Sie sind auf Seite 1von 4

COLORIMETRY /

SPECTROPHOTOMETRY
Experiment 3
Ebru AKHARMAN, Gebze Technical University, Turkey

AIM:
While materials absorb a portion of light in our environment, they reflect a portion of light.
There is a diferent place of this situation in biochemical experiments. If the concentration of
used solution or material is not know, the concentration of this solution or material should be
determined by colorimetry / spectrophotometry.

These two apparatus are used for calculating absorbance. Absorbance is linear proportion with
concentration. Thus, if absorbance is known, concentrations can calculated. The concentration of
methyl red will calculated in this experiment. Also, this experiment is a sample for using
colorimetry / spectrophotometry.

INTRODUCTION: light to be determined. Colorimeters hence


make it possible to as certain the
Most test substances in water are colorless concentration of a known solute since it is
and undetectable to the human eye. To test proportional to hte absorbance.
for their presence we must find a way to
see them. A colorimeter or A spectrophotometer is a photometer (a
spectrophotometer can be used to measure device for measuring light intensity ) that
any test substance that is itself colored or can measure intensity as a function of the
can be reacted to produce a color. In fact a color or more specifically the wavelenght of
simple definition of colorimetry is the light. Important features of
measurement of color and a colorimetric spectrophotometers include the spectral
method is any technique used to evalute an bandwidth and linear range. The most
unknown color in reference to known common application of spectrophotometers
colors. In a colorimetric chemical test the is the measurement of light absorbtion.
intensity of the substance being tested.
Some reactions have limitations or
variances inherent to them that may give
misleading results. Most limitations or
variances are discussed with each particular
test instruction.

A colorimeter is generally and tool that


characterizes color samples to provide an
objective measure of color characteristics.
In chemistry, the colorimeter is an
apparatus that allows the absorbance of a
solution at a particular frequency of visual Image.1:Basic structure of spectrophotometer
As light passes through a material, it is methyl red and buffer is measured with
absorbed y molecues in the material. spectrophotometer.
Consequently, the intensity of light dcreases
exponentially with distance as the light 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, 0.5 ml, 0.1 ml
passes through the material. Transmittance and 0.0 ml methyl reds are measured and
through a sample solution is easily completed volume to 2.5 ml with buffer.
measured by measuring the intensities of Then, every sample as replica1 and replica2
incident and transmitted light. Using the is added every well with micropippets. to 96
value for transmittance, it is then possible to well plate adding amount of sample is 200
calculate the absorbance of the sample. l.
Transmittance(T) is a measurement of Blank is buffer and added in penultimate
how much light passes through a substance. well. Unknown protein is added in the last
The higher the amount of light that passes well.
through, the larger transmittance is defined
as the ratio of the intensity of incident light. Save datas .
If the intensity of incident light is 0 and the
intensity of transmitted light is , then Concentration of unknown protein is found.

RESULTS:
=

avr.-
= = REP1andREp2 blank
REP1 REP2 avr. avr.
A: Absorbance A 0,529 0,461 0,495 0,463
: Molar extinction B 0,234 0,242 0,238 0,206
coefficient
C 0,153 0,157 0,155 0,123
: Lenght of the light path
: Concentration D 0,266 0,141 0,204 0,172
E 0,258 0,053 0,156 0,124
F 0,054 0,047 0,051 0,019
There is a linear relationship between the
Blank G 0,026 0,038 0,032 0
absorbance and the concentration of a
sample. unknown H 0,35 0,367 0,359 0,327

The relationship between concentration and


transmittance is logarithmic. Table.1: The Table of Absorbance

MATERIAL AND METHODS: The average of the obtained


absorbances is calculated. The
Spectrophotometer average of blank is decreased from
Methyl Red other average of absorbance.
Tubes
Pipettes
96 well plate The concentrations of samples of
methyl red are calculated.
1 ml methyl red and buffer is measured and
put in the cuvette. Then, absorbance of
According to datas, absorbance - This chart shows us that the experiment is
concentration graph is created ve fail. Because, the absorbance and
slope is found. concentration are not directly proportional
at each point of the graph.
Methyl Concentr
red ation of
amounts Buff absorba methyl
(ml) er nce red
A 2,5ml 0 ml 0,463 100
0,5
B 2 ml ml 0,206 81,48
C 1,5 ml 1 ml 0,123 62,96
1,5
D 1 ml ml 0,172 44,44
2,0 Image.2: Graph Formula

E 0,5 ml ml 0,124 25,92


2,4
F 0,1 ml ml 0,019 11,11 According to the this Formula;
2,5
Absorbance of unknown protein = 0,327
G 0 ml ml 0 7,41
Table.2: Showing Values Table
The slope of curve = 0,0035
If you want to show this data graphically:
0,327 = 0,0035 * X

0,327
X= 93,43
0,0035

So that unknown have approximately %


93,43 methyl red. Its concentration is
0,93 mg/ml.

DISCUSSION:

Colorimetry is the measurement of the


absorbtion of light. Spectrometry is the
measurement of a spectrum, that is, the
distrubution of light produced or absorbed
by a substance. Spectrometer consists of
two devices; a spectrometer and a
photometer. Spectrometer: It produces a
desired range of wavelength of light. First a
lens transmits a straight beam of light that
passes through a prism to split it into
several component wavelengths. Then a
Graph.1:Absorbance Concentration Graph wavelength slit transmits only thedesired
wavelengths. Photometer: After the desired
range of wavelength of light passes through
the solution of a sample in cuvette, the
photometer detects the amount of photons RESOURCES:
that is absorbed and then sends a signal to a
galvanometer or digital display. http://cellbiologyolm.stevegallik.org/node/
7
Methyl red was used as a sample solution.
The spectrophotometer is used to measure http://pediaa.com/difference-between-
the absorbance of solutions containing absorbance-and-transmittance/
methyl red and buffer in different amounts. http://www.differencebetween.com/differe
The obtained data is saved and graphics are nce-between-absorbance-and-vs-
created. The concentration of unknown transmittance/
protein is calculated using Beer-Lambert
Law. The resulting graph is not a regularly http://www.globalw.com/support/colorim
growing graph. In this case, individual eter.html
errors may have been made in the
experiment. The test materials may be
defective in the damaged part. The person
who made the calculations may have made
an incorrect account.

Das könnte Ihnen auch gefallen