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MIDLANDS STATE UNIVERSITY
DEPARTMENT OF CHEMICAL TECHNOLOGY
HCT 204: CHEMICAL INSTRUMENTATION 1
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EXPERIMENT 1:
Determination of the distribution of trace heavy metals in urban roadway dust in Gweru.
INTRODUCTION
Dust is useful as an indicator for pollution. Various parameters can be measured in dust samples chief
among which are the heavy metals like Pb, As, Cu, Zn, Ni, Fe, Mn, Al to name a few.When introduced
into the human body by inhalation, ingestion or through diffusion via the skin(in the form of salts like
ZnCrO4 ) these metals cause various maladies ranging from skin lesions through gastro tract problems to
severe brain damage.
People working in dusty environments are exposed to high levels of various toxic metals. The dust that
enters the body through inhalation is mostly that which is airborne. In protected areas like open
verandas and shades at garages, flea markets and bridges , this air borne dust gradually settles down at
a rate dependent on their densities. For dust particles of similar composition, the density should vary
directly with particle size. Small particles are air –borne for longer periods than larger ones and end up
being inhaled into the human body.
The ease with which dust samples can be taken enhances its use as a pollution index. It is found almost
everywhere and can be sampled using very simple equipment.
Dusts along roadways are due to a number of sources. Because of the presence of metals in the earth’s
crust, the existence of these metals in roadway dust is to be expected. Automobiles contribute direct
exhaust emission and tyre wear particles which probably come from body rusting and ablation from the
interior of the exhaust system. It is apparent that streets with high traffic densities should also have high
metal levels.
The construction and demolition of structures or buildings that has been painted with heavy metal –
based paints contributes a wide range of metal s into the vicinity of roadways. Some industrial activities
like welding, spray painting, metal casting emit toxic metals into the environment.
LITERATURE REVIEW
A literature review is a general survey of the work that has been carried out elsewhere and published in
journals by other workers on levels of trace heavy metals in roadway dust.
You are supposed to carry out a literature review and report your findings in less than two pages.
Remember it is very important to take note of references in your literature review.
REAGENTS AND CHEM ICALS REQUIRED
Analytical grade (AR) reagents are to be used throughout.
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Nitric Acid SG 1.40
Perchloric Acid 72% (w/w)
Hydrochloric Acid SG 1.34
Standard solutions for various metals (1000ppm, spectrosol)
PROCEDURES
Sampling
Identify a sampling site of your choice and prepare an appropriate sampling plan.
Carry out your sampling according to your sampling plan by scooping the dust with clean spatulas into
well labeled polythene bags and tie with strings.
Samples should be collected at 2m intervals starting from the edge of the road going away from the
road.The total number of samples to be collected should be enough to allow one to draw a good graph
of the metal distribution.
Standard Solution Preparation
Using 1000ppm commercial stock solutions transfer 10ml into a 100ml volumetric flask and dilute to the
mark with distilled water. This gives a 100ppm working standard for each metal.
Prepare from the working standard appropriate calibration standards by serial dilution measuring 1ppm;
2ppm; 4ppm; 6ppm; and 8ppm. Prepare composite standards. As and Hg standards should be prepared
according to the requirements of the methods used to analyse these metals i.e. hydride vapour
generation and mercury vapour generation.
Sample Treatment

Air-dry the dust samples at room temperature, i.e 24-30oC.

Weigh the dry sample on an analytical balance and sieve the samples through a series of
stainless steel of diameter 600,400,200,100 and 50 microns.

Weigh each fraction and put it in a well labeled bag.

To 0.4000g portions of the prepared dust in 250ml beakers add distilled water to form a sherry.
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
Add HNO3 (5ml) and HCLO4 (0.5ML)

Heats the mixture on a hot plate until a residue remains. Add 2M HCL (2.5) to the residue and
transfer quantitatively the contents to a 100ml volumetric flask bringing to the mark using
distilled water.

Analyse the samples by Flame AA or Graphite furnace AA depending on the level of each
element in the samples. Use the following instrumental condition in your analysis by FAA.
METAL
WAVELENGTH(nm) BAND PASS(nm)
LAMP
CURRENT(mA)
FLAME TYPE
Cd
228.8
0.5
4
Oxidising
Cr
357.9
0.2
5
Reducing
Cu
324.7
0.5
4
Oxidising
Fe
248.3
0.2
5
Oxidising
Mn
279.5
0.2
5
Oxidising
Ni
232.0
0.2
4
Oxidising
Pb
217.0
1.0
5
Oxidising
Zn
213.9
1.0
5
Oxidising
Express each fraction as a percentage of the bulk sample and the metal levels as mg metal/kg dry
sample.
Draw graphs showing the relationship between;
i.
Weight of fraction and particle size
ii.
Metal levels and particle size
iii.
Metal level and distance from the road
Discuss and conclude your results.
References
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References are expected to come from your literature review.
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EXPERIMENT 2:
Ultraviolet spectrophotometric determination of Aspirin, Phenacetin and caffeine and Anadin tablets
using solvent extraction.
PRINCIPLE
Caffenol and Anadin tablets are a mixture of aspirin, phenacetin and caffeine. Each of these substances
has characteristic absorption in the ultraviolet region, with the principle maximum lying at 227 for
aspirin, 275nm for caffeine and 250nm for phenacetin.In the procedure, a powdered tablet is dissolved
in methylene chloride and the aspirin is separated from the phenacetin and caffeine by extracting it into
aqueous sodium bicarbonate solution. The separated aspirin is back –extracted into methylene chloride
by acidifying the aqueous layer and is then measured spectophotometically at 277nm. The phenacetin
and caffeine that remain in the original methylene chloride layer are determined in a mixture as
described in analytical texts in Chapters on UV/VIS spectrometry according to Beer’s law. According to
Beer’s law, when two absorbing species in solution have overlapping peaks, the total absorbance. A is
the sum of two absorbances. For two absorbing species,
A=axbcx + aybcy
OR A= Exbcx + Eybcy
Where A is the total absorbance observed ax and ay are the absorptivities of X and Y, Ex and Ey are the
molar absorptivities of X and Y in grams per litre of the solution and b is the path length. For two
unknowns, two measurements have to be made and absorbencies at each peak will be,
A1=Ax1 +Ay1 =Ex1bcx + Ey1bcy
And Ax2 + Ay2 = Ex2bcx + Ey2bcy
Concentration of x and y can be obtained by solving these equations.
Equations
A
HCO-3
A CH2CI2
A (277nm)
P
CH2CH2
P (250nm)
+ C (275nm)
c
Reagents and Chemicals
Provided CH2CI2, 4% (wt /vol) NaHCO3 solution (chilled), concentrated. HCl, 1M H2SO4.
To prepare
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Standard solutions. Prepare individual standard solutions of about 100mg/L, 20mg/L and 10mg/L each of
aspirin, phenacetin and caffeine in methylene chloride as follows:
Weigh about 25mg (to the nearest 0.1mg) of each, transfer to 250ml volumetric flasks, and dissolve to
volume with methylene chloride. Dilute 10 and 5ml of this solution to 50ml in 50ml volumetric flasks to
prepare the 20 and 10mg/h solutions, respectively.
PROCEDURE
Weigh accurately and record the weight of one tablet. This should be equivalent to about 220mg aspirin,
160mg phenacetin, and 30mg caffeine. To minimize required dilutions and save on solvents, cut the
tablet into quarters and weigh out one –quarter portion to be analysed. Crush to a fine powder in a
beaker. Add, with stirring, 20ml methylenechloride; then transfer the mixture quantitatively to a 60ml
separatory funnel, rinsing all particles in with a little more methylene chloride. Extract the Aspirin from
the methylene chloride solution with two 10ml portions of chilled 4% sodium bicarbonate to which has
been added two drops hydrochloric acid, and then with one 5ml portion water. Wash the combined
aqueous extracts with three 10ml portions of methylene chloride (to remove traces of water) into a
50ml volumetric flask and dilute to the mark with methylene chloride. Then dilute further a 1ml aliquot
of this solution to 50ml with methylene chloride in a volumetric.
Acidify the bicarbonate solution (aqueous extract), still in the separatory funnel, with 6ml
of1MH2SO4.This step should be performed without delay, to avoid hydrolysis of aspirin. The acid must
be added slowly in small portions. Mix well only after the most of carbon dioxide evolution has ceased.
The pH at this point should be 1 to 2 (pH test paper). Extract the acidified solution with eight separate
10ml portions of methylene chloride and filter through an ethylene chloride wet paper into a 100ml
volumetric flask. Dilute to volume. Then, Dilute further a 5ml portion of this solution to 25ml with
methylene chloride in a volumetric flask.
Record absorbance versus wavelength curves for the standard solutions and unknown solutions
between 200 and 300nm. (This step may be deleted if you do not have a recording ultraviolet
spectrophotometer). Does the wavelength of 277nm appear to be the most suitable wavelength for the
determination of aspirin? Do the wavelengths of 250 and 275nm appear to be the best wavelengths for
the measurement of the absorbance for the mixture of phenacetin and caffeine? Explain. Using the
absorbencies of the standard and the unknown aspirin solution at 277nm) calculate the percent aspirin
in the caffeine and /or Anadin tablets and the number of milligrams of aspirin per tablet keeping in mind
the dilutions.
To calculate the concentrations of phenacetin and caffeine, the absorbencies of phenacetin and caffeine
standards and of the methylene chloride extract of the sample must all be read at both 250 and 275nm.
/using these absorbencies, calculate the percentage phenacetin and caffeine in the APC tablets and the
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milligrams of each per tablet. See Chapter 13 for the spectrophotometric determination of mixtures.
(G.D Christian, Analytical Chemistry)
Note: Aspirin tends to decompose in solution, and analysis should be performed as soon possible after
preparing solutions.
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EXPERIMENT 3:
Qualitative Gas Chromatographic analysis of a multicomponent mixture of pesticides.
Introduction and Overview
Gas Liquid chromatography (GLC) is a type of chromatography in which the mobile phase is a gas, such
as nitrogen, helium, etc, and the stationary phase in an inert liquid. The sample is usually in liquid form,
but is flash vapourised as it is injected into the instrument and is maintained in the gaseous state
through the instrument. The major components of a gas chromatograph are shown in block diagram
below:
Carrier
Gas
Flow
Controller
Injection
Part
(heated)
Column
Oven
Detector
(heated)
Electrometer
Recorder
Carrier Gas or Mobile Phase
The carrier gas chosen depends; on the detector to be employed; nitrogen or argon is used with the
most popular detector, the flame ionization detector (FID). The carrier gas is supplied at a reduced
pressure from a large gas cylinder equipped with a pressure regulator. Often the carrier gas is filtered
through tubes containing a drying agent, a molecular sieve and oxygen scrubber to remove moisture,
impurities and oxygen from the prior to its entering the column. The flow of controller is needle valve or
other device used to control the gas flow rate. In some instruments a rotometer is used to measure the
the actual flow rate. A flow rate of 75cm3 per min, is most often used for 6,4mm o.d. columns, while a
flow rate of 25cm2 per min is used for 3,2 o.d. Columns.
The injection Part
The sample is introduced into the GC through the injection Part, a small hearted chamber capped with a
septum. The sample is introduced by means of a small calibrated syringe. The septum is pierced by the
Syringe needle and reseals when the syringe needle is withdrawn /typical volumes injected into packed
columns are 1-3ul.
The injection part temperature part temperature should be high enough (e.g. 2200C) to vapourise the
sample instantly (i.e. flash vapourisation). Sample vapourisaton should be rapid so that the vaporized
sample is swept by the carrier gas into the column as a discrete’’plug’’
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The column
The column where the actual chromatographic separation occurs is enclosed in an oven that maintains
the desired temperature.
A conventional ‘’packed’’ column is filled with a granular solid support coated with a thin layer of liquid
stationary phase. Separation occurs by differences in the distribution of the various sample components
between the carrier gas and the liquid stationary phase. (s/p).
The s/p is normal coated evenly on the surface of the solid support with a solution of the liquid phase
and then evaporating off the solvent. The solid support must have a uniform pore diameter and a large
surface area. These properties are needed to support an adequate coating of stationary liquid phase and
provide good contact with the mobile phase. The particles should be of regular shape with good
mechanical strength to permit an efficient, well-packed column. The solid support can be made from
silica and other materials. Columns can be metal or glass. Glass is preferred because it is inert towards
most organic compounds. After packing a column is conditioned before its use by passing carrier gas
through it at elevated temperatures to remove volatile impurities.
The Detector
The function of the detector is to sense when a compound is leaving the column and to provide a signal
that is proportional to the concentration of the compound of the compound in the carrier gas stream.
Several types of detectors are available. The FID is the most common and responds to all organic
compounds. The detector is heated to the temperature needed to keep the sample compounds from
condensing.
The Electrometer
The output from the detector is a very small electric current. This is fed into an electrometer which
amplifies and converts the detector output to a voltage that is large enough to be recorded.
The Recorder
The recorder records the voltage from the electrometer as a function of time to give a chromatogram
showing the separated sample components as peaks in the chromatogram.
Theory of Gas liquid chromatography
Chromatographic Efficiency
The width of chromatographic peaks is a measure of the efficiency of chromatographic system. The
system includes the entire instruments and not just the column. The ability to obtain sharp, narrow
peaks is often expressed in terms of a plate number. A large value for a plate number indicates high
chromatographic efficiency and excellent separation ability.
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The plate number, N, is related to the length of the chromatographic column, L, by the equation,
H = L/N
Where H is the plate height.
Effect of flow rate on chromatographic efficiency:
The Van Deemter equation is the classical statement of the effect of flow rate on chromatographic
efficiency. The simplified form of this equation is
H = A + B/u + Cu
Where u is the average linear gas flow rate in cm/sec, and A, B and C are constants. A plot of H versus u
shows that H has a minimum value at a certain value of u. This is the optimum value of u for
chromatographic separation.
H
----------------
u opt
Temperature and temperature programming
Temperature is a major factor in adjusting conditions for a satisfactory separation. When sample
components elute rapidly and are incompletely resolved, lowering the column temperature will slow the
elution and probably improve the peak resolution. If a mixture containing both high and low a
component is to be separated, the temperature needed to separate the boiling compounds may slow
the separation of the high –boiling components too much. Late eluting peaks will be broader and
resolution often poor. In such cases temperature programming can be used. In this technique, the
column temperature is increased linearly with time at a preset rate. The more volatile sample
components are separated at the lower temperatures while the higher- boiling compounds gradually
move at a faster rate through the column so that their peaks appear earlier on the chromatogram.
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Principle of method
An acetone extract of the specimen is partitioned between hexane and saturated brine. The hexane,
containing the non polar pesticides (organophosphorus, organochlorine and carbaryl), plus fat, is
cleaned up on a wood’s column and then examined by the GLC,TLC Brine solution containing the polar
pesticides is extracted by ethyl acetate and extract examined by GLC,TLC, e.t.c. If only one type (polar or
non-polar) is being sought the full procedure need not be followed, the appropriate parts are selected.
Procedure
Put 10g of soil sample and 10g anhydrous sodium sulphate and 50ml acetone in a250ml beaker and
homogenize by stirring. Filter the contents through a fluted filter paper and place on water bath with fan
to evaporate to approximately 5- 10ml.
Transfer the residue to a 250ml separating funnel containing 100ml of saturated sodium chloride
solution and rinse the beaker with 2 x 5 ml hexane and 1 x 5 ml acetone adding the rinsing to the
separating funnel. Shake vigorously for 2 minutes, allow the layers to separate and run the aqueous
layer into the original beaker. Transfer the hexane to a 250ml separatory funnel.
Re-extract the aqueous layer twice or more, each with 10ml hexane. Wash the combined hexane with 2
x 10 ml brine solution by gentle shaking, and add the washings to the main brine solution, which is then
reserved for the extraction of the polar pesticides.
If necessary, dry the combined hexane with a little solid anhydrous sodium sulphate and then decant
onto 1.5g celite in a 50ml beaker, washing the sodium sulphate with a little hexane. Evaporate the
hexane at low temperature, with occasional stirring until a homogenous hexane-free friable powder is
obtained and transfer to a small wood’s column for clean up, Fill in the large wood’s columns with
Florasil and saturate with hexane. Elute from the small columns into the large columns using dimethyl
sulphaxide (DMSO) (6ml).Elute from the large columns with 50ml hexane containing 15% of ether.
Concentrate the eluate to 10ml and reserve for GLC analysis.
Extract the combined brine solution with 3 x 50 ml ethyl acetate (vigorous shaking for 5 minutes) drying
each extract successively with the small quantity of anhydrous sodium sulphate and evaporating
successively in a 150ml beaker, finally to almost dryness. Dissolve in acetone and make up to 10ml, and
reserve for GLC.This solution contains the polar organophosphorus pesticides.
GLC ANALYSIS
It is advisable to inject first on FID, then NPD and possibly after appropriate dilution, on ECD.
Confirmation of identity can sometimes be partly accomplished by injection on more than one column,
but if possible, TLC confirmation should be done.
FID – Flame Ionisaton Detector, non selective, detects both polar and non polar pesticides.
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NPD- Nitrogen Phosphorus Detector; selective, detects polar pesticides.
ECD- Electron capture Detector; selective, detects non polar pesticides
Stationary Phase: 10% OV101 and 2% OV 17
GLC CONDITIONS
Inject Pot temperature: 2300C
Column temperature:
2200C
Detector temperature: 2500C
INJECTION PROCEDURE
Inject 1ul of standard pesticide, parathion until there is a consistent retention tin. Inject 1ul of blank
prepared the same way as the sample extracts. Inject your unknown sample and record the retention
time. Calculate the relative retention time. Inject the various available standards and identify the
unknowns by calculating the relative retention time.
Questions
1. Why is anhydrous sodium sulphate used in the extraction procedure.
2. Comment on the use of the two solvents; acetone and hexane.
3. Why is it important to make sure that no samples in acetone are injected while using ECD as
detector?
Discuss and conclude your results.
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EXPERIMENT 4:
Quantitative Gas Chromatographic Analysis of alcohol in a multicomponent mixture (Internal standard
technique)
Principle
The basis for quantitative analysis by Gas Chromatography is that the area under each peak is
proportional to the concentration of the sample compound, assuming constant conditions of column
temperature, flow rate, etc
The areas of peaks are easily measured using modern electronic integrators. Manual methods include
triangulation and cutting and weighing. These manual methods are naturally less accurate.
Most instrumental errors can be eliminated by using the internal standard method. In this method, a
constant amount of a pure compound (the internal standard) is added to a specific volume of the
unknown sample and to several synthetic mixtures containing different amounts of the compound to be
measured.
The synthetic mixtures are chromatographed first and a plot is made of percent compound versus ratio
of peak area of the compound to the peak area of the standard. The unknown is then chromatographed,
the peak areas of the compound and internal standard are measured, and their ratio is calculated. The
percentage (or concentration) of the compound in the unknown sample is then read from the graph.
Results from this technique can be satisfactory reproduced even if operating conditions (such as flow
rate and temperature) have varied slightly from run to run.
Apparatus and Reagents
1. A gas chromatograph with a Flame ionization detector will be used for this experiment. Please
become familiar with the operating instructions for the particular instrument available,
including, the direction of the power supply control unit and the record recorder paper available
for the recorder. (A good chromatographic column for the experiment uses Porapak Q
stationary phase. The column is operated at approximately 1800C giving retention times around
5 minutes).
PROCEDURE
Preparation of calibration curve solutions
1. Internal standard (n- propanol) – Dilute 0.4ml n-propanol in distilled water and reconstitute to
one litre volumetric flask. This solution is 0.04% n-propanol in water.
2. Prepare a 5 %( m/v) ethanol solution in distilled water. The specific gravity of this solution
should be 0.9759 at 15.60C at 200C
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3. Intermediary Standards
Prepare as follows:
1ml of stock (solution) standard to 100ml water= 50mg/dL
a) 2ml of stock standard to 100ml water gives 100mg/dL
b) 3ml of stock standard to 100ml water gives 150mg/ dL
c) 4ml of stock standard to 100ml water gives 200mg/dL
d) 5ml of stock (solution) standard to 100ml water= 250mg/dL
e) 6ml of stock standard to 100ml water gives 300mg/dL
f)
7ml of stock standard to 100ml water gives 350mg/ dL
g) 8ml of stock standard to 100ml water gives 400mg/dL
4.
WORKING STANDARDS
Dilute each intermediary standard (5.0ml in a volumetric flask using the 0.04% n- propanol.Store
in a refrigerator.
SAMPLE PREPARATION
a) Heparin anti-coagulated blood is preferred, although any other anti-coagulant can be
used.
b) Using a pipette add 5ml of sample (blood) into a 50ml-volumetric flask.
c) Into the same volumetric flasks dilute the sample to the mark using the aqueous
internal standard
7. GLC Setting
a) Connect the column according to instruction in the manual and optimize for about 30 to 60
minutes.
b) For Pye Unicam 304, the following would be a general guide line and can be used as a guide line
for other instrument type.
Column temperature
:
1800C
Detector temperature
:
2500C
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Injector temperature
:
2300C
Hydrogen pressure
:
18psi
Air pressure
:
5psi
Nitrogen pressure
: 26psi
Chart recorder speed
: 300mm/hr
Chart range
: 10mµ
8. Analysis of Standards and Samples
a) Inject the highest standard and adjust the instrument for the best resolution.
b) Inject each standard and each sample and record peak areas of both ethanol and n-propanol
c) Calculate the ratios of ethanol peak areas versus those of n-propanol.
d) Calculate the concentration of alcohol in your samples in mg/dL
NB: A standard curve can be used to calculate the concentration in each sample, or one can use
the standard that has the nearest ratio to that of sample.
Questions
What are the advantages of using the internal standard technique?
Why did you choose your method for area determination?
Could you have predicted the exit order of the components without determining their retention
times? What factors determined this.
Do your chromatogram exibit ‘tailing’ what causes it and what conditions could be changed to
prevent it?
Reference:
1. Fritz and Schenk ‘Quantitative Analytical Chemistry’ 5th Edn., 1987.
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EXPERIMENT 5
UV-VIS Spectroscopy: Colorimetric Determination of Phosphorus in Cola Drinks
Phosphorus is a form of phosphate can be determined calometrically by formation of a complex with a
molybdate compound in acid solution. The reaction proceeds as follows:
7H3PO4 + 12(NH4)6Mo7O24.4H2O
7(NH4)3[ PO4(MoO3)12] + 51NH4+ 51OH- = 33H20
The colourless hexavalent molybdenum phosphate complex is reduced to a blue pentavalent form by
ascorbic acid in acidic medium and can be measured spectrophotometically at 830nm.
REAGENTS
Make a stock solution (1 litre) of 1000ppm phosphate using potassium dihydrogen phosphate in
deionised water. From the stock solution, prepare a working standard solution (100ml) of 10ppm
phosphate.
Dissolve 1.00g of ammonium heptamolybdate (NH4)6Mo7O24.4H2O in 50ml deionised water.
Dissolve 1.75g of ascorbic acid in 100ml of deionised water.
Dilute 17ml of concentrated sulphuric acid in 200ml of deionised water.
Prepare a reducing solution by mixing 39ml of the ammonium molybdate solution, 60ml of ascorbic acid
solution, and 125ml of the diluted sulphuric acid solution in a 250ml volumetric flask and filling to the
mark with deionised.
Sample
A cola soft drink is provided. The sample has been let to stand at atmospheric pressure for at least 24h
prior to the practical (why is it important?). Prepare a diluted sample solution (20 x dilution) in a
volumetric50ml volumetric flask. Fill to the mark the deionised water.
Procedure
Pipette in 50ml volumetric flasks suitable quantities of the working standard solution in order to obtain
calibration solutions with final concentrations of 0, 0.8, 1.6, 2.4, 3.2, 4.0 and 6.0 ppm PO43Pipette 1ml portions of the diluted sample solution into the three 50ml volumetric flasks. To each of the
nine flasks; add 20ml of the reducing solution. Fill up to the mark the deionised water. Heat the
calibration and test solutions for 45mins in a water bath at 500C. In the meantime, ensure yourself that
the colour of the cola sample will not interfere in the spectrophotometric quantification of the blue
pentavalent molybdenum phosphate compound at 830nm. How is this done? Is there any interference
from the food colouring?
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Measure the absorbance of each solution at 830nm.Plot a calibration curve with the absorbance as a
function of the phosphate ion concentration. Calculate the phosphorus (P) content in your sample.
Evaluate the precision of the analysis (assuming no error in the calibration procedure), by reporting the
95% confidence limits for the result. Assuming an average consumption of 35bottles (at 300ml) of cola
drink per person and year, how much phosphorus is needed annually to meet the production demand in
the cola industry in Zimbabwe?
ACKNOWLEDGEMENTS
D.Lozano-Calero, P.Martin-Palomeque, S.Madueno-Loriguillo, J.CHEM.Educ.1996, 73, 1173-1174
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