spectrophotometry

Kusumo hariyadi (KSH)

Blok 3
 Absorption spectrophotometry is the measurement
of the absorption of electromagnetic radiation with a
certain wavelength narrow, monochromatic
approach.
Spectrophotometric analysis can be used for
qualitative and or quantitative analysis.
Measurements can be made on the absorption
wavelength:
Ultraviolet (UV) at a wavelength of 190-380 nm
Visible light (visible) at a wavelength of 380-780 nm
 1 Wavelength ()
The wavelength of light is defined as the distance
between two peaks of a wave, usually expressed
with the symbol lambda ()
Unit wavelength UV - VIS is a nano-meter (nm)
1 nm = 1 mu = 10-9 m = 10-6 mm
The light is a mixture of radiations having different
wavelengths, which can didespersikan by a
monochromatic beam monochromator become.
 Transmittance and absorbance
Light transmittance is forwarded (in the transmit beam)
While absorbance is light absorbed
Suppose a beam through the cuvette (sample containers of
glass) then there is a fraction of the light in the forward and
partly absorbed (absorbed)
Mathematically so that there is a relationship between the
transmittance (T) and absorbance (A)
Relationship as follows:
A = 2 - log T A = absorbance
T = transmittance (in%)
 When T = 100%
Then A = 0
 Legal Lambert - Beer
When monochromatic light through a solution, the amount
of light absorbed is proportional / proportional to the
concentration of substances in solution.
Mathematically laws Lambert - Beer can be formulated as
follows:
A = k. c. l
A = absorbance / absorbance / optical density, ie the amount of
light absorbed
K = coefficient ekstinsi / constant
C = concentration of sample examined
Cuvette L = diameter / length of the solution through which the
light, eg = 1 cm
Because k and l fixed number, it means that A is proportional to c
 5. How to run the spectrophotometer:
Detailed operating instructions can be followed in accordance with the
instructions / manual of the instrument:
The general procedure in running the spectrophotometer are:
1.Check that there is no cuvette in the cuvette
2.Turn the power switch ON
3.Select the appropriate lamp: Deuterium (D2) to the UV or visible region for the
Tungsten (visible light)
4.Set the desired wavelength by rotating the wavelength regulator (such as
selecting a radio wave)
5.Check the% T must designate the number 100 by turning the button on the tool /
A on the number 0
6.Place the cuvette containing the blank: may contain akuadest / solvent / solvent
without the sample
7.And measuring A / A made 0
8.Fill cuvette with raw substance / standard, measuring A
9.Replace the contents of the cuvette with a sample of raw substance, measuring A
10.Compute sample concentration by comparing sample A with a standard
Multiply the standard levels
HOW I WORK

1.Fill
cuvette with raw substance / standard,
measuring A
2.Replace the contents of the cuvette with a
sample of raw substance, measuring A
3.Compute sample concentration by
comparing sample A with A standard
Multiply the standard levels
example

Standard levels made = 10 ppm

A standard = 0.5
A sample = 0.7
content of the sample
0.7 / 0.5 X 10 = 14 ppm
HOW II

Standardcurve was made standard

according to the Lambert-Beer law
A substitute samples on standard curve
The obtained sample concentration
 A

0,5

C ppm

40
 Make linier function
Ex: Y = 0,5 X + 50
Y=A
X=C
If A =0,5 X = 50,5 / 0,5 = 101 ppm
HOW TO WORK III?
For samples that do not have standard

A 1% 1cm x substances sought in the

literature
Suppose A 1% 1 CM substance x = 2000
A sample of the substance x = 0.05
The levels of substance x in the sample is
0.05 / 2000%