Liquid Standards in Fluorescence Spectrometry

C. B u r g e s s a n d K.D. M i e l e n z ( E d i t o r s ) , Advances

in Standards

and Methodology

in

Spectrophotometry

1 9 8 7 Elsevier S c i e n c e P u b l i s h e r s B.V., A m s t e r d a m — P r i n t e d in T h e N e t h e r l a n d s

LIQUID STANDARDS IN FLUORESCENCE SPECTROMETRY

R.A. VELAPOLDI Norsk Hydro Research Centre, Box 4313, N-5001 Bergen, Norway (On leave from: Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, MD. 20899 USA.) ABSTRACT The use of spectrofluorimetric measurements has expanded tremendously in all fields during the last few decades. Because of this expansion, it has become of paramount importance to develop well-characterized secondary standards to produce reliable data, to interpret this data accurately, and to compare data among laboratories. In this paper, the current state of solution standards for fluorescence spectrometry including uses, requirements, and documentation is reviewed. The standards are used for instrument calibration or correction for wavelength accuracy, sensitivity, stability, source variation, detection system spectral responsivity, and measurement veracity. In turn, the values assigned to these standards are used to determine directly the same parameters for the chemical systems under study including the corrected excitation and emission spectra, quantum yields, and decay times. Included also are lists of standards, new literature values for the various fluorescence parameters, and a brief discussion of some of the problems and pitfalls in the use of the standards. INTRODUCTION

The objective of

this

paper

is

to

review

standards used in fluorescence spectrometry. in Fluorescence

Spectrometry",

Miller,

briefly

editor,

(ref.l)

Photoluminescence", Mielenz, editor, (ref.2) have addition to the seminal volume

by

general considerations, absolute

as

specific information.

Although

current state of

and

"Measurement of

recently appeared.

These, in

Parker (ref.3), provide detailed coverage of and

relative

measurement procedures, errors,

and available standards in fluorescence spectrometry. these volumes as well

the

Two excellent volumes, "Standards

literature the

mentioned main

The reader is referred to

therein

focus

of

or

in this paper for

this paper is on solution

standards and their fluorescence parameters, some comments will be made on other luminescence addition,

standards

comments

on

such

as

fluorophores

measurement

procedures

in

polymers

and

errors

or are

glasses. given

In where

relevant.

The fluorescent solutions are considered primary standards determine procedures) literature.

the are

are

the

fluorescent discussed

independently

to be secondary standards since the calibrated

characteristics.

These

elsewhere

volume

in

this

instrumentation primary or

in

used to

standards

(or

the referenced

176

Use of Fluorescence Measurements The use of fluorescence measurements few decades since the

introduction

has

by

increased tremendously in the last

Bowman

spectrofluorimeter using two monochromators. inherent sensitivity and selectivity of optical components and computers, spectrometry in diverse

fields

the

includes

The information obtained

cellular

make-up

and

almost endless.

(ref.4)

of the first

has been due to the

cytology

of users of fluorescence

and cellular biology, bio-,

mail delivery systems, safety wearing in

these areas is extremely varied and

interactions,

chemical component determinations, conversion/transformation.

ingenuity

as

analytical, physical, and geochemistries, apparel, etc.

al.

growth

the technique, the advances in electro-

and such

et This

qualitative

photochemical/kinetic

The lists

of

areas

and

quantitative

processes, and energy

and obtainable information are

This explosive growth in use underscores the need for reliable,

accurate fluorescence measurements to eliminate inaccuracies and confusion.

Absolute vs Relative Measurements Absolute fluorescence

measurements

are

instrumentation not readily available are

concerned

with

the

in

applications

measurements on various systems and

difficult most

and

perform

interpretations

and require

Most researchers of

fluorescence

want

to become involved in the all-

too-time-consuming effort of standards research.

Thus the availability of well-

characterized

secondary

characteristics would

standards

be

science in many fields. determine instrumental

The

the

a

secondary such

direct

chemical

in turn, are used for the studied.

chemical

can

wavelength

stability, effect

on

including

the

fluorescence advancement of

be used to check or

accuracy, sensitivity,

and instrument measurement the

measured

corrected

fluorescence excitation and

times, and polarization values.

These,

and physical characterization of the system

Finally, the values obtained

relative to secondary standards

to

standards

as

systems

emission spectra, quantum yields, decay

determined

contribution

responsivity, has

accurately

invaluable

These

latter

characteristics of

an

with

parameters

detection system spectral veracity.

don't

to

laboratories.

can

for secondary standards or other systems be

used to compare corrected fluorescence

data among laboratories.

General Requirements for Standards The general

requirements

numerous times (e.g., Demas,

for

fluorescence

ref.5;

standards

Velapoldi,

have been delineated

ref.6) and include: stability;

ease of purification; little overlap between excitation and emission spectra; no oxygen quenching; and a high, constant wavelength.

solubility in different included in

quantum

yield as a function of exciting

Other requirements such as broad, featureless fluorescence spectra,

general

solvents,

requirements,

isotropic relate

emission, to

the

ease

etc., although usually of

application and probably shouldn't be included in this category.

use

or specific

177

Documentation of Standards The availability of potential

standards

is

tremendous, especially with the

development and purification of chemicals in many areas including laser dyes and efficient energy conversion.

The

material for which the specific been accurately determined.

word

standard connotes a well characterized

physical,

Procedures

chemical, or mechanical property has

for

the purification or preparation of

the material, the experimental procedures, and the instrumental conditions in the measurement of the

fluorescence

used

parameter should be well documented and

readily available.

Although

many

materials

have

documentation is often scarce.

been

The

included by Chapman et al.

(ref.7)

of Reporting

Emission

Fluorescence

refs.5,8,9) still hold

for

the

proposed

as

recommendations

standards,

in "Proposal for Standardization of Methods Spectra"

reporting

and

of

expanded

appropriate

wavelength

"featureless" spectra)

reporting or

graphical interpretations. to guess a

maximum

of

intervals

easy

comparison

data

There is

10)

that

others (e.g.,

One further entreaty

corrected spectra, provide digital data at

wavenumber

for

by

fluorescence properties and the

proposal of materials as secondary fluorescence standards. has to be mentioned: when

the required

for the reporting of data

a

(at

least

and

to

every

5

nm for

reduce errors from

dearth of publications (I would venture

have

digital

values

reported for proposed

fluorescence standards.

With the appropriate documentation, potential quality indicator and as their specific measurements

measurements. and

interpretations

and must know the limitations

of

If standard systems are chosen system

being

on

The

the

users

different

researcher

use this information as a

must

chemical

keep

in

systems

mind that are

often

must select standards with care

the standards used and their instrumentation.

that have different fluorescence parameters from

measured

knowledge of the instrument or

could

guide in selecting suitable standards for

However,

measurement system dependent.

the chemical

a

people

(e.g.,

measurement

narrow-

vs

broad-band spectra),

effects is essential (in this case,

potential bandpass errors).

FLUORESCENCE STANDARDS

Wavelength Standards Externally used lasers or low pressure discharge sources with narrow spectral lines, fluorescing substances

in

solution,

and

the

excitation source of the

instrument have been recommended for calibrating monochromator wavelength scales (e.g., refs.10,11,12,13).

For calibrations with

accuracies

approaching

0.1 nm,

the

use of spectral

178

lines from lasers or

low

pressure

discharge

sources

is the method of choice

although the geometrical arrangement of the instrument and physical placement of the external source has to be considered if calibration errors are to be avoided (refs.10,12).

Velapoldi

and

calibrate the emission and of 2 0 0 to 9 0 0 nm.

Mielenz

excitation

Selected

sources

others are certainly available.

(ref.14)

34

used

spectral

lines to

monochromators over the wavelength range lines are listed in Table 1,

and

although

The monochromators are calibrated individually

if space is available; if not,

one

such as glycogen or

colloidal

silica

monochromator is calibrated and a scatterer

second monochromator

(refs.12,15).

in

a

cuvette

is used to calibrate the

TABLE 1 Spectral Lines from External Sources Used for Monochromator Wavelength Calibration X, n m

a

Source

Reference

Line

Laser

Zn Hg Hg

_

2 0 2 .. 5 5 2 5 3 ., 6 5 2 9 6 .. 7 3 3 2 5 .. 0 3 3 3 4 .. 1 5 3 6 5 .. 0 1 4 0 4 .. 6 6 4 0 7 .. 7 8 4 3 5 .. 8 4 5 1 4 .. 5 4 5 4 6 .. 0 7 5 7 6 .. 9 6 5 7 9 .. 0 7 6 3 2 .. 8 2 6 9 2 .. 9 5 7 2 4 .. 5 2 7 5 2 .. 5 5 7 9 9 .. 3 0 8 5 2 .. 1 1 8 9 4 .. 3 5

(ref.16) (ref.12)

-

u

-

(ref.13) (ref.12)

He-Cd

-

Hg Hg Hg Hg Hg

-

II

(ref.13) (ref.12)

Ar

-

Hg Hg Hg

II

-

-

(ref.13) (ref.16)

He-Ne

Ne Ne

-

-

-

Kr Kr

(ref.13)

Cs Cs

-

(ref.16)

-

II

II

a

Wavelengths in air. For ease of use, organic

materials

proposed as wavelength standards.

or

inorganic ions in solution have been

Melhuish (ref.17) gave absorbance maxima for

zone-refined, anthracene-free phenanthrene in cyclohexane; Reisfeld (ref.18) and Velapoldi et al. (ref.19)

have

maxima of inorganic ions in

given

glasses;

(ref.10) have suggested using

organic

values for the peak maxima of the some of

which

are

listed

calibration accuracy of 1-2

in

values and

for West

species

the excitation and emission and Kemp (ref.20) and Miller

in

polymer blocks (although no

latter type have been given). Table

2,

nm, although it

will must

wavelength maxima are matrix dependent

(e.g.,

^DQ to

silicate,

transitions for Eu(III)

in

probably be

The materials,

provide a wavelength

kept in mind that: a) the

the peak maxima for the strong phosphate, and borate glasses

179

and water are 610, 612,

617,

and 617 nm, respectively); b) narrow instrumental

bandpasses are necessary (i.e., single doublets at

bandpasses

of

1-5 nm.

peaks Fig.

at bandpasses of 7-14 nm 'become'

1);

c)

no

single

source of these

materials is available; and d) the wavelength values have not been certified.

TABLE 2 Luminescence Wavelength Standards - Organics in Solution and Inorganic Ions in Glass Matrices

Material

Matrix

Phenanthrene

n - C f iH 12

λ, e x

Borate Glass

Tb(III)

Borate Glass

Sm(III)

Phosphate Glass

Eu(III)

Phosphate Glass

λ, e m

211 220 251 292 330 346 248 254 274 220 303 341 352 369 379 -

w

Gd(III)

a

a

Reference

-

(ref.17)

312

(réf.18)

486 541

562 597 645 707 578 592 612

318 363 384 394 416 526

"

(réf.19)

a

Wavelengths rounded to nearest nm. The wavelength position and stability

of the instrument may

be

variable

of

with

'lines' from the excitation source

time

and

are

difficult to use with

certainty for the calibration of monochromators.

Excitation and Emission Spectra In general, excitation and emission technical, or true spectra (refs.11,14). obtained Technical

directly spectra

(detector system

from refer

the to

responsivity,

spectra may be presented as uncorrected, Uncorrected spectra refer to spectra

spectrofluorimeter spectra source

corrected variations

with for

no

corrections

instrumental

with

time

made.

parameters

and wavelength,

monochromator bandpass and wavelength, photomultiplier non-linearity, e t c ) .

180

-τ

1

1

1

ι —

1

IbJUü O-O-ff**

ι

460

1

500

1

540

1

580

~ I

620

660

~'

WAVELENGTH. NM Fig. 1. Tb(III) and Eu(III) doped silicate glass showing effect of monochromator bandpass: = 7.0 nm; = 3.5 nm.

True spectra refer

to

refractive

cell

index,

spectra

corrected

window

for

sample

transmittance,

parameters (eg, solvent

etc).

Specific

errors

are

discussed extensively by various authors in the two volumes mentioned previously (refs.1,2).

Excitation Spectra. excitation

spectrum

Melhuish for

(ref.17)

dilute

gave

equation

(absorbance

<10

1

),

to represent the

isotropic

emitting,

fluorescent materials in solution:

Χ ( λ χ) = X a ( X x ) k [ p ( X x ) / T ( X x ) ] N ( X x ) [ T ' ( X x ) ] "

where X a is the

measured

excitation

radiation; k is a constant;

of the detector window;

and

(1)

spectrum

ρ(Χχ)/τ(λχ)

Ν ( λ χ) is the reference detector

1

including corrections for stray

is the beam-splitter correction factor;

wavelength response including the transmittance

τ'(λ^)

is

the

transmittance

of the sample cell

window.

Several methods have spectra including the quantum counters,

transmittance,

summarized

of

and

to if

determining corrected excitation pyroeletric detectors, bolometers,

scatterers

common

with

procedure

a

calibrated detection

is use of a quantum counter

accurate measurements, corrections for polarization,

reflectance

spectrofluorimeter have reduced or eliminated

and

The most

For the most

for

thermopiles,

actinometers,

system (refs.8,17,24). (refs.3,25).

been use

be the

made

of

the

optical

(refs.11,22,23).

following

conditions

components Potential

are

used:

in

the

errors are a) a quantum

counter with non-viscous solvent; b) the beam splitter intercepts the excitation

181 beam at an angle <15°; c) rear viewing with a minimum of 2 cm of quantum counter solution between the excitation

radiation

and

the

reference detector; and d)

appropriate filters to eliminate stray

radiation

(refs.11,17,22,26) .

surface viewing of the quantum counter

by the photomultiplier

If front

(pmt) is used, an

appropriate filter can be placed between the quantum counter and the pmt so that only the long wavelength emission is measured, thus averting absorbance-emission overlap errors (ref.26).

Two ways can be used to

check

the

compare the corrected, normalized determine

if

any

peaks

veracity of the correction procedure: 1)

values

(from

the

with

source)

absorbance spectrum and 2)

the are

observed

in

the corrected

excitation spectrum (ref.27).

Melhuish (refs.9,17) listed several materials and their normalized absorbance ranges 220-680 nm including:

spectra that cover the wavelength (220-325 n m ) ,

quinine

(280-380 n m ) ;

sulfate

2-aminopyridine

3-aminophthalimide (340-425 nm) ;

proflavine (390-470 n m ) ; fluorescein (450-510 n m ) ; rhodamine Β (490-570 n m ) ; and methylene blue (570-680 n m ) .

Most of these materials also have been proposed as

potential emission standards (e.g., refs.6,9).

The corrected excitation and

emission

spectra for quinine sulfate dissolved

in 0.1 mol/L perchloric acid were determined by 10 laboratories in a round robin test, see Fig.

2

and

3

Table

for

values

2/10 power points, decreasing to approximately power points, absorbance

respectively.

spectrum

These

(ref.17)

9/10

the 'red edge' of the spectrum, the is 14, 7, and 1%, respectively.

power CV's

measurement conditions.

6%

values 10%

within

approximately 3% at the 1/2 and

and

The

spectrum vary from about 7% at the

coefficients of variation for the excitation

2% at the 5/10 and 9/10

and agree

at

the

points.

with 2/10

the power

'blue edge' point

and

For these power points on

are 11, 7, and 3% while the agreement

These slightly larger differences are probably

due to the well-known, quinine sulfate 'red-edge' shift observed when excitation occurs at wavelengths greater that 360 nm (ref.28).

Emission Spectra.

The general

equation for an uncorrected emission spectrum

given by Costa et al. (ref.23) is:

where E O ^ , ^ ) is the measured

spectrum

[called J i ^ / ^ )

(ref.23)], E ^

is the

spectral irradiance of the sample at λχ, αίλχ) is the absorptance of the sample at λ^· R O ^ ) is the spectral the correction due to

sample

responsivity

of the detection system; C O ^ , ^ )

is

effects (refractive index, reabsorption, emission

anisotropy); and y p ^ ( λ χ ) is the spectral photon yield of luminescence.

182 TABLE 3 Average Corrected Excitation Spectrum, Χ ( λ ) , for Quinine Sulfate Ex Spec (RR)

Lambda, nm 270 275 280 285 290 295 300 305 310 315 320 325 330 335 a

X

m

0 0 0 0 0 0 0 0 0 0 0 0 0 0

Coeff of Var

151 158 200 257 335 424 530 634 728 792 801 785 817 891

0 0 0 0 0 0 0 0 0 0 0 0 0 0

Lambda, nm

Ex Spec (RR)

340 345 350 355 360 365 370 375 380 385 390 395 400

0 957 0 994 0 986 0 920 0 803 0 658 0 497 0 340 0 217 0 129 0 108 0 059 0 .031

088 071 069 052 055 064 055 042 039 031 019 020 027 026

3

Coeff of Var 0.015 0.006 0.014 0.023 0.033 0.055 0.076 0.100 0.113 0.131 1.246 1.058 1.095

= 450 nm, solvent = 0.1 mol/L H C 1 0 4, bandpasses = 5 nm, [QS] = 1.0 ppm.

WAVELENGTH, nm Fig. 2. Corrected fluorescence spectra of quinine sulfate from a round robin test with ten participating laboratories: excitation spectrum, -•-; emission spectrum, and coefficient of variation at each λ, + + +. = 347.5 nm, ^ = 450 nm, solvent = 0.1 mol/L HCIO4, monochromator bandpasses = 5 nm.

Methods for determining the

spectral responsivity/correction factors for the

detection system include: a) radiance standards (e.g., calibrated tungsten strip lamps; position

b)

irradiance

plus

a

standards

calibrated

(e.g.,

barium

quartz-halogen

sulfate lamp;

c)

reflector calibrated

at sample source-

183

monochromator (use quantum counter to fluorescence

standard

(e.g.,

calibrate source-monochromator); and d) a

quinine

sulfate,

determination of the various quantities on

the

spectral responsivity are difficult and time procedure has errors obtained.

that

must

be

A detailed discussion of

cresyl

violet,

etc).

The

right of equation 2 such as the

consuming using methods a-c.

considered

before

Each

accurate values can be

the various emission spectra and correction

procedures can be found in Costa et al. (ref.23).

Generally, the use of a fluorescence standard, method d, is easiest. materials

have

been

proposed

as

fluorescence

combination of the spectral responsivity

and

standards

to

Various

determine

a

various correction factors of the

detection system according to equation 3:

(3)

where: E g is the units

and

corrected

R'i)^)

is

correction factors. et al. (ref.29),

spectrum

detection

of

system

the standard in appropriate

responsivity

including

various

Melhuish (refs.9,30), Velapoldi and Mielenz (ref.6), Magde,

and

compounds that cover spectra for three

emission

the

Ghiggino the

of

et

emission

these,

al.

(ref.31)

range

quinine

from

sulfate,

have 300

to

given digital data for 800 nm.

The emission

3-aminophthalimide, and cresyl

violet, are given in Fig. 3.

Υ . U T

,

,

4 0 0

,

5 0 0

!

6 0 0

7 0 0

1

Τ

8 0 0

WAVELENGTH, nm Fig. 3. Three corrected emission spectra quinine sulfate, 3-aminophthalimide,

Digital data are digital data are

given

given

in

for the

sulfate emission spectrum (given mentioned round robin

test,

quinine

for which digital data are available: cresyl violet, - φ - .

sulfate

respective

in

Table

references.

in

Fig.

2

and

agrees

well

with

4, while the other The average quinine

Table 4) from the previously the

values from Velapoldi and

184 Mielenz (ref.14) (summarized in Table 4) 9/10 power points of approximately 9%,

with 3%,

laboratory's data were not used since the

agreements at the 1/10, 1/2, and

and 0.5%, respectively.

(Note: One

deviations were more than 4 times the

calculated standard deviations.)

Wolfbeis, et al. (refs.31-33) have proposed several compounds with structures similar to quinine various pH's.

sulfate

as

fluorescence

standards

in aqueous solution at

Ghiggino, et al. (ref.34) have recently investigated and proposed

ß-carboline as a replacement for

quinine

sulfate

as a standard.

The emission

spectrum closely resembles that of quinine sulfate. Fig. 4, and, as noted later, the quantum yield is high

and

the

decay

time

is a single exponential.

This

Table 4 Average Corrected Emission Spectra, Ε ( X ) , from the Round Robin T e s t NBS

X, nm

375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450 455 460 465 470 475 480 485 490 495 500 505 510 515

a

and

for Quinine Sulfate

E p( X )

Coeff

E p( X )

(RR)

of Var

(NBS)

0.006 0.013 0.025 0.056 0.101 0.161 0.239 0.338 0.448 0.561 0.669 0.767 0.847 0.914 0.963 0.989 0.996 0.985 0.962 0.924 0.872 0.820 0.763 0.699 0.641 0.582 0.525 0.471 0.423

0.468 0.240 0.332 0.196 0.153 0.093 0.077 0.067 0.052 0.046 0.039 0.029 0.024 0.017 0.015 0.009 0.006 0.012 0.019 0.028 0.039 0.035 0.040 0.052 0.056 0.058 0.063 0.073 0.080

0.004 0.010 0.024 0.049 0.090 0.150 0.229 0.324 0.430 0.542 0.650 0.750 0.837 0.911 0.965 0.990 0.999 0.995 0.970 0.929 0.877 0.826 0.768 0.709 0.649 0.596 0.540 0.487 0.438

RSE

0.019 0.006 0.003 0.003 0.002 0.002 0.003 0.003 0.003 0.003 0.003 0.002 0.002 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.002 0.003 0.002 0.001 0.003 0.003

X, nm

520 525 530 535 540 545 550 555 560 565 570 575 580 585 590 595 600 605 610 615 620 625 630 635 640 645 650 655 660

E p( X )

Coeff

E p( X )

(RR)

of Var

(NBS)

0.375 0.332 0.292 0.257 0.226 0.199 0.173 0.152 0.133 0.117 0.102 0.089 0.078 0.067 0.060 0.051 0.046 0.040 0.036 0.033 0.032 0.030 0.027 0.025 0.024 0.022 0.021 0.012 0.009

0.085 0.099 0.114 0.119 0.135 0.150 0.145 0.164 0.171 0.184 0.202 0.235 0.248 0.284 0.323 0.365 0.397 0.514 0.535 0.481 0.506 0.590 0.680 0.754 0.765 0.902 0.902 0.048 0.023

0.392 0.349 0.308 0.272 0.239 0.211 0.185 0.162 0.143 0.126 0.110 0.096 0.085 0.074 0.065 0.056 0.049 0.043 0.038 0.033 0.029 0.025 0.022 0.019 0.016 0.015 0.013 0.011 0.010

RSΕ

0.002 0.001 0.003 0.003 0.002 0.002 0.001 0.001 0.001 0.002 0.002 0.003 0.003 0.001 0.003 0.004 0.006 0.002 0.006 0.003 0.011 0.003 0.015 0.014 0.037 0.015 0.027 0.035 0.073

f*X^ = 347.5 nm, solvent = 0.1 mol/L HClO^, bandpass = 5 nm, [QS] = 1 . 0 ppm. b O f f i c e of Standard Reference Materials, National Bureau of Standards Gaithersburg, Md. 20899 USA; Velapoldi and Mielenz (réf.6).

185 material should be further investigated as a fluorescence standard, although for the

present,

experimental

the

fluorescence

conditions

values

remain

the

for

best

quinine

sulfate

documented

and

under

specific

probably

the most

accurate.

^

^

,

I " " · u

1

1

/ :

:

·ΐ υ

3 5 0

1

\ X

7

:

1

:

1

1

1

1

1

1

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

Τ

7 0 0

WAVELENGTH, n m Fig. 4. Corrected emission carboline (réf.34).

spectra

for

quinine

sulfate

- # - (ref.14) and β-

Quantum Yield and Quantum Counter Standards The definition of quantum

yield

(fraction

of

molecules that emit a photon

after excitation by a radiation source) and its importance in various scientific areas (chemical analysis,

photochemical/photophysical

processes, assignment of

electronic levels, fluorescent material evaluation, energy convertors, etc) have been discussed extensively (see, e.g.,

refs.5,35).

quantum counter

constant

is

that

excitation wavelength

it

(be

have

a

quantum

flat).

fluorescence properties and accurately

A prime prerequisite for a

quantum

Thus,

measured

yield

independent of

materials with appropriate

quantum yields that are useful

as quantum counters are discussed in this section.

Quantum Yields. Miller (ref.l) have

Demas

(ref.5)

summarized

has

the

critically reviewed and contributors in

determination

of quantum yields including

measurement methods, corrections based on instrument and sample characteristics, quantum counters, luminescence photon data presentation.

As summarized in

be exercised in choosing function.

a

standard

yield the and

standards, and recommendations for

Introduction of this paper, care must using

it for a specific measurement

Several widely used and accepted standards, operating conditions, and

pertinent references are listed in Table 5.

280-315 330-370 210-400

2-Aminopyridine 3-Carboline DANgS DPA DPH1 Coumarin 440 535 Fluorescein [ R u ( b i p y ) 3] 2+ Cresyl Violet Rhodamine Β 575 590(6G) HITCn Nile Blue A 0 0.95 - 1.00 0.52±0.02 k 0.92±0.03 0.54±0.03 0.68±0.04 0.95±0.03 0.75±0.15 0.59±0.23

0.51±0.03 f 0.55±0.03 0.60±0.04 0.66±0.05 0.60±0.02

QYb

2.5 1.44 0.58 10.0 5.0 1.32 2.83 2.5 1.30 2.2 1.75 1.50 4.1 6.0 8.0

QC Conc. , g/L 5 3 2 2 2 2 8 5 2 2 4 4

X Flatnessu

(ref.29) (refs.9,25,26,39) (ref.26) (refs.26,43) (ref.44) (ref.26) (ref.44) (ref.26) (ref.44) " (ref.27)

(ref.5)

(refs.28,38,39) (ref.39) (ref.14) (ref.40) (ref.34) (refs.36,41) (ref.5) (ref.42) (ref.26)

Reference

U s e laser grade materials; ^Quantum Yield, use dilute solutions (A < 0.01); c0ver wavelength range; *%ote red edge shift; [ H + ] concentration; Uncertainties based on literature or recommendations (ref.5); ^Sodium l-dimethylaminonaphthalene-51 ,6-disulphonate; 9,10-diphenylanthracene; quantum yield uncertain, needs more work if warranted due to narrow bands; 1 phenyl-1,3,5-hexatriene; JPerfluoro-n-hexane; Value based on quantum yield of 0.83 for 9,10-DPA in cyclohexane; should be m e0H 0.60 (corrected using 0.95 for 9,10-DPA, ref.5); -"-Fluorescein is relatively unstable in NaOH; more stable in NaHCO^; M n , 1 ' , 3 , 3,3',3'-hexamethylindotricarbocyanine; °2recommended to avoid polarization problems in viscous solvents (ref.45); l amino-7-(dimethylamino)-3,4-benzophenazoxonium Perchlorate; P Q C ' S can be used at longer wavelengths, comparator used r ,7-bisRhodamine Β as reference standard (ref.26); ^2,7-bis-(ethylamino)-6-methyl-3,4-benzophenazoxonium Perchlorate; 2 t (diethylamino)phenazoxonium chloride; sBenzopyrillium salts; Used with Rhodamine Β, gave 200-780 nm range.

e

a

Oxazine 725 170e* Basic Blue 3 r CZ144S CZ682

H 2 S 0 4 , 0.1 e " , 1.0 H C 1 0 4, 0.1, 1.0 H 2 S 0 4 , 0.1, 1.0 H 2S 0 , , 1.0 NaOH, 0.1 hexane PFN^ MeOH " NaOH, 0 . 1 1 MeOH MeOH MeOH m " MeOH, EtOH MeOH MeOH Ethylene Glycol MeOH Ethylene Glycol " C H 2 C 12 "

200-400 d

Quinine Sulfate

290-390 360-400 360-490 400-520 280-560 510-635 360-590 " " 350-700 360-590 p 280-700 360-590 p 255-700 240-700 220-700 485-780 r

Solvent; Cone. mol/L

Range λ, nm

Material a

Quantum Yield and Quantum Counter Standards

TABLE 5

oo

°*

187

Although many compounds have been suggested as standards, the values reported for the quantum yields of individual compounds have varied tremendously.

In his

review, Demas has suggested that quantum yields should be checked by independent measurement procedures - for example, by (or modified version) and The standards with

calorimetry

values

determined

although this should be rectified more widely used.

The

most

use of the Weber-Teale method (ref.36) or thermal blooming techniques (ref.29). according

as

to

this

suggestion are few,

the various independent procedures become

widely accepted materials include quinine sulfate,

fluorescein, 9,10-DPA and rhodamine 6 G , although some questions still remain for some of these compounds (ref.5).

Several

other

materials listed in Table 5,

2+

including ß-carboline, DPH, [ R u ( b i p y ) 3] , and the much needed red emitters such as cresyl violet, oxazines, and benzopyriIlium salts, are potentially standards, however verification of

the

reported

excellent

fluorescence values should be

done.

Lavallee

et

al.

potential quantum

(ref.37)

yield

investigated

standards

with

material in the red spectral region research on red

emitting

methyltetraphenylporphine, Zn(II)

complex

of

-

systems

various

the

porphyrin

objective

of

materials

getting

as

one good

especially for people who are performing

such

as

chlorophyl

a.

The compounds N-

chloro-N-methyltetraporphinato-zinc(II),

methyltetra(p-sulfophenyl)porphine

potential standards; however quantum yields

are

were

and

recommended

the as

quite low, varying from -0.008

to 0.014. Quantum Counters.

Bowen

(ref.46),

based

on

the

earlier work of Vavilov

(ref.47), developed a 'quantum flat' detector (which he named a quantum counter, QC) by placing an optically dense,

luminescent

phototube viewed only the emission by directly proportional to the photon time, many publications have

the flux

provided

counters, proposed new quantum counter materials.

In addition to

the

incident

on

the screen.

on

the flatness of quantum

materials,

and

accepted rhodamine Β quantum counter

Table

quantum counters to

more

for

use

systems

as

well

or

porphyrin (cytochemical) type

Since that

and found new uses for these

fluorescein

been investigated and are reported in 800 nm

The

information

currently

(and the earlier quinine sulfate

screen before a phototube.

screen and the phototube current was

Q C ' s ) , several new dyes have

5.

Extending the useful range of in

research

as

on chlorophyl and

for efficient 'sun' energy

conversion has seen extensive activity.

Demas et al. (ref.26) measured

several

old

built as

well

a

computerized as

coumarins, oxazines, nile blue A, versus

the

well

characterized

new

and

QC

to

materials

methylene

rhodamine

'flatness' could only be determined

quantum counter comparator and

Β

blue.

including

(refs.9,39,48,49);

-590 nm.

rhodamines,

All dyes were measured thus quantum

Kopf and Heinz (ref.44) and

188

Brecht

(ref.27) m e a s u r e d

including basic blue these last three (ref.45)

several

3 and the

studies

to

avoid

equilibrated

excited

old

new

were

and

non-viscous

"polarization, state

being

polarization

dye

reactions"

the

concentration,

g/L g a v e a

from

formation of dye aggregates quantum counter material

(see

'thick' enough)

-

The

The

former

or

the m a i n

stability/purity,

large

and

(2%)

were

over

concentration showing

related

to the

or absorbance m i n i m a of the

who did

by

compared the

Stability/purity:

from

use

the

to

further

if

the Q C cell w e r e n o t

flatness

d u e to p o l a r i z a t i o n ,

effects.

These

effects

o f rear v i e w i n g p m t ' s ,

were

non-viscous

solution, and appropriate

QC where

photochemical

rhodamine Β quantum

counters

is p r o b a b l y a

quantum

and Mielenz

filters

reabsorption-reemission

and

roles.

Due

recommendation

Cehelnik

f r o n t a n d rear v i e w i n g p m t

latter

counter

by

by

in q u a n t u m c o u n t e r s h a s b e e n

deviations

emitted

radiation played major

The use of impure

g/L w h i l e a

recommendations pmt's

(as

reduced or eliminated

that

flat response

excursions

reabsorption/reemission

the radiation

This

dye

refs.43,50)

sufficient depth of quantum

recommended use.

al.

showed

radiation,

substantially

stray

partially

(ref.9).

(ref.52)

systems.

These

also

supported by Ostrom et

to exclude

and

w i t h w a v e l e n g t h s a t 425 a n d 480 n m

flatness.

geometry/polarization:

solvents,

geometry,

d y e c o n c e n t r a t i o n o f 1.3

viewing

stray

in

by Taylor and Demas

emission,

showed a relatively

(ref.51) o n t h e u s e o f rear

detection

suggested

from flatness were noted w i t h

QC

' f l a t n e s s ' o f -10%

the largest excursions

- QC

Most solvents used

effects:

360 t o 590 n m r a n g e a t a 5.0

three n e w red e m i t t e r s

(ref.5).

- Dye concentration: Nile blue A

of

as

anisotropic

In t h e s e a n d e a r l i e r w o r k , e x c u r s i o n s reasons

recommended

benzopyriIlium dyes.

counter

fluorescence

characteristics of

availability

of laser

grade

good practice

materials,

the

QC,

materials

source availability problems that

decomposition, be changed after

have

has

been of

t o f o l l o w for o t h e r Q C ' s .

of c o u r s e , changes the m e a s u r e d

thus

may

it

three months

giving

help

plagued

uncertain

results.

The

the purity, composition, and

researchers

in g e t t i n g

reliable

dyes.

Solid Quantum Yield and Quantum Counter as sodium salicylate

(ref.53), lumogen

organic materials dissolved

in

plastic

s t a n d a r d s or q u a n t u m c o u n t e r s .

The

d i s c u s s e d here because the concepts by McKinnon

(ref.55).

as the organic collectors potential

species

for s o l a r

Note

is

dissolved energy

in

Many solid materials

and other been

and

suggested as quantum

powder

yield

materials will not be

been discussed earlier

plastics, by

investigated

Weber

(ref.20).

and

such

t y p e s of p o w d e r s , a n d

in t h i s

volume

h o w e v e r , of easily useable m a t e r i a l s

conversion

standards by West and Kemp

have

solid have

made,

Standards.

(ref.54)

Lambe

such

for p r o p e r t i e s

as

(ref.56) a n d a s

A worrisome problem with

these

189 materials, however, is emission either 'roughing up' the grinding into a powder suggested that these

anisotropy.

surface

of

(ref.57).

the

The

anisotropy

plastics

Additionally,

materials,

used

wavelength range and are not as

as

by Mandai

quantum

'quantum

can be reduced by

careful grinding or by et al. (ref.58) have

counters,

do not cover the

flat' as the non-viscous solutions of

the quantum counters discussed above.

Fluorescence Decay Time Standards Another parameter

useful

fluorescence decay time.

in

molecular energy manifolds and rate of

energy

reactions,

and

characterizing

Fluorescence

transfer

two

species,

radiative

and

in characterizing micelle systems.

values.

materials

is the

are useful in describing

Decay

the

rates

non-radiative

intersystem crossing and internal conversion.

phase/modulation techniques.

fluorescent times

molecular interactions including determining the

between

determining

decay

excited state such

as

They have also found use recently

times

Quantum yields

of

processes

are

can

measured by pulsed or by

also be calculated from these

Probably the most widely used decay time values were those assembled by

Birks and Munro (ref.59), Birks (ref.60), were proposed and widely used

as

materials recommended as standards for yields, and quantum counters. bis(2-phenylozazolyl)benzene

and Berlman (ref.61).

decay

As

excitation and emission spectra, quantum

expected, these included quinine sulfate, p-

(POPOP),

2,5-diphenyloxazole

9,10-diphenylanthracene, phenanthrene, fluorescein, Chen (ref.62) suggested the use

of

pyrenebutyrate quenched with [I~]

Many materials

time standards, especially most of the

quinine to

sulfate

give

(PPO),

anthracene,

acridine, rhodamine B, etc.

standards

quenched with [Cl~] or ywith decay times ranging

from 0.2 to 115 ns.

A recent paper by Lampert

et

these collections of works were

al.

error, basically because "relatively were in operation at the

(ref.63), however, suggests that although

monumental,

time

".

should be re-evaluated using advanced mode-locked, frequency

optoacoustic

doubler,

They

argon

excitation

autocorrelated pulse width of ~6 ps. wavelength were obtained using a

included in Table 6 are

few

ion-pumped

Decay

were

Using a pulsed, dye

laser

reached

with

with an

time data as a function of emission

detection system consisting of a monochromator

values) a

technology.

wavelengths

and conventional photon counting equipment. (which agree with earlier

of the values listed were in

suggest that most of the values

measurement

cavity-dumped,

various

many

crude techniques for lifetime measurements

plus

Several of their decay time values

others

inorganic

are

listed

in Table 6.

Also

species (solid and liquid solutions)

that have luminescence decay times in the με and ms range.

Quinine sulfate was not

recommended

decay time data were well fit

by

a

sum

as

a standard (refs.63,70) because the of

two exponentials rather than by a

190

single exponential.

This

different conformers as Lentz

(ref.64)

used

component decay.

indicated suggested

a

a

two-component

earlier

by

phase/modulation

However,

(excitation wavelength of fluorescence decay time

they

of

that

and

quinine

homogeneous (can be fit by a

system

found

480-500 nm,

and

in

a

possibly

sulfate

single

decay - possibly due to

Fletcher

in

(ref.28). substantiated

limited

Barrow and the two-

spectral window

extending to 520 n m ) , the

0.1

Ν H 2 S 0 4 is 'effectively'

exponential with τ = 19.3 ns) and thus can

be used as a fluorescence decay time standard. Additionally, they found, as Chen had suggested (ref.62), that quenching to give decay times

ranging

from

of

3 ns

the fluorescence with C l ~ was viable to

19.2 ns.

On

the other hand, (3-

carboline which was recently proposed as a decay time standard (ref.34), has a τ of 22.03 ns with a single exponential

decay across the emission band and should

be considered as an alternative to quinine sulfate.

Imasaka et al. (ref.71)

TABLE 6 Materials Used as Fluorescence Decay Time Standards

Substance 2-Aminopyridine Anthracene ß-Carboline 1-Cyanonaphthalene DMNA DPH

C

II

0.1,1.ON H 2 S 0 4 C

C

C

H

C

F

H

~ 6 12 1.0N H 2 S 0 4

6 12 κ gas phase C H 2C 1 2 6 12 H 6 12 C H 6 6 0.1N NaOH C

II

Fluorescein 3-Methylindole II

1-Methylindole 1,2-Methy1indole POPOP PP09 ρρΟρΠ

γ-Pyrenebutyrate Quinine sulfate II II

" It

Rhodamine Β Eu(III) Gd(III) _ +2 [ R u ( b i p y ) 3] Sm(III) a

Solvent

C

C

H

~ 6 12 H OH 2 5 C - C H 6 12 C 2H 5O H

c

c

H

OH

2 5 C C H ~ 6 12 C _ C H 6 12 H 2 0 + KI 0.1N H 2 S 0 4 + KCl 1.0N H 2 S 0 4 10.ON H 2 S 0 4 0.1N H 9 S 0 4 4 11

c

-

H

2 5

*

O H

Silicate glass Borate glass Phosphate glass

τ, ns 9..6 a 5..23±0.05 22..03±0.12 a 18..23 a 24.. l a 2..40 a 32..510.05 15..7±0.02 6..1+0.01 e 4..5±0.03 a 4..36 a 8.. 1 7 a 6.. 2 4 a 5..71 1..35±0.2 a 1.. 4 2 a 1..10±0.02 1 18..0-115 . f fl 0.. 2 - 1 9 . 2 20. 4 • f 21. 8 • t 19.. K 3.63 f 19.. 3 2..8810.06 2..68±0.03 ms 4..1010.01 ms 0..6410.02 με 1..9110.03 ms

Reference (ref.40) (ref.63) (ref.34) (ref.63)

(ref.42) II

(ref.64) (ref.63) II II

" (ref.65) (ref.63) (ref.66) (ref.62) (ref.67) II

(ref.63) (ref.64) (ref.68) (ref.19) (ref.18) (ref.69) (ref.18)

V a l u e s for degassed solutions; ith one atmosphere cyclohexane; d N,N-Dimethyl-l-naphthylamine; Trans-l,6-diphenyl-l, 3,5-hexatrie ene; Average of 13 literature values (see ref.67); ^Values from many authors agree for single exponential, however, appears to be two exponentials that show wavelength dependence; ^2,5-Diphenyl1 oxazole; "p-Bis(2-phenylozazolyl)benzene; D e c a y time dependent on C l ~ or I concentration. c

191

have measured the decay times for in the

near

infrared

region

a polymethine dye (NK427) in various solvents (850 nm)

environmental hydrophobicity probe. of decay times from 0.6 to

1.3 ns

to

show

its

potential

as a micro-

No digital data were given, however, values obtained for NK427 in various solvents could

be interpolated from a plot of solvent dielectric constant vs decay time.

Lakawicz

et

al.,

using

phase

modulation

techniques

(ref.65), recommend

several standards with τ values that agree with those in Table 6.

Additionally,

they suggest that a reference emitter be used instead of the normal scatterer to reduce any wavelength and time dependencies spatial differences of the radiation must be taken with the τ value chosen

from

of the

the pmt photocathode as well as scatterer.

In this case, care

for the reference and the geometry of the

excitation beam.

REFERENCES 1

2

3 4 5

6 7 8 9 10

11

12 13

14 15 16 17

18

J.N. Miller (Ed.) "Techniques in Visible and Ultraviolet Spectrometry, Vol. 2: Standards in Fluorescence Spectrometry", Chapman and Hall, London, 1981, 117 pp. K.D. Mielenz, (Ed.) "Optical Radiation Measurements, Volume 3, Measurement of Photoluminescence", Academic Press, New York, 1982, 319 pp. C A . Parker, "Photoluminescence of Solutions", Elsevier Publishing Co., Amsterdam, 1968, 544 pp. R.L. Bowman, P.A. Caulfield, and S. Udenfriend, Science, 122, (1955) 32. J.N. Demas, Measurement of Photon Yields in "Optical Radiation Measurements, Vol. 3, Measurement of Photoluminescence." Κ. D. Mielenz (ed.) Academic Press, New York, 1982, pp. 195-248. R.A. Velapoldi, J. Res. Nat. Bur. Stds., 76A, (1972) 641. J.H. Chapman, Th. Förster, G. Kortüm, C A . Parker, Ε. Lippert, W.H. Melhuish, and G. Nebbia, Appl. S p e c t r o s c , 17, (1963) 171. J.N. Demas and G.A. Crosby, J. Phys. Chem., 75, (1971) 991. W.H. Melhuish, J. Res. Nat. Bur. Std., 76A, (1972) 547. J.N. Miller, Monochromator Wavelength Calibration in "Techniques in Visible and Ultraviolet Spectrometry, Vol. 2: Standards in Fluorescence Spectrometry" J. N. Miller (ed.) Chapman and Hall, London, 1981, pp. 814. K.D. Mielenz, Photoluminescence Spectrometry in "Optical Radiation Measurements, Volume 3, Measurement of Photoluminescence", Academic Press, New York, 1982, pp. 1-87. ASTM "Annual Book of ASTM Standards, Section 14, General Methods and Instrumentation", ASTM, Philadelphia, 1984, pp. 476-478. R.W. Burke, and R.A. Velapoldi, in IUPAC Recommended Reference Materials for Realization of Physicochemical Properties, Wavelength and Transmittance, submitted, 1985. R.A. Velapoldi and K.D. Mielenz, NBS Spec. Publ. 260-64, Gaithersburg, Md. 20899, 1980, 122 pp. G.G. Guilbault, "Practical Fluorescence; Theory, Methods, and Techniques", Marcel Dekker, New York, 1973, p. 150. C R . Harrison, M.I.T. Wavelength Tables, Vol 2, M.I.T. Press, Cambridge, Massachusetts, 1983. W.H. Melhuish, Correction of Excitation Spectra in "Optical Radiation Measurements, Vol. 3, Measurement of Photoluminescence", Academic Press, New York, 1982, pp. 115-138. R. Reisfeld, J. Res. Nat. Bur. Stds., 76A, (1972) 613.

192 19 20 21 22

23

24

25 26 27 28 29 30 31 32 33 34 35

36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

60

R.A. Velapoldi, R. Reisfeld, and L. Boehm, Phys. and Chem. of Glasses, 14, (1973) 101. M.A. West and D.R. Kemp, American Laboratory, March, (1977) 37. J.B. Birks, J. Luminescence, 9, (1974) 311. W.H. Melhuish, Sample-Induced Errors in Fluorescence Spectrometry, in "Optical Radiation Measurements, Vol. 3, Measurement of Photoluminescence", Academic Press, New York, 1982, pp. 175-193. L.F. Costa, K.D. Mielenz, and F. Grum, Correction of Emission Spectra in "Optical Radiation Measurements, Vol. 3, Measurement of Photoluminescence", Academic Press, New York, 1982, pp. 139-174. G.K.C. Roberts, Correction of Excitation and Emission Spectra in "Techniques in Visible and Ultraviolet Spectrometry, Vol. 2: Standards in Fluorescence Spectrometry", J. N. Miller (Ed.) Chapman and Hall, London, 1981, pp. 49-67. W.H. Melhuish, New Zealand J. Sei. Tech., 37, (1955) 142. J.N. Demas, T.D.L. Pearson, and E.J. Cetron, Anal. Chem., 57, (1985) 51. E. Brecht, Anal. Chem., 58, (1986) 384. A.N. Fletcher, Photochem. Photobiol., 9, (1969) 439. D. Magde, J.H. Brannon, T.L. Cremers, and J. Olmsted, III, J. Phys. Chem., 83, (1979) 693. W.H. Melhuish, Appl Opt., 14, (1975) 26. O.S. Wolfbeis and E. Urbano, J. Heterocyclic Chem., 18, (1982) 841. O.S. Wolfbeis and E. Furlinger, Z. Phys. Chem. N.F., 129, (1982) 171. O.S. Wolfbeis, E. Furlinger, and R. Wintersteyer, Monatsch. Chem., 113, (1982) 509. K.P. Ghiggino, P.F. Skilton, and P.J. Thistlethwaite, J. Photochem., 31, (1982) 171. J.W. Bridges, The Determination of Quantum Yields in "Techniques in Visible and Ultraviolet Spectrometry, Volume 2T~ Standards in Fluorescence Spectrometry", J. N. Miller (Ed.) Chapman and Hall, London, New York, 1981, pp 68-78. G. Weber and F.W.J. Teale, Trans. Faraday S o c , 53, (1957) 646. D.K. Lavallee, T.J. McDonough, Jr., and L. Cioffi, Appl. S p e c t r o s c , 36, (1984) 430. ~~ J.E. Gill, Photochem. Photobiol., 9, (1969) 313. W.H. Melhuish, J. Phys. Chem., 72, (1961) 793. S.R. Meech and D. Phillips, J. Photochem., 23, (1983) 193. C M . Himel and R.T. Mayer, Anal. Chem., 42, (1970) 130. E.D. Cehelnik, R.B. Cundall, and T.F. Palmer, Chem. Phys. Letters, 27, (1976) 586. ~~ K.H. Drexhage, J. Res. Nat. Bur. Std., 80A, (1976) 421. U. Kopf and J. Heinz, Anal. Chem., 56, (1984) 1931. D . C Taylor and J.N. Demas, Anal. Chem., 51, (1979) 717. E. Bowen, J. P r o c Royal Soc. London, 154, (1936) 349. S.J. Vavilov, Z. Phys., 42, (1927) 311. W.H. Melhuish, J. Opt. S o c Am., 52, (1962) 1256. W.H. Melhuish, Rev. Sei. Instrum., 33, (1962) 1213. J.E. Selwyn and J.I. Steinfeld, J. Phys. Chem., 76, (1972) 762. E.D. Cehelnik and K.D. Mielenz, Appl. Opt., 15, ΤΊ976) 2259. C S . Ostrom, J.N. Demas, and B.A. DeGraff, Anal. Chem., Accepted for Publication, (1986). N. Kristianpoller, J. Opt. Soc. Am., 54, (1964) 1285. A. Bril and A.W. de Jager-Veenis, J. Res. Nat. Bur. Stds., 80A, (1976) 401. R. McKinnon, This Volume W.H. Weber and J. Lambe, Appl. Opt., 15, (1976) 2299. K.D. Mielenz and R.A. Velapoldi, Unpublished Results. K. Mandai, T.D.L. Pearson, and J.N. Demas, Anal. Chem., 52, (1980) 2184. J.B. Birks and I.H. Munro, The Fluorescence Lifetimes of Aromatic Molecules in "Progress in Reaction Kinetics, Volume 4", Pergamon Press, New York, 1967, pp. 239-303. J.B. Birks, "Photophysics of Aromatic Molecules", Wiley-Interscience, New York, 1970.

193

61 62 63 64 65 66 67 68 69 70 71

I.B. Berlman, "Handbook of Fluorescence Spectra of Aromatic Molecules", Academic Press, London, 1970. R.F. Chen, Anal. Biochem., 57, (1974) 593. R.A. Lampert, L.A. Chewter, D. Phillips, D.V. O'Connor, A.J. Roberts, and S.R. Meech, Anal. Chem., 55, (1983) 68. D.A. Barrow and B.R. Lentz, Chem. Phys. Letters, 104, (1984) 163. J.R. Lakawicz, H. Cherek, and A. Baltur, J. Biochem. Biophys. Methods, 5, (1981) 131. D.M. Raynor, A . E . McKinnon, A.G. Szabo, and P.A. Hackett, Can. J. Chem. 54, (1976) 3246. R.F. Chen, J. Res. Nat. Bur. Std., 76A, (1972) 593. J.M. Harris and R.E. Lytle, Rev. Sei. Instrum., 48, (1979) 1469. J. Van Houten, J. Am. Chem. Soc. 98, (1976) 4853. D.V. O'Connor, S.R. Meech, and D. Phillips, Chem. Phys. Letters, 88, (1982) 22. T. Imasaka, A. Yoshitake, K. Hi rata, Y. Kawabata, and N. Ishibashi, Anal. Chem., 57, (1985) 947.