Hitachi F 2500 Manual

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Fig. 4-5  Calculation Method 
 
 
2) Peak area  
Input the peak wavelength and its accuracy.   
Measurement is made in the wavelength range 
specified at the Instrument tab.     
Peak wavelength is obtained and a calibration 
curve is prepared using the peak area of relevant 
peak.    Select an integration method among 
Rectangular, Trapezoid and Romberg.    Also set 
a threshold value and sensitivity for defining the 
peak.  
 
 
 
Fig. 4-6    Quantitation Tab (peak area) 
(c) Three wavelength 
calculation  
I = I2  - 1 - 3  1 - 2 × I
3 + 2 - 3 × I1
WL 1 I  I
2 
WL 2  I
1 I
3 
WL 3
(a) One wavelength 
calculation   
 WL 1 I  I
1 
I = I
1 
(b) Two wavelength 
calculation   
WL 1I  I
2 
WL 2I
1 
I = I
2  - I1
4.2    Creating an Analysis Method  
						

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3) Peak height  
Input the peak wavelength and its accuracy.   
Measurement is made in the wavelength range 
specified at the Instrument tab.    Peak is 
determined within the accuracy set here and 
calculation is made in the height of that peak.     
 
 
Fig. 4-7    Quantitation Tab (peak height) 
 
 
4) Derivative  
Input a peak wavelength (or wavelength to be 
used for calibration curve preparation).   
Measurement is made in the wavelength range 
specified at the Instrument tab and a calibration 
curve is created using the derivative value at the 
peak wavelength.   
Set the derivative order, smoothing order and 
data points as derivative parameters.   
 
 
Fig. 4-8    Quantitation Tab (derivative) 
4.2  
						

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5) Ratio  
Upon selecting Ratio, an input column for three 
factors (K0, K1, K2) and a setting column for 
calculation method (+, -, *) are displayed.   
 
NOTE:  By this quantitation method, a 
calibration curve cannot be created.   
 
 
 
Fig. 4-9    Quantitation Tab (peak ratio) 
 
 
Calculation is made by the following equation.   
 
 
RATIO =   
 
 
# (calculation method):    +, -, *   
WL1, 2, 3 :  Specified wavelengths   
WL3  :  Background correcting wavelength   
A( )  :  Measured value at specified 
wavelength  
 
(b) Calibration type  
Set a calibration type.    When selecting factor input 
(when standard calibration curve and its factors are 
already known), put a mark in the check box and enter 
values for A
0, A1, A2 and A3.   
K2 (A (WL2) – A (WL3))  K1 (A (WL1) – A (WL3)) 
#K0 
4.2    Creating an Analysis Method  
						

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1) None  
Mode in which calibration curve is not used.   
When this mode is selected with Wavelength set 
for Quantification type, multi-wavelength 
measurement at up to six wavelengths is possible.   
However, no spectrum can be displayed.   
2) 1st order  
This specifies the most generally used calibration 
curve.    A linear calibration curve is to be created.   
The formula of calibration curve is as follows.   
 
x = A
1y + A0  
 
Here, x is sample concentration and y is sample 
data.  
 
 
 
 
 
Fig. 4-10  1st Order 
 
 
Data 
Ko Conc 4.2  
						

							4 - 10 
3) 2nd order  
A calibration curve is created using a quadratic 
curve.    The formula of calibration curve is as 
follows.  
 
x = A
2y2 + A1y + A0  
 
Here, x is sample concentration and y is sample 
data.  
 
 
 
 
Fig. 4-11  2nd Order 
 
 
Data 
Ko Conc  4.2    Creating an Analysis Method  
						

							4 - 11 
4) 3rd order  
A calibration curve is created using a cubic curve.   
The formula of calibration curve is as follows.   
 
x = A
3y3 + A2y2 + A1y + A0  
 
Here, x is sample concentration and y is sample 
data.  
 
 
 
 
Fig. 4-12  3rd Order 
 
 
 
 
Data 
Ko Conc 4.2  
						

							4 - 12 
 
5) Segmented  
A calibration curve created by connecting 
standard samples linearly according to the 
measured values of respective samples or input 
data.  
 
 
Fig. 4-13  Segmented 
 
 
NOTE:  A proper calibration curve can be 
created when the photometric value 
increases or decreases monotonously 
versus the concentration value.    With a 
monotonic increase, the calibration 
curve is created with standard 1 (STD1) 
as origin.    And with a monotonic 
decrease, the calibration curve is 
created with STD1 plotted on the 
photometric value axis and STD5 on the 
concentration axis.    In these examples, 
the number of standards is set at 5.     
Max. No. of standards:    20 
 For the part which 
exceeds the measuring 
range, extend the line 
just before it.   
 
Data 
STD 1 
Conc  STD 2 STD 3 STD 4STD 5STD 6STD 7  4.2    Creating an Analysis Method  
						

							4 - 13 
 
 
 
Calibration curve with monotonous increase 
 
 
 
 
 
Calibration curve with monotonous decrease 
 
 
(c)  Number of wavelengths   
When “Calibration type” is “None,” up to six 
wavelengths are selectable, and with other calibration 
types, 1 to 3 wavelengths are selectable.   
 
(d) Concentration unit  
Input a concentration (in mg/mL for example) from the 
keyboard.  
 
(e) Manual calibration  
Put a check mark in this box for creating a calibration 
curve using factors.    The number of factors (A
0, A1, A2, 
A
3) varies with the calibration type selected.   
 
 
 
Data 
STD 1
Conc  STD 2STD 3STD 4 STD 5 
Data 
STD 1
Conc  STD 2
STD 3
STD 4
STD 5 4.2  
						

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(f)  Force curve through zero   
By putting a check mark in this box, a calibration curve 
is created so that its factor A
0 passes through “0” 
automatically.   
 
(g)  Digit after decimal point   
Set the number of digits after decimal point for the 
calculated concentration of the measured sample.   
The concentration value is rounded off to the set 
number of digits.     
Input range:    0 to 3   
 
(h)  Lower concentration limit/Upper concentration limit   
This is used to set a normal value range for the 
concentration of the measured sample.    If the 
concentration is higher than the upper limit set here, 
“H” will appear in the Avg Conc column, and if lower 
than the lower limit set here, “L” will appear in the Avg 
Conc column.   
 
(3) Instrument  
 
When you click the Instrument tab, the window in Fig. 4-14 
appears.   
 
 
 
Fig. 4-14  Instrument Tab 
 
 
4.2    Creating an Analysis Method  
						

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(a) Data mode  
Select one of the following data modes.   
The Luminescence mode is for measuring 
luminescence in the biological/chemical field and only 
the sample side signal is acquired without performing 
ratio photometry.   
• Fluorescence  
• Luminescence  
• Phosphorescence  
 
(b) Wavelength mode  
Select a wavelength for creating a calibration curve.   
•  EX WL Fixed   
•  EM WL Fixed   
•  Both WL Fixed   
 
(c)  Wavelength (1 to 6)   
Input the number of wavelengths specified at the 
Quantitation tab.   
 
Model Input Range  
F-4500  0, 200 to 900 nm   
F-2500  0, 220 to 800 nm 
 
 
(d) EX Slit  
Select a slit width for the excitation side.    Unit:    nm   
 
F-4500 F-2500  
1.0 2.5 
2.5 5.0 
5.0 10.0 
10.0 20.0 
20.0  
 
 
(e) EM Slit  
Select a slit width for the emission side.    Unit:    nm   
 
F-4500 F-2500  
1.0 2.5 
2.5 5.0 
5.0 10.0 
10.0 20.0 
20.0  
 
4.2