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 sample  99.0 ± 0.1% 
Difference (proportional to sample concentration)  1.0 ± 0.2% 
 
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, the measurement accuracy is 
as follows: 
 
Output signal level at sample measurement  100 ± 0.1 
Value corresponding to blank  0 ± 0.1 
Difference (proportional to sample concentration)  100 ± 0.2 
 
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 sample concentration. 
Although actual practice may involve some error factors which increase 
as the sample concentration becomes extremely low, the fluorometry is 
capable of measuring low concentrations 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 
I
o and the quantity of transmitted radiation It is represented by signal Is.  
A level at which the signal I
s 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.  
						

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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 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. 
High 
concentration Low 
concentrationHigh 
concentration Low 
concentration
(a)  Absorptiometry (b)  Fluorometry 
Amplified  
						

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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 properly.    Thus, each component can be separated in 
measurement. 
 
 
 
Fig. E-3    Measurement of Multi-component Sample 
 
 
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 
 
 
 
Absorption spectra
Component A
Component BExcitation spectra Fluorescence spectra
Component A Component B
(a)  Absorptiometry (b)  Fluorometry  
						

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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).    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. 
 
 
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 for the fluorescence peak of interest.    Table E-2 
presents the Raman spectral peak position at each excitation 
wavelength for the purpose of reference. 
 
E.3.1 Temperature 
Dependency of 
Fluorescence 
Intensity 
E.3.2 Chemical 
Change in 
Sample due to 
Radiation 
E.3.3 Raman 
Scattering 
  
						

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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. E-5    Sample Having an Extremely High Concentration 
 
 
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 Fig. E-6, this condition occurs due to 
overlapping between the short-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 
E.3.4 Handling of 
High-
concentration 
Samples 
Excitation beam 
Fluorescence
Fluorescence is 
reflected here. 
Relative intensity 
Fluorescence 
spectrum  Excitation 
spectrum 
Re-abso
rption 
occurs here. Wavelength  
						

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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, care should be exercised not to mistake the second-order 
and third-order scattered radiation 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 radiation 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 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 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 
the cell with tissue paper and then set it into 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 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 identify a fluorescence peak of sample. 
 
E.3.7 Measurement 
Example of 
Fluorescence 
Spectrum 
Relative intensity 
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 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 
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. 
 
WARNING 
Rhodamine B can cause injury if directly touched or accidentally 
ingested.    When handling it, be sure to wear proper protective 
gear such as safety gloves and safety mask.    If Rhodamine B 
adheres to the skin, wash it off with soap and plenty of water.   
Consult a physician when needed.    If accidentally ingested, 
immediately consult physician. 
 
 
Pour Rhodamine B into a triangular cell in the procedure illustrated in 
Fig. F-1.    The triangular cell filled with Rhodamine B should be in 
principle stored at a dark place. 
F.1.1 Handling of 
Rhodamine B 
  
						

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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 Spectrophotometer 
menu to calibrate the zero point. 
 
Rhodamine B
Cut the supplied 
ampoule of 
Rhodamine B with 
a cutter. 
Syringe 
(F649090)
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. 
F.1.2 Operating 
Procedures