Luminescence Standards for Macro- and ... - ACS Publications

Chapter 7

Luminescence Standards for Macro-and Microspectrofluorometry

Downloaded by UNIV OF MONTANA on April 7, 2015 | http://pubs.acs.org Publication Date: December 30, 1989 | doi: 10.1021/bk-1989-0383.ch007

Rance A. Velapoldi and Michael S. Epstein Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, MD 20899 Requirements for standards used in macro- and micro spectrofluorometry differ, depending on whether they are used for instrument calibration, standardization, or assessment of method accuracy. Specific examples are given of standards for quantum yield, number of quanta, and decay time, and for calibration of instrument parameters, including wavelength, spectral responsivity (determining correction factors for luminescence spectra), stability, and linearity. Differences in requirements for macro- and micro-standards are consid ered, and specific materials used for each are compared. Pure compounds and matrix-matched standards are listed for standardization and assessment of method accuracy, and existing Standard Reference Materials are discussed. The i n h e r e n t s e n s i t i v i t y and s e l e c t i v i t y o f s p e c t r o f l u o r o m e t r y , c o u p l e d w i t h the advances i n e l e c t r o - o p t i c a l components and compute r s , has l e d t o the wide use o f s p e c t r o f l u o r o m e t r y d u r i n g the l a s t t h r e e decades i n d i v e r s e a r e a s o f a n a l y s i s , i n c l u d i n g a n a l y t i c a l c h e m i s t r y , c e l l u l a r b i o l o g y , g e o c h e m i s t r y , and m a i l s o r t i n g systems The i n f o r m a t i o n o b t a i n e d from t h e s e measurements has had a p r o f o u n d impact on a l l o f t h e s e a r e a s , f o r example, i n a s s e s s i n g m o l e c u l a r i n t e r a c t i o n s used f o r q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s e s , c h r o m a t o g r a p h i c s e p a r a t i o n s , c e l l u l a r make-up and i n t e r a c t i o n s , m o l e c u l a r volume, r o t a t i o n and d i f f u s i o n c o e f f i c i e n t s f o r l a r g e m o l e c u l e s , d e t e r m i n a t i o n o f p o r o s i t i e s o f g e o l o g i c samples on a m i c r o - and m a c r o - s c a l e , p h o t o c h e m i c a l and k i n e t i c p r o c e s s e s , and energy t r a n s f o r m a t i o n / c o n v e r s i o n . I n g e n e r a l , luminescence measurements a r e r e l a t i v e r a t h e r t h a n a b s o l u t e , s i n c e the i n s t r u m e n t c h a r a c t e r i s t i c s and sample p r o p e r t i e s t h a t determine the f l u o r e s c e n c e i n t e n s i t i e s are o f t e n n o t w e l l defined. A b s o l u t e luminescence measurements are d i f f i c u l t t o p e r f o r m and r e q u i r e time and i n s t r u m e n t a t i o n n o t a v a i l a b l e i n most l a b o r a t o ries. Thus, luminescence measurements r e l y h e a v i l y on s t a n d a r d s t o determine i n s t r u m e n t responses and p a r a m e t e r s , the c h e m i c a l composit i o n o f samples, and the c h a r a c t e r i s t i c s o f c h e m i c a l s y s t e m s . To This chapter not subject to U.S. copyright Published 19S9 American Chemical Society

In Luminescence Applications; Goldberg, M.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7.

VELAPOLDI AND EPSTEIN

Macro- and Microspectrofluorometry

99


a v o i d i n a c c u r a c y and c o n f u s i o n i n luminescence measurements and d a t a i n t e r p r e t a t i o n , r e l i a b l e s t a n d a r d s a r e needed. Definition and Uses of Standards. I n the c o n t e x t o f t h i s p a p e r , the term " s t a n d a r d " denotes a w e l l - c h a r a c t e r i z e d m a t e r i a l f o r w h i c h a p h y s i c a l parameter o r c o n c e n t r a t i o n o f c h e m i c a l c o n s t i t u e n t has been d e t e r m i n e d w i t h a known p r e c i s i o n and a c c u r a c y . These s t a n d a r d s can be used t o check o r determine (a) i n s t r u m e n t a l parameters such as w a v e l e n g t h a c c u r a c y , d e t e c t i o n - s y s t e m s p e c t r a l r e s p o n s i v i t y , and s t a b i l i t y ; (b) the i n s t r u m e n t response t o s p e c i f i c f l u o r e s c e n t s p e c i e s ; and (c) the a c c u r a c y o f measurements made by s p e c i f i c i n s t r u m e n t s o r measurement p r o c e d u r e s ( a s s e s s whether the a n a l y t i c a l measurement p r o c e s s i s i n s t a t i s t i c a l c o n t r o l and whether i t e x h i b i t s bias). Once the luminescence i n s t r u m e n t a t i o n has been c a l i b r a t e d , i t can be used t o measure the luminescence c h a r a c t e r i s t i c s o f c h e m i c a l systems, i n c l u d i n g c o r r e c t e d e x c i t a t i o n and e m i s s i o n s p e c t r a , quantum y i e l d s , decay t i m e s , e m i s s i o n a n i s o t r o p i c s , energy t r a n s f e r , and, w i t h a p p r o p r i a t e s t a n d a r d s , the c o n c e n t r a t i o n s o f c h e m i c a l c o n s t i t u e n t s i n complex samples. S e v e r a l books and symposium p r o c e e d i n g s on l u m i n e s c e n c e s t a n dards and measurements have been p u b l i s h e d i n the l a s t s e v e r a l y e a r s , i n c l u d i n g : "Advances i n Standards and Methodology i n S p e c t r o p h o t o metry" ( I ) , "Measurement o f P h o t o l u m i n e s c e n c e " ( 2 ) , "Standards i n Fluorescence Spectrometry" ( 3 ) , and "Modern F l u o r e s c e n c e S p e c t r o scopy" (Volumes 1-4) ( 4 ) . These b o o k s , the r e f e r e n c e s w i t h i n them, and the c l a s s i c i n the f i e l d , "Photoluminescence o f S o l u t i o n s " by C . A . P a r k e r ( 5 ) , p r o v i d e the r e s e a r c h e r w i t h e x t e n s i v e i n f o r m a t i o n about luminescence s t a n d a r d s and measurements. I n t h i s c h a p t e r , we w i l l r e v i e w luminescence s t a n d a r d s f o r m a c r o s p e c t r o f l u o r o m e t r y and e x t e n d the d i s c u s s i o n t o s t a n d a r d s f o r microspectrofluorometry. The term " l u m i n e s c e n c e " i n c l u d e s b o t h f l u o r e s c e n c e and phosphorescence; however, we w i l l c o n t i n u e t o use g e n e r a l l y a c c e p t e d t e r m i n o l o g y such as " s p e c t r o f l u o r o m e t r y " o r " m i c r o s p e c t r o f l u o r o m e t e r , " even though " s p e c t r o l u m i n i m e t r y " might be more r e p r e s e n t a t i v e o r "luminescence s p e c t r o m e t e r f o r measurements on a m i c r o s c a l e " might be more c o r r e c t ( 6 ) . A l t h o u g h m a c r o s p e c t r o f l u o r o m e t r i c s t a n d a r d s are d i s c u s s e d f i r s t , the comments b a s i c a l l y h o l d f o r m i c r o s p e c t r o f l u o r o m e t r i c s t a n d a r d s , e x c e p t i n the s p e c i a l a r e a s o f s t a b i l i t y and s i z e and shape, w h i c h w i l l be d i s c u s s e d l a t e r . Macrospectrofluorometric

Standards

Requirements of Standards. The g e n e r a l r e q u i r e m e n t s f o r luminescence s t a n d a r d s have been d i s c u s s e d e x t e n s i v e l y (3,7-9) and i n c l u d e s t a b i l i t y , p u r i t y , no o v e r l a p between e x c i t a t i o n and e m i s s i o n s p e c t r a , no oxygen q u e n c h i n g , and a h i g h , c o n s t a n t quantum y i e l d independent o f e x c i t a t i o n wavelength. S p e c i f i c system p a r a m e t e r s - - s u c h as the b r o a d or narrow e x c i t a t i o n and e m i s s i o n s p e c t r a , i s o t r o p i c o r a n i s o t r o p i c emission, s o l u b i l i t y i n a s p e c i f i c solvent, s t a b i l i t y (standard r e l a t i v e t o s a m p l e ) , and c o n c e n t r a t i o n - - a l m o s t r e q u i r e the s t a n d a r d t o be i n the same c h e m i c a l and p h y s i c a l environment as the sample. I n the i d e a l c a s e , the s t a n d a r d and the sample s h o u l d be the same c h e m i c a l i n the same m a t r i x . The b e s t we c a n hope f o r i s a j u d i c i o u s c h o i c e o f w e l l - c h a r a c t e r i z e d s t a n d a r d s t o c o v e r most measurement situations. A t the same t i m e , the r e s e a r c h e r and u s e r must be aware


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LUMINESCENCE APPLICATIONS

o f the c o r r e c t a p p l i c a t i o n o f the s t a n d a r d s and the l i m i t a t i o n s i n h e r e n t i n luminescence s p e c t r o m e t e r s and s t a n d a r d s .


Functions of Standards. F l u o r e s c e n t s t a n d a r d s c a n be u s e d f o r t h r e e basic functions: c a l i b r a t i o n , s t a n d a r d i z a t i o n , and measurement method assessment. I n c a l i b r a t i o n , the s t a n d a r d i s used t o check o r c a l i b r a t e i n s t r u m e n t c h a r a c t e r i s t i c s and p e r t u r b a t i o n s on t r u e spectra. F o r s t a n d a r d i z a t i o n , s t a n d a r d s a r e used t o determine the f u n c t i o n that r e l a t e s chemical c o n c e n t r a t i o n to instrument response. T h i s l a t t e r use has been expanded from pure m a t e r i a l s t o q u i t e complex s t a n d a r d s t h a t are c a r r i e d t h r o u g h the t o t a l c h e m i c a l measurement p r o c e s s ( 1 0 ) . These more complex s t a n d a r d s a r e now used t o a s s e s s the p r e c i s i o n and a c c u r a c y o f measurement p r o c e d u r e s . C a l i b r a t i o n . I n g e n e r a l , standards used f o r instrument c a l i b r a t i o n a r e p h y s i c a l d e v i c e s ( s t a n d a r d lamps, f l o w m e t e r s , e t c . ) o r pure c h e m i c a l compounds i n s o l u t i o n ( s o l i d o r l i q u i d ) , a l t h o u g h some combined forms c o u l d be used ( e . g . , T b + E u I n g l a s s f o r w a v e l e n g t h c a l i b r a t i o n ) . C a l i b r a t e d i n s t r u m e n t parameters i n c l u d e wavelength accuracy, detection-system s p e c t r a l r e s p o n s i v i t y (to determine c o r r e c t e d e x c i t a t i o n and e m i s s i o n s p e c t r a ) , and s t a b i l i t y , among o t h e r s . F l u o r e s c e n c e d a t a such as c o r r e c t e d e x c i t a t i o n and e m i s s i o n s p e c t r a , quantum y i e l d s , decay t i m e s , and p o l a r i z a t i o n t h a t a r e t o be compared among l a b o r a t o r i e s a r e dependent on t h e s e c a l i brations. The i n s t r u m e n t and f l u o r e s c e n c e parameters and v a r i o u s s t a n d a r d s , r e v i e w e d r e c e n t l y (1,2,11), are d i s c u s s e d b r i e f l y b e l o w . 3 +

3 +

Wavelength. Three types o f s t a n d a r d s used f o r w a v e l e n g t h c a l i b r a t i o n a r e n a r r o w - s p e c t r a l - l i n e l o w - p r e s s u r e d i s c h a r g e s o u r c e s and l a s e r s ; o r g a n i c and i n o r g a n i c s p e c i e s i n s o l u t i o n ; and narrow bands from the e x c i t a t i o n s o u r c e s ( X e , Hg) ( e . g . , see R e f s . 11-16). For wavelength a c c u r a c i e s a p p r o a c h i n g 0 . 1 nm, n a r r o w - l i n e s o u r c e s a r e recommended; s e v e r a l are l i s t e d i n T a b l e I . A few l i n e s o r many l i n e s c o v e r i n g the w a v e l e n g t h r e g i o n o f i n t e r e s t can be used ( 1 7 ) . P r o c e d u r e s f o r c a l i b r a t i n g b o t h monochromators i n a f l u o r e s c e n c e s p e c t r o m e t e r u s i n g narrow l i n e s o u r c e s have been d i s c u s s e d ( 1 5 , 1 8 ) ; c a r e must be t a k e n w i t h placement o f the c a l i b r a t i o n s o u r c e . For ease o f use and w a v e l e n g t h a c c u r a c i e s o f 1-2 nm, o r g a n i c m a t e r i a l s o r i n o r g a n i c i o n s i n s o l u t i o n have been recommended as standards (Table I I ) . However, t h e s e must be u s e d c a r e f u l l y because (a) the peak maxima are m a t r i x dependent, (b) narrow i n s t r u m e n t a l bandpasses are n e c e s s a r y , ( c ) i m p u r i t i e s may a f f e c t peak l o c a t i o n , and (d) the peak w a v e l e n g t h v a l u e s have g e n e r a l l y n o t been c e r t i f i e d (11). As mentioned p r e v i o u s l y ( 1 1 ) , the w a v e l e n g t h p o s i t i o n and s t a b i l i t y o f s p e c t r a l l i n e s from xenon o r mercury e x c i t a t i o n s o u r c e s o f s p e c t r o f l u o r o m e t e r s may be v a r i a b l e w i t h time-, and such s o u r c e s a r e d i f f i c u l t t o use w i t h c e r t a i n t y f o r the c a l i b r a t i o n o f monochromators . Spectral Responsivity Standards (for Corrected Spectra). Depending on the c o n d i t i o n s , many d i f f e r e n t o r g a n i c and i n o r g a n i c compounds i n v a r i o u s s o l v e n t s have been used as s t a n d a r d s f o r d e t e r m i n i n g the s p e c t r a l r e s p o n s i v i t y of instruments. S e v e r a l measurement p r o c e -


7.


101


Table I . S p e c t r a l L i n e s from E x t e r n a l Sources Used f o r Monochromator Wavelength C a l i b r a t i o n


Wavelength (nm)

Source 3

Line

Laser

202.55 253.65 296.73 325.03 365.01 404.66

Zn Hg Hg

_

435.84 514.54 546.07 576.96 579.07 632.82

Hg

692.95 724.52 752.55 799.30 852.11 894.35

Ne Ne

a

Hg Hg

3+

_

15 24 25 25 25 24

Ar Hg Hg Hg

-

-

He-Ne

Cs Cs

27 27 24 24 27 27

Kr Kr

-

Luminescence Wavelength S t a n d a r d s - - O r g a n i c s and I n o r g a n i c Ions i n G l a s s M a t r i c e s

Phenanthrene Gd Tb Sm Eu A n t h r a c e n e , Napht h a l e n e , Ovalene 3 +

-

He-Cd

Matrix

3 +

27 15 15 24 25 15

-

Wavelengths i n a i r .

Table I I .

3+

Reference

n-CeHi2 Borate Glass Borate Glass Phosphate G l a s s Phosphate G l a s s Polymer B l o c k s

m

Wavelength < > * 211- 346 248- 274 220- 379 318- 526

i n Solution

Reference

_

312 486 562-707 578-612 300-550

Wavelengths rounded t o n e a r e s t nanometer.


19 20 20 20 21 22

102


d u r e s , i n c l u d i n g a b s o l u t e measurements, c a n be used t o determine v a r i o u s c o r r e c t i o n f a c t o r s g i v e n i n e q u a t i o n s 1 and 2 . Excitation Spectra (11,19;.· 1

*(λ )

- Χ (Χ )ηρ(Χ )/τ(Χ )]Ν(Χ )[τ'(Χ )Γ

χ

χ

Α

χ

χ

(1)

χ

χ

the

where Χ(Χ ) i s the c o r r e c t e d spectrum, Χ i s the measured e x c i t a t i o n spectrum i n c l u d i n g c o r r e c t i o n s f o r s t r a y r a d i a t i o n ; k i s a c o n s t a n t ; ρ(Χ )/τ(Χ ) i s the b e a m - s p l i t t e r c o r r e c t i o n f a c t o r ; # ( λ ) i s the r e f e r e n c e d e t e c t o r w a v e l e n g t h response i n c l u d i n g the t r a n s m i t t a n c e o f the d e t e c t o r window; and τ' (Χ ) i s the t r a n s m i t t a n c e o f the sample c e l l window. For e x c i t a t i o n s p e c t r a , the a c c u r a c y o f the c o r r e c t i o n p r o c e d u r e can be checked by comparing the c o r r e c t e d , n o r m a l i z e d v a l u e s w i t h the absorbance spectrum (19) o r d e t e r m i n i n g i f any peaks from the s o u r c e are o b s e r v e d i n the c o r r e c t e d e x c i t a t i o n spectrum (23). Emission Spectra (11,24;: χ

χ

Λ

χ

χ


χ

E(X ,X ) x

m

- Ε

λ χ

α10 s ) r e s u l t e d i n p h o t o b l e a c h i n g o f the o r g a n i c f l u o r o p h o r . F i g u r e 3 shows the p o r o s i t y on a m i c r o s c a l e f o r a g e o l o g i c t h i n s e c t i o n from a d r i l l i n g core. Haaijman (53) took a d i f f e r e n t approach f o r the s t a n d a r d i z a t i o n o f m i c r o s c o p i c immunofluorescence measurements u s i n g F I T C . Rather t h a n u s i n g s p e c i a l i n s t r u m e n t a t i o n o r p r o c e d u r e s t o reduce p h o t o b l e a c h i n g , he d e s i g n e d a s t a n d a r d u s i n g FITC bound t o a m i n o e t h y l Sephadex beads, i n w h i c h the amount o f bound f l u o r o p h o r was q u a n t i f i e d u s i n g absorbance measurements. Though the response o f the FITCl a b e l e d beads t o changes i n p h y s i c o c h e m i c a l v a r i a b l e s ( i . e . , pH and d u r a t i o n o f e x c i t a t i o n ) was n o t i d e n t i c a l t o t h a t o f F I T C - l a b e l e d c e l l s , the d i s c r e p a n c i e s were n o t c o n s i d e r e d major and the beads were deemed to be adequate f o r immunofluorescence m i c r o s c o p y and s u p e r i o r to other a v a i l a b l e standards. These problems are n o t as s i g n i f i c a n t i n the macroenvironment, where many s t a n d a r d s can be used f o r b o t h c a l i b r a t i o n and s t a n d a r d i z a t i o n , s i n c e p h o t o d e g r a d a t i o n o f the sample i s u s u a l l y n o t s i g n i f i c a n t , r u n n i n g around 5-10%. However, i f measurements o f the h i g h e s t a c c u r a c y o r o f s m a l l d i f f e r e n c e s are r e q u i r e d , t h e s e u n c e r t a i n t i e s



7. VELAPOLDI AND EPSTEIN

0

4 0

Macro- and Mwwspectrofluorometry

. 45

, 90

109

135

Exposure time (s) F i g u r e 2 . I r r a d i a t i o n o f u r a n y l g l a s s m i c r o s p h e r e s (A) and FITCl a b e l e d microbeads (B) demonstrate p h o t o b l e a c h i n g and s t a b i l i t y o f both materials.

F i g u r e 3 . P o r o s i t y measurements on a m i c r o s c o p i c s c a l e f o r a s m a l l (2 cm χ 2 cm χ 30 μιη) t h i n s e c t i o n o f sandstone impregnated w i t h f l u o r o p h o r - d o p e d p o l y m e r . Average p o r o s i t y - 16.3%; range o f p o r o s i t i e s - 1.8-42%.


110


may be too l a r g e , and as d i s c u s s e d p r e v i o u s l y , new w o r k i n g s t a n d a r d s s h o u l d be p r e p a r e d .


Size and Shape. The dimensions o f the s t a n d a r d are more c r i t i c a l i n the m i c r o e n v i r o n m e n t than i n the macroenvironment, s i n c e m i c r o s c o p i c measurements commonly r e q u i r e changes i n f i e l d a p e r t u r e s and m a g n i f i cation. I f a m i c r o s c o p i c s t a n d a r d has a s m a l l ( / i m - s i z e d ) , w e l l d e f i n e d shape, such as a sphere o r c y l i n d e r , an a c c u r a t e i n t e n s i t y / volume r e l a t i o n s h i p can be e s t a b l i s h e d , w h i c h s h o u l d be independent o f the microscope o p t i c s . S t a n d a r d i z a t i o n i s thus v a l i d no m a t t e r what m i c r o s c o p e parameters are employed, as l o n g as the s p e c t r a l c h a r a c t e r i s t i c s o f the s t a n d a r d and the sample are q u i t e s i m i l a r o r identical. Use of Macrostandards in the Microenvironment. M a c r o s t a n d a r d s , such as u r a n y l i o n - d o p e d g l a s s microscope s l i d e s , have been used as s t a n dards f o r m i c r o s p e c t r o f l u o r o m e t r i c measurements by s e v e r a l a u t h o r s (59,60). The a p p e a l o f such an approach i s o b v i o u s , because w e l l c h a r a c t e r i z e d and s t a b l e s t a n d a r d s are r e a d i l y a v a i l a b l e and easy t o use. However, as d i s c u s s e d by S e r n e t z and Thaer ( 6 1 ) , the d i a m e t e r o r t h i c k n e s s o f a macrostandard w i l l be g r e a t e r t h a n the e x c i t a t i o n and e m i s s i o n f i e l d d i a m e t e r s o f the m i c r o s p e c t r o f l u o r o m e t e r , and thus the f l u o r e s c e n c e e m i s s i o n w i l l be a f u n c t i o n o f m i c r o s c o p e parameters t h a t d e f i n e the volumes i l l u m i n a t e d and o b s e r v e d , such as o b j e c t i v e m a g n i f i c a t i o n and n u m e r i c a l a p e r t u r e , e m i s s i o n and e x c i t a t i o n a p e r t u r e s i z e , and f o c a l p o i n t . These parameters w i l l be d i f f i c u l t t o reproduce on two o r more d i f f e r e n t m i c r o s c o p e s and w i l l be s u b j e c t t o f o c u s i n g e r r o r s when u s i n g the same m i c r o s c o p e . Use o f a macros t a n d a r d w i l l be f u r t h e r c o m p l i c a t e d by e r r o r s due t o p r e f i l t e r and p o s t f i l t e r e f f e c t s and would be i m p o s s i b l e f o r nonphotometrie ( i . e . , v i s u a l ) d e t e c t i o n schemes. Use of Microstandards in the Microenvironment. The most common approach t o p r e p a r i n g m i c r o s t a n d a r d s i s t o p l a c e a f l u o r o p h o r i n o r to b i n d i t to a s t a b l e , n o n f l u o r e s c i n g h o s t o f w e l l - d e f i n e d shape. Ploem (62) u s e d m i c r o d r o p l e t s o f FITC s o l u t i o n i n m i c r o - w e l l s i n a p e r s p e x s l i d e , b u t found s i g n i f i c a n t p h o t o b l e a c h i n g under i l l u m i n a tion. More s u c c e s s f u l were m i c r o c a p i l l a r i e s (61-63) o f about 6 μιη i n s i d e d i a m e t e r and 50 mm l e n g t h f i l l e d w i t h f l u o r o p h o r s o l u t i o n . These s t a n d a r d s d i d n o t p h o t o b l e a c h as much as the m i c r o d r o p l e t s , s i n c e the e x c i t e d p o r t i o n o f the f l u o r o p h o r s o l u t i o n , w h i c h was l e s s than 40 μια i n l e n g t h , was c o n t i n u o u s l y renewed by c o n v e c t i o n a l f l o w and d i f f u s i o n . However, a s p e c i f i c l e n g t h o f c a p i l l a r y had t o be i s o l a t e d u s i n g the microscope measuring a p e r t u r e . V e l a p o l d i e t a l . (64) used a s i m i l a r approach b u t p r e p a r e d f i b e r s o f u n i f o r m diameter (5-45 μπι) from i n o r g a n i c i o n - d o p e d glasses. The f l u o r e s c e n c e parameters o f t h e s e m a t e r i a l s can be changed by s u b s t i t u t i n g v a r i o u s i o n s , such as T b , S m , E u , M n , U 0 , C u , and S n . They show e x c e l l e n t s t a b i l i t y under i r r a d i a t i o n u s i n g i n c i d e n t e x c i t a t i o n (measurement i m p r e c i s i o n o f 1% under c o n t i n u o u s i r r a d i a t i o n i n the microscope f o r 24 h) and have a f l u o r e s c e n c e f l u x d e n s i t y p r o p o r t i o n a l t o the f i b e r l e n g t h . R e c e n t l y , m i c r o s p h e r e s (5-40 μιη d i a m e t e r ) o f t h e s e i o n - d o p e d g l a s s e s have been p r e p a r e d and e v a l u a t e d ( 6 5 ) . Ions can be s e l e c t e d t o produce e i t h e r sharp ( T b , Sm , E u ) o r b r o a d ( M n , U 0 , C u , 3 +

2 +

+

3+

3 +

2+

2 +

2

3+

3+

3+

2+

2 +

2


+


7.


111


Sn ) s p e c t r a l d i s t r i b u t i o n s . The spheres e x h i b i t a f l u o r e s c e n c e f l u x d e n s i t y p r o p o r t i o n a l t o the r a d i u s cubed ( F i g u r e 4 ) . They a r e s i m i l a r i n s i z e and shape t o c e l l s and have a d i s t i n c t advantage over f i b e r s o r c a p i l l a r i e s i n t h a t no p h y s i c a l parameters ( i . e . , c a p i l l a r y l e n g t h ) need t o be d e f i n e d b y the m i c r o s c o p e o p t i c s . F i g u r e s 5a and b i l l u s t r a t e homogeneously and nonhomogeneously uranium-doped g l a s s microspheres. The luminescence i n t e n s i t y d i s t r i b u t i o n was o b t a i n e d u s i n g an automated m i c r o s p e c t r o f l u o r o m e t e r system w i t h a s c a n n i n g m i r r o r o r s t a g e ( 0 . 1 t o 2 . 5 μιη s t e p r e s o l u t i o n ) , w h i c h was scanned i n a r a s t e r mode (100x100), y i e l d i n g 10,000 d a t a p o i n t s o f f l u o r e s c e n c e f l u x d e n s i t y as a f u n c t i o n o f x j p o s i t i o n . Many i n o r g a n i c i o n - d o p e d g l a s s e s l u m i n e s c e , g i v i n g coverage from the u l t r a v i o l e t t o the r e d ( T a b l e V I ) . I n a d d i t i o n , i o n s t h a t undergo i n t e r c o n f i g u r a t i o n a l e l e c t r o n i c t r a n s i t i o n s upon a b s o r p t i o n and e m i s s i o n o f r a d i a t i o n , such as P b , show e m i s s i o n maximum s h i f t s as l a r g e as 150 nm upon c h a n g i n g m a t r i c e s ( F i g u r e 6 ) , whereas i o n s 2 +

Table V I .

Element

Tl Pb Ag Ce Eu Sn Cu Bi U Mn Cd Fe Cr Mo Gd Tm Dy Tb Sm Eu Pr Nd

C h a r a c t e r i s t i c s o f Luminescence from Ion-Doped G l a s s e s

Spectra Type* Broadband Broadband Broadband Broadband Broadband Broadband Broadband Broadband Broadband Broadband Broadband Broadband Broadband Broadband Narrowband Narrowband Narrowband Narrowband Narrowband Narrowband Narrowband Narrowband

Luminescing Species +

Tl Pb Ag,Ag Ce Eu Sn Cu Bi (U0 ) Mn CdS Fe Cr Mo Gd Tm Dy Tb Sm Eu Pr Nd 2 +

3+

2 +

+

+

3 +

2 +

2

2+

3 +

3 +

3+

3+

3+

3+

3 +

3+

3 +

3 +

3+

+

Excitation Range (nm) 215-260 241-295 250-320 215-317 230-400 265 254-445 319 340-480 356-422 350-470 458 585 980 220-280 265-350 340-380 250-378 403-469 340-395 440-475 530-585

Inorganic

Useful Emission Range ( n m ) c

250-370 270-480 290-430 310-420 325-650 370-450 385-600 390-525 490-620 500-680 560-790 570-650,800-880 710-1000 990-1030 311 350,383,455 475,576 495,544,580,620 563,572,599,647,705 578,591,611,653,700 610,710,890 890,1060

Shape o f the s p e c t r a l d i s t r i b u t i o n ; broadband s p e c t r a have a band w i d t h a t h a l f h e i g h t o f g r e a t e r t h a n 5 nm. The w a v e l e n g t h range o b s e r v e d t o e x c i t e luminescence i n the i o n - d o p e d g l a s s . T h e approx imate w a v e l e n g t h range o v e r w h i c h luminescence has been o b s e r v e d (broadband) o r the approximate wavelengths a t w h i c h l u m i n e s c e n c e maxima o c c u r (narrowband). A l l wavelengths a r e approximate s i n c e they r e p r e s e n t an average v a l u e t a k e n from d i f f e r e n t r e f e r e n c e s u s i n g v a r i o u s e x c i t a t i o n s o u r c e s and base g l a s s c o m p o s i t i o n s ( 6 5 ) . c




112


Macro- and

Microspectrofluorometry

113



Figure 5. Two- and three-dimensional representations of r e l a t i v e luminescence f l u x of uranyl ion-doped glass beads, measured i n a 100 x 100 raster with 10,000 data points. Depicted are a homogeneously doped bead (a) and a nonhomogeneously doped bead (b).



114


2+

F i g u r e 6. C o r r e c t e d s p e c t r a f o r P b - d o p e d phosphate ( 1 ) , b o r a t e ( 2 ) , and s i l i c a t e (3) g l a s s e s . E x c i t a t i o n shown as r e l a t i v e i n t e n s i t y ; e m i s s i o n shown as r e l a t i v e q u a n t a / u n i t b a n d w i d t h .


7.


Macro- and Micwspectrofluorometry

115

t h a t undergo i n t r a c o n f i g u r a t i o n a l e l e c t r o n i c t r a n s i t i o n s , such as E u * , show e m i s s i o n maximum s h i f t s o f o n l y a few nanometers ( β ) . Thus the c h o i c e o f the r i g h t i o n and m a t r i x g i v e s some v a r i e t y o f luminescence c h a r a c t e r i s t i c s . A l t h o u g h i n o r g a n i c i o n - d o p e d g l a s s e s are i d e a l as s t a n d a r d s f o r c a l i b r a t i o n , they have l i m i t e d a p p l i c a t i o n f o r r e l a t i n g i n s t r u m e n t s i g n a l to a n a l y t e c o n c e n t r a t i o n because o n l y a s m a l l number o f d i f f e r e n t s p e c t r a l d i s t r i b u t i o n s are a v a i l a b l e ( r e l a t i v e to the m y r i a d of o r g a n i c s p e c i e s ) . There are l i k e l y to be o n l y a few cases i n w h i c h the s p e c t r a l d i s t r i b u t i o n o f a s p e c i f i c c l a s s i s s i m i l a r enough to the a n a l y t e spectrum to be u s e f u l . The use of u r a n i u m doped g l a s s e s f o r the q u a n t i f i c a t i o n o f F I T C - l a b e l e d s p e c i e s (6*5) i s one such c a s e . The f a c t r e m a i n s , though t h a t a s t a n d a r d o f the same m a t e r i a l as the sample i s p r e f e r r e d f o r s t a n d a r d i z a t i o n . The approach t o s t a n d a r d i z a t i o n u s e d b y Haaijman (53) and o t h e r s (66,67), i n w h i c h the f l u o r o p h o r i s i n c o r p o r a t e d w i t h i n o r bound t o the s u r f a c e o f a p l a s t i c s p h e r e , i s more v e r s a t i l e t h a n the use o f i n o r g a n i c i o n - d o p e d s p h e r e s , s i n c e the s t a n d a r d c a n be t a i l o r e d e x a c t l y t o the s p e c i f i c a t i o n s r e q u i r e d b y the a n a l y t e s p e c i e s . However, t h i s approach i n c r e a s e s the u n c e r t a i n t y o f the measurement because the p h o t o b l e a c h i n g c h a r a c t e r i s t i c s o f b o t h the s t a n d a r d and the sample must be c o n s i d e r e d . The i d e a l approach i s t o employ b o t h types o f s t a n d a r d s . The g l a s s m i c r o s p h e r e s c a n be u s e d t o c a l i b r a t e i n s t r u m e n t s and s e t i n s t r u m e n t o p e r a t i n g parameters on a d a y - t o - d a y b a s i s , and the f l u o r o p h o r - d o p e d polymer m a t e r i a l s can be used t o determine the c o n c e n t r a t i o n - i n s t r u m e n t response f u n c t i o n .


3

Applications

ot Macrospectrofluorometric

Standards

Standardization. Standardization i n a n a l y t i c a l chemistry, i n which s t a n d a r d s a r e u s e d t o r e l a t e the i n s t r u m e n t s i g n a l t o compound c o n c e n t r a t i o n , i s the c r i t i c a l f u n c t i o n f o r d e t e r m i n i n g the r e l a t i v e c o n c e n t r a t i o n s o f s p e c i e s i n a wide v a r i e t y of m a t r i c e s . Environ m e n t a l Standard Reference M a t e r i a l s (SRM's) have been developed f o r v a r i o u s p o l y n u c l e a r a r o m a t i c hydrocarbons ( P A H ' s ) . I n f o r m a t i o n on SRM's can be o b t a i n e d from the O f f i c e o f S t a n d a r d R e f e r e n c e M a t e r i a l s , N a t i o n a l Bureau o f S t a n d a r d s , G a i t h e r s b u r g , MD 20899. Summa r i z e d i n T a b l e V I I , t h e s e SRM's range from "pure compounds" i n aqueous and o r g a n i c s o l v e n t s t o " n a t u r a l " m a t r i c e s such as s h a l e o i l and urban and d i e s e l p a r t i c u l a t e m a t e r i a l s . P u r e Compound SRM's. Pure compound SRM's ( o r s t a n d a r d s ) a r e conve n i e n t t o use f o r e n v i r o n m e n t a l samples such as w a t e r , where l i t t l e sample e x t r a c t i o n i s n e c e s s a r y . G e n e r a t o r columns (SRM 1644) were d e v e l o p e d t o p r o v i d e d i f f e r i n g c o n c e n t r a t i o n s o f pure P A H ' s b y v a r y i n g the temperature of the b a t h i n w h i c h the g e n e r a t o r column i s p l a c e d (68). F i g u r e 7a i l l u s t r a t e s the raw f l u o r e s c e n c e s i g n a l o b t a i n e d d u r i n g the SRM c e r t i f i c a t i o n p r o c e s s u s i n g the s t a n d a r d a d d i t i o n method f o r d e t e r m i n i n g benzo [a] pyrene c o n c e n t r a t i o n s by an "on stream" f l u o r e s c e n c e t e c h n i q u e . F i g u r e 7b i s a p l o t of the i n t e n s i t i e s , showing the b e s t l e a s t - s q u a r e s f i t s t o d a t a c o l l e c t e d a t f o u r d i f f e r e n t t e m p e r a t u r e s . I n t h i s c a s e , the i n s t r u m e n t response as a f u n c t i o n o f c o n c e n t r a t i o n can be d e t e r m i n e d f o r a s p e c i f i c PAH w h i l e the unknown sample i s measured by the same p r o c e s s (assuming



116


Column 215 1.2X10NÛ»

-5.0

0.0 [Benzo(a)pyrene], ng/g

5.0

Figure 7. (a) Multichannel analyzer CRT display of raw data using standards-addition technique and (b) raw data (x) and f i t t e d least-squares l i n e (—) f o r the c a l c u l a t i o n of the benzo[a]pyrene concentration i n the generator column e f f l u e n t at four temperatures.


7.


T a b l e V I I . S t a n d a r d Reference M a t e r i a l s (SRM's) f o r the D e t e r m i n a t i o n o f P o l y c y c l i c A r o m a t i c Hydrocarbons ( P A H ' s ) S RM #


1587 1596

1582 1588 1597

Anthracene, benzo[a]anthracene, benzo[a]pyrene 16 PAH's

P r i o r i t y p o l l u t a n t PAH i n acetonitrile N i t r a t e d PAH i n m e t h a n o l Dinitropyrenes i n solvent 5

Natural Matrix Materials: 1580 Organics i n shale

oil

6 n i t r a t e d PAH's 4 n i t r a t e d pyrenes (3 d i n i t r o and 1 n i t r o )

c

5 PAH's + 4 oxygennitrogen- containing compounds 5 PAH's + d i b e n z o t h i o p h e n e

c

P e t r o l e u m crude o i l Cod l i v e r o i l ' Complex PAH m i x t u r e from coal t a r Urban p a r t i c u l a t e m a t t e r Urban d u s t / o r g a n i c s Diesel particulate material c

a

C e r t i f i e d Compounds

SRM Name

Pure S o l u t i o n s : 1644 G e n e r a t o r columns f o r PAH 1647

117


d

9 PAH's

c

1648 1649 1650

c , e

c

c

5 PAH's 6 PAH's

a

0 f f i c e o f S t a n d a r d Reference M a t e r i a l s , N a t i o n a l Bureau o f S t a n d a r d s , G a i t h e r s b u r g , MD 20899. ^ N i t r a t e d PAH's do n o t f l u o r e s c e b u t can be made t o f l u o r e s c e by p a s s i n g t h r o u g h a Zn o r P t column t o reduce n i t r o groups t o amino g r o u p s , i n f o r m a t i o n values for other organics i n c e r t i f i c a t e . C e r t i f i e d v a l u e s f o r p e s t i c i d e s and P C B ' s . C e r t i f i e d values for inorganic constituents.

o n l y t h a t the same s p e c i f i c PAH i s p r e s e n t i n the unknown s a m p l e ) . L i q u i d chromatographic t e c h n i q u e s can be used t o s e p a r a t e complex m i x t u r e s o f PAH's (See, f o r example, R e f s . 6 9 , 7 0 ) , and the PAH o f i n t e r e s t d e t e r m i n e d by t h a t mode. Another u s e f u l s t a n d a r d i s SRM 1647, p r i o r i t y p o l l u t a n t p o l y n u c l e a r a r o m a t i c hydrocarbons ( i n a c e t o n i t r i l e ) . I t c a n be used t o c a l i b r a t e l i q u i d chromatographic i n s t r u m e n t s ( r e t e n t i o n t i m e s , i n s t r u m e n t r e s p o n s e ) , t o determine p e r c e n t r e c o v e r i e s , and t o f o r t i f y aqueous samples w i t h known PAH c o n c e n t r a t i o n s . Figure 8 i l l u s t r a t e s the HPLC s e p a r a t i o n and UV d e t e c t i o n ( f l u o r e s c e n c e i s a l s o u s e d e x t e n s i v e l y ) f o r the 16 p r i o r i t y p o l l u t a n t s . Matrix SRM's. M a t r i x SRM's can be used f o r a v a r i e t y o f f u n c t i o n s . T a y l o r (71) r e v i e w e d the uses o f m a t r i x s t a n d a r d s , w h i c h i n c l u d e : (a) method development and e v a l u a t i o n , (b) e s t a b l i s h m e n t o f measurement t r a c e a b i l i t y , and (c) a s s u r a n c e o f measurement c o m p a t i b i l i t y . D u r i n g c e r t i f i c a t i o n o f these m a t r i x SRM's, the s e l e c t i v i t y o f f l u o r e s c e n c e


118



υ< c

TIME

(min)

F i g u r e 8. Reversed-phase HPLC s e p a r a t i o n o f SRM 1647, p r i o r i t y p o l l u t a n t p o l y n u c l e a r a r o m a t i c hydrocarbons ( i n a c e t o n i t r i l e ) , u s i n g UV d e t e c t i o n .



7.



119

d e t e c t i o n was used t o s i m p l i f y a n a l y t i c a l p r o c e d u r e s . F o r example, i n the c e r t i f i c a t i o n p r o c e s s e s o f SRM's 1648 (urban p a r t i c u l a t e m a t t e r ) and 1649 (urban d u s t / o r g a n i c s ) , UV d e t e c t i o n and q u a n t i f i c a t i o n o f s e l e c t e d PAH's gave q u i t e u n i n t e r p r e t a b l e r e v e r s e d - p h a s e HPLC chromatograms ( F i g u r e 9) u n l e s s normal-phase f r a c t i o n s e p a r a t i o n s were done b e f o r e the r e v e r s e d - p h a s e d e t e r m i n a t i o n s ( F i g u r e 10) ( 7 2 , 7 3 ) . Reversed-phase HPLC s e p a r a t i o n s u s i n g f l u o r e s c e n c e d e t e c t i o n w i t h w a v e l e n g t h programming p r o v i d e d " s i n g l e s t e p " q u a n t i f i c a t i o n ( F i g u r e 1 1 ) . These do n o t t r u l y r e l a t e c o n c e n t r a t i o n t o i n s t r u m e n t response b u t i n s t e a d r e l a t e c o n c e n t r a t i o n t o the t o t a l c h e m i c a l measurement p r o c e s s , i n c l u d i n g e x t r a c t i o n , s e p a r a t i o n , and instrument response. T h i s response f u n c t i o n may be b i a s e d compared t o the one o b t a i n e d f o r the pure compound s o l u t i o n s , s i m p l y because the c h e m i c a l measurement p r o c e s s response i s i n c l u d e d . I f the sample and s t a n d a r d have e s s e n t i a l l y the same m a t r i c e s ( e . g . , a i r p a r t i c u l a t e s o r r i v e r s e d i m e n t s ) , one can go t h r o u g h the t o t a l measurement p r o c e s s w i t h b o t h the sample and the s t a n d a r d i n o r d e r t o (a) check the a c c u r a c y o f the measurement p r o c e s s u s e d (compare the c o n c e n t r a t i o n v a l u e s o b t a i n e d f o r the s t a n d a r d w i t h the c e r t i f i e d v a l u e s ) and (b) o b t a i n some c o n f i d e n c e about the a c c u r a c y o f the c o n c e n t r a t i o n measurements on the unknown sample s i n c e both have gone through the same c h e m i c a l measurement p r o c e s s ( e x c e p t sample c o l l e c t i o n ) . I t i s n o t recommended, however, t h a t pure s t a n d a r d s be used to s t a n d a r d i z e the t o t a l c h e m i c a l measurement p r o c e s s f o r n a t u r a l m a t r i x type samples: c h e m i c a l c o n c e n t r a t i o n s i n the n a t u r a l m a t r i c e s c o u l d be s e r i o u s l y m i s r e a d , e s p e c i a l l y s i n c e the pure PAH p r o b a b l y would be t o t a l l y e x t r a c t e d i n a g i v e n s o l v e n t , whereas the PAH i n the m a t r i x m a t e r i a l p r o b a b l y w o u l d n o t b e . A l l the parameters and m a t r i x e f f e c t s , i n c l u d i n g e x t r a c t i o n e f f i c i e n c i e s , a r e c a r e f u l l y checked i n the c e r t i f i c a t i o n p r o c e s s l e a d i n g t o SRM's.

TIME (mln)

F i g u r e 9. Reversed-phase HPLC a n a l y s i s o f PAH's e x t r a c t e d from SRM 1649, urban d u s t / o r g a n i c s , w i t h UV d e t e c t i o n , not p r e c e d e d by normal-phase HPLC clean-up, ( R e p r i n t e d from r e f e r e n c e 72. C o p y r i g h t 1984 American Chemical S o c i e t y . )




14 Aromatic Carbon Fraction

U V -U 254 nm

16 Aromatic Carbon Fraction UV J 254 n m ι

1

1

1

1

1

0

10

20

30

40

50

Time (min) Figure 10. Reversed-phase H P L C analysis of PAHs extracted from S R M 1649, urban dust/organics, with U V detection, preceded by normal-phase H P L C fractionation based on ring carbon number. (Reprinted from reference 72. Copyright 1984 American Chemical Society.) Continued on next page.






uv±4_ 254 nm


254 nm

22 Aromatic Carbon Fraction UV

j l

254 nm

ο

10

20

30

40

50

Time (min) Figure 10. Continued.


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Bidimensional Fluorescence Wavelength Programming LC Analysis of Air Particulate Extract

0

10

20

30

40

60

60

TIME (minutes)

F i g u r e 1 1 . Reversed-phase HPLC a n a l y s i s o f t o t a l PAH's e x t r a c t e d from SRM 1648, urban p a r t i c u l a t e m a t t e r , w i t h programmed f l u o r e s c e n c e d e t e c t i o n . ( R e p r i n t e d from r e f e r e n c e 7 2 . C o p y r i g h t 1984 American Chemical S o c i e t y . )





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Standards for the Future. I n g e n e r a l , more s t a n d a r d s t h a t c o v e r a w i d e r w a v e l e n g t h range a r e needed f o r c a l i b r a t i o n , and n a t u r a l m a t r i x s t a n d a r d s are needed f o r s t a n d a r d i z a t i o n and measurement assessment. A n a l y s t s should advise s u p p l i e r s of standards ( e . g . , n a t i o n a l l a b o r a t o r i e s ) about w h i c h m a t r i c e s a r e b e i n g commonly c o l l e c t e d and w h i c h compounds need t o be c e r t i f i e d . In microspectrofluorometry, more f l u o r o p h o r - t a g g e d m a t e r i a l s a r e needed, as w e l l as i n c r e a s e d s t a b i l i t y o f the o r g a n i c - t a g g e d s p e c i e s . U n t i l we have s t a b l e o r g a n i c s p e c i e s t h a t w i l l n o t p h o t o b l e a c h under i n c i d e n t i r r a d i a t i o n , we w i l l need b e t t e r c o r r e l a t i o n o f the f l u o r e s c e n c e f l u x o f the s t a b l e , i n o r g a n i c i o n - d o p e d spheres t o the c o n c e n t r a t i o n o f the o r g a n i c s p e c i e s . T h i s r e q u i r e m e n t may n o t h o l d f o r some new t y p e s o f automated i n s t r u m e n t a t i o n ( e s p e c i a l l y m i c r o s p e c t r o f l u o r o m e t e r s ) o r f o r f l o w systems, s i n c e the r e s i d e n c e times o f any i n d i v i d u a l f l u o r o p h o r under the microscope o b j e c t i v e s h o u l d be s h o r t enough t o preclude photobleaching. Nevertheless, s t a t i c i n v e s t i g a t i o n s of samples such as c e l l s t o g i v e l o c a t i o n a l i n f o r m a t i o n c e r t a i n l y w i l l c o n t i n u e t o be o f i n t e r e s t , and the s t a b l e s t a n d a r d s w i l l c o n t i n u e t o s e r v e a u s e f u l c a l i b r a t i o n f u n c t i o n i n the f u t u r e .

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