Hitachi F7000 Instruction Manual

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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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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. 
 
 
 
 
Fig. E-2    Comparison between Absorptiometry and Fluorometry 
 
 
In addition to high sensitivity, the fluorometry is advantageous in that 
more information is attainable.    An emission spectrum is also available 
besides an...

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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...

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E.3  Remarks on Measurement in   
Fluorescence Analysis 
 
For most kinds of samples, an increase of 1 °C in sample temperature 
causes the fluorescence intensity to decrease by 1 to 2%. 
It is also reported that for some kinds of biochemical samples, the 
fluorescence intensity 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)....

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Fig. E-4    Raman Spectrum of Water 
 
 
Table E-2    Raman Peak Positions at Respective Excitation Wavelengths 
 
 (excitation 
wavelength) Water Ethanol CyclohexaneCarbon 
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 
 
Relative intensity 
Excitation wavelength Raman 
scattering  

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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 the 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....

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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. 
 
 
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,...

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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 Fig. E-7, other peaks than a fluorescence peak of sample 
appear in measurement of fluorescence spectrum.    With reference to 
this example, it is necessary to...

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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 detector (photomultiplier).    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...

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Fig. F-1    Handling of Rhodamine B 
 
 
(1) Click the 
  (analysis method) button on the Measurement 
toolbar.    A box for setting your analysis method will appear. 
 
(2)  Select the General tab.    On the General tab page, specify 
“Wavelength scan” for the measurement mode. 
 
(3)  Select the Instrument tab. 
 
(4)  Set “Fluorescence” for the data mode, “400 V” for the 
photomultiplier voltage and “Excitation” for the scan mode. 
 
(5)  Execute the Zero Adjust command from the...