Hitachi F 2500 Manual

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And, eventually the molecule returns to the ground state while 
emitting fluorescent radiation.    Also, if radiationless transition to 
the triplet state takes place, then phosphorescence is emitted 
during triplet-to-singlet transition (from the excited triplet state to 
the ground singlet state).    Generally phosphorescence persists 
for 10
-4 sec or longer due to the selection rule imposed on the 
triplet-to-singlet transition.    In contrast, fluorescence persists for 
a period of 10
-8 to 10-9 sec in most cases. 
 
As mentioned above, part of the radiation absorbed by the 
substance is lost as vibration energy, etc.; therefore, the 
fluorescence wavelength emitted from it is longer than the 
excitation wavelength (Stokes’ law). 
The ratio of the number of photons emitted during fluorescence 
to the number of photons absorbed is called the quantum 
efficiency of fluorescence.    The larger the quantum efficiency a 
substance has, the more fluorescence it emits.    Also, the 
intensity of florescence emitted from a substance is proportional 
to the quantity of light absorbed by it.    When a dilute solution 
sample is measured, the intensity of fluorescence is expressed 
by  
F = KI
0clεφ 
where, 
F  :  Intensity of fluorescence 
K :  Instrumental constant 
I
0  :  Intensity of exciting radiation 
c  :  Concentration of substance 
l  :  Cell path length (distance in the cell through which the   
  radiation travels) 
ε  :  Absorptivity of substance 
φ  :  Quantum efficiency of substance 
  
						

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E.2  Advantages of Fluorometry 
 
As contrasted with fluorometry, absorptiometry for a 
low-concentration sample is explained in the following : 
A sample having 99% transmittance to blank is taken as an 
example.    In the absorbance measurement of such a substance, 
inaccuracies must always be taken into consideration.     
Here, the inaccuracy is assumed to be 0.1%. 
Since it has an effect on both the blank and sample, 
Percent transmittance of blank  100.0 ± 0.1% 
Percent transmittance of sample  99.0 ± 0.1% 
Difference (proportional to  1.0 ± 0.2% 
concentration of sample) 
 
In this example, the uncertainty in concentration measurement is 
±20%.    While in the fluorometry, a difference from zero level 
corresponds to the concentration of sample. 
Accordingly, measurement accuracy is as follows : 
Output signal level at measurement of sample  100 ± 0.1 
Value corresponding to blank  0 ± 0.1 
Difference (proportional to  100 ± 0.2 
concentration of sample) 
 
As can be seen from the above, the fluorometry is very 
advantageous for analyzing a low-concentration sample since its 
uncertainty is in most cases theoretically independent of the 
concentration of sample.    Although actual practice may involve 
some error factors which increase as the concentration of 
sample becomes extremely low, the fluorometry is capable of 
measuring low concentration with an accuracy 100 times higher 
than in absorptiometry. 
Figure E-2 is an explanatory illustration of the foregoing 
description.    In absorptiometry, a difference between the 
quantity of incident radiation Io and the quantity of transmitted 
radiation is represented by signal Is.    A level at which the signal 
Is becomes almost equal to a noise level is used as a detection 
limit.   
In fluorometry, however, since the quantity of fluorescence I
F 
itself is represented as a signal, just a small amount of 
fluorescence can be amplified electrically for enabling detection.   
Still more, since a fluorescence wavelength of a substance is 
different from its excitation wavelength (incident light 
wavelength), the fluorescence wavelength is not readily affected 
by the exciting radiation, thereby contributing to ensuring high 
sensitivity.   
						

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 Amplified 
 
Fig. E-2    Comparison between Absorptiometry and Fluorometry 
 
In addition to high sensitivity, fluorometry is advantageous in that 
more information is attainable.    A fluorescence spectrum is also 
available besides an excitation spectrum which corresponds to 
an absorption spectrum in absorptiometry. 
The two kinds of wavelengths can be selected as desired, and a 
fluorescence spectrum can be recorded using a properly 
selected excitation wavelength (or vice versa).    Thus, 
quantitative and qualitative analyses can be made for a sample 
containing plural components. 
Figure E-3 shows a simplified spectral graph of measurement of 
a sample containing multiple components.    In absorptiometry, 
since only the absorption spectrum can be measured, two or 
more component wavelengths are presented.    If the absorption 
wavelengths are similar to each other, each component cannot 
be separated in measurement.    In fluorometry, even if the 
absorption wavelengths are similar, a difference in fluorescence 
makes it possible to select each fluorescence wavelength 
properly.    Thus, each component can be separated in 
measurement. 
 
 
Fig. E-3    Measurement of Mult-Component Sample 
Low 
concentrationHigh 
concentrationLow 
concentration
(b)  Fluorometry (a)  Absorptiometry
High 
concentration
 
Amplified
Absorption spectra 
Component B 
(b)  Fluorometry 
Fluorescence spectra
(a)  Absorptiometry
Component A
Excitation spectraComponent A
Component B 
						

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Table E-1 compares information attainable in absorptiometry and 
that in fluorometry. 
 
Table E-1  Comparison of Information Attainable in 
Absorptiometry and Fluorometry 
Absorptiometry Fluorometry 
  Absorption spectrum only 
(Corresponding to excitation 
spectrum in fluorometry)  Excitation spectrum 
 Fluorescence spectrum 
 
E.3  Notes of Fluorescence Analysis Measurement 
 
For most kinds of samples, an increase of 1°C in the 
temperature of sample causes the intensity of fluorescence to 
decrease by 1 to 2%.    It is also reported that for some kinds of 
biochemical samples, the intensity of fluorescence decreases as 
much as 10% as the temperature increases by 1°C. 
When analyzing a sample having a temperature-dependent 
property, it is advisable to use the constant-temperature cell 
holder (P/N 650-0150).    Constant-temperature measurement 
can be carried out by circulating constant-temperature water 
through this cell holder. 
 
Some kinds of samples may be susceptible to a chemical 
change due to exciting radiation.    In analysis of such a sample, 
keep the shutter closed to cut off an excitation beam until 
measurement is started, and then open the shutter immediately 
before measurement.    If any chemical change due to exciting 
radiation is observed still, determine a signal level at the start 
time through extrapolation according to variation in signal level. 
  E.3.1 Temperature 
Dependency of 
Fluorescence 
Intensity 
E.3.2 Chemical 
Change in 
Sample due to 
Radiation
  
						

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In fluorescence measurement, spectra having different natures 
from that of fluorescence may be observed.    These are called 
Rayleigh scattering spectrum and Raman scattering spectra; the 
former appearing at the same wavelength position as the 
excitation spectrum, and the latter appearing at the 
longer-wavelength side near Rayleigh scattering. 
In a fluorescence spectrum, when the excitation wavelength is 
shifted, only the peak height is changed while the peak 
wavelength position remains intact.    In a Raman scattering 
spectrum, when the excitation wavelength is shifted, the peak 
wavelength position is also changed accordingly.    Both the 
Rayleigh scattering and Raman scattering are caused by a 
solvent which may be contained in the sample.     
When examining the spectral plot, be careful not to mistake 
these scattering effects of the fluorescence peak of interest. 
Table E-3 presents the Raman spectral peak position at each 
excitation wavelength for the purpose of reference. 
 
 
Fig. E-4    Raman Spectrum of Water 
E.3.3 Raman 
Scattering
 
Relative intensity
Excitation wavelength
Raman scattering 
						

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Table E-3  Raman Peak Positions at Respective Excitation 
Wavelengths
 
 
(Excitation 
wavelength)
 WaterEthanolCyclohexane Carbon 
Tetrachloride
 Chloroform 
248  271  267  267                    
313 350 344  344  320  346 
365 416 405  408  375  410 
405 469 459  458  418  461 
Excitation 
wavelength 
and 
Raman 
peak 
position 
(nm)
 436 511 500  499  450  502 
 
 
In measurement of a high-concentration sample, a variety of 
error factors may be involved.    The most significant error factor 
consists in that an excitation beam is absorbed at the entrance of 
a cell to prevent a sufficient level of excitation at the center of 
cell. 
Figure E-5 illustrates an extreme case of this condition.     
Although fluorescence is emitted in the vicinity of the entrance 
for the excitation beam, it is not taken into the emission 
monochromator. 
 
 
 
 
 
 
 
 
 
Fig. E-5    Sample Having an Extremely High Concentration 
 
  E.3.4 Handling of 
High- 
Concentration 
Samples
 
Fluorescence is reflected here. Excitation beam
Fluorescence
  
						

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If only the incident point of excitation beam is bright, it is 
necessary to dilute the sample properly for measurement. 
The second significant error factor consists in extinction due to 
concentration.    This condition is caused by preventing activation 
through interaction of molecules. 
The third significant error factor consists in re-absorption of 
fluorescence.    As shown in Figure E-6, this condition occurs 
due to overlapping between the shot-wavelength side of 
fluorescence spectrum and the long-wavelength side of 
excitation spectrum.    Therefore, it seems that the fluorescence 
spectrum has been shifted toward the long-wavelength side to 
some extent.   
In measurement of an ordinary kind of sample, however, this 
condition will not impede quantitative determination significantly. 
 
 
 
Fig. E-6    Explanatory Illustration of Re-absorption 
 
In any case, if there is a possibility of a measurement error due 
to high concentration of a sample, dilute the sample properly or 
carry out surface fluorescence measurement using a solid 
sample holder. 
 
Wavelength 
Fluorescence spectrum
     Excitation spectrum
Re-absorption occurs here.
 
Relative intensity
  
						

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Where the excitation and emission wavelengths are plotted near 
each other, care should be exercised not to mistake the Raman 
and Rayleigh scattering for the fluorescence spectrum as 
mentioned in E.3.3.    Where the excitation and emission 
wavelengths are plotted apart from each other, care should be 
exercised not to mistake the second-order and third-order 
scattered radiations for the fluorescence spectrum. 
The second-order scattered radiation appears at a wavelength 
two times longer than the excitation wavelength, and the 
third-order scattered radiation occurs at a wavelength three 
times longer. 
For instance, if the excitation wavelength is 240 nm, the 
second-order and third-order radiations take place at 480 and 
720 nm, respectively.    For eliminating these scattered radiations, 
insert a short-wavelength cutoff filter in the path of fluorescing 
radiation (before the emission monochromator).    It is advisable 
to use the filter set (P/N 650-0157) which is available as an 
optional accessory. 
 
Since the fluorescence spectrophotometer provides high 
sensitivity, just a slight amount of contamination on a cell may 
have an adverse effect on results of measurement.    To prevent 
this, treat the cell properly after its use. 
Do not leave the cell containing say sample.    In evaporation of a 
solvent, a residue of sample may adhere to the wall of the cell to 
cause contamination. 
In measurement of a very dilute sample, contamination on the 
inner and outer walls of the cell may cause a problem.    If a 
droplet of sample solution is put on the outer wall of the cell in 
sample injection into it, wipe off the cell with tissue paper and 
then set it on the cell holder. 
 E.3.5 Second-Order 
Scattered 
Radiation
 
E.3.6 Contamination 
of Cell
  
						

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Figure E-7 shows a measurement example of fluorescence 
spectrum. 
 
 
 
 
(1)  Scattering of exciting radiation 
(2)  Raman spectrum of solvent 
(3)  Fluorescence of impurities, solvent, etc. 
(4)  Fluorescence of sample 
(5)  Second-order spectrum of exciting radiation 
Fig. E-7    Measurement Example of Fluorescence Spectrum 
 
As shown in Figure E-7, other peaks than a fluorescence peak of 
sample appear in fluorescence spectral measurement.     
With reference to this example, it is necessary to identify a 
fluorescence peal of sample. 
 
 
E.3.7 Measurement 
Example of 
Fluorescence 
Spectrum
 
(1) 
(5)  
(4)
 
(3)
 
 
(2)
 Relative intensity
EX EX × 2 Wavelength 
						

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APPENDIX F  MEASUREMENT OF INSTRUMENTAL 
RESPONSE (CORRECTED SPECTRA) 
 
 
Spectrum correction is performed to enable measuring a true 
spectrum by eliminating instrumental response such as 
wavelength characteristics of the monochromator or detectors.     
The measurement of instrumental response is needed to perform 
spectrum correction.    “Instrumental Response” is the function to 
measure and save the instrumental response.     
 
 
F.1  Measurement of Instrumental Response on Excitation Side 
 
This is the function to obtain the instrumental response on the 
excitation side such as wavelength characteristics of the 
excitation monochromator using Rhodamine B as a standard 
(quantum counter).   
The instrumental response is automatically read with a single 
wavelength scan operation.    A spectrum is correctable within a 
range of 200 to 600 nm.   
 
(1)  Handling of Rhodamine B 
 
Pour Rhodamine B into a triangular cell in the procedure 
illustrated in Fig. F-1.    The triangular cell filled with   
Rhodamine B should in principle be stored at a dark place.     
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. F-1    Handling of Rhodamine B 
Syringe F649090 
 
Rhodamine B 
 Cut the supplied 
ampoule of 
Rhoda mine B 
with a cutter.     Suck the solution 
into a syringe.   Open the cover 
of triangular cell 
and pour the 
solution into it.   Fill the cell with the 
solution in a volume 
at least half the 
capacity and close 
the cover.