Group 5

Anisya Dwi Meli Amelia Ihsan Fajar Rhiza Fitri Salsabilla Az Zahra
1807908 1802096 1805893 1808000 1804849
Today's
Highlight
1. Spectrometric and Spectrometer
2. Principle and Structure
3. Type of Spectrophotometer
4. Absorbance and transmittance
5. Lamberts beer’s law Limitation of Lambert Beer’s law
Part I: Preparation of 4. Add the hydroxylamine hydrochloride (NH2OH HCl),

standard solution add one mL to each flask using volumetric pipet

(Don't shake it, just let it drain naturally)
5. Add one mL of ammonium acetate (NH4C2H3O2) to
1. Prepare the standard solution, that is
each flask using volumetric pipet
solutions with a known concentration of
6. Use 10 mL volumetric pipet to add 10 mL of
our iron (II) complex.
1,10-phenanthrolineto each flask
2. Label five 50 mL volumetric flasks with
7. Dilute it with distilled water until halfway (Careful
the numbers 1 until 5
add distilled water, not surpass the calibration mark)
3. Add iron (III) stock solution to each
8. Mix your solution thoroughly, by inverting until
flask (Pipetting 1 until 5 mL into each
several minutes
50 mL volumetric flasks.
9. Waiting for 45 minutes to see the concentration of
iron complex
 Part II : Production of Absorption
Spectrum and Part III : Production of Beer-Lambert Plot
1. Pair the Spectrovis Plus with a 4. Put the cuvet into the Spectrovis Plus
LabQuest, it’s like the detector that 5. Click red area in Lab Quest to calibrate, wait until
will the absorbance of each sample. warm up. After done click finish
2. Placed 5 standard solution into the 6. Put the solution number 5, the most concentrated
cuvets solution
3. Callibrate it, notice the cuvet has a 7. To collect an absorbance spectrum press “play’’ the
cloudy side and a transparent side, Lab Quest
because the light pass through the 8. After spectrum appears you can press “Stop’’
transparent side of the cuvet 9. The rainbow display indicates what color light
sample mostly absorb
1. Repeat it for each solution
 Part IV : Determination of iron percentage in the unknown
1. Prepare the unknown iron(III) stock
solution
2. Add approximately 20 milliliters of
distilled water to dissolve, you can
using beaker
3. Add 5 drops of concentrated sulfuric
acid to help dissolved the solid
4. Stir it until dissolved
5. Quantitatively transfer the solution to a
50 milliliter volumetric flask
6. Mix halfway
7. Dilute to calibration mark and mix once by
inverting for several minutes
8. The meniscus should lie right on the calibration
mark
9. We can use it to perform the same reaction as
in part I, and that can be analyzed using
spectrophotometry
 Spectroscopy Spectrometer
Field of science that learns about the Instrument or tools to measure the
theory of interaction between matter and matter-energy interactions
energy

Spectrometric
Field of science that learns about the
measurement methods and
instrumentation to measure the matter-
energy interactions.
 Spectrophotometer
A spectrophotometer is an instrument that measures
the amount of photons (the intensity of light)
absorbed after it passes through sample solution.

Spectrophotometric
Spectrophotometric is a method to measure
how much a chemical substance absorbs light
by measuring the intensity of light as a beam
of light passes through sample solution.
Spectrophotometric UV-VIS Analysis
ultraviolet–visible
spectrophotometry refers to
absorption spectroscopy or
reflectance spectroscopy in
part of the ultraviolet and the
visible light. This means it uses
light in the visible and
ultraviolet range.
Principle
The Principle of Spectrophotometer is based on the absorption of ultraviolet light or visible light by chemical
compounds, which results in the production of distinct spectra.
.
When the matter absorbs the light, it undergoes excitation and de-excitation, resulting in the production of a
spectrum. When matter absorbs ultraviolet radiation, the electrons present in it undergo excitation. This causes
them to jump from a ground state (an energy state with a relatively small amount of energy associated with it)
to an excited state (an energy state with a relatively large amount of energy associated with it).
 Structure
Structure The basic structure of
spectrophotometers. It consists of
a light source, a collimator, a
monochromator, a wavelength
selector, a cuvette for sample
solution, a photoelectric detector,
and a digital display or a meter.

It produces a desired range of wavelength of light. First a collimator (lens) transmits a

straight beam of light (photons) that passes through a monochromator (prism) to split it into
several component wavelengths (spectrum). Then a wavelength selector (slit) transmits only
the desired wavelengths..
TYPE OF SPECTROPHOTOMETER
UV/VIS

Single Beam Double Beam

A single beam Double beam

spectrophotometer has spectrophotometer has
only one beam of light. two beams of light.
Single
Single Beam
Beam Spectrophotometer
Spectrophotometer
A sample is placed in the UV/VIS beam and
absorbance versus wavelength is measured.
A single beam spectrophotometer utilizes one
beam of light that passes through the sample
and the intensity of the light reflected from a
reference is measured without the sample.
Low cost, cheaper, less part and less
complicated
Have a larger dynamic range and are optically
simpler and more compact.
Double Beam
Double Beam Spectrophotometer
Spectrophotometer

A double beam spectrophotometer compares the light intensity between two light paths,
one path containing a reference sample and the other the test sample.
ADVANTAGE VS DISADVANTAGE
The Advantage :
High stability because
reference
Sample are measured virtually
at the same moment in time.
Easier and more stable to use
The disadvantages :
higher cost
Lower sensitivity because through
put of light is poorer because of
the more complex optics
Lower reliability because of the
greater complexity.
Transmittance
Transmittance
is defined as a ratio of the intensity of
incident light (I0) to the amount of intensity
passes through the object (I). The
transmittance is denoted as T.

The
amount of energy which is being transmitted
Examples : T = 0,4 , so the %T = 40
during the process.
Transmittance example
If you pass the light from a semi-transparent block
of glass. Let say 30% of light is reflected from the
surface of the glass. Remaining 70% of the light will
try to pass the block of glass. But, some amount of
light will be absorbed by the molecules of light.
Let say it is 35% of light.

Now the remaining 35% light will pass from the other side of the block. It means that the glass block
has a transmittance of 35%.
Absorbance
Absorbance
The amount of light that is absorbed
when it travels through a material.

%T = 40, so the Absorbance is 2- log(40) = 0.398

When white light passes through or is
reflected by a colored substance, a
characteristic portion of the mixed
wavelengths is absorbed. The
remaining light will then assume the
complementary color to the
wavelength(s) absorbed.
Lambert
Lambert state..
state.. Beer
Beer state..
state..
Absorbance of light in homogenous solution is Absorbance of light in sample/solution is
DIRECTLY PROPORTIONAL to the length of DIRECTLY PROPORTIONAL to the concentration
sample in which light passes of solution in which light travels
 Lambert-Beer’s Law
The Beer-Lambert law relates the attenuation of light to the properties
of the material through which the light is traveling.

The Beer-Lambert law states that there is a linear relationship between the concentration
and the absorbance of the solution, which enables the concentration of a solution to be
calculated by measuring its absorbance.

The law states that:

A(λ) = e(λ) l c.
Example
Limitation of Lambert-Beer’s Law

Derivations in absorptivity coefficients. At high concentration

(>0.01M) due to electrostatic interactions between molecules in
close proximity
Scattering of light. Due to particulates in the sample
Fluorescence or Phosphorescence of the sample
Change in refractive index at high analysis concentration
Shifts in chemical equilibria as a function of concentration
Stray light
for your attention