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Spectrophotometry

Essay by   •  March 21, 2017  •  Lab Report  •  2,052 Words (9 Pages)  •  1,183 Views

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Introduction

Experiment 1

Spectrophotometry is used to measure the intensity of light by measuring amount of photons absorbed as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. Spectrophotometry is the quantitative measurement of the interaction of ultraviolet (UV, 200 – 400 nm), visible (VIS, 400 - 700nm) and infrared (IR, 700 – 15000nm) radiation with a material (Marczenko & Balcerzak, 2000). Spectrophotometer is a device, which measures the absorbance of solution as light of specified wavelength is passed through it.

Experiment 2

UV-Vis spectrophotometry uses the light in UV and visible part of the electromagnetic spectrum. Light of this wavelength is able to effect the excitation of electron in the atomic and molecular ground state to higher energy levels, giving rise to an absorbance wavelength specific to each molecule. UV-Vis spectrophotometry is used in this experiment to determine the maximum absorption spectrum of oxygenated haemoglobin, deoxygenated haemoglobin, protein (serum albumin, BSA) and nucleic acid (RNA). The absorption spectrum of haemoglobin is measured at the visible region, which is at the wavelength range of 400-700nm while the absorption spectrum of BSA and RNA is observed in the UV region, which range between 200-400nm. Beer-Lambert Law equation is used to calculate the concentration of a substances. Beer-Lambert Law states that the amount of light of a particular wavelength absorbed by a substance across a constant distance is proportional to the concentration of that substance (Parnis & Oldham, 2013). Beer’s Law is written as:

A =               where: A = absorbance[pic 1]

                            = constant (the extinction of coefficient)[pic 2]

                           l  = path length

                           c = concentration

Aim

Experiment 1

To study the concentration of phosphorus in the unknown sample using the colorimetric analysis technique and standard curve plotted.

Experiment 2

To study the maximum absorption spectrum of oxygenated and deoxygenated haemglobin, protein (BSA) and nucleic acid (RNA) by using UV-Vis spectrophotometry analysis.

Results

Experiment 1

Table 1. The absorbance reading of 5 standard solutions with different mass of phosphorus (μg) at the wavelength of 660nm.

Solution

Mass of Phosphorus (μg)

Volume of standard solution (mL)

Volume of distilled water (mL)

Absorbance at 660nm

Average absorbance

Blank

0

0

5.0

0.000

0.000

Standard 1

10

0.1

4.9

0.036

0.0355[pic 3]

[pic 4]

0.035

Standard 2

20

0.2

4.8

0.063

0.064

0.065

Standard 3

30

0.3

4.7

0.087

0.0915

[pic 5]

0.096

Standard 4

40

0.4

4.6

0.119

0.125

0.131

Standard 5

50

0.5

4.5

0.148

0.1455

[pic 6]

0.143

Sample calculation:

Concentration of standard solution = 100 μg/mL

Mass of Phosphorus

Volume of standard solution

100 μg

1mL

10 μg

[pic 7]

[pic 8]

Figure 1. The standard curve of the absorbance at 660nm against the mass of phosphorus (μg).

Table 2. Absorbance at 660nm, mass of phosphorus (μg), concentration of phosphorus (μg/mL), average of concentration of phosphorus (μg/mL) and standard deviation of the sample solutions of X and Y.

Solution

Absorbance at 660nm

Mass of phosphorus (μg)

Concentration of phosphorus (μg/mL)

Average of concentration of phosphorus (μg/mL)

Standard deviation

Sample X (1)

0.054

18.00

2.571

2.571

0

Sample X (2)

0.054

18.00

2.571

Sample Y (1)

0.127

42.33

6.047

6.072

0.035

Sample Y (2)

0.128

42.67

6.096

Sample Calculation:

Mass of phosphorus in solution sample Y (1):

[pic 9]

[pic 10]

[pic 11]

[pic 12]

Concentration of phosphorus (μg/mL) in sample Y (1):

Concentration (μg/mL) = Mass of phosphorus (μg) ÷ Volume (mL)

                          = 42.33μg ÷ 7mL

                          = 6.047 μg/mL

Average Concentration of phosphorus (μg/mL):

[pic 13]

[pic 14]

 [pic 15][pic 16]

...

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