Posted: February 3rd, 2015

How do you determine the wavelength at which to set the spectrophotometer?

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

 

  1. Set the spectrophotometer to read at 425 nm. Fill a cuvette with deionised water and use this as a blank to zero the instrument. Load the cuvette (do not touch the optical surface i.e. the sides through which the light will pass) with each solution in turn and record the absorbance readings. Determine the absorbance of the given solution X of unknown concentration.

 

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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.

 

  1. Take six clean 50.00 cmP3P volumetric flasks and label them G to L. Prepare solutions as given in the table below. Use pipettes to measure volumes.
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

 

  1. To each flask add 2.5 cmP3P acidic molybdate solution and 1.0 cmP3P ascorbic acid solution. Mix gently and make up to the 50.00 cmP3P mark with deionised water.

 

  1. Allow the flasks to stand for at least 10 minutes for the colour to develop and then measure the absorbance at 690 nm of solutions H to L against the blank (solution G).

 

_____________________________________________________________________

 

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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