Hitachi F 2500 Manual

							8 - 4 
For remeasuring any of the standards after their measurements, 
click its data as shown in Fig. 8-6.    Then click the Remeasure 
button.  
 
 
 
 
Fig. 8-6 
 
 
A message box will open as in Fig. 8-7, so press the sipping 
lever.  
 
 
 
Fig. 8-7 
 
 
When a standard is remeasured, the calibration curve is 
recalculated and sample data is also recalculated based on the 
curve and then displayed.    To saved the recalculated data, click 
the End button or the          (save) button on the toolbar.     
 
A message box is displayed in both standard and sample 
measurement sessions.    So operate according to the messages.   
When the message box appears, press the sipping lever of the 
sample sipper.   
 
NOTE:  When the sample sipper is used, blank measurement 
cannot be made.  8.1.2  When Using a 
Sample Table 
8.1    Use of Sample Sipper  
						

							8 - 5 
Select the Measure command from the Spectrophotometer menu 
or click the          (measurement) button on the toolbar, and the 
window in Fig. 8-8 will open.   
 
 
 
Fig. 8-8 
 
 
Press the sipping lever and measurement starts.     
For measurement of the 2nd and subsequent standards, a 
message box as in Fig. 8-9 will open.    For remeasurement or 
for skipping measurement of the next standard, select the 
relevant item with the mouse (put a bullet in its radio button) and 
then click Yes.  When clicking No, the current measurement 
sequence will be terminated.     
For restarting measurement, click the     button.  
Measurement then starts from the 1st standard.   
 
 
 
Fig. 8-9 
 
 
8.1.2  
						

							8 - 6 
On completion of standard measurement, the window in Fig.   
8-10 will open.   
 
 
 
Fig. 8-10 
 
 
By clicking OK with the mouse, a calibration curve is drawn and 
then samples can be measured.     
By clicking Cancel, the measurement is terminated, and the 
standards’ data is not saved.     
For remeasurement of the final standard, select “Repeat the last 
measurement” and click OK.    Set the final standard in place and 
press the sipping lever.    When repeat measurement has been 
set, measurement is started from the 1st one (Repl.No.1) for that 
standard. 
 
In the case of sample measurement also, a message box as in 
Fig. 8-11 will appear.    Press the sipping lever, and 
measurement of the 1st sample starts.     
 
 
 
Fig. 8-11 
 
8.1    Use of Sample Sipper  
						

							8 - 7 
For measurement of the 2nd sample, a message box as in   
Fig. 8-12 opens.   
 
 
 
Fig. 8-12 
 
 
Press the sipping lever for measuring the 2nd sample.     
For skipping this sample (and measuring the 3rd sample), select 
“Skip this sample?” and click the Yes button with the mouse.   
For remeasurement, select “Repeat the last measurement?” and 
click Yes button with the mouse.    When repeat measurement 
has been set, measurement is started from the 1st one 
(Repl.No.1) for the 1st sample.   
In case of interrupt measurement, select “Insert a new sample 
into the sequence” and click the Yes button with the mouse.     
A message box as in Fig. 8-13 opens, so click the OK button.   
 
 
 
Fig. 8-13 
 
 
8.1.2  
						

							8 - 8 
For remeasurement after a series of measurements, click the 
data to be remeasured as in Fig. 8-14.    Then click the 
Remeasure button.   
 
 
Fig. 8-14 
 
 
A message box will open as in Fig. 8-15.    Press the sipping 
lever, and measurement starts.    When remeasuring a standard, 
the calibration curve is recalculated and sample data is also 
recalculated based on the curve and then displayed.    To save 
the recalculated data, click the     (Save) button. 
 
 
Fig. 8-15 
 
 
(1)  When not Using Sample Table   
 
Set your analysis method, and when the monitor window 
appears, press the sipping lever.    Measurement starts.     
 
(2)  When Using Sample Table   
 
Select the Measure command from the Spectrophotometer 
menu or click the     (measurement) button on the toolbar.  
A message box opens.    Follow the messages given.    8.1.3 In Wavelength 
Scan or Time 
Scan Mode   
8.1    Use of Sample Sipper  
						

							8 - 9 
8.2    Use of AS-3000 Intelligent Autosampler   
 
Set the rack parameters, injection volume and other items with 
the AS-3000 intelligent autosampler.    For details, refer to the 
instruction manual attached to the AS-3000.     
 
Select the Measure command from the Spectrophotometer menu 
or click the     (measurement) button on the toolbar.   
Then, press the START key on the AS-3000 intelligent 
autosampler.  Measurement will start.  
 
NOTE:  In measurement without using a sample table, 
remeasurement, interrupt measurement and blank 
measurement cannot be done. 
 
NOTE:  In measurement when using a sample table, 
remeasurement, interrupt measurement and blank 
measurement cannot be done.    But remeasurement of 
standards is possible.    The standard to be 
remeasured should be set in the initial test tube 
position.  
 
For measurement after completion of settings: 
 
(1)  Select the Measure command from the Spectrophotometer 
menu in the FL Solutions program or click the 
(measurement) button on the toolbar.     
The window in Fig. 8-16 opens.   
 
 
Fig. 8-16    For Start from Calibration Curve Creation 
 
 
(2) Press the START key on the AS-3000.   
 
(3)  Measurement is started.    8.2.1  When Not Using   
a Sample Table   
8.2.2  When Using a 
Sample Table   
 
8.2.1  
						

							8 - 10 
8.3  For Analog Output  
 
For connection, use the analog output terminal.    Select the 
Analog Output Gain command from the Utility menu.     
 
 
 
Fig. 8-17 
 
 
This is a setting of data value corresponding to analog output of 
1 V.   
Analog output value (V) becomes as follows.   
 
 Model F-4500 
Low :  (Data value - photometric value lower limit)/1024   
High :  (Data value - photometric value lower limit)/8192   
 
  Model F-2500   
Low :  (Data value)/1024   
High :  (Data  value)/8192   
 
8.3  For Analog Output  
						

							 A -
1
APPENDIX A    DETAILS ON QUANTITATION 
 
 
A.1 Foreword 
In the Photometry mode of the F-2500 spectrophotometer, there 
are four calibration types. 
 
  Linear working curve 
  Quadratic working curve 
  Cubic working curve 
  Multiple segment working curve 
 
These are explained in more detail below. 
 
A.2  Linear Working Curve (1st order) 
 
Regression line is determined via least squares method from a 
maximum of 20 data.    The calculation formula is as follows : 
x = A1· y + A0 
A1 = 
()
yx
nyx
y
nyii i i
ii−
∑⋅
∑ ∑
−
∑ ∑1
1
22, A0 = x
nAy
nii∑
−⋅∑
1 
where,  x :    Concentration of each sample (input value) 
y :    Data on each sample (measured value) 
n :    Number of samples 
 
A.3  Quadratic Working Curve (2nd order) 
 
A quadratic curve is determined via least squares method from a 
maximum of 20 data.    The calculation formula is as follows : 
x = A2 · y
2 + A1 · y + A0 
A2 = 
()() ()()
()()()}
Sy xSyy SyxSyy
SyySy y Syy22
22 22−
−

 S(yy) = ()yy
nii
22∑−∑ 
A1 = 
()()()()
()()()}
SyxSy y Sy xSyy
SyySy y Syy22 2 2
22 22−
−

 S(yx) = yxyx
niiii∑−⋅
∑ ∑ 
A0 = 
x
nAx
nAy
nii i∑
−∑
−∑
122
 S(yy2) = yyy
niii 32∑−⋅
∑ ∑ 
 S(y
2x) = yxyx
niiii 22∑−⋅
∑ ∑ 
 S(y
2y2) = ()yy
nii
422∑−∑  
						

							 A -
2
where,  x :    Concentration of each sample (input value) 
y :    Data on each sample (measured value) 
n :    Number of samples 
 
A.4  Cubic Working Curve (3rd order) 
 
A cubic curve is determined via least squares method from a 
maximum of 20 data.    The calculation formula is as follows : 
X = A3 · Y
3 + A2 · y2 + A1 · y + A0 
where,  x :    Concentration of each sample (input value) 
y :    Data on each sample (measured value) 
n :    Number of samples 
 
A.5  Multiple Segment Working Curve (Segmented) 
 
The calibration curve is apt to bend when measuring turbid 
samples or the like.    Use of a quantification program allows 
correcting the calibration curve by using up to 20 standards.   
Figure A-1 shows an example of curve correction.    For the part 
which exceeds the measuring range of the standards, simply 
extend the line as it is. 
 
 
Fig. A-1    Correction of Curve Bending 
 
STD5
STD4
STD1
STD6 
STD3
STD2
CONC
Data  
						

							 A -
3
 
 
(1)  A correct calibration curve can be created only when the 
measured value increases or decreases monotonically 
versus the concentration value.      Especially when the 
inclination is negative-going, be sure to measure a blank 
having a data value larger than the other standards.     
A curve not showing a monotonic increase will appear as in 
Fig. A-2.    And when the data value of the blank is small, 
regardless of a negative-going inclination, the curve will 
appear as in Fig. A-3. 
 
 
Fig. A-2    Example of Curve without Monotonic Increase 
(unsuitable curve) 
 
 
STD5
CONC 
Data
STD4
STD1
STD2
STD3
STD6
Data
STD1
CONC
STD2
STD3 
STD4 
STD5
Fig. A-3  Example of Negative-going Curve where 
Measured Value of Blank is Small   
(unsuitable curve)