Method for the substantial reduction of quenching effects in luminescence spectrometry

Method for reducing quenching effects in analytical luminescence measurements. Two embodiments of the present invention are described which relate to a form of time resolution based on the amplitudes and phase shifts of modulated emission signals. In the first embodiment, the measured modulated emission signal is substantially independent of sample quenching at sufficiently high frequenices. In the second embodiment, the modulated amplitude and the phase shift between the emission signal and the excitation source are simultaneously measured. Using either method, the observed modulated amplitude may reduced to tis unquenched value.

BACKGROUND OF THE INVENTION 
Luminescence spectrometry is a pervasive analytical procedure having 
exceptionally high sensitivity and selectivity. Luminescence measurements 
are, however, subject to errors caused by quenching species present in the 
sample under investigation. Such species reduce the luminescence signal 
and, if not compensated for, result in low concentration estimates for the 
luminescent species. Problems of quenching are especially severe in 
complex media such as biological, mineralogical, or environmental samples 
where the nature and amounts of quenching species are often unknown and 
not readily controlled. 
There are several approaches to the elimination of luminescence quenching 
effects: dilution, standard addition, and back extrapolation of 
luminescence decays or correction for the degree of quenching. In the 
dilution approach the sample is diluted until the quenching species 
concentration is too low to affect the luminescence intensity. In the 
standard addition method a known concentration of the luminescent species 
is added, and the emission intensity is remeasured. Since the luminescence 
of the standard is quenched to the same extent as that of the unknown, the 
concentration of the unknown can be readily inferred. In the decay method 
the sample is excited with a short-duration light pulse, and the decay 
curve is extrapolated back to the time of the exciting flash. The 
extrapolated signal may be related directly to the concentration of the 
luminescent species and is independent of quenching. Alternatively, the 
observed intensity is corrected for quenching from the measured lifetime. 
All three methods have weaknesses and merits. All fail if the quenching is 
too large. Sample purification before analysis is then mandated. Dilution 
is simple, but requires additional sample handling and is suitable only if 
the concentrations of the luminescent species are sufficiently high so 
that dilution does not reduce the signal intensity below the analytically 
useful range. Finally, without a priori knowledge of the degree of 
quenching, the degree of dilution must be established experimentally. 
Standard addition does not depend on knowledge of the extent of quenching. 
However, sample handling is relatively labor intensive since it requires 
at least two measurements, and the method does not readily lend itself to 
on-line processing. 
The decay methods avoid additional sample handling and eliminate errors 
arising from sample scatter and from short-lived luminescence. However, 
the instrumentation is elaborate and expensive, and the significant 
computational requirements often prevent its use for real-time analytical 
procedures. 
Phase-resolved spectroscopy has been used to reduce interfering signals in 
the determination of the concentration of one or more components of 
interest by means of direct supression or resolution of the unwanted 
signal such as scattered light or fluorescent background. For mixtures of 
emitters having different fluorescent lifetimes, the total phase-resolved 
intensity is the sum of the individual contributions. The advantage of 
phase-resolved fluorimetry is that multiple detector phase angles can be 
used instead of or in addition to multiple wavelengths to generate 
sufficient information for the determination of multiple unknown 
concentrations. Even components with identical spectra can be 
simultaneously determined by the use of different detector phase angles, 
provided the fluorescing species have sufficiently different fluorescence 
lifetimes. The basis of this technique is the use of an excitation beam 
that is modulated at a high frequency. If a mixture of two emitting 
species A and B is measured with the detector 90.degree. out-of-phase (in 
quadrature) with component A, for example, the individual spectrum of 
component B can be obtained. Essentially all of the work performed with 
phase-resolved fluorescence spectroscopy has involved the resolution of 
the individual spectra in multicomponent systems. Recently, however, J. N. 
Demas and R. A. Keller in "Enhancement of Luminescence and Raman 
Spectroscopy by Phase-Resolved Background Suppression," Anal. Chem. 57, 
538 (1985) have suppressed both fluorescence interference in Raman spectra 
and scattered light interference in fluorescence spectra for cases in 
which one signal component is very weak relative to the other. There has 
been no work to date on the use of modulation methods for reducing the 
effects of chemical species which quench luminescence. 
Accordingly, it is an object of the present invention to substantially 
reduce the effects of quenching species on the luminescence of species of 
interest. 
Another object of the present invention is to provide a method for the 
quantitative analysis of species by luminescence spectrometry in the 
presence of quenching species. 
Additional objects, advantages and novel features of the invention will be 
set forth in part in the description which follows, and in part will 
become apparent to those skilled in the art upon examination of the 
following or may be learned by practice of the invention. The objects and 
advantages of the invention may be realized and attained by means of the 
instrumentalities and combinations particularly pointed out in the 
appended claims. 
SUMMARY OF THE INVENTION 
To achieve the foregoing and other objects and in accordance with the 
purpose of the present invention, as embodied and broadly described 
herein, the method of this invention includes preparing a series of 
solutions each of which includes a chosen, fixed concentration of the 
luminescent species of interest and a known concentration of the quenching 
species, there being solutions with several different concentrations of 
quenching species, exciting each of the samples in turn with a modulated 
source of electromagnetic radiation having a chosen wavelength and a 
chosen modulation frequency, observing the modulated luminescence emission 
signal from each of the excited samples resulting from the interaction of 
the modulated electromagnetic radiation with the sample as a function of 
the chosen frequency, increasing the chosen frequency until the observed 
emission becomes independent of the extent of quenching and substantially 
the same for all of the prepared solutions having the same concentration 
of the luminescent species, observing the modulated emission signal from 
the sample of interest at the frequency at which the observed modulated 
luminescence emission becomes substantially independent of the extent of 
quenching, preparing a series of solutions containing known quantities of 
luminescent species in the absence of quenching species which covers the 
range of concentration expected for the sample of interest, determining 
the unquenched modulated luminescence signal therefrom at the modulation 
frequency at which the observed modulated luminescence was found to be 
substantially independent of quenching effects for samples containing 
quenching species, and determining the concentration of the luminescent 
species in the sample of interest according to the algorithm: 
EQU S=S.sub.0f {(1+A.sup.2)/[(1/.phi.).sup.2 +A.sup.2 ]}.sup.1/2, 
where S is the observed modulated emission amplitude and S.sub.0f is the 
desired unquenched value thereof at the chosen modulation frequency f, 
A=2.pi.f.tau..sub.0, and .phi. is the extent of qunching and is equal to 
.tau./.tau..sub.0, where .tau. is the quenched excited-state lifetime and 
.tau..sub.0 is the unquenched value thereof, by comparing S.sub.0f 
determined for the sample of interest with the unquenched signal values 
thereof as a function of concentration of luminescent species. The 
calibration for the determination of the appropriate modulation frequency 
and the analytical curve for unquenched samples need be performed at most 
one time for a particular luminescent species/quenching species system. 
Moreover, it is to be observed from the relationship between S and 
S.sub.0f set forth hereinabove, that if A&gt;&gt;1/.phi., 
EQU S.apprxeq.S.sub.0f [(1+A.sup.2)/A.sup.2 ].sup.1/2 .apprxeq.S.sub.0f 
for A&gt;&gt;1, so that if conditions are chosen conveniently, the measured value 
for the modulated luminescence emission amplitude is close to the value of 
the desired unquenched value thereof. Additionally, the modulation 
frequency appropriate for the substantial reduction of the effects of the 
quenching species may be determined either from a knowledge of the 
quenched lifetime of the luminescent species or from an experimental 
determination thereof as described hereinabove. Finally, it is important 
to recognize that the S.sub.0f values measured from the analytical curve 
generated from the prepared unquenched samples must be measured at some 
modulation frequency in order that a meaningful comparison can be 
achieved. 
In a further aspect of the present invention, in accordance with its 
objects and purposes, the method hereof also includes preparing a series 
of solutions having known concentrations of luminescent species in the 
range expected for the unknown sample under investigation and measuring 
the unquenched modulated luminescence emission amplitude therefor by 
irradiating each solution in turn with modulated electromagnetic radiation 
having a chosen wavelength at a modulation frequency determined by the 
relationship 2.pi.f.tau..sub.0 .apprxeq.1 in order to generate an 
analytical curve for the luminescent species, exciting the sample of 
interest with a modulated source of electromagnetic radiation at the 
appropriate modulation frequency, simultaneously measuring the modulated 
amplitude of the emission signal, and the phase shift, .delta., of the 
emission relative to the excitation source, determining .tau..sub.0 from a 
measurement of .delta..sub.0, the unquenched phase shift, using one of the 
solutions prepared for said step of measuring the unquenched modulated 
amplitude according to the relationship .delta..sub.0 =tan.sup.-1 
(2.pi.f.tau..sub.0), and determining the concentration of luminescent 
species in the sample of interest according to the algorithm: 
EQU S=(P.sup.2 +Q.sup.2).sup.1/2, and .delta.=tan.sup.-1 (Q/P), 
where P and Q are the in-phase and quadrature signals measured using a 
phase-sensitive detector, and .delta. is the phase shift of signal 
relative to the reference signal and is equal to tan.sup.-1 (2.pi.f.tau.), 
S is the modulated amplitude which is related to the unquenched modulated 
amplitude according to: 
EQU S=S.sub.0 /[(1/.phi.).sup.2 +A.sup.2 ].sup.1/2, 
where S.sub.0 is the desired unquenched modulated emission amplitude for 
the sample of interest, A=2.pi.f.tau..sub.0, and .phi. is the extent of 
quenching and is equal to .tau./.tau..sub.0, where .tau. is the quenched 
excited-state lifetime and .tau..sub.0, is the unquenched value thereof, 
by comparing the calculated value of S.sub.0 for the sample under 
investigation with the unquenched modulated luminescence emission values 
as a function of concentration of luminescent species determined from the 
analytical curve as described hereinabove. Again, as for the first 
embodiment of the present invention, the analytical curve need be 
performed only once, and similarly for the evaluation of .tau..sub.0. It 
is also to be noted that a simple calculation is required to extract 
S.sub.0 from the measurements on the quenched sample, since the algorithm 
contains the measured value for S, the value for .tau. obtained from the 
measurement of .delta., and the value of .tau..sub.0 obtained from the 
measurement of .delta..sub.0 for an unquenched sample of luminescent 
species. 
Benefits and advantages of the present invention include the reduction in 
complexity over pulsed methods, and the avoidance of extra sample handling 
steps required for the dilution and the standard addition methods.

DETAILED DESCRIPTION OF THE INVENTION 
Reference will now be made in detail to the present preferred embodiments 
of the invention, examples of which are illustrated in the accompanying 
drawings. Briefly, the present invention includes two methods for 
eliminating the quenching of a luminescent species (uranyl ion as an 
example) by a quenching species (chloride ion as an example). The first 
method takes advantage of the inverse relationship between luminescence 
lifetime and the percentage of signal modulation at a chosen modulation 
frequency. If the modulation frequency is sufficiently high, the observed 
modulated amplitude, S, is essentially independent of the extent of 
quenching. In the second method, the modulated amplitude S and the phase 
delay for the quenched sample are measured and the unquenched signal, 
S.sub.0, computed from these quantities. 
If a sample is excited with a sinusoidally varying source, the sample 
emission will vary sinusoidally. The emission will, however, be 
phase-shifted from the excitation, and the degree of modulation of the 
emission will be lower than the degree of modulation of the excitation 
source. These differences arise from the inability of the excited-state 
concentration to follow the excitation. The phase shift, .delta., and the 
degree or percentage of emission modulation, m, are given by: 
EQU .delta.=tan.sup.-1 (2.pi.f.tau.), and (1) 
EQU m=M/[1+(2.pi.f.tau.).sup.2 ].sup.1/2, (2) 
where f is the modulation frequency, .tau. is the excited-state lifetime, 
and M is the degree of excitation modulation. If the excitation is not 
sinusoidal, the waveform is decomposed into its Fourier components and a 
series of equations similar to Equations 1 and 2 is derived for the 
fundamental and harmonics. Since it is possible to discriminate 
instrumentally against the harmonics, only the fundamental frequency will 
be considered hereinbelow. Either of Equations 1 and 2 may be used to 
evaluate .tau.. 
FIG. 1 shows a schematic representation of the apparatus of the present 
invention. For a full description of the apparatus, kindly refer to 
"Enhancement of Luminescence and Raman Spectroscopy by Phase-Resolved 
Background Suppression," by J. N. Demas and R. A. Keller, Anal. Chem. 57, 
538 (1985), the disclosure of which is hereby incorporated by reference 
herein. Basically, the excitation beam is modulated before it enters the 
sample, and the photomultiplier tube signal is processed using a lock-in 
amplifier referenced to the modulation frequency. More specifically, the 
output beam 10 from laser 12 is focused using lens 14 into acoustooptic 
modulator 16 producing thereby a square-wave modulated output 18. At low 
frequencies, the internal oscillator circuitry of driver 20 was found to 
be adequate to control modulator 16 and provide a reference signal for 
dual-channel phase-sensitive detector 22. However, at higher frequencies, 
excessive phase noise developed, and an optical triggering arrangement was 
employed using a portion of the laser beam 24 which was reflected from 
beam splitter 26 onto photomultiplier tube 28 from which the triggering 
signal was derived. Lens 30 was employed to focus the modulated laser 
output beam 18 into sample 32 contained in sample holder 34. Modulated 
luminescence emitted light 36 emerging from the sample was analyzed using 
monochrometer 38 and detected by photomultiplier tube 40. The signal 
therefrom was directed to phase-sensitive detector 22. Data acquisition 
system 42 received the output from phase-sensitive detector 22 for 
processing. 
The analysis of the emission signal according to the teachings of the 
present invention rests on the assumption that the quenching arises from 
diffusional (dynamic) quenching and not from ground-state associational 
quenching. Under these conditions .tau. and the total emission intensity 
are related by: 
EQU I/I.sub.0 =.tau./.tau..sub.0, (3) 
where I is the total luminescence intensity at dc or very low modulation 
frequencies, and the subscript 0 denotes the unquenched value of the 
quantity. 
The first embodiment of the present invention relies on the opposing effect 
between .tau. and the percentage of signal modulation. Increased quenching 
decreases .tau. while m increases. Therefore, as the quenching process 
decreases the total emission yield, some of the lost intensity is regained 
in the detected modulated amplitude because of the higher percentage 
modulation. Moreover, operation at high frequencies can largely eliminate 
the detrimental effects of even extensive quenching. 
The observed modulated emission signal as a function of f and the extent of 
dynamic quenching is given by: 
EQU S=S.sub.0 /[(1/.phi.).sup.2 +A.sup.2 ].sup.1/2, (4) 
EQU S=S.sub.0f {(1+A.sup.2)/[(1/.phi.).sup.2 +A.sup.2 ]}.sup.1/2, (5) 
EQU A=2.pi.f.tau..sub.0, and (6) 
EQU .phi.=.tau./.tau..sub.0, (7) 
where S is the observed modulated amplitude and S.sub.0 is the modulated 
amplitude for an unquenched sample at very low modulation frequencies, f. 
S.sub.0f is the unquenched signal at f. .phi. is the extent of quenching, 
which is also the low-frequency emission intensity of a quenched sample 
relative to that of an unquenched one, since at very low frequencies, 
S/S.sub.0 =I/I.sub.0. 
Turning now to FIG. 2 hereof, S/S.sub.0 is plotted versus the 
dimensionless, normalized frequency, A, for several values of .phi.. It is 
readily observed that at very low frequencies (A.apprxeq.0), S decreases 
most with changes in the extent of quenching. As f increases, however, the 
differences between the successive curves for decreasing values of .phi. 
become progressively smaller, and all of the curves approach one another. 
Thus, by modulation of the signal at sufficiently high frequencies, the 
measured signal becomes essentially independent of the extent of 
quenching. The S.sub.0 derived therefrom for a sample having unknown 
luminescent species concentration can then be related to the actual 
concentration thereof using an independently generated calibration curve. 
The second embodiment of the present invention relies on the fact that in 
the use of a dual-channel phase-sensitive detector, one can simultaneously 
determine the modulated amplitude, S, and the phase shift .delta., of the 
emission signal relative to the excitation source. Once S and .delta. are 
known, the unquenched modulated intensity at zero modulation frequency, 
S.sub.0, is calculated using Equations 1-7 hereof. This procedure does 
require a determination of .tau..sub.0 in the analysis medium but this may 
readily be accomplished using a reference sample. Once .tau..sub.0 is 
obtained, it is generally unnecessary to remeasure it for every 
experiment. 
Both embodiments of the present invention rely on the measurement of the ac 
signal amplitude with noise or variations in the phase angle reduced as 
much as possible. Dual-channel lock-in amplifiers provide this capability. 
Such an amplifier can be simply described as containing two normal lock-in 
amplifiers. One amplifier processes the in-phase signal; that is, in phase 
with the reference signal, while the other amplifier processes the signal 
relative to a 90.degree. phase-shifted reference. The two outputs are 
denoted by the in-phase, P, and quadrature, Q, signals, respectively. The 
vector sum of the P and Q signals is S. The phase shift, .delta., relative 
to the reference and S are given by: 
EQU S=(P.sup.2 +Q.sup.2).sup.1/2, and (8) 
EQU .delta.=tan.sup.-1 (Q/P), (9) 
where S is the modulated amplitude in Equations 4 and 5 hereof. It should 
be pointed out that the first embodiment of the present invention requires 
only a single channel amplifier. 
The method of the present invention is further illustrated by the following 
example. 
EXAMPLE 
As an example of the advantages of the present invention and a specific 
situation in which it is utilized, results based on the quenching of the 
UO.sub.2.sup.+ luminescence are presented. Details of the measurements 
are described in "Elimination of Quenching Effects in Luminescence 
Spectrometry by Phase Resolution," by J. N. Demas, W. M. Jones, and R. A. 
Keller, Anal. Chem. 58 1717 (1986), the disclosure thereof being hereby 
incorporated by reference herein. Uranyl solutions were prepared by the 
dissolution of high-purity U.sub.3 O.sub.8 in concentrated HNO.sub.3 and 
subsequently in 1.00M H.sub.3 PO.sub.4. All measurements were at 80 ppm of 
U. KCl was used as a source of chloride ion quencher. Quenched solutions 
were identical to the unquenched solutions except for the addition of the 
quencher. Phase angle and vector amplitude measurements were made at 0.1, 
0.8, 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, and 30 kHz. The determination of the 
.tau.'s from Equation 1 hereof required phase-angle shifts between the 
excitation and the emission radiation to be measured. Since the 
oscillator, the modulator, and the amplifier introduce phase shifts, it 
was necessary to obtain a reference phase shift for a zero-lifetime 
emitter. This was accomplished using aqueous rhodamine 6G which has a 
fluorescence lifetime of 3-ns, which is essentially instantaneous relative 
to that for the UO.sub.2.sup.+ which has a multimiscrosecond .tau.. 
Because the phase shift of the amplifier varied with the sensitivity range 
and the frequency, phase shifts were measured for all frequencies and 
ranges used. A comparison using intensity and .tau. Stern-Volmer plots for 
quenching of UO.sub.2.sup.+ by chloride showed the two results to be 
indistinguishable. Thus, significant static quenching was ruled out. 
Returning now to FIG. 3 hereof, the experimentally measured plots of the 
normalized signal S/S.sub.0f versus .phi. and f and the solid lines, which 
represent theoretical curves calculated from Equation 5 hereof and the 
measured value of .tau..sub.0, agree without the introduction of 
adjustable parameters. The performance of the method can be evaluated by 
the variation in corrected luminescence amplitudes for a number of samples 
with different extents of quenching. For the first embodiment hereof the 
directly obtained intensities, S's, were used, while for the second 
embodiment the measured values of the S's and .delta.'s for each frequency 
were used to generate intensities S.sub.0 corrected to zero frequency 
using Equations 1 and 4 hereof. The data are summarized in the following 
Table. 
Mean and standard deviations were calculated for nine data points ranging 
from unquenched to 87% quenched samples. All data were taken at 80 ppm U 
in 1.00M phosphoric acid. As can be seen from the Table, embodiment 1 
hereof improves as f increases. The .sigma.'s improve significantly for 
f.gtoreq.8.00 kHz and represent systematic errors rather than noise. This 
is expected from the use of Equation 5 hereof, since S is only insensitive 
to A when A&gt;.phi..sup.-1. As a result of the significant Cl.sup.- 
quenching (.phi..sup.-1 max=7.4), A exceeded the maximum .phi..sup.-1 only 
above 8 kHz. Above 8 kHz the reduction of quenching effects becomes quite 
dramatic due to the sum-of-squares form of Equation 5 hereof. For less 
heavily quenched samples, the necessary frequency is correspondingly 
reduced. This reduction in sensitivity to quenching is accompanied by a 
loss of signal amplitude. For example, at 30 kHz the signal is reduced to 
9% of the low-frequency value. However, the relative standard deviation is 
improved by a factor of 100. 
TABLE 
______________________________________ 
Effect of Modulation Frequency on the Elimination of Quenching 
Effects 
S .+-. .sigma. So .+-. .sigma. 
(Embodiment 1) (Embodiment 2) 
f(kHz) 
(.sigma./S, %) 
(mV) (.sigma./S.sub.o, %) 
(mV) 
______________________________________ 
0.100 3.31 .+-. 2.54 
(77) 8.06 .+-. 2.71 
(34) 
0.800 2.97 .+-. 1.87 
(63) 9.46 .+-. 0.33 
(3.5) 
1.000 2.82 .+-. 1.65 
(59) 9.37 .+-. 0.23 
(2.5) 
2.000 2.28 .+-. 0.93 
(41) 9.61 .+-. 0.13 
(1.4) 
3.000 1.89 .+-. 0.56 
(30) 9.69 .+-. 0.21 
(2.2) 
4.000 1.60 .+-. 0.36 
(23) 9.67 .+-. 0.17 
(1.8) 
5.000 1.38 .+-. 0.24 
(18) 9.70 .+-. 0.17 
(1.8) 
6.000 1.22 .+-. 0.17 
(14) 9.57 .+-. 0.10 
(1.0) 
8.00 0.965 .+-. 0.091 
(9) 9.63 .+-. 0.12 
(1.3) 
10.00 0.807 .+-. 0.052 
(6) 9.64 .+-. 0.12 
(1.2) 
15.00 0.560 .+-. 0.019 
(3.4) 9.61 .+-. 0.08 
(0.8) 
20.00 0.429 .+-. 0.009 
(1.9) 9.67 .+-. 0.11 
(1.1) 
30.00 0.289 .+-. 0.002 
(0.7) 9.68 .+-. (1.7) 
______________________________________ 
The second embodiment hereof yields excellent recovery of the true S.sub.0 
(.apprxeq.9.6 mV) as judged by the mean and standard deviations except at 
0.1 kHz where .delta. is very small. As a general rule, analyses should be 
performed at a frequency where 2.pi.f.tau..gtoreq.1. The .sigma.'s for the 
second embodiment largely reflect noise in the measured .delta.'s and 
amplitude measurements since there are no inherent systematic errors. 
There are large .sigma.'s at low f values, which decrease as f increases. 
In conclusion the embodiments of the present invention are well-suited for 
extracting quenching-corrected intensities from heavily quenched samples. 
Errors approaching an order of magnitude in estimated concentrations are 
reduced to a few percent by either method. With currently available 
phase-sensitive detectors and modulators, .tau..sub.0 's as short as 30 ns 
yield 2.pi.f.tau.=9.4, which is adequate even for heavily quenched 
samples. Cross-correlation techniques can extend this method to the 
gigaHertz frequency range even with the use of low-frequency lock-in 
amplifiers. The first embodiment hereof requires no computations beyond 
those performed in a dual channel lock-in amplifier. The vector output of 
the amplifier is the desired signal. Alternatively, the information can be 
obtained from a single channel amplifier by manually choosing the phase 
angle to maximize the signal. Moreover, knowledge of .tau..sub.0 is not 
required. The sole requirement is that A&gt;.phi..sup.-1. That this condition 
is satisfied is readily determined from the signals from the lock-in 
amplifier. This permits the frequency to be adjusted to satisfy the 
requirements of the particular system under investigation. The second 
embodiment requires more elaborate computations than the first embodiment 
hereof and requires the independent measurement of the unquenched .tau., 
.tau..sub.0, in the analytical medium and the instrumental zero-lifetime 
phase angle. However, these measurements would only have to be made 
occasionally. The required calculations can easily be performed by use of 
a microprocessor. 
The foregoing description of two preferred embodiments of the invention has 
been presented for purposes of illustration and description. It is not 
intended to be exhaustive or to limit the invention to the precise form 
disclosed, and obviously many modifications and variations are possible in 
light of the above teaching. The embodiment was chosen and described in 
order to best explain the principles of the invention and its practical 
application to thereby enable others skilled in the art to best utilize 
the invention in various embodiments and with such various modifications 
as are suited to the particular use contemplated. It is intended that the 
scope of the invention be defined by the claims appended hereto.