Spectrophotometry

HANIFA FATHIYA ALBARTY

RAHMAD CATUR BAYU RAGIL

DEFINITION
Spectrophotometer is a device consisting of a spectrometer

and a photometer. Spectrometer produces light of a specific

wavelength spectrum, while the photometer is gauge the
intensity of the transmitted light or in absorption.
Spectrophotometer is an analysis method based on the
measurement of monochromatic light that absorp by the
solutions in the specific wavelenght.

FUNCTION
Spectrophotometer is an instrument for measuring the

transmittance or absorbance of a sample as a function of

wavelength. Each media will absorb light at specific
wavelengths or color depending on the solution formed.

Each transparent objects can absorb light at specific wavelengths,

depending on the structure-forming. Before we use the

spectrophotometry, first we have to know the characteristic of the
solution we will observe. So we can use the appropiate type of
spectrophotometry.

Type of spectrophotometry

UV-visible
spectrophotometry

1. UV-visible spectrophotometry

The spectrophotometer is a combination of UV and

Visible spectrophotometer. Using two different light
sources, UV light source and a light source visible. For
spectrophotometric system, UV-Vis most widely available
and most popularly used.

2. IR(Infra Red) spectrophotometry

This Spectrofotometer using method of observing the interaction of
molecules with electromagnetic radiation is in the wavelength range from
180 360 nm. Usually used for qualitative analysis. Generally
spectrophotometry IR widely used to identify functional groups in a
compound, particularly organic compounds.

Atomic Absorption Spectrophotometer (AAS)

This spectrophotometer used in the method of analysis for the

determination of metallic elements.

Work Principle

WORK PRINCIPLE

Light from the lamp is polikromatis passed through the lens toward the monochromator.
Monochromator then will change polikromatis light into monochromatic light (single).
Rays with a certain length and then be passed to a sample containing a substance in a certain
concentration.
Therefore, there is a light that is absorbed and some are forwarded.
The light that is passed is then received by a detector.
Detector then will calculate and determine the light received light absorbed by the sample.
Light absorbed is proportional to the concentration of substances contained in the sample so that it
will be known concentration of a substance in the sample quantitatively.

Example
Maybe you want to know how much the vitamin C levels in your
suplement, you can use the spectrophotometry, but first you have to
know thr characteristic of vitamin. The light tat absorb by the vitamin C is
equal with the level of vitamin C in your suplement.

How to operate the device

1. Connect the spectrophotometer to the current source
2. Turn on the spectrophotometer by pressing the ON button on the spectrophotometer.
3. Display program will appear and tell you that the initiation process is underway, wait until the
process is complete characterized by the appearance of a green color and a written status ready.
4. Let stand for 15 minutes to warm up, after the spectrophotometer is ready for use.
5. Set the wavelength.
6. After the spectrophotometer is ready to use for the measurement of the sample absorbance at
a particular wavelength.
7. Cuvette inserted after wipe with a paper towel. The bright side of the cuvette facing skylight of
the spectrophotometer.
8. After finished, cuvette removed and cleaned of the solvent and then dried.
9. Spectrophotometer is turned off by clicking on the button OFF

1. The solution being analyzed is colored solution

If the solution to be analyzed is a colorless solution, then the solution should be changed first into a
colorless solution. Except when measured using a UV lamp.
2. The maximum wavelength
The wavelength used is the wavelength of maximum absorbance having. This is because the sensitivity
is also maximal at these wavelengths, the absorbance change for each unit of concentration is greatest.
In addition, around the wavelength maximum, will form the absorbance curve is flat so the LambertBeer law can be fulfilled. And if performed repeated measurements, the error rate will be small.
3. Calibration Wavelength and Absorbance
Spectrophotometer is used to measure the intensity of light emitted and absorbed light. It relies on the
electromagnetic spectrum that is absorbed by the body. Each media will absorb light at specific
wavelengths depending on the compound formed. Therefore, it is necessary to calibrate the wavelength
and absorbance in a spectrophotometer so that the measurement is in getting more thoroughly.

Safety
1. Avoid any light that comes into the appliance, usually at the time of
closing a cuvette, if there is another light, amount of light that is
measured automatically be increased.
2. Spectrophotometer device is quite safe because the device just use light
to measure the sample.