Posted: February 3rd, 2015
Paper, Order, or Assignment Requirements
Given that the standard phosphate solution has a POB4PB3-P concentration of 200 µg ml-1, calculate the concentration of phosphate in flask H to K. Plot a graph of absorbance (A) versus concentration and use this graph to determine the phosphate concentration of river water.
Please draw all graphs on graph paper or use a computer program such as Excel or OfficeHD both result tables.
answer these questions :
QUESTIONS
1. How do you determine the wavelength at which to set the spectrophotometer?
2. What should you do if your unknown solution shows an absorbance reading of 2.3?
EXPERIMENT 4
ULTRAVIOLET/VISIBLE (UV/VIS) SPECTROSCOPY
USafety information
The chemicals listed below will be used in this experiment. The likely hazards associated with each of the chemicals are noted and recommended procedures for handling are given. You must read this page and the experimental description carefully before starting the experiment and before coming into the laboratory. Note any potential hazards and adopt precautions as your safe lab practice. When you are satisfied that you understand any possible difficulties that might arise and the recommended procedures for dealing with them, sign the declaration and have it initialled by a demonstrator. This must be done prior commencing lab work. At the beginning of the lab session demonstrators will quiz you about the safety information and experimental procedure in order to identify your ability to work safely and efficiently. If you fail to prove the ability for safe and efficient work you will not be allowed to start lab practical. Please note that it is you own responsibility to complete the lab practical during time that is allocated to you. Be sure to request information or help if you are in doubt on any point.
Chemical | Hazard | Precautions |
Phosphate solution | Irritant | Do not ingest, avoid skin/eye
contact, wear gloves |
Acid molybdate solution | Corrosive | Do not ingest, avoid skin/eye
contact, wear gloves. |
Ascorbic acid solution | Irritant | Do not ingest, avoid skin/eye
contact, wear gloves. |
UNOTE:
Carefully observe the instructions regarding clean-up of the apparatus. Take great care when handling materials to avoid spillage, splashing etc. Do not ingest and do not breathe chemical vapours. In case of accident or if you feel unwell, report to a member of staff and seek medical advice immediately.
Declaration – I have read and understood the contents of the safety information sheet and the script for the experiment
Signed (student): ……………………………………………………..
Checked (demonstrator): ………………………………………….. Date: ………………………
EXPERIMENT 4
ULTRAVIOLET/VISIBLE (UV/VIS) SPECTROSCOPY
Demonstration of the Beer-Lambert Law
The colorimetric estimation of inorganic phosphate
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LEARNING AIMS
To gain familiarity with the use of laboratory UV/VIS equipment
To verify the Beer-Lambert Law of light absorption by coloured compounds in solution, and to use the result to determine the concentration of a given solution.
LEARNING OUTCOMES
To critically discuss the use of UV/VIS in Chemical Analysis
To manipulate UV/VIS instrumentation
DIRECTED READING
Pharmaceutical Analysis 2PndP Ed; Watson (2005) Elsevier, ISBN: 0443074453; Chapter 4, pp: 87-112 (introduction to UV)
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INTRODUCTION
Molecules of many substances absorb light of characteristic wavelength in the infra-red, visible and ultra-violet regions of the spectrum. When light is passed through these substances, which can be in the form of a solid or solution, the intensity of the light transmitted (I) is reduced compared to the incident light intensity (IB0B).
Beer-Lambert Law: (for monochromatic light only i.e. light of a single wavelength). The intensity of light transmitted (I) by a homogenous liquid decreases exponentially according to the number of absorbing molecules in the optical path. It depends upon the thickness of the solution (L) (Lambert’s Law) and on the concentration (c) of the absorbing molecules present (Beer’s Law):
I/IB0B = 10P- εcL
ε is known as the molar absorption coefficient, molar absorptivity or extinction coefficient.
Transmittance T = I/IB0B (×100 to give %T)
Absorbance A = logB10B(IB0B/I) = logB10B(1/T) = -logB10BT
Hence,
A= εcL The Beer-Lambert Law
Units:
A [dimensionless]
c [mol dmP-3P] SI: [mol mP-3P]
L [cm] SI: [m]
ε [dmP3P molP-1P cmP-1P] SI: [mP2P molP-1P]
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EXPERIMENTAL
Task 1. Demonstration of the Beer-Lambert Law
If the Beer-Lambert Law is obeyed for the dye solution used in this experiment over the range of concentrations used, then a plot of the absorbance (A) against concentration of the dye in solution should give a straight line. Thus by measuring the absorbance of the given solution (X), its concentration can be determined from the graph. Scatter in the calibration plot is usually due to errors in the preparation of solutions or, in single-beam instruments, fluctuations in the light source.
Use the dye stock solution (10 mg dm-3) to make up the mixtures shown in the table below. Use the burettes or pipettes to measure volumes. These measurements must be accurate:
Solution | A | B | C | D | E | F |
Dye (cmP3P) | 2 | 3 | 4 | 6 | 8 | 10 |
Water (cmP3P) | 8 | 7 | 6 | 4 | 2 | 0 |
Absorbance | 0.091 | 0.139 | 0.188 | 0.289 | 0.381 | 0.449 |
1st repeated Absorbance | 0.091 | 0.139 | 0.188 | 0.289 | 0.380 | 0.450 |
2nd repeated Absorbance | 0.091 | 0.137 | 0.185 | 0.288 | 0.378 | 0.449 |
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EVALUATION OF RESULTS
Calculate the concentrations of dye in each solution (A to F) and plot a graph of the absorbance versus this concentration. Draw the best straight line through the points. Determine the concentration of dye solution X.
Please draw all graphs on graph paper or use a computer program such as Excel or OfficeHD
Insert your figure and concentration value into the box below.
Task 2. The colorimetric estimation of inorganic phosphate
Most biochemical compounds are colourless and can only be analysed colorimetrically after reacting them with a specific chemical reagent to give a coloured product. Inorganic phosphates (colourless) react with ammonium molybdate in an acid solution to form phosphomolybdic acid. Addition of a reducing agent (ascorbic acid in this experiment) reduced the molybdenum in the phosphomolybdate to give a blue complex.
Flask | Standard phosphate solution | River water | Absorbance | 1st repeated Absorbance reading | 2nd repeated Absorbance reading |
G (blank) | 0 cmP3 | 0 cmP3P | 0.438 | 0.436 | 0.434 |
H | 0.25 cmP3P | 0 cmP3P | 0.450 | 0.454 | 0.451 |
I | 0.50 cmP3P | 0 cmP3P | 0.550 | 0.550 | 0.553 |
J | 0.75 cmP3P | 0 cmP3P | 0.589 | 0.589 | 0.589 |
K | 1.00 cmP3P | 0 cmP3P | 0.648 | 0.647 | 0.647 |
L | 0 cmP3 | 10 cmP3P | 0.452 | 0.454 | 0.455 |
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EVALUATION OF RESULTS
Given that the standard phosphate solution has a POB4PB3-P concentration of 200 µg ml-1, calculate the concentration of phosphate in flask H to K. Plot a graph of absorbance (A) versus concentration and use this graph to determine the phosphate concentration of river water.
Please draw all graphs on graph paper or use a computer program such as Excel or OfficeHD in the box below.
_____________________________________________________________________
QUESTIONS
How do you determine the wavelength at which to set the spectrophotometer?
What should you do if your unknown solution shows an absorbance reading of 2.3?
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