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...

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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...

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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,...

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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...

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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...

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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...

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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...

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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...

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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...

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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...