Lecture Iii

Analytical spectroscopy
Analytical spectroscopy is the science

of determining how much of a
substance is present in a sample by
accurately measuring how much light
is absorbed or emitted by atoms or
molecules within it.
Instrumentation
An instrument that measures
the intensity of light absorbed
by atoms or molecules is called
a spectrophotometer.
Instrumentation
An instrument that measures the intensity of light absorbed by atoms or
molecules is called a spectrophotometer.
Light source
Light source
a. A tungsten lamp emits light of wavelengths

350–2000 nm and is adequate for
colorimetry.
b. A deuterium lamp is a high-energy source

that emits light of approximately 200–370
nm and is used for all spectroscopy in the
ultraviolet region of the spectrum.
Monochromator
For most quantitative measurements, light

must be monochromatic, i.e. of one particular
wavelength. This is achieved by passing the
polychromatic light (i.e. light of many
wavelengths) through a monochromator.
There are two types of monochromator in
modern spectrophotometers: prisms or
diffraction gratings.
Prisma
Diffraction gratings
A diagram of a diffraction grating.

Sel atau Kuvet
Ada berbentuk silinder (tabung

reaksi) dan ada bentuk persegi. Sel
berbentuk persegi lebih baik.
Detector
After light has passed through the

sample, any decrease in intensity, due
to absorption, is measured by a
detector. This is usually aclever piece of
electronics called a photomultiplier tube
A diagram of a photomultiplier tube.
Experimental measurement of
absorbance
The sequence of events in making a measurement

with a spectrophotometer is as follows.
1. The monochromator is set to the wavelength of

measurement.
2. The solvent (or ‘blank’) is placed in the light path.
3. The instrument is set to zero absorbance.
4. The sample solution (or ‘test’) is placed in the light
path .
5. The absorbance is read directly by the instrument.
Dilutions
The most important part of any

spectroscopic assay is not the performance
of the spectrophotometer (although the
accuracy of the instrument is checked
periodically). The crucial part of any
experiment is the accurate preparation of
the test and standard solutions.
PENGOLAHAN DATA STATISTIK
1. Kecermatan (accuracy) adalah ukuran tingkat kedekatan hasil analisis dengan
kadar analit yang sebenarnya.
2. Keseksamaan (precision) adalah ukuran tingkat kecocokan antara hasil uji
tunggal dengan rata-rata hasil uji berulangkali pada sampel yang diambil dari
campuran yang homogen.
Worked example
1. You are presented with a stock solution

containing a 50 µg mL–1 solution of a drug.
Prepare 100 mL of solution to contain 5,
10, 20 and 30 µg mL–1 of drug. The first
step is to calculate how much of the 50 µg
mL–1 stock solution will be required for
each dilution.
1 Spektrum absorpsi theophyllin dalam air
Gambar: Plot Hk Beer pada Theophyllin dalam larutan air
This can be done by using the relationship below
where [ ] represent the concentrations of drug.

2. Pada penetapan kadar rifampicin dengan alat
Kolorimeter Dubosq sbb:
Sampel sejumlah 100 ml dipipet 5 ml
dipindahkan kdl labu ukur 100 ml ad kan vol 100
ml. Pipet 5 ml cukupkan vol 100. Pipet 5 ml
cukupkan vol 50 ml. Sampel diukur dengan alat
kolorimeter dengan pembanding 100 ml kadar 1
ug/ml.
Hitung kadar rifampisin dalam sampel apabila
setelah warna pengamatan sama, diperoleh
data tinggi pembanding 10 skala dan sampel 8
skala.
This relationship may bemore easily remembered as
Using this relationship, the 30 µg mL–1 solution is prepared from

(30/50) x100 = 60 mL of stock solution made up to 100 mL with
solvent. The 20 µg mL–1 solution is prepared from (20/50) x 100 =
40 mL of stock made up to 100 mL with solvent, and so on for all
the dilutions.
3. Suatu larutan obat 6,4 x 10-5 M mempunyai
absorban 0, 847 dalam sel 1 cm pada 255 nm.
a) Hitung absortivitas molar senyawa tsb.
b) Sampel obat tersebut ditimbang secara
tepat 10 mg(BM 200) dilarutkan dalam
air sampai 1 liter. Pada 255 nm absorban
dalam sel 1 cm adalah 0,556. Hitung
kemurnian sample
4. Q Five standard solutions of a drug (relative molecular mass
288.4) were prepared in spectroscopy-grade ethanol and the
absorbance of each solution was measured at 285 nm in 1
cm cells.
a) Is Beer’s law obeyed for this
drug at this wavelength?
1%
b) Calculate the E1cm and molar
absorptivity for this drug at 285
nm.
c) Calculate the % transmittance
given by a 0.5 mg/100 mL
solution in a 2 cm cell.
1%
b) E
Calculate the 1cm and molar absorptivity for
this drug at 285 nm.
Konsentrasi (%) :
1 mg/100 mL= 0,001 % g/100 mL
= 0,001 % w/v
1%
A = E1cm x cxl
Jadi:
1%
0,562 = E 1cm x 0,001 x 1
1%
E 1cm = 562
The molar absorbtivity ε can be calculated as follow:
-1
1 mg/100 mL = 0,01 g/L
0,01
=
288,4 mol L-1
A = εcl
Hence,
0,01
0,562 = ε x -1 x 1
288,4 mol L
Therefore,
ε = 16210
c) Calculate the % transmittance given by a 0.5 mg/100 mL
solution in a 2 cm cell.
Answer:
A = 562 x 0,0005 x 2
A = 0,562
log Io/I = 0,562
Therefore,
Io/I = 3,648
and
% Trasmittance = 100 x Io/I = 100 x 1/3,648 = 27,4% i.e.
For this solution, 72.6% of available light is absorbed and
27.4% is transmitted.

Lecture Iii

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Lecture Iii

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Analytical spectroscopy

Analytical spectroscopy is the science

a. A tungsten lamp emits light of wavelengths

b. A deuterium lamp is a high-energy source

For most quantitative measurements, light

A diagram of a diffraction grating.

Ada berbentuk silinder (tabung

After light has passed through the

The sequence of events in making a measurement

1. The monochromator is set to the wavelength of

The most important part of any

1. You are presented with a stock solution

where [ ] represent the concentrations of drug.

Using this relationship, the 30 µg mL–1 solution is prepared from

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