Praktijkvoorschriften pw6
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Praktijkvoorschriften pw6
Determination of zinc in multivitamin tablet using atomic
absorption spectrophotometry
Determination of ethanol in beer using GC method: internal
standard
Determination of phosphate in diet Coca Cola using visible
spectrophotometry
Determination of caffeine in diet Coca Cola using HPLC
Determination of fat content of coffee cream using extraction
Determination of citric acid in Hubba-Bubba chewing gum
using acid-base titration
Determination of total acid content of fruit juice using acidbase
titration
Determination of phosphoric acid in diet Coca Cola
using potentiometry.
Determination of sugar using polarimetry
Het onderdeel calculation is bij de voorschriften weggelaten.
Dit wordt behandeld in de theorielessen.
Praktijkvoorschriften pw6
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Determination of zinc in multivitamin tablet using atomic
absorption spectrophotometry
1. Scope
The method is suitable to determine the zinc content of effervescent
multivitamin tablets (max. 8 mg/tablet) that usually contain several metal
ions and other substances (sugar, sweeteners, etc.) as well.
2. Principle
The tablets under investigation are dissolved in water. The zinc content is
measured by atomic absorption spectrometer (AAS) after adding hydrochloric
acid to the solution. A calibration curve is made by using zinc solutions with
known concentration and then the zinc concentration of the sample solution
can be determined by using the calibration curve. The zinc content of the
sample (effervescent tablets) is calculated from the concentration of the
sample solution.
3. Apparatus
3.1. Equipment
3.1.1. Instruments
• Calibrated analytical balance
• Atomic absorption spectrometer (Type e.g.: SHIMADZU AA-680)
3.1.2. Glassware
• 100 mL volumetric flasks (calibrated as Class ‘A’)
• 200 mL volumetric flasks (calibrated as Class ‘A’)
• 1000 mL cylinder
• 5 mL pipette (calibrated as ‘Class A’)
• 10 mL pipette (calibrated as ‘Class A’)
• 10 mL burette
• 1 mL syringe
• 200 mL beaker (tall)
• Weighing funnels
• Funnels
• Beaker
• Watch-glass
3.1.3. Other equipment
• Filtering stand
• Filtering ring
3.2. Materials and their CAS numbers: see Table 1.
Table 1: Materials and their CAS numbers
Name CAS
Zinc oxide 1314-13-2
(e.g.:Fluka zinc oxide, pss. p. a.)
Concentrated hydrochloric acid
(e.g.:Fluka hydrochloric acid fuming, pss. p. a.)
7647-01-0
3.3. Reagent solutions
3.3.1. ZnCl2 standard solution (1 mg/ mL Zn2+ content)
Weigh 0.1245 g of ZnO on an analytical balance as precisely as
possible and dissolve it in 5 mL concentrated hydrochloric acid. Wash
this solution into a 100 mL volumetric flask using distilled (or ion
exchanged) water, fill it to the mark and mix well.
Note: Working with concentrated hydrochloric acid is very dangerous.
The operator must know and obey the safety rules!
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3.3.2. ZnCl2 solution (0.05 mg mL Zn2+ content)
This is made with a 20 times dilution of the ZnCl2 standard solution:
pipette 5 mL of the ZnCl2 standard solution, wash it into a 100 mL
volumetric flask using distilled water, fill it to the mark and mix well.
3.3.3. Hydrochloric acid solution (0.02 mol/ L)
About 1 mL concentrated hydrochloric acid (using a 1 mL syringe) is
diluted to 600 mL, in a cylinder, with distilled water and mixed well.
4. Preparation
4.1. Preparation of apparatus
4.1.1. Calibration of the analytical balance
Calibrate the analytical balance according to the instruction manual.
4.1.2. Calibration of the volumetric flasks and pipettes
See on a separate part of the web site.
4.2. Preparation of sample
Weigh each of the tablets in the box on an analytical balance and
calculate the average mass (m). Put one of the tablets (make a note
about its mass) into a tall 200 mL beaker, fill the beaker up to 1/3 of
its height with distilled water, cover it with a watch-glass and wait until
it is completely dissolved. Wash all the material spattered on the
watch-glass into the solution with distilled water, mix the solution and
wash it into a 200 mL volumetric flask. Fill the flask, to the mark, with
distilled water and mix well.
If the solution is completely clear and sediment-free, pipette 10 mL of
it into a 100 mL volumetric flask and then fill it up to the mark with
0.02 mol/L hydrochloric acid. (This way we model the effect of the
hydrochloric acid found in the stomach). In this case the preparation of
the sample is finished. If the solution is not completely clear and
sediment-free shake the contents of the 200 mL volumetric flask
thoroughly and pour about half of the solution into a 3 200 mL beaker
put it onto a magnetic stirrer. While stirring, pipette 10 mL of the
solution into a 100 mL volumetric flask, fill it up to the mark with 0.02
mol/L hydrochloric acid and mix well. (Thus we model the case when
the consumer drinks the stirred solution.)
Let the remaining solution settle in the 200 mL beaker. Carefully
pipette 10 mL of the solution into a 100 mL volumetric flask (so that
the sediment is not stirred up), fill it up to the sign with 0.02 mol/L
hydrochloric acid and mix well the solution. (This way we model the
case when the consumer drinks the settled solution without its
sediment.)
4.3. Preparation of the series of the calibrating solutions
This is done by diluting the 0.05 mg/mL ZnCl2 solution, so that we have
a series of calibrating solutions with the mass concentration of 1, 2, 3,
and 4 mg/L Zn2+ respectively. This means that using a 10 mL burette
we have to measure 2, 4, 6 and 8 mL parts of the 0.05 mg cm-3 ZnCl2
solution into 100 mL volumetric flasks fill them up to the mark with
0.02 mol/L hydrochloric acid, mixing well.
5. Procedure
The atomic absorption spectrometer is switched on and the zinc lamp is
adjusted if necessary. After the warming up time and the self-test of the
instrument, the necessary parameters (e.g. lamp number, lamp current,
wavelength, slit, measuring method, integration time, concentrations of the
calibration solutions, etc.) are set and then the measurement is started.
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Atomise (vaporize) the 0.02 mol/L hydrochloric acid solution as BLANK, the
calibrating solutions as STANDARDs and then (once the calibration is finished)
the UNKNOWN solution(s) (the one with sediment and the other without
sediment in the latter case of the point 4.2). As well the absorbance, the
instrument gives the measured mass concentration (ρ [mg/L]).
7. Expression of results
The results of the measurements are given in [mg Zn2+/tablet] units, e.g. 3.6
mg Zn2+/tablet.
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Questions
Choose the correct answer!
1. The temperature of the acetylene – air flame is
a) 1500 - 2000 K
b) 2000 - 2500 K
c) 2500 - 3000 K
d) 3000 - 3500 K
2. How does the solution under investigation get into the flame?
a) A pump presses it.
b) Its flow is helped by gravity.
c) The gases feeding the flame suck it.
d) An auxiliary gas sucks it.
3. The lamp used in atomic absorption spectrometers is a
a) tungsten bulb
b) deuterium lamp
c) mercury vapour lamp
d) hollow cathode lamp
4. The wavelength of the absorbed light
a) depends on the type of flame.
b) is characteristic of the quality of the substance under investigation.
c) is characteristic of the quantity of the substance under investigation.
d) does not depend on the quality of the substance under
investigation.
5. The absorbance
a) depends only on the concentration
b) is proportional to the concentration
c) is inversely proportional to the concentration
d) does not depend on the concentration
Choose the answer that is incorrect!
6. Among the following events which is the one that is not caused by the heat
of the flame
a) The evaporation to dryness of the solution.
b) The evaporation of the substance.
c) The atomisation.
d) The excitations of the atoms.
7. Choose the statement that is not true! Using metal halogenide compounds
in atomic absorption spectrometry is advantageous, because
a) they are not corrosive substances.
b) they usually dissolve easily.
c) they evaporate easily.
d) they can be easily atomised.
8. Choose the incorrect statement! It is not good if large drops get into the
flame, because
a) the flame cools down.
b) the evaporation and the atomisation will be uneven.
c) too many atoms get into the flame.
d) the substance is atomised to an unsatisfactory extent.
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Determination of ethanol in beer using GC
method: internal standard
1. Scope
The goal of this analytical procedure is to determine the percentage ethanol in
beer and other beverages. Dutch normal beer must contain no more than 5
vol% ethanol.
2. Principle
We add the same amount of 1-propanol to each solution to be measured. The
analyte is injected into a GC with polar column. The height of two peaks for
ethanol and propanol is measured. By calculating the ratio (height ethanol :
height propanol), the result does not depend on the injection volume or any air
bubbles present in the needle during injection. Comparing the ratio with a
calibration of a known ethanol concentration will give the exact amount of
ethanol in the sample.
3. Apparatus
3.1. Equipment
3.1.1. Instruments
Gas chromatograph with polar column Carbowax or Tenax. Nitrogen
carrier gas and FID detector. (capillary column could also be used)
3.1.2. Glassware and other equipment
6x25 mL volumetric flasks (calibrated as Class ‘A’)
pipettes
GC injection syringe 0-5 micro litre
3.2. Materials and their safety codes
Name No.
Remarks
ethanol 64-17-5
absolute or 100% ethanol is not recommended
95 vol% is satisfactory
1-propanol 71-23-8
99 vol% is satisfactory free of other alkanoles
acetone 67-64-1
4. Preparation
Prepare the gas chromatograph as described in the user manual. Make sure
that temperatures of oven, injector and detector are stabilised.
Column 1.6 metre Carbowax 20 M by GC- use:
Carrier gas Nitrogen 20 mL per minute
Oven 100 oC
Detector FID 150 oC
Injector 150 oC
Range 10-9
Attenuation 1-1024 to give max peak heights
Note: Use the attenuator to give the largest peaks possible. This will give
better measurements of the peak heights on paper. Since both peaks will be
equally larger or smaller at different degrees of attenuation, this will have no
effect on the ratio.
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5.Procedure
Preparation of calibration curve:
Take 4 volumetric flasks of 25 mL and pipette into each flask 1 mL
1-propanol.
Put respectively 0.5 ; 1 ; 2 and 3 mL ethanol in the flasks. Fill up with
distilled water and mix well.
Preparation of the beer sample:
De-gas 30 cL Heineken beer (in can or small flask) by adding a few
drops of acetone.
Pipette 2x 20 mL into two volumetric flasks of 25 mL
Add 1 mL 1-propanol to each flask and fill up with distilled water.
Inject 1 μL of the calibration solutions from low to high ethanol vol%
Measure the height of both peaks.
Inject 1 μL of the duplo sample Heineken Beer.
Measure the height of both peaks.
6. Expression of results
The results will be given in the % by volume (v/v%) of beer sample.
A conclusion should be drawn: whether the average of the duplo does not
exceed 5 vol%
7. Precision
The precision of this method is 10 % based on 20 student results.
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8. Questions
1
The main goals of gas chromatography are to:
I Separation of mixtures into their components
II quantative determination of components
a Normally first I followed by II
b Normally II first, followed by I
c Its doesn’t matter in what order I,II or II,I
d I and II both are not main goals.
2
For good separation the injector must be heated:
a For better gas flow
b To evaporate the sample
c To remove any fluctuations in temperature
d For warmer components.
3
When the sample contains polar components then:
a A polar stationary phase will lead to better separation
b A polar stationary phase will decrease retention times.
c A non-polar stationary phase will increase retention times.
d Polar or non-polar it doesn’t make any difference.
4
The injector temperature and detector temperature must be set :
a Lower than the column temperature to avoid condensation
b Equal in temperature to the column.
c Higher than the column temperature to avoid condensation.
d to any temperature lower of higher.
5
I Using a FID any water in the sample will cause an extra peak.
II Using a catharometer (TCD) will give a air-peak from any air in the needle.
a I is right but II is wrong.
b II is right and II is wrong.
c Both I and II are right.
d Both I and II are wrong
6
A sample containing both ethanol and 1-butanol is analysed on a polar
column.
a Ethanol will come out first
b 1-butanol will come out first
c They will come out at the same time.
d It cannot be predicted.
7
The use of the internal standard method is often used because
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a Standards for chromatography are cheap.
b It will save the amount of used chemicals
c It doesn’t make any difference when injecting less or more sample
d It is rarely used because it is a bad method.
8
When drawing the calibration graph with horizontally concentration ethanol
and vertically is set to the quotient of areas
a buthanol : ethanol
b area ethanol
c it doesn’t make any difference, all are fine.
d ethanol : butanol
9
A sample liquid is analysed by GC using internal standard. The measurement
is performed using 10 mL liquid and put it into a volumetric flask of 25 mL
adding the internal standard and filling up to the mark. From this solution a
chromatogram is used to determine the ethanol concentration. In the
calibration graph the student reads 5.00 vol % ethanol. The original liquid will
contain:
a 2.00 vol %
b 5.00 vol %
c 10.0 vol %
d 12.5 vol %
10
When the ethanol used for the standard, is common denaturised ethanol this
means that :
a The ethanol is produced chemically and not in a natural way.
b Men cannot drink it because butanol is added.
c Extra methanol is added to make it undrinkable
d It is a brand name like Merck or Sigma.
11
When denaturised ethanol is used an extra peak might appear.
a This extra peak is due to air bubbles
b This is caused by some methanol in the ethanol
c This means the student has not injected rapidly enough.
d This is due to a electrical failure.
12
The best injection technique is to: Fill the syringe with the sample solution and
press it out. Repeat this three times to clean the inside of the syringe.
Then:
a Fill the syringe with sample, clean the needle, suck some air in,
bring slowly into the injector, press rapidly.
b Fill the syringe with sample, suck some air in, bring in rapidly and
inject slowly.
c Fill the syringe with sample, bring in the needle slowly and inject
rapidly.
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d Fill the syringe with sample, bring in the needle rapidly and inject
slowly.
13
When a peak is drawn on paper using the attenuator on 32 (actually 2^32) the
peak is to small. To get the peak twice as large the next injection should be
taken at:
a 2^64
b 2^32
c 2^16
d neither of the above is correct.
14
When using the internal standard method, a student injects, by accident, 50%
more sample, so instead of 2 micro litre he injects 3 micro litre.
a This effects the ethanol and butanol area so it will be a bad analyses.
b This effects the ethanol and butanol heights so it will be a bad analyses
c This doesn’t effect the analyses since the quotient ethanol/butanol will stay
the same.
d This doesn’t effect the analyses as long a the peaks stay separated.
15
I To give acceptable results the peaks must be clearly separated (no
overlap).
II When peaks are symmetrical and thin, the peak heights can be used
as well as peak areas.
a I is true but II is not
b II is true but I is not
c Both I and II are true
d Both I and II are false.
16
I When operating the gas chromatograph, the gas flow should be optimised
for better separation.
II When the separations of the alcohols is performed on a longer column
the separation will be worse and retention times will be longer.
a I is true and II is false
b II is true and I is false
c Both I and II are true
d Both I and II are false
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Determination of phosphate in diet Coca Cola using
visible spectrophotometry
1. Scope
This method is used for the determination of phosphate in diet Coca Cola.
2. Principle
Phosphate is an example of phosphorus derivatives that most of us use
everyday of our lives. Phosphates are of great importance: they are used to
make animal skeletons i.e. bone and teeth, they are used to make ribonucleic
acids, the genetic code, they are used as pH buffers both in body fluids such
as blood and in the laboratory. They are important ingredients in fertilizer.
Refreshing cola drinks contain phosphoric acid. It adds tartness to their
flavour.
Phosphate ions, with iron(II) and molybdate ions, form a blue coloured
complex absorbing around 750 nm.
The phosphate content in the diet Coca Cola is determined by interpolation
from a calibration curve.
3. Apparatus
3.1 Equipment
3.1.1 Instruments
Analytical balance; accuracy = 0.1 mg
Spectrophotometer (VIS),1 cm glass cuvette
Apparatus for degassing and filtering
3.1.2 Glassware and other equipment
Volumetric flasks (class A)
Transfer or one-bulb pipettes
Graduated cylinders
3.2 Materials
Name Grade CAS-No.
Ammonium heptamolybdate tetrahydrate reagent 12054-85-2
Ammonium iron(II) sulphate hexahydrate reagent 7783-85-9
Sulphuric acid reagent 7664-93-9
Potassium dihydrogen phosphate p.a. 7778-77-0
3.3 Reagent solutions
3.3.1 10 g/100 mL ammonium heptamolybdate tetrahydrate in 4 mol/L
sulphuric acid
3.3.2 Dissolve 5 g ammonium iron(II) sulphate hexahydrate in 8 mL
1 mol/L sulphuric acid; dilute the solution to 100 mL
3.3.3 Reagent R : one (1) volume of ammonium heptamolybdate
solution
(3.3.1) and nine (9) volumes of iron(II) solution (3.3.2).
This solution should be freshly prepared.
3.3.4 Potassium dihydrogen phosphate (KH2PO4) p.a.
4. Preparation
4.1 Preparation of apparatus
Switch on the spectrophotometer
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4.2 Preparation of sample
Degass the diet Coca Cola sample by shaking and filtering,
boiling, or use an ultrasonic bath.
5. Procedure
5.1 Preparation of the calibration curve and sample
Process the sample and the calibration curve solutions simultaneously.
Calibration curve
Prepare a standard solution using potassium dihydrogenphosphate
(KH2PO4) p.a.
The calibration curve solutions should contain 0 – 10 mg/L phosphate
(PO43- ). Use 50 mL volumetric flasks.
Do not make up to the mark yet.
Sample preparation
The Coca Cola sample contains approximately 500 mg/L phosphate
(PO43-).
Transfer the correct amount of the degassed Coca Cola sample in a 50
mL volumetric flask. The absorption of the sample should be in the
midpoint of the calibration curve
Do not make up to the mark yet.
Add to all the volumetric flasks : 10 mL reagent R (3.3.3), then make up to the
mark with distilled water and mix well.
5.2 Measurement
Measure the absorbance of the sample and the solutions of the calibration
curve with the spectrophotometer using a wavelength of 750 nm.
6. Quality requirements
All glassware must be rinsed well, some detergents contain phosphate.
7.Expression of results
The results will be given in mg.L-1 PO438.Precision
The standard deviation of the results of 10 students is 2.8%
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9.Questions
1. 300.0 mg potassium dihydrogenphosphate is dissolved in 500.0 mL.
25.00 mL is transferred in a 100.0 mL volumetric flask
The concentration of the potassium dihydrogenphosphate is then :
a. 15 mg/L
b. 150 mg/L
c. 75 mg/L
d. 125 mg/L
2. 1.0971 g ammonium iron(II) sulphate hexahydrate is dissolved in 250.0 mL
Of this solution 10.00 mL is transferred to a 250.0 mL volumetric flask.
From this volumetric flask 5.00 mL is transferred to a 50.0 mL volumetric
flask.
The concentration of the iron(II) ions is then :
a. 9.82 mg/L
b. 5.00 mg/L
c. 2.50 mg/L
d. 4.91 mg/L
3. How much potassium dihydrogen phosphate do you weigh for a 1000.0 mL
standard solution, when a dilution of 5.00 mL of this standard solution in
100.0 mL has a concentration of 1.0 mg/L phosphor?
a. 87.9 mg
b. 93.4 mg
c. 6805 mg
d. 879 mg
4. If the absorption of a solution is 0.5, the transmission is :
a. 3.16%
b. 1.70%
c. 31.6%
d. 50.0%
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Determination of caffeine in diet Coca Cola using HPLC
1. Scope
This method is used for the determination of caffeine in diet Coca Cola.
It is also applicable for the determination of caffeine in other beverages.
2. Principle
Caffeine (1,3,7-trimethylxanthine) is a stimulant that is commonly found in
many foods and drinks that we consume. Caffeine has a mildly addictive
effect on the body; it is therefore interesting to know exactly how much
caffeine is in certain beverages.
One way to analyse caffeine content in beverages is by using highperformance liquid chromatography (HPLC).
Caffeine, an alkaloid from the group of xanthine derivates, can be determined
with HPLC. The cola sample is, after dilution and degassing, suitable for
HPLC analysis on a C-18 reversed phase column.
In this experiment a calibration curve is used; the peak height or the peak
area is measured and plotted against the concentration of caffeine in the
standard solutions.
The caffeine content is determined from the plot.
3. Apparatus
3.1 Equipment
3.1.1 Instruments
Analytical balance; accuracy = 0.1 mg
HPLC with a C-18 reversed phase column like Hypersil C18, Zorbax
C18, 250x4 mm,particle size 3-5 μm
a 20 L sample loop
and UV-detector 254 nm.
Integrator or recorder
Apparatus for degassing and filtering
3.1.2 Glassware and other equipment
100 mL and 1000 mL volumetric flasks (class A)
transfer or one-bulb pipettes (5 , 10 , 20 and 50 mL)
0.45 m porosity syringe filter (nylon, PVDF)
3.2 Materials
Name Grade CAS-No.
Caffeine Reagent 58-08-2
Methanol HPLC 67-56-1
Acetic acid HPLC 64-19-7
Water HPLC 7732-18-5
3.3 Reagent solutions
3.3.1 500 mg/L caffeine solution
Weigh out accurately about 500 mg reagent grade caffeine, and
dissolve in eluent.
3.3.2 Eluent
methanol = 10% in acetic acid, 1 mol.L-1
4.Preparation
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4.1 Preparation of apparatus
HPLC
Flow eluent : 1 mL.min-1
UV detector : detection range : to be determined
4.2 Preparation of sample
Degass the diet Coca Cola sample by shaking and filtering.
5. Procedure
Sample preparation
Transfer 50.00 mL of the degassed Coca Cola sample into a 100 mL
volumetric flask.
Make up to the mark with distilled water and mix well.
Preparation of the calibration curve
Transfer 5.00, 10.00 and 20.00 mL of the standard caffeine solution
into 100 mL volumetric flasks.
Make up to the mark with distilled water and mix well.
Measurement
Use 0.45 m syringe filters (nylon, PVDF) to filter the solutions before
injection. Record the chromatograms of the calibration curve solutions
and the sample solution.
6.Expression of results
The results will be given in mg.L-1.
7.Precision
The standard deviation of the results of 10 students is 13 %
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8.Questions
1. An non-polar component in a sample is separated on a reversed-phase
column. The mobile phase is 30% (m/m) acetone in petroleum ether.
Increasing the percentage of acetone in the mobile phase will:
a. lengthen the retention time of the non-polar component
b. shorten the retention time of the non-polar component
c. not change the retention time of the non-polar component
2. Predict the order of elution for a normal-phase separation :
a. benzene, n-hexanol, n-hexane
b. n-hexanol, benzene, n-hexane
c. n-hexane, benzene, n-hexanol
3. For a HPLC separation, the distribution constant for component A is 3.5,
for B 1.5 and for C 2.5.
The component that will first pass at the end of the column is ;
a. A
b. B
c. C
4. For the HPLC determination of vitamin C in a soft drink we have the
following results:
Calibration results :
Concentration (g.L-1)
Area
Vitamin C
1.25 6.32
Saccharin
1.44 30.4
Sample preparation : 100.0 mg saccharin is added to 25.0 mL soft drink,
the total volume is made up to 100.0 mL
Sample results :
Area
Vitamin C 4.66
Saccharin 19.5
The concentration vitamin C in the soft drink is :
a. 1.0 g.L-1
b. 3.99 g.L-1
c. 3.68 g.L-1
Literature
Skoog,Holler,Nieman : Principles of Instrumental Analysis ,5th edition,
Saunders College Publishing
Chapter 28, High performance Liquid Chromatography.
Internet page: ugrad - www.cs.colorado.edu/pop/facts.html
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Determination of fat content of coffee cream
using extraction
1. Scope:
The method is suitable for the extraction and quantitative determination of fat
in coffee creamers using dichloromethane and methanol. It is in accordance
with the method by Bligh and Dyer (1959).
2. Principle: The extraction of fat from several (food) products under mild
conditions.
3. Apparatus:
3.1 Equipment:
3.1.1 Instruments
Turrax (Ultra turrax )
Centrifuge
Rotary evaporator
3.1.2 Glassware and their equipment
Folding filters (S&S, Ø 150 mm, 595½)
Separation vessel 250 ml
Round bottom vessel 250 ml
Laboratory glass
3.2 Materials and their safety codes
Name CAS no.
Dichloromethane 75-09-2
Methanol 67-56-1
Potassium chloride 7447-40-7
Sodium sulphate 7757-82-6
4. Procedure:
Check beforehand that all equipment meets current regulations.
Weigh in a 250 ml cup, a min. of 10 g and max. of 50 g of the sample
(depending on the expected fat content). Add 50 ml 10% potassium chloride
and mix.
Add successively 100 ml dichloromethane and 50 ml methanol.
Homogenise with the turrax for 3 minutes at appr.12.000 rpm.
Directly after homogenisation, pour the mixture into a separation vessel and
wait for a visible separation. Collect the dichloromethane (lower layer) in a
250 ml cup, add some sodium sulphate and stir.
Filter the dichloromethane over a folding filter into a weighed round bottom
vessel of 250 ml. Evaporate the dichloromethane in a rotor evaporator at 45
°C, discard the dichloromethane from the collection vessel and extend the
evaporation for 30 minutes. Weigh the vessel with the fat (m3 g).
Determine the oil extracted and fat content of the sample by gravimetry.
Remarks:
If no separation occurs in the separation vessel, pour the mixture into some
centrifuge vessels. Centrifuge for 5 minutes at 1500 rpm. Separate the upper
layer and filter the lower layer over a folding filter into a cup. Add some
sodium sulphate and filter the solution into a round bottom vessel.
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Proceed with evaporating the dichloromethane as described above.
5. Expression of results
The results will be given in mass%.
6. Precision
The relative standard deviation of the results of 4 students is 10%.
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7. Questions
1. Which solvents are non-polar:
a. cyclohexane
b. methanol
c. acetonitrile
d. tetrahydrofuran
2. The density of dichloromethane is:
a. equal
b. higher
c. lower
than water.
3. Fat is a:
a. di-ester of a fatty acid and glycerol
b. di-ester of a fatty acid and glycol
c. tri ester of an unsaturated fatty acid and glycerol
d. tri ester of a saturated fatty acid and glycerol
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Determination of citric acid in Hubba-Bubba chewing gum using
acid-base titration
1. Scope
The aim of this analytical procedure is to determine the citric acid content in
Hubba Bubba bubble gum. This bubble gum is available in the U.K. and most
parts of Europe. It is manufactured by Wrigley in Plymouth, UK. The method
described here is based on an analytical procedure used by the Wrigley company
in their Plymouth laboratories. For more information about Hubba Bubba products
see http://www.wrigley.co.uk/HubbaBubba/Index.cfm.
2. Principle
The determination is based on an acid/base reaction between the citric acid in
the bubble gum and standard sodium hydroxide. The citric acid content of the
bubble gum can be calculated from titration results.
3. Apparatus
3.1 Equipment
� kitchen pastry roller
� 250 cm3 conical flask
� 250 cm3 graduated flask
� 100 cm3 graduated flask
� magnetic stirrer and follower
� 10 cm3 burette (reading to nearest 0.02 cm 3)
� top pan analytical balance
3.2 Materials and their CAS numbers
Orange flavoured Hubba Bubba was used (‘Awesome Orange: It's an orange
attack! Let your mouth go wild with this awesome flavour’).
� Sodium hydroxide 1310-73-2
� Phenolphthalein 77-09-8
3.3 Reagent solutions
� Standard 0.100 mol/L sodium hydroxide. If this is not available, dissolve
10.00 g of sodium hydroxide in about 100 mL of pure water. Wash carefully to a
250 mL graduated flask and make up to the graduation mark. Homogenise the
solution. Standardise by titration with 0.100 mol/L hydrochloric acid, itself
standardised against solid potassium hydrogencarbonate.
� Phenolphthalein indicator. Weigh out 0.20 g of phenolphthalein and dissolve in
about 50 mL of methanol. Transfer solution to a 100 mL graduated flask and
make up to the graduation mark with methanol and homogenise the solution.
4. Procedure
� Take one orange flavoured Hubba Bubba bubble gum piece, unwrap it and
place onto a wood block.
� With a ‘kitchen rolling pin’, roll the bubble gum into a very thin strip
approximately
160 x 30 x 0.5 mm.
� Cut the thin strip into small pieces about the size of long grain rice.
� Weigh out 1.00 g of orange flavour Hubba Bubba bubble gum bits.
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� Add to 100 mL of pure water contained in a 250 mL conical flask. Add a
magnetic follower and stopper.
� Stir vigorously for 30 minutes making sure bubble gum bits don’t lump together.
� Add 0.5 mL of phenolphthalein indicator and titrate with 0.1 mol dm -3 sodium
hydroxide contained in a 10 mL burette. End point is pink.
� Repeat twice more and average all three results.
5. Expression of results
Give the mass of citric acid monohydrate in Hubba Bubba bubble gum in
percentage by mass (mass of citric acid monohydrate in 100 g of bubble gum).
The manufacturer’s allowed range is 1.9 – 2.1 percentage by mass.
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Determination of citric acid in Hubba-Bubba chewing gum using
acid-base titration: Questions
1. Citric acid is a tribasic acid. What is the correct formula for citric acid
monohydrate?
a. CH2COOH.CH(OH)CH2COOH
b. CH2COOH.CH(OH).CH2COOH.H2O
c. CH2COOH.C(OH)(COOH)CH2COOH.H2O
d. CH2COOH.CH2.CH2COOH.H2O
2. In what mole ratio do sodium hydroxide and citric acid monohydrate react?
a. 1:1
b. 2:1
c. 3:1
d. 4:1
3. In the reaction between sodium hydroxide solution and citric acid solution
which pair are the spectator ions (i.e. ions which do not change during the
reaction)
a. sodium ions and hydroxide ions
b. sodium ions and hydrogen ions
c. sodium ions and citrate ions
d. hydrogen ions and hydroxide ions
4. 25 mL of sodium hydroxide required 23.8 mL of 0.108 mol/L hydrochloric
acid. What is the concentration of the alkali (in mol/L)?
a. 0.094
b. 0.099
c. 0.103
d. 0.113
5. The calculation says “Using the following to calculate the percentage by mass
of citric acid monohydrate in the Hubba Bubba bubble gum:
Each cubic centimetre of 0.1 mol/L sodium hydroxide is equivalent to 7.0 mg of
citric acid monohydrate.”
Explain how this statement is obtained by answering these questions:
a. How many moles of sodium hydroxide are there in 1 mL of 0.1 mol/mL
sodium hydroxide solution?
b. In what mole ratio do citric acid and sodium hydroxide react?
Note: This is not the same as Question 2, but you can use that answer
to help you.
c. How many moles of citric acid will react with 1 mL of 0.1 mol/L
sodium hydroxide solution?
d. What is the relative molar mass of citric acid monohydrate?
e. What mass of citric acid monohydrate will react with 1 mL of
0.1 mol/L sodium hydroxide solution?
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Determination of total acid content of fruit juice using
acidbase titration
1. Scope
The total acid content in a sample of the fruit juice is determined by titration
with a standardized sodium hydroxide solution. The sample could be
pineapple or grape fruit juice. The acid content of fruit juices consists of
organic acids as citric acid, malic acid, ascorbic acid (Vitamin C) and others.
We take all acids as if they were monobasic.
2. Principle
Total acidity as the sum of monoprotic acids in a sample is determined by
gradually adding sodium hydroxide solution to produce sodium salts of all
fruit acids and water:
HA(aq) + NaOH(aq) → NaA(aq) + H2O
HA means all fruit acids NaA means sodium salts of all fruit acids
3. Apparatus
3.1. Equipment
- ordinary laboratory equipment
3.2. Glassware
- conical flask 250 mL, 3 pieces
- transfer pipette 20 mL, 1 piece
- burette 50 mL
3.3. Materials and their safety codes
Name CAS No. R/S codes
Sodium hydroxide
solution c(NaOH)
0.1 mol/L(fixanal)
1310-73-2 R:36/38
S:26-37
Phenolphthalein
indicator 2%
solution in
methanol
77-09-8 R:11-23/25
S: 7-16-24
4. Procedure
Measure 20.00 mL of fruit juice. Transfer it in a 250 mL conical flask. Add
roughly 75 mL distilled water and three drops of phenolphtalein indicator and
titrate the analyte with the sodium hydroxide solution to the permanent
pinkish -red colour.
Attentions:
1. NaOH standard solution must be free of carbonate and the water used for
dilutions must be boiled (and cooled) freshly before use to eliminate CO2
content.
2. The measurement cannot be performed in strongly coloured or heavily
turbid fruit juices with pulp because the appearance of the pink colour
cannot be seen properly.
5. Result
Sample c(acids) mol/L
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Questions
1. Which of the laboratory glassware listed below is not used with the
titration?
a desiccator
b volumetric flask
c pipettes
d burettes
2. The sodium hydroxide solution could be standardised with:
a hydrochloric acid
b sodium hydrogen carbonate
c standardised hydrochloric acid
3. FIXANAL is an ampoule of solution
a with the same concentration as sample
b with an accurate amount of titrant in ampoule
c which must be standardised
4. Which of these acids is not an acid in fruit juice?
a acetic acid
b citric acid
c ascorbic acid
d oxalic acid
5. During a titration a 20.00 mL sample of fruit juice consumed 12.32 mL of
sodium hydroxide solution of c(NaOH) = 0.1020 mol/L.
The total acidity of sample is:
a 0.063 mol/L
b 0.06283 mol/L
c 0.06282 mol/L
6. The total acidity of a fruit juice is 0.075 mol/L. Calculate the consumption
of sodium hydroxide solution c(NaOH) = 0.0980 mol/L for a 20.00 mL
sample.
The correct answer is:
a 15.31 mL, b 15.30 mL, c 15.35 mL
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Determination of phosphoric acid in diet Coca Cola
using potentiometry.
1. Scope
The determination of phosphoric acid is based on the acid – base reaction of
phosphoric acid with sodium hydroxide. All types of Coca Cola/Pepsi Cola
contain phosphoric acid.
2. Principle
The determination is based on the 1 : 1 mole reaction of phosphoric acid with
sodium hydroxide. The equation of the reaction is:
H3PO4 + OH- → H2PO4- + H2O
3. Apparatus
3.1. Equipment
3.1.1 Instruments
Calibrated analytical balance; accuracy = 0.1 mg
Potentiometer with glass and reference (Ag/AgCl/Cl-) electrode
3.1.2 Glassware and other equipment
beaker 150 mL
magnetic stirrer
motor driven burette or burette
3.2 Materials and their safety codes
Name CAS no.
Sodium hydroxide 1310-73-2
Potassium hydrogen phthalate 877-24-7
3.2 Reagent solutions
Standard sodium hydroxide solution, 0.04 mol/L.
4. Procedure
Sample preparation
Take about 150 mL Coca Cola in a round bottomed flask of 250 mL ,
equipped with a reflux condenser, and heat the flask for 2 hours. After cooling,
pipette 50 mL refluxed Coca Cola into a beaker and place a glass and a
reference electrode in the solution.
Stir the mixture and titrate with 0.04 mol/L sodium hydroxide solution until the
first equivalent point. Titrate further to measure the S – shape. Titrate with 0.1
mL increments around the equivalent point.
Standardisation of sodium hydroxide solution
Weigh accurately about 100 mg potassium hydrogen phthalate on an
analytical balance, transfer it to a glass beaker and dissolve it in ca. 50 ml
water that has been boiled and allowed to cool. Put a glass and reference
electrode into the solution. Stir the mixture and titrate with 0.04 mol/L sodium
hydroxide solution. Titrate with 0.1 mL increments around the equivalent
point.
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5. Expression of results
The results will be given in mg H3PO4 /L Coca Cola.
6. Precision
The relative standard deviation of the results of 3 students is 10%.
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7. Questions
1. 50 mL Coca Cola, which contains 950 mg H3PO4 /L, needs ............. mL
NaOH, c(NaOH) = 0.04204 mol/L to reach the first equivalent point
a. 35.68
b. 13.86
c. 9.540
d. 11.53
2. The pH in the equivalent point of the solution see question 1. is about
Ka (H3PO4/H2PO4- ) = 10-2.13
Ka ( H2PO4-/HPO42- )= 10-7.21
a. 4.7
b. 7.0
c. 8.7
d. 9.9
3. The pH of Coca Cola is about:
a. 7.0
b. 3.2
c. 8.2
d. 10.2
4. Coca Cola must be refluxed:
a. To remove CO2
b. To remove caffeine
c. To remove low boiling acids
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Determination of sugar using polarimetry
1. Scope
The quality (sugar content) of commercial sugar products: semi-white sugar,
sugar or white sugar, extra-white sugar, is determined using polarimetry. The
method is widely used in sugar trade. Also the name saccharimetry is used
when determining the quality of sugar. We adopted it from the Institute for
Public Health, Ljubljana, Slovenia.
2. Principle
In polarimetry we make use of one of the phenomena occurred when light
strikes a matter. Here we observe the passage of polarized visible light
through the solution of a substance (sugar), which rotates it. Namely, certain
compounds, mostly organic (notably those containing asymmetric carbon
atoms) rotate the plane of polarized light. The phenomenon is called optical
rotation and such substances optically active compounds.
Measuring angle of rotation the concentration of a substance in a solution is
determined.
How is a polarized light produced? Most of the light we encounter every day is
a chaotic mixture of light waves vibrating in all planes, which are
perpendicular to the direction of propagation. Such a combination of light
waves is known as unpolarized light. If the light passes through certain
materials (example: calcite), which shows the phenomenon of double
refraction (you see double line when you put a crystal on a line), two beams
are leaving crystal and both are composed of polarized waves. That means
waves of light are now vibrating in only one plane. Specially cut into a prism
(Nicole prism) calcite functions as polarizer giving a polarized beam of light in
a polarimeter. Polarized beam travels through our solution with an optically
active substance and is absorbed by analyser, a second Nicole prism,
depending on the relative position of both prisms to each other and on the
substance in between.
So, using a polarimeter we detect and measure a change in the plane of
polarisation (rotation), induced by optical active samples.
The measured angle of rotation depends upon many variables:
• The type or nature of sample (example: sugar solution)
• Concentration of the optical active components
• The length of the sample tube
• The wavelength of the light source
• Temperature of the sample
We describe the nature of a sample by introducing the specific optical rotatory
power (or specific rotation) of a substance, defined as

[ ] 
 l


in SI units: rad m2 kg-1 (Notice: 2π rad = 360 0 (deg),
where α is the angle of rotation in rad,
γ is the mass concentration in kg/m3,
and l is the length of the sample tube in m. Specific rotation is determined at
a specified temperature Θ (usually 20 oC) and a wavelength of light source
(usually sodium lamp with its D line at 589 nm).
Some substances rotate the light to the right (or clockwise) as viewed looking
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towards the light source, we sign this rotation and α as +, some to the left (or
anticlockwise), signing α as -.
In practical measurements readings are taken at different units:
α in o (deg),
γ in g/cm3,
l in dm
and so
[ ]20
D 

 l
is usually tabulated in o cm3 /g dm.
For example:
Sucrose (cane sugar) solution
[ ]20
D 
= + 66.5
o

 l
[ ] 20
0
/dm at a concentration of 1 g/cm3.
3. Polarimetry of sugar solutions
Polarimetry is frequently used for determining the quality of sugar products.
Measurements are made by polarimeters or saccharimeters with the scale in
angle degrees (o) and sugar degrees (oZ). Angle of rotation depends linearly
on concentration of sugar in the solution other parameters (temperature, light
source, length of the tube) being the same.
Sugar industry with its International Commission for Uniform Methods of
Sugar Analysis (ICUMSA) introduces International Sugar Scale (ISS) in oZ
units. 100.00 oZ units (sugar degrees) belong to Normal Sucrose Solution
prepared from exactly 26.000 g of sucrose dissolved in pure water to 100
cm3. At 20 oC and D sodium lamp rotation for this solution in a tube of 200
mm will be α = +34.626 o (deg). The ISS is linearly divided, i.e. a rotation of
+17.313 o (13 g/100 cm3) equals to a reading of 50.00 oZ.
The 0 oZ point in ISS is fixed by the indication given by the saccharimeter for
pure water.
Normal Sucrose Solution was used to calibrate and standardize polarimetric
methods and instruments. Sugar solutions are not very stable and have to be
renewed regularly.
Today quartz control plates are used as a standard for the calibration of
polarimeters. More find in Techniques (Polarimetry).
Interrelation between both scales is defined from a straight line (y = a.x)
equation:
oZ = 100.00/34.626 o (deg) = 2.889 o (deg)
4. Apparatus
4.1. Saccharimeter graduated for the normal 26 g sucrose, or polarimeter.
- The instrument should be installed in a room where the temperature is
maintained close to 20 0C. Calibrate the instrument against standard quartz
plates.
- Light source consisting of sodium vapour lamp.
Precision polarimeter tubes length 200 mm, error does not exceeded ± 0.02
mm.
- Analytical balance, accurate to within 0.1 mg.
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- Individually calibrated 100 mL volumetric flask with stopper. A flask with
real capacity in the range 100.00 ± 0.01 mL may be used without correction.
Flask with a capacity outside those limits is used with an approximate
correction to adjust the capacity to 100 mL.
- Water-bath, controlled thermostatically to 20 ± 0.1 oC.
4.2. Materials in their safety codes*
Name CAS No. R/S codes
Lead acetate trihydrate
Pb(CH3COO)2 · 3 H2O
Mr = 379.39
6080-56-4 R:61-33-48¸48/22.1-50/53-62 S: 53.1-45-60-61
Diethyl ether
(C2H5)2O
60-29-7
R:6-12,16,19,20
S:1,7,9,15,16,33
*These chemicals are needed to clarify the sugar solution, which is not
always necessary.
4.3. Reagents
- Clarification agent: lead acetate solution (poisonous solution!).
Add 560 g of dry lead acetate trihydrate to about 1000 mL of freshly boiled
water. Boil the mixture for 30 minutes and then leave it to stand overnight.
Decant the supernatant liquid and dilute with freshly boiled water to obtain a
solution with density 1.25 g/mL at 20 oC.
Protect this solution from a contact with the air.
- Diethyl ether (very inflammable!).
5. Procedure
5.1. Preparation of sample solution
Weigh as quickly as possible 26 ± 0.002 g of the sample and transfer it
quantitatively into a 100 mL volumetric flask with approximately 60 mL of
water.
- Dissolve by swirling but without heating
- Where clarification is necessary, add 0.5 mL of lead acetate reagent. Mix the
solution by rotating the flask and wash the walls until the meniscus is about
10 mm below the calibration mark.
- Place the flask in the water-bath controlled to 20 ± 0.01 oC until the
temperature of the sugar solution is constant.
- Eliminate any bubbles formed at the surface of the liquid with a drop of
diethyl ether.
- Make up to volume with water.
- Stopper and mix thoroughly by inverting the flask at least three times.
- Allow to stand for five minutes
5.2. Measurement of rotation
- Maintain temperature 20 ± 0.2 0C for all subsequent operations.
- Obtain the zero correction of the apparatus.
- Filter the sample through the filter paper. Discard the first 10 mL of the
filtrate. Collect the next 50 mL of the filtrate.
- Wash the polarimeter tube by rinsing twice with the sample solution.
- Fill the tube carefully at 20 ± 0.1 oC with the sample solution.
- Remove all bubbles when sliding the end plate in position. Place the tube in
the cradle of the instrument.
- Read the rotation to within 0.05 oZ or 0.02 angular degrees. Repeat four
times. Take the mean of the five readings.
6. Calculation
6.1. The results are expressed in oZ to nearest 0.1 oZ
To convert the angular degrees into degrees Z the following formula is used
o (deg)
0Z = 2.889.
Details are explained in Polarimetry under Techniques.
6.2. Repeatability
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The difference between the two results of two determinations when carried
out simultaneously or in rapid succession on the same sample by the same
analyst, under the same conditions and each representing the mean of five
readings, must not exceed 0.1 oZ.
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Questions
1. When light passes through a material and, on leaving it, vibrates in only
one plane, it is said to be:
a) polarized
b) reflected
c) refracted
2. Look at the two displayed formulae for glucose in its non-cyclic form.
Number the asymmetric C-atom which is typed in bold.
C-atoms are numbered from the aldehyde group –CHO on.
CHO
|
H-C-OH
|
HO-C-OH
|
H-C-OH
|
H-C-OH
|
CH2OH
D(+)-glucose
CHO
|
HO-C-OH
|
H-C-OH
|
HO-C-OH
|
HO-C-OH
|
CH2OH
L(-)-glucose
Which of the following is the correct answer?
a) 2
b) 3
c) 4
d) 5
3. What is the mass concentration of sucrose in a solution at 20 oC if the
length of the tube is 100 mm and the measured angle is +66.5 o ?
a) 1 g/mL
b) 0.5 g/mL
c) 100 mg/mL
4. What angle will be measured with the sample prepared in this experiment?
The length of the polarimeter tube is 200 mm.
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a) 34.58 o
b) 17.3 o
c) 34,6 o
5. What is the name of the phenomenon that a substance exhibits and which
is used to determine the concentration of that substance in a solution?
a) refraction
b) polarization
c) optical rotation
d) absorption of light