Spectrophotometry:

An Analytical Tool
 What is Spectroscopy?
• Spektroskopi dapat didefinisikan sebagai
ilmu yang mempelajari interaksi antara
cahaya (sumber radiasi) dan materi

PGCC CHM 103 Sinex

 Spectroscopy

Spectral Distribution of Radiant Energy

Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)

PGCC CHM 103 Sinex

The process of light being absorbed by a solution
concentration 2 with sample
concentration 1
I < Io
blank where Io = I

light
source detector

Io I
As concentration
b increased, less
light was
Cell with
transmitted (more
Pathlength, b,
light absorbed).
PGCC CHM 103 Sinex containing solution
 Some terminology
I – intensity where Io is initial intensity

T – transmission or %T = 100 x T
(absorption: Abs = 1 – T or %Abs = 100 - %T)
T = I/ Io

A – absorbance
A = - log T = -log I/ Io
PGCC CHM 103 Sinex
 Beer’s Law

A = abc
where a – molar absorptivity, b – pathlength,
and c – molar concentration

PGCC CHM 103 Sinex

 Analyze at what wavelength?
Scan visible wavelengths from 400 – 650 nm
(detector range) to produce an absorption
spectrum (A vs. l)
Crystal Violet Absorption Spectrum
1.4

1.2

1
Absorbance

0.8

0.6

0.4 lmax
0.2

0
200 250 300 350 400 450 500 550 600 650 700 750
wavelength, nm
phototube detector range
lmax - wavelength where maximum absorbance occurs
PGCC CHM 103 Sinex
 The BLANK
The blank contains all substances
expect the analyte.
Is used to set the absorbance to zero:
Ablank = 0
This removes any absorption of light
due to these substances and the cell.
All measured absorbance is due to
analyte.
PGCC CHM 103 Sinex
 The relationship between absorbance and
transmittance is illustrated in the following
diagram:

PGCC CHM 103 Sinex

 The components of a Spec-20D

Light source - white light of constant intensity

slits

filter occluder

Grating
slits Separates white light
Phototube
into various colors
detects light &
measures intensity Rotating the grating
Sample
changes the wavelength
When blank is the sample going through the sample
Io is determined
PGCC CHM 103 otherwise
Sinex I is measured
Gugus kromofor dan auksokrom
Gugus kromofor : gugus yang mengabsorbsi radiasi
elektromagnetik UV-VIS
Contoh : karbonil, alken, azo, nitrat dan karboksil

Gugus Auksokrom : gugus fungsional yang mempunyai

elekron bebas
Contoh : –OH, O-NH2, dan –OCH3

Gugus auksokrom yang terikat pada gugus kromofor

akan menyebabkan pergeseran panjang gelombang ke
arah yg lebih besar dan kenaikan intensitasnya

PGCC CHM 103 Sinex

 STEPS IN DEVELOPING A
SPECTROPHOTOMETRIC
ANALYTICAL METHOD

1. Run the sample for

spectrum
2. Obtain a monochromatic
wavelength for the
maximum absorption
wavelength.
3. Calculate the concentration
of your sample

PGCC CHM 103 Sinex

 A
Slope of Standard Curve =
C
Absorbance at 280 nm

1.0

0.5

1 2 3 4 5
Concentration (mg/ml)

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where
becomes
PGCC CHM 103 Sinex non-linear.
 SPECTROMETRIC ANALYSIS USING
STANDARD CURVE
Absorbance at 540 nm
1.2

0.8

0.4

1 2 3 4
Concentration (g/l) glucose

Avoid very high or low absorbencies when drawing a

standard curve. The best results are obtained with 0.1 < A
< 1. Plot the Absorbance vs. Concentration to get a
straight line
 • Every instrument has a useful range for a
particular analyte.
• Often, you must determine that range
experimentally.
• This is done by making a dilution series of
the known solution.
• These dilutions are used to make a
working curve.
PGCC CHM 103 Sinex
 Make a dilution series of a known quantity
of analyte and measure the Absorbance.
PGCCPlot
CHM 103 concentrations
Sinex v. Absorbance.
Berapa konsentrasi dalam sampel (%b/b) jika
ditimbang 25 mg sampel dilarutkan dalam aquades
ad 25 ml, kemudian diambil 1 ml dan di adkan 10 ml
kemudian diukur absorbansinya !
In this graph, values above A=1.0 are not linear. If we
use readings above A=1.0, graph isn’t accurate.
Contoh Analisis Vitamin C dengan spektrofotometer
UV-Vis
Preparasi standar vitamin C :
Dibuat beberapa macam konsentrasi standar vitamin C
yaitu 4 ppm, 8 ppm, 12 ppm dan 16 ppm

Preparasi sampel :
Ditimbang sampel sebanyak 2 gram kemudian dilarutkan
dalam 250 ml aquadest.Setelah itu larutan tersebut dipipet
sebanyak 2 ml dan diencerkan sampai 100 ml.
Larutan kedua dipipet lagi 25 ml dan diencerkan sampai 100 ml.

Pertanyaan : Hitunglah kadar vitamin C

PGCC CHM 103 Sinex

 Data absorbansi standar dan sampel pada
Panjang gelombang 271 nm

PGCC CHM 103 Sinex

PGCC CHM 103 Sinex
 Contoh Analisis Parasetamol dengan
spektrofotometer UV-Vis

Preparasi standar parasetamol:

Dibuat beberapa macam konsentrasi standar parasetamol
yaitu 6 ppm, 7 ppm, 8 ppm, 9 ppm dan 10 ppm

Preparasi sampel :
Ditimbang sampel sebanyak 50 mg kemudian dilarutkan
dalam 100 ml NaOH 0,1 N.Setelah itu larutan tersebut dipipet
sebanyak 1 ml dan diencerkan sampai 50 ml.

Pertanyaan : Hitunglah kadar parasetamol

PGCC CHM 103 Sinex

 Data absorbansi standar dan sampel pada
Panjang gelombang 257 nm

Absorbansi sampel = 0,351

PGCC CHM 103 Sinex

PGCC CHM 103 Sinex