Spectrocolorimetry Measurements – SPEKOL
Introduction:
SPEKOL is a single-beam spectrophotometer with a grating monochromator working
in the wavelength range 190-1100 nm. Together with special countershafts, this is the
multifunctional device designed to measure: absorption, transmission, fluorescence and
phosphorescence emission, reemission, turbidy (cloudiness) and also photometric,
fluorimetric and turbidymetric of various solution.
Absorbancy (absorption) is the physical quantity servant to describe amount of
absorbed (rapt) radiation. As shows figure 1 intensity of bundle after the passage through the
matter decrease. We can describe absorbancy by equation:
A  log
I0
I
.
x
I
I0
Fig. 1
Transmittance is the physical quantity which describe amount of bulk radiation. It is
definite by the relationship:
T
I
I0
or in percentage:
T
I
100% .
I0
Relationship between absorbancy and transmittance shows equation:
log
or exspress in percentage:
where:
1
A
T
A  2  log T
A -- the absorbancy
I 0 - intensity of incident beam
I - intensity of beam which pass through the solution
T – transmittance.
The absorption spectrum describe the relationship between amount of absorb radiation
and wavelength of incidental radiation. In the visible range it could be dark lines or band on
the constant emission spectrum and it is characteristic for every substance.
Wavelength λ – distance between two neighboring points in the same phase.
wavelengh
t
The most popular units express wavelength are :
1nm  10 9 m
1 m  10 6 m .
1A  10 10 m
Range of wavelength of electromagnetic radiation
Range of
radiation
Wavelenhgt
[ nm]
UV-C
UV
UV-B
UV-A
100-280
280-315
315-380
VIS
380-780
IR-A
IR
IR-B
780-1400
1400-3000
IR-C
300010000
Others physical quantity characteristic for electromagnetic wave are:


1
Frequency ν. Frequency’s unit is Hz  . It define number of vibration in time unit.
s
Wave number k, which describe number of wavelengths in length unit. It’s unit is Kejzer.
1K 
1
cm
Relationship between wave number and wavelength describe equation:
k
1

.
Absorption laws:
Lambert law
I  I 0 e  x
where :
A - the absorbancy
I 0 - intensity of incident beam
I - intensity of beam which pass through the thickness x
α – absorption coefficient which is characteristic for every substance
x - thickness of the layer
Hence, I of electromagnetic radiation exponentially decreasing with the increasing length of
absorber.
x
I0
I
Fig. 1
Beer`s Lambert law- refer multicomponents solutions:
I  I 0e  cx
where c is concentration of solution.
α
A
c
c
Absorption coefficient is independent of concentration of absorber. Absorption increase with
the increasing concentration..
Absorption Additivity law
Absorption value of several component is equal to sum of absorption values of each
components.
Ac  A1  A2  ...  An
Application:
NMR spectroscopy, detection of various matters
1
2
3
1. Display and Button display of data’s and control buttons for use of internal software
function.
2. Sample compartment to insert accessories and samples cells.
3. Cell selector arm of the Cell change the position of he manual cell changer.
Positions of the 4 cell changer
Position 5
Sample
nr 3
Position 4
Sample
nr 2
Position 3
Sample
nr 1
Position 2
Position 1
Distilled
water
Golas:
 Determine the absorption spectrum of chosen substance
 Check the relationship between absorption ant concentration of the substance
 Find concentration of the substance with the help of curve A=f(c)
Procedure:
Absorbance measurement
1. Set the wavelength 450 nm with the up and down button.
2. Select the operating mode ABS by using MODE button.
3. Place the cell with distilled water into the path of the beam.
4. Place the sample in the other position of cell changer.
5. Close the sample compartment cover.
6. Pull the cell holder with the distilled water in the measurement beam.
7. Measure the blank value by pressing 100%T button.
8. Using the cell holder cover the beam (position 2).
9. To calibrate device use the 0%T button.
10. Place the cell with sample into the path of beam.
11. Read the absorbance.
12. Increase the wavelength by 10 nm until 780 nm will be achieved.
Make graphs A=f(λ) and T=f(λ).
λ
T
A
Repeat the measurements for other 10 different concentrations solutions. Read and
tabulate only the extreme values of absorbance and transmittance.
Amax
Tmin
c
Make graphs Amax=f(c) and Tmin=f(c).
Mark the value of Amax and using the graphs read it’s concentration.
With the help of the SPEKOL study the substance with unknown concentration.
1. Press MODE button until the absorbance ABS is displayed.
2. Place the distilled water in the sample compartment and close the sample compartment
cover.
3. Press 100%T button to measure the blank solution.
4. Cover the measurement beam (position 2).
5. Calibrate the device by using 0%T.
6. Place the standard sample into the measurements beam.
7. Press MODE button until the concentration CONC is displayed.
8. Press the FUNC button until the Input Std. CONC is displayed.
9. Use the up and down button to set the concentration of the standard on the digital
display and confirm with ENT.
10. Insert the sample solution into the cell holder or cell changer.
11. Close the sample compartment cover.
12. Read the results.
.
Compare the received value with the value read from calibration curve.