperat-ore p h o s p h o r e s c e n c e

Short-Wave E x c i t a t i o n

Material

T a b l e 1.



19.

JONES

Photoluminescence

Techniques

189

f o r t h e s e two i o n s ; i t a l s o p r o v i d e s t h e c a p a b i l i t y o f s i m u l t a n e o u s l y a n a l y z i n g f o r b o t h i o n s (23,2*0. A s shown i n F i g s . 3 a n d h, t h e e m i s s i o n s p e c t r u m f o r l e a d ( I I ) p e a k s a t 390 nm, a n d f o r a n t i m o n y ( I I I ) t h e e m i s s i o n p e a k s a t 620 nm. The b a n d p e a k i n g a t k25 nm ( F i g . 3 ) i s a c o m b i n a t i o n o f s c a t t e r e d l i g h t a n d h y d r o g e n c h l o r i d e i m p u r i t y e m i s s i o n . The e x c i t a t i o n s p e c t r a ( F i g s . 3 a n d h) p e a k a t 276 nm f o r l e a d ( I I ) a n d a t 250 nm a n d 300 nm f o r a n t i m o n y ( I I I ) . These e m i s s i o n s p e c t r a have b e e n c o r r e c t e d f o r t h e v a r i a t i o n w i t h w a v e l e n g t h o f t h e response o f our p h o t o m u l t i p l i e r and g r a t i n g . The e x c i t a t i o n s p e c t r a have n o t b e e n c o r r e c t e d f o r t h e v a r i a t i o n i n t h e lamp i n t e n s i t y versus wavelength. T h u s , t h e e x c i t a t i o n maxima c a n d i f f e r f o r d i f f e r e n t lamps. F o r l e a d , however, t h e band i s s o s h a r p t h a t no dependence upon t h e lamp i s e x p e c t e d ( i f we assume t h a t t h e s p e c t r a l o u t p u t o f t h e s o u r c e does n o t v a r y r a p i d l y w i t h wavelength). R a p i d , c o n v e n i e n t d e t e c t i o n o f g u n s h o t r e s i d u e o n t h e hands of a suspect, f o l l o w i n g a shooting, can thus be accomplished by the photoluminescence determination o f t h e presence o f l e a d and antimony. F o l l o w i n g t h e f i r i n g o f a gun, t h e b a c k s o f b o t h hands a r e washed i n a s t r e a m o f d i s t i l l e d w a t e r . Each handwashing i s f i l t e r e d , a n d t h e r e s i d u e , c o l l e c t e d o n a membrane f i l t e r , i s d i s solved i nhydrochloric acid. Upon e x c i t a t i o n o f t h e s o l u t i o n , c o o l e d t o 77 K, t h e l e a d a n d a n t i m o n y c o m p l e x e s e m i t l i g h t w i t h maxima a t w a v e l e n g t h s c h a r a c t e r i s t i c f o r t h e t w o m e t a l l i c e l e ments . By t h e u s e o f t h i s p r o c e d u r e , i t i s p o s s i b l e t o d e t e c t a s l i t t l e a s 1 . 0 n g o f l e a d a n d 10 n g o f a n t i m o n y o n t h e hand. The t o t a l t i m e f o r sample c o l l e c t i o n a n d a n a l y s i s i s l e s s t h a n 30 m i n . Glass. Glass f r e q u e n t l y p r o v i d e s evidence i n c r i m i n a l cases i n v o l v i n g b u r g l a r i e s , h i t - a n d - r u n d r i v i n g , and auto a c c i d e n t s . C r i m i n a l i s t s c u r r e n t l y u s e p h y s i c a l p r o p e r t i e s such as d e n s i t y , r e f r a c t i v e i n d e x , and d i s p e r s i o n f o r comparison purposes t o d e t e r mine i f g l a s s p a r t i c l e s f o u n d o n a s u s p e c t may h a v e o r i g i n a t e d from g l a s s b r o k e n a t t h e scene o f a c r i m e . U n f o r t u n a t e l y , because o f t h e c l o s e c o r r e l a t i o n , measurement o f more t h a n one o f t h e s e p h y s i c a l p r o p e r t i e s p r o v i d e s l i t t l e a d d i t i o n a l i n f o r m a t i o n . One method t h a t o f f e r s p o t e n t i a l l y more p r o m i s e i n e s t a b l i s h i n g common o r i g i n o f g l a s s samples i s t h e c o m p a r i s o n o f t h e t r a c e e l e m e n t a l c o m p o s i t i o n , b u t i t i s t i m e consuming and e x p e n s i v e . C u r r e n t l y , we a r e s t u d y i n g t h e l u m i n e s c e n c e o f g l a s s a s a means o f c o m p a r i s o n . Luminescence i n g l a s s a r i s e s from t h e p r e s ence o f i o n i c i m p u r i t i e s o r a d d i t i v e s s u c h a s a l u m i n u m a n d c o p p e r . There i s evidence t h a t t h i s luminescence i s a l s o s e n s i t i v e t o t h e heat treatment o f g l a s s . Our p r e l i m i n a r y e x p e r i m e n t s s u g g e s t e d that t h e luminescent properties o f glass could provide a r a p i d , r e l i a b l e , i m p r o v e d method f o r d e t e r m i n i n g t h e o r i g i n o f g l a s s . We t h e r e f o r e c o l l e c t e d a p p r o x i m a t e l y ^00 g l a s s samples f r o m c r i m e l a b o r a t o r i e s i n C a l i f o r n i a a n d Canada. We m e a s u r e d t h e r e f r a c t i v e i n d e x o f t h e 1**3 C a l i f o r n i a samples t h a t h a d p a r a l l e l


FORENSIC

SCIENCE


9r

WAVELENGTH,

nm

Figure 3. Analysis of three handwashing samples, re ceived as unknowns, for antimony (Sb). The solid line and broken line spectra refer to the right and left handwashings, respectively, of a person who had fired two rounds from a .380 Browning automatic pistol with his right hand. The dashed-dotted line spectrum is from the right hand of a second person at the scene of the shooting, who did not fire a weapon. The solid, broken, and dashed-dotted line spectra indicate 0.18 pg, 0.03 μg, and no detectable anti mony, respectively. See text for a definition of excitation spectra.

9 RIGHT HAND 8 -

L E F T HAND N

0

SHOOTING

7 EXCITATION

200

300

400 500 W A V E L E N G T H , nm

Figure 4. Analysis of three handwashings for lead. The three samples are the same unknowns analyzed for antimony in Figure 3. Analysis of the right hand (shooting hand) of the person who fired the gun yielded 0.60 μ-g lead. See text for a defini tion of excitation spectra.



19.

JONES

Photoluminescence

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191

surfaces. Seventeen percent of the samples were i n d i s t i n g u i s h a b l e w i t h the experimental p r e c i s i o n of ±0.0002. F i f t y percent of the samples had a r e f r a c t i v e index between 1.5l60 and I.518O. These data demonstrate the need f o r improved methods o f comparison. We are c u r r e n t l y i n v e s t i g a t i n g the luminescence p r o p e r t i e s of the same g l a s s samples, and, t o date, we have s t u d i e d 13 samples that were i n d i s t i n g u i s h a b l e by measurements of t h e i r r e f r a c t i v e index. A l l samples e x h i b i t phosphorescence with two broad bands that peak i n the green (5^0 nm) and the r e d (730 nm). The r a t i o of the green t o the red band i s dependent upon the wavelength o f e x c i t a t i o n , but f o r a given wavelength of e x c i t a t i o n , t h e r a t i o o f the phosphorescence bands v a r i e d among the samples. Indeed, twelve of the t h i r t e e n samples ( p r e v i o u s l y i n d i s t i n g u i s h a b l e ) were d i s t i n g u i s h a b l e by t h i s measurement. This i s a tremendous improve ment i n the i n d i v i d u a l i ζ at i o n o f glass by a simple procedure. H a i r . U n t i l r e c e n t l y , the a p p l i c a t i o n of luminescence spe c i f i c a l l y to the a n a l y s i s o f human h a i r has not been attempted i n any systematic manner. I t has been shown that three of the amino a c i d s , i . e . , phenylalanine, t y r o s i n e , and tryptophan, found i n h a i r p r o t e i n both f l u o r e s c e and phosphoresce (15). I t has been e s t a b l i s h e d that f o r other p r o t e i n s that contain a l l three of the amino a c i d s , the luminescence (both fluorescence and phosphores cence) i s predominately the r e s u l t of the tryptophan chromophores, with p o s s i b l y some c o n t r i b u t i o n from the t y r o s i n e (15 ). More d i r e c t l y r e l a t e d to the luminescence of h a i r are the studies of Konev (25) i n v o l v i n g the luminescence o f wool k e r a t i n . He observed both fluorescence and phosphorescence from wool f i b e r s t h a t were c h a r a c t e r i s t i c o f tryptophan. Some researchers s t a t e t h a t the energy i n i t i a l l y absorbed by the t y r o s i n e chromophores i n p r o t e i n i s t r a n s f e r r e d to the tryptophan chromophores b e f o r e the former have a chance t o lumi nesce. This energy t r a n s f e r process would e x p l a i n the predominant emission from the tryptophan. However, i t i s known that the t y r o sine and tryptophan fluorescence i s r e a d i l y quenched by i n t e r a c t i o n s with the environment, i . e . , by proton t r a n s f e r , hydrogen bonding, or charge t r a n s f e r ; and i t has been suggested t h a t these i n t e r a c t i o n s favor the tryptophan emission. Konev (26) has argued t h a t because o f the high s e n s i t i v i t y of tryptophan fluorescence to the micro-environment of a c e l l , the fluorescence acts as an i n d i cator o f perturbations i n the molecular o r g a n i z a t i o n of the c e l l . There i s evidence t h a t d i s u l f i d e bonds, such as those present i n h a i r k e r a t i n , can a f f e c t the p r o t e i n emission ( 2 6 ) . This s e n s i t i v i t y of the tryptophan and t y r o s i n e emission to the microscopic environment suggests that i t should be p o s s i b l e to d i s t i n g u i s h h a i r samples from d i f f e r e n t i n d i v i d u a l s by the use of i n d i v i d u a l luminescence p r o p e r t i e s . Studies of the d i f f e r e n c e s i n the kera t i n s forming h a i r c l e a r l y i n d i c a t e t h a t no constant chemical com p o s i t i o n of k e r a t i n s can be expected. Indeed, the process of k e r a t i n i z a t i o n probably depends upon such p h y s i o l o g i c a l and



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environmental v a r i a t i o n s as n u t r i t i o n a l supply, temperature, and s o l a r r a d i a t i o n ( 2 7 ) . Our p r e l i m i n a r y work i n d i c a t e d that h a i r s phosphoresce when e x c i t e d by u l t r a v i o l e t l i g h t , at 7 7 K, as a r e s u l t o f the presence o f amino acids i n the p r o t e i n o f the h a i r . D i f f e r e n c e s i n e x c i t a t i o n and emission spectra as w e l l as phosphorescence decay times e x i s t f o r h a i r s from d i f f e r e n t i n d i viduals. Examples o f the luminescence r e s u l t s are given i n F i g . 5 . The t y p i c a l phosphorescence spectra f o r the h a i r o f two d i f f e r e n t i n d i v i d u a l s and f o r three d i f f e r e n t wavelengths o f e x c i t a t i o n with u l t r a v i o l e t r a d i a t i o n are shown. In a d d i t i o n t o the s l i g h t d i f f e r e n c e s i n the spectra f o r d i f f e r e n t i n d i v i d u a l s , a s i g n i f i c a n t v a r i a t i o n i n the r e l a t i v e i n t e n s i t i e s i s evident. Of p a r t i c u l a r i n t e r e s t i s the v a r i a t i o n i n the r a t i o o f phosphores cence i n t e n s i t i e s f o r 2 5 0 versus 3 5 0 nm e x c i t a t i o n . The v a r i a t i o n i n the spectra f o r d i f f e r e n t wavelengths o f e x c i t a t i o n i n d i c a t e s that more than one species i s phosphorescing. This i s a l s o e v i denced by the r a t e o f decay o f the luminescence upon e x t i n g u i s h i n g the e x c i t a t i o n . I f t h e molecules emitting were the same type and i f a l l o f these had the same environment, the emission would decay e x p o n e n t i a l l y with time. The decay curves as shown i n F i g 6 a r e , i n f a c t , nonexponential. A n a l y s i s o f the phosphorescence decay curves i n F i g . 6 i n d i cates a v a r i a t i o n i n the decay curves f o r d i f f e r e n t i n d i v i d u a l s and suggests the p o s s i b l e use o f the decay curves f o r i n d i v i d u a l i z a t i o n o f h a i r samples. We t h e r e f o r e undertook a more exten s i v e i n v e s t i g a t i o n o f the phosphorescence decay curves. Because f r e q u e n t l y only a l i m i t e d number o f h a i r samples are a v a i l a b l e i n a c r i m i n a l case, we r e f i n e d our techniques so that we could observe the phosphorescence spectra and decay curves f o r s i n g l e strands o f h a i r . The emphasis o f our studies t o date has been t o i n v e s t i g a t e the use o f the phosphorescence technique as an adjunct t o micro scopic examination ( 2 8 ) . H a i r s from l i g h t - h a i r e d i n d i v i d u a l s , a l l approximately the same c o l o r , were examined m i c r o s c o p i c a l l y . Hair from eight i n d i v i d u a l s that c o u l d not be d i f f e r e n t i a t e d on the b a s i s o f c o l o r , diameter, morphology o f t h e h a i r r o o t , presence or l a c k o f medulla, and c u t i c u l a r s c a l e p a t t e r n was s e l e c t e d . We measured the phosphorescence decay times at 7 7 Κ f o r t e n s i n g l e strands o f h a i r from each o f the eight i n d i v i d u a l s . The decay time ( t ) i s d e f i n e d here as the time r e q u i r e d f o r the phos phorescence i n t e n s i t y t o drop from t h e i n i t i a l steady-state value ( I ) t o I / 5 . In F i g . 7* average values o f t f o r 2 5 0 nm e x c i t a t i o n f o r each i n d i v i d u a l ' s h a i r are given as v e r t i c a l b a r s . The bars incorporate a ±1 standard d e v i a t i o n i n the mean value of t f o r the ten h a i r s o f each donor. The amount o f overlap i n decay time d i d not make i t f e a s i b l e t o make p o s i t i v e i d e n t i f i c a t i o n o f an i n d i v i d u a l from h i s h a i r on the b a s i s o f t alone. However, i n s e v e r a l cases, h a i r s w i t h approximately the same t can be d i s t i n g u i s h e d by t h e i r s t r u c t u r e d phosphorescence spectra. Thus, f o r t h i s group o f eight i n d i v i d u a l s , whose h a i r was i n d i s t i n g u i s h a b l e by microscopic Q

0


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Photoluminescence

Techniques


19.

Figure 5. Phosphorescence spectra at 77 Κ of the human (head) hair from two different individuals for different excitation wavelengths


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Figure 7. Phosphorescence decay times (t) for hair samples from eight blondhaired donors. The error bars represent ±1 standard deviation from the mean value of t for 10 hair samples from each donor.


19.

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examination, d i f f e r e n t i a t i o n through phosphorescence p r o p e r t i e s was p o s s i b l e . For very l a r g e p o p u l a t i o n s , luminescent p r o p e r t i e s alone are not expected t o be s u f f i c i e n t to i n d i v i d u a l i z e a h a i r sample. However, with a proper s t a t i s t i c a l a n a l y s i s , the c e r t a i n t y t o which phosphorescent examination o f h a i r can be used f o r i t s i n d i v i d u a l i z a t i o n can be p r o p e r l y evaluated.


Conclusion Considerably more work i s r e q u i r e d before these techniques can be introduced i n c o u r t , but our s t u d i e s and the work o f others show t h a t photoluminescence techniques have p o t e n t i a l f o r wide a p p l i c a t i o n i n f o r e n s i c s c i e n c e . Indeed, because o f the s i g n i f i cant advances demonstrated i n recent y e a r s , one can expect t o see spectrophotofluorometers become as commonplace as i n f r a r e d spec trometers i n a crime l a b o r a t o r y . Although luminescence spectrom e t r y i s not, i n g e n e r a l , as s p e c i f i c as i n f r a r e d spectrometry, i t i s c o n s i d e r a b l y more s e n s i t i v e and convenient. Ac knowledgments The e f f o r t o f the e n t i r e s t a f f of the Laboratory at The Aerospace Corporation i s l a r thanks are extended t o A. R. Calloway, and R. N e s b i t t . The author a l s o b e n e f i t e d sions with Dr. S. S i e g e l .

F o r e n s i c Science acknowledged. P a r t i c u D. J . Carre, Q. Kwan, from numerous d i s c u s

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