SUMMARY/ABSTRACT

This experiment is about the usage of the UV/Vis Spectrophotometer. The parameters
involved in this experiment are absorbance readings, concentration of solution, path length,
wavelengths and max. The purposes of this experiment are the determination of wavelength
of maximum absorbance, max, and to prepare a serial dilution and to generate a standard
calibration graph. All of those can be obtained by undergoing wavelength scan and
photometric scan through the Perkin - Elmer UV/Vis Spectrophotometer Lambda EZ210. In
the UV/Vis Spectrophotometer, radiation will be emitted and the radiation will be absorbed
by the chromophores in the chromogen. The absorbance, concentration, and max of the
sample can then be obtained. The experiment first started by preparing a serial dilution with
concentrations of 5 ppm, 15 ppm, 25 ppm, 35 ppm, and 45 ppm. The series was then brought
to the UV/Vis Spectrophotometer to undergo two scans; wavelength scan and photometric
scan. The max, absorbance readings, concentrations of unknown samples, and the standard
calibration graph were then obtained. A max of 516.0 nm was obtained. Concentrations for
unknown samples #1 and #2 were obtained which were 21.515 ppm and 41.500 pm
respectively. Absorbance readings of 0.777 and 1.519 were also obtained for unknown
samples #1 and #2 respectively. Meanwhile for concentrations 5 ppm to 45 ppm, absorbance
readings of 0.188, 0.575, 0.768, 1.313, and 1.668 were obtained in order. It was observed that
the concentration of the sample increases proportionally with the absorbance readings
obtained, thus supporting the theory of Beers Law; which states that the relationship of
absorbance with the path length and concentration is directly proportional. Based on the
results, the theory and objectives were achieved in this experiment. In conclusion, by learning
to use the UV/Vis Spectrophotometer, a person can improve his or her analytical skills in the

laboratory and will definitely prove beneficial in the future.

OBJECTIVE
To determine max for food dye sample (wavelength scan)
To prepare a serial dilution and generate a standard calibration graph for sample
quantitation (photometric scan)

INTRODUCTION
In this experiment, two things are focused on; which are wavelength scanning and
photometric scanning. In the wavelength scan, the wavelength of maximum absorbance, max,
can be obtained. Whereas for the photometric scan, the concentration and absorbance of each
individual component in an unknown sample can be determine.
Beginning with a 100 ppm Carmoisine stock, it was then diluted into 5 volumetric
flasks (5 ppm, 15 ppm, 25 ppm, 35 ppm, 45 ppm). The volumetric flasks will then be brought
to undergo wavelength scan and photometric scan. A cuvette filled with 45 ppm dilution and
another cuvette filled with a blank solution were then put through a wavelength scan to obtain
the max. Next, each of the serial dilution was put through a photometric scan one by one to
acquire the standard calibration graph and as well to obtain the absorbance and concentration

readings of an unknown sample.

THEORY
The Beer-Lambert Law (or Beers Law) states the relationship between the
absorbance of a solution and the concentration of the absorbing species. The absorbance is
directly proportional to the path length of the radiation through the solution and the
concentration of the absorbing species. In this experiment, the Beer-Lambert Law Equation
was applied to obtain the needed results :

GENERAL BEER-LAMBERT LAW EQUATION

A = abc
Where :
A = Absorbance

a = Wavelength-dependent

b = Path length

c = Concentration

absorptivity coefficient

(cm)

(M or mol/L)

= Wavelength-dependent molar

b = Path length

c = Concentration

absorptivity coefficient

(cm)

(M or mol/L)

(M-1cm-1)

WHEN WORKING WITH MOLARITY

A = bc
Where :
A = Absorbance

(M-1cm-1)

Experimental measurements are usually made in terms of Transmittance (T). The

Transmittance (T) of the solution is the fraction of incident radiation transmitted by the
solution, which is defined as :

T = I / Io
Where :

T = Transmittance

I = Light intensity after passing through Io = Initial light intensity

the sample

Figure 1

The absorbance (A) of a solution is defined as the negative log of the transmittance (T) of the
solution.

A = - log T = - log (I / Io)

Transmittance is often expressed as a percentage, which is :

A = 2 - log %T

MATERIALS

Apparatus
Perkin - Elmer UV/Vis

Spectrophotometer Lambda EZ210

Sample cuvettes, path length 1 cm
Volumetric flask 50 ml (five)
Pipette 5 ml, 10 ml and 25 ml (one each)
Rubber bulb (three)
Beaker 100 ml (one)
Graduated cylinder 50 ml (one)
Dropper (one)
Labelling sticker
Tissue paper

Chemicals
100 ppm Carmoisine stock (100 ml)
Unknown concentration of Carmoisine
sample (two)
Distilled water

PROCEDURE
1. Serial dilutions (5 ppm, 15 ppm, 25 ppm, 35 ppm and 45 ppm) were prepared from
the 100 pm food dye stock.
2. Dilution formula was used to calculate the volume needed for all dilutions from the
100 ppm food dye stock.
3. The dilutions were added into 5 respective 50 mL volumetric flasks.
4. Distilled water was added to the mark for all the volumetric flasks.
5. All 5 volumetric flasks containing different dilutions were shut with rubber stoppers
and were shaken properly.
6. Perkin Elmer UV/Vis Spectrophotometer Lambda EZ210 standard operating
procedure was briefed by the technician.
7. Two cuvettes were filled with 45 ppm dilution and a blank solution respectively and
were inserted into the sample compartment.
8. Wavelength scan was done.
9. Photometric scan was done by filling up two cuvettes with 45 ppm dilution and a
blank solution and was repeated for the serial dilutions prepared and was scanned one

by one.
10. Concentrations of Unknown #1 and Unknown #2 were determined.
11. Data was completely recorded.

RESULTS
A. WAVELENGTH SCAN
i.
Instrument Parameters
Starting Wavelength :
Ending Wavelength :
Path Length :

200.0

nm.
nm.

mm.

Result for Wavelength Scan

Sample used :

1 (Carmoisine)

Concentration of the sample :

max obtained :

ii.

10.0

700.0

516.0

nm.

.
45

ppm.

B. PHOTOMETRIC SCAN
i.
Instrument Parameters
Wavelength, =
516.0
nm.
Path Length =
10.0
mm.

ii.

Result for Photometric Scan

Record the absorbance readings and the unknown concentrations on the table below.
No.
1
2
3
4
5
6
7

Sample
Std 1
Std 2
Std 3
Std4
Std 5
Unknown #1
Unknown #2

Concentration (ppm)
5
15
25
35
45
21.515
41.500

By referring to the standard calibration graph obtained, the r2 value is

Absorbance
0.188
0.575
0.768
1.313
1.668
0.777
1.519

DISCUSSION
The experiment was first started by obtaining five volumetric flasks. Calculations
were then made, using the dilution equation, to decide on how much distilled water should be
added to each volumetric flask to make a series of dilutions with different concentrations (5
ppm, 15 ppm, 25 ppm, 35 ppm, 45 ppm). After the calculations were made, an amount of
distilled water was added to each volumetric flask and was shaken properly. The series of
dilutions were then brought to the Perkin - Elmer UV/Vis Spectrophotometer Lambda EZ210
to undergo two scans; wavelength scan and photometric scan.
For the wavelength scan, a cuvette was filled with the 45 ppm dilution and another
cuvette was filled with a blank solution. Both cuvettes were put into the UV/Vis
Spectrophotometer and the wavelength scan was started. For the photometric scan, the series
of dilutions were put through the scan one by one. The wavelength of maximum absorbance,
max, was obtained through the wavelength scan. On the other hand, the standard calibration
graph acquired (through photometric scanning the serial dilution) was used to obtain the
concentration and absorbance readings of unknown samples #1 and #2.
It was observed that through the wavelength scan, a max of 516.0 nm was obtained.
Next, it was observed that through the photometric scan, an absorbance reading of 0.777 and
concentration of 21.515 ppm were obtained for unknown sample #1, and an absorbance
reading of 1.519 and concentration of 41.500 ppm were obtained for unknown sample #2. As
for the serial dilution samples 1 to 5 (concentrations 5 ppm to 45 ppm), absorbance readings
of 0.188, 0.575, 0.768, 1.313, and 1.668 were obtained.
According to theory, Beers Law states that the relationship between absorbance and
the path length and concentration is directly proportional. It was observed that the results
obtained agreed with the theory stated. Based on the results, an absorbance reading against
concentration of sample graph was plotted and the trend was observed. It was observed that
the trend of the graph was directly proportional, therefore supporting the theory even more.
The purposes of this experiment had been achieved, which was to determine max, as well as

to prepare a serial dilution and generate a standard calibration graph.

Several errors might have occurred during the experiment. Some possibilities were
taken into consideration. Firstly, parallax error, the observer might not have read the
measurements properly thus leading to abnormal deviations in the results. Secondly, physical
error (caused by experimenter), the experimenter might have added the wrong amount of
solution in the sample and claims that it is the right amount but will still show abnormality in
the results. Lastly, the cleanliness of the apparatus, the materials used might not have been
washed properly, leaving residue or sediments behind that will interrupt the scanning process
that will also lead to abnormality in the results.

PLOT GRAPH

CONCLUSION
I have learned that the max of a sample can be obtained by scanning a sample with
radiation using the UV/Vis Spectrophotometer. The wavelength of maximum absorbance,
max, was obtained through wavelength scan; a serial dilution was also prepared and a
standard calibration graph was generated through photometric scan. Doing so, the objectives
of this experiment have been achieved. The flow of the experiment can be improve even
more by reading the manual beforehand, so that nothing unwanted occurs during the
experiment. The other thing is to focus at all times of the experiment, so that the results
obtained will be at best. It is safe to conclude that the purposes of this experiment have been
achieved.

REFERENCES
1) [Brian M. Tissue, 1996], Spectroscopy, http://elchem.kaist.ac.kr/vt/chemed/spec/beerslaw.htm, [13th February 2013]

2) [Mohd Zulkhairi Abdul Rahim], Analytical Chemistry Laboratory Guide, 2011, UniKL
MICET, [14th February 2013]
3) [Anonymous, 2013],
http://wiki.answers.com/Q/What_is_the_purpose_of_wavelength_scan, [14th February
2013]

4) [UW-Madison, 1995-1996],
http://www.uwplatt.edu/chemep/chem/chemscape/labdocs/catofp/measurea/concentr/spec
20/whywipe.htm, [14th February 2013]

5) [mr_notlucky, 2009], http://answers.yahoo.com/question/index?

qid=20090213230835AA0aLTe, [14th February 2013]

APPENDIXES

Pre Laboratory Question

1. State the Beers Lambert Law.
Beer-Lambert Law states that the relationship between absorbance (A) with
absorptivity coefficient (a), path length (b), and concentration of species (c) is directly
proportional.
A = abc / A = bc

2. What is the purpose of wavelength scan?

The purpose is to measure the absorbance and emission of light through a sample. It
can also be used to determine concentrations of a sample.

3. What is the volume needed to prepare a 50 ppm of Carmoisine from a 100 ppm of
Carmoisine in 100 ml volumetric flask?
MiVi = MFVF

[100 ppm] Vi = [50 ppm][100 ml]

Vi = [50 ppm 100 ml] / 100 ppm
Vi = 50 ml

Volume needed is 50 ml.

Post Laboratory Question

1. Why we need to wipe the sides of the cuvette clear surface?
To remove unwanted materials or fingerprints off the outside of the cuvette. Those
unwanted materials could bend or prevent the emitted radiation from being absorbed
by the sample. This will lead to inaccurate and inconsistent readings of a sample.

2. Why UV/Vis need to be warmed up for at least half an hour before starting the analysis?
To warm up the lamps because the emission wavelengths are proportional to
temperature. Half an hour is enough to allow the bulbs and detectors to power up and
reach a constant operating temperature. The reason it needs to have a constant
operating temperature is so that the emission will be constant. If the temperature is not
constant, the emission intensity will vary thus not knowing what contains in the

spectrum produced.