DEPARTMENT OF PHARMACY
UNIVERSITY OF MALTA
MEDICINAL CHEMISTRY
PRACTICALS PHR 2028
PRACTICALS HANDBOOK
1
Claire Shoemake; GZ
2016
PRACTICAL SESSIONS
BSc.(Hons) Pharm Sci
PHR 2028 Summary of
Sessions
1. Calibration of volumetric glassware
This scope of this session is to demonstrate the importance of eliminating readings
from non-calibrated glassware as a source of error in analytical laboratories. This
particular session involves the calibration of a glass burette using distilled water
or deionised water as a suitable calibration liquid.
Your write up should include information on the methodology used, results
obtained and a discussion on the importance of using calibrated glassware in a
chemical laboratory.
2. Moisture content in Flour
Flour is a worldwide staple foodstuff. Studies indicate that its stability and quality
are compromised in the presence of high humidity. The scope of this practical
session is an estimation of the moisture content of a provided sample of flour
using two different methodologies- specifically the hot air oven and the moisture
analyzer.
Your write up should contain details of the specific methodologies used and
respective references. A comparison of the results obtained from both
methodologies should be carried out using appropriate statistical tools.
3. Water for injection
Water for injections is water used for the preparation of medicines for parenteral
administration, when water is used as a vehicle for dissolving or diluting
substances of preparations for parenteral administration. A sample of water is
provided for the determination of the following parameters:
Acidity and Alkalinity
Oxidisable substances
Sulphates
Conductivity
Residue on evaporation
pH
2
Your write up should include the methodologies used and a discussion of the
result obtained. Comment on other tests which should be carried out to determine
the suitability of use of water for injections.
4. Determination of the sugar content in honey
You have been provided with a honey sample for the determination of its reducing
sugar content.
Your write up must include information on the principle of the test, the
methodology used and calculation of results.
5. Dry Run
Nitrite and Nitrate Analysis in Foods
Nitrites and Nitrates occur naturally in foods but may also be present as a result of
their use as preservatives. A copy of the results obtained in a local survey
conducted to measure the residual level of nitrite and nitrate in a variety of meat
samples is attached.
Describe the methodologies for testing of nitrites and nitrates. Discuss their main
use and importance in preservation. Compare these results to the permitted levels
according to local regulations on the use of preservatives in foodstuffs and
discuss.
3
TABLE OF CONTENTS
PRACTICAL 1
CALIBRATION OF VOLUMETRIC GLASSWARE
Marking Scheme
1.1
Introduction
1.1.1
To Contain vs To Deliver
1.1.2
The Analytical Balance
1.1.3
Volumetric Glassware
1.1.3.1 Direct Calibration
1.1.3.2 Indirect Calibration
1.1.3.3 Relative Calibration
1.1.4
In Conclusion
1.2
Methodology
1.2.1
Calibration of Burette
1.3
Results
1.4
Definitions & Formulæ
1.5.
Further Data Manipulation
1.5.1
Calculation of Standard Deviation
1.5.2
Calculation of Absolute Error
1.5.3
Calculation of Relative Error
1.5.4
Significance of Results
1.0
PRACTICAL 2
CALCULATION OF THE PERCENTAGE MOISTURE
IN FLOUR USING THE HOT AIR OVEN METHOD
Marking Scheme
Introduction
Methodology
Data Manipulation
2.0
2.1
2.2
2.3
3.0
3.1
3.2
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.3
4.0
4.1
4.2
4.3
4.4
4.5
PRACTICAL 3
WATER FOR INJECTIONS
Marking Scheme
Introduction
Methodology
Acidity or Alkalinity
Oxidisable Substances
Sulphates
Residue on Evaporation
pH
Data Manipulation
PRACTICAL 4
DETERMINATION OF THE SUGAR CONTENT IN
HONEY
Marking Scheme
Introduction
Preparing a Sugar Standard Solution – 1%
Standardisation
Results
Calculations
4
p. 5
p. 5
p. 6
p. 6
p. 6
p. 6
p. 7
p. 7
p. 7
p. 7
p. 7
p. 7
p. 9
p. 11
p. 11
p. 11
p. 11
p. 11
p. 11
p. 12
p. 12
p. 13
p. 13
p. 14
p. 15
p. 15
p. 16
p. 17
p. 17
p. 17
p. 17
p. 18
p. 18
p. 18
p. 19
p. 19
p. 20
p. 20
p. 21
p. 21
p. 21
4.6
4.7
4.8
4.9
5.0
5.1
5.2
5.3
Determination
Results
Calculations
Data Manipulation
p. 21
p. 21
p. 21
p. 21
PRACTICAL 5
DRY RUN: NITRITE AND NITRATE ANALYSIS IN
FOOD
Introduction
Supplied Data
Data Manipulation
p. 22
p. 22
p. 22
p. 25
p. 26
READING LIST
5
Practical 1 Marking Scheme
Calibration of the Volumetric Glassware
Name of Student
Carrying out the Experiment
6 marks
Marks
obtained
Criteria
Maximum
marks
2
handles equipment correctly and with skill
observes/ measures systematically and accurately
2
records the results accurately
2
6
Calculations
6 marks
Marks
Obtained
Criteria
Maximum
marks
2
calculates the standard deviation accurately
calculates the standard error accurately
2
calculates the relative error accurately
2
6
Evaluation
8 marks
Mark
obtained
Criteria
comments on the significance of the results obtained.
Maximum
marks
2
shows scientific knowledge and understanding of the results obtained
2
comments on the quality of the experiment and the results obtained
2
suggests improvements to the experiment
2
8
Total Mark
/ 20 marks
Name of Demonstrator: _____________________________
Signature of Demonstrator: __________________________
6
Experiment 1
1.0
Calibration of Volumetric Glassware
1.1
Introduction:
Calibration is the process by which a stated measure such as the volume of a container is
checked for accuracy. In general, measurements of mass can be determined more precisely
and accurately than measurements of volume. Therefore, the mass of the liquid contained or
dispensed by the glassware will be measured and the corresponding volume calculated using
the density of the liquid. However, a relatively small change in temperature causes a change
in the liquid’s volume and thus its density.
1.1.1 To Contain vs To Deliver
Volumetric glassware is calibrated either to contain (TC) or to deliver (TD) the stated
volume. Beakers and graduated cylinders are generally calibrated to contain while most
pipettes and burettes are calibrated to deliver.
1.1.2
The Analytical Balance
The basic measuring device in the laboratory is the analytical balance. The accuracy of the
counterweights inside the balance is much better than one part per thousand and the balances
are serviced and calibrated at regular intervals to ensure their accuracy.
In the most accurate work two corrections are required. One is to correct for difference
between an object weighed in air and the same object weighed in vacuum. According
Archimedes’ principle an object is buoyed up by a force equal to the weight of air it
displaces. Second, is the fact that glass expands with increasing temperature, so the volume
of a container also increases. By convention, volumetric glassware is always calibrated at
20°C. Since the temperature at which the calibration is done may be somewhat different
there is a small correction for the cubic coefficient of expansion of glass. Fortunately the
correction is very small within a few degrees of 20°C and can be neglected in ordinary work.
1.1.3
Volumetric Glassware
Volumetric glassware is calibrated at a specific temperature, usually 20°C, but quite often it
is used to deliver or contain volumes at a different temperature. The temperature variations
make it necessary to adjust samples and/or standards to the calibration temperature before
measurement, or to apply temperature corrections to the volumes measured. Glassware that
is designed to deliver specific volumes also may have specific drain time associated with its
calibrated volume and must be scrupulously cleaned to drain properly. Therefore it can be
seen that variances in volumetric measurements can be a major, if not the chief, source of
error in an analytical laboratory.
There are three general methods commonly employed to calibrate glassware:
1.
2.
3.
Direct, absolute calibration
Indirect, absolute calibration
Relative calibration
7
1.1.3.1
Direct Calibration
A volume of water delivered by a burette or contained in a volumetric flask is obtained
directly from the weight of the water and its density. For example, if at 25°C, a 20.00 ml
pipette delivered 19.970 g of water then the volume delivered at 25°C would be 19.970 g x
1.0040 ml/g = 20.05 ml. At 20°C the volume would be 19.970g x 1.0037 ml/g = 20.04mL.
(See table in Section 3.0 on page 3 for temperature specific values for water density)
1.1.3.2
Indirect Calibration
Volumetric glassware can be calibrated by comparison of the mass of water it contains or
delivers at a particular temperature with that of another vessel which had been calibrated
directly. The volumes are directly related to the masses of water. This method is convenient
if many pieces of glassware are to be calibrated.
1.1.3.3
Relative Calibration
It is often necessary to know only the volumetric relationship between two items of
glassware without knowing the absolute volume of either. The situation arises, for example,
in taking an aliquot portion of a solution. Suppose that it is desired to titrate one-fifth of an
unknown sample, the unknown sample might be dissolved and diluted to volume in a 250 ml
volumetric flak. A 50 ml pipette would then be used to withdraw an aliquot for titration.
For the calculation in this analysis, it would be necessary to know the exact volume of the
flask or the pipette, but it would not be necessary to know the exact volume of the flask or
the pipette, but it would be required that the pipette deliver exactly one-fifth of the contents
of the flask. The method used for the relative calibration in this case would be to discharge
the pipette five times into the flask and marking the level of the meniscus on the flask.
1.1.4
In Conclusion
The use of calibrated glassware is of great importance in a chemical laboratory in order to
ensure that any volumes measured are known with a certain degree of accuracy.
1.2
1.2.1
Methodology
Calibration of Burette
Record the temperature in the laboratory
Wash the supplied burette thoroughly with deionised water such that no drops of
distilled water are left on the internal surface of the burette.
If droplets of water are still observed to adhere to the inner surface of the burette after
delivering deionised water, the burette must be considered as still dirty and the
cleaning process repeated.
Allow the burette to drain for some time.
Clean and dry a small flask using paper towels and a stream of warm air. Handle the
flask using the paper towels since fingerprints and sebaceous oils may cause the
calibration process to be imprecise.
Fit the flask with a stopper.
Weigh the flask on an analytical balance to the nearest 0.1 mg.
Using a transfer pipette, rinse the burette with distilled water at room temperature.
8
Drain the burette completely into a beaker.
Place the burette into the burette clamp.
Ensure that the clamp is mounted tightly and is level.
Using a funnel, re-fill the burette with deionised distilled temperature equilibrated
water.
Ensure that the burette is not over-filled. (The burette must be filled to slightly past
the 0.00ml mark.)
Carefully wipe off any solution spilled on the outside of the burette.
Open the stopcock and drain the burette to the 0.00ml mark.
Ensure that no air bubbles are present in the burette or in the stopcock tip. If any are
present, the burette must be re-drained slightly to force out the air bubbles and refilled.
Touch the beaker to the tip of the burette to remove any adhering drops of water.
Allow the burette to stand for 5 minutes.
If the level of the burette has changed, more water must be added slowly, to reach the
0.00ml mark.
Place the flask under the burette.
Open the stopcock and slowly transfer 10ml into the flask
Touch the tip of the burette to the wall of the flask.
Allow the flask to stand for 30 seconds such that the film of liquid on the walls of the
burette to drain.
Stopper the flask in order to prevent evaporation.
Record the apparent volume of liquid extracted from the burette to the nearest
0.01ml.
Weigh the flask to the nearest 0.1mg.
Repeat the procedure in 10ml increments, all the way to 50ml.
Refill the burette was re-filled and repeat the entire procedure for a further 2 times.
Record your results onto the tables provided in Section 1.3 below. Refer to the
definitions included at the end of Section 1.4 prior to recording your results.
Estimate the Standard Deviation, Absolute and Relative Error of these results.
Comment on the significance of these results
9
1.3
Results
Interval 0-10ml
Final Reading (ml)
Initial Reading (ml)
Apparent Volume (ml)
Mass
Actual Volume (ml)
Correction
Average Correction for interval 0-10ml:
Consequently, the burette delivers ------------ml less than indicated by the burette reading.
Interval 10-20ml
Final Reading (ml)
Initial Reading (ml)
Apparent Volume (ml)
Mass
Actual Volume (ml)
Correction
Average Correction for interval 10-20ml:
Consequently, the burette delivers ------------ml less than indicated by the burette reading.
Interval 20-30ml
Final Reading (ml)
Initial Reading (ml)
Apparent Volume (ml)
Mass
Actual Volume (ml)
Correction
Average Correction for interval 20-30ml:
Consequently, the burette delivers ------------ml less than indicated by the burette reading.
10
Interval 30-40ml
Final Reading (ml)
Initial Reading (ml)
Apparent Volume (ml)
Mass
Actual Volume (ml)
Correction
Average Correction for interval 30-40ml:
Consequently, the burette delivers ------------ml less than indicated by the burette reading.
Interval 40-50ml
Final Reading (ml)
Initial Reading (ml)
Apparent Volume (ml)
Mass
Actual Volume (ml)
Correction
Average Correction for interval 40-50ml:
Consequently, the burette delivers ------------ml less than indicated by the burette reading.
Total average correction over the 0-50ml range =
11
ml
1.4
Definitions & Formulae:
The apparent volume is calculated by finding the difference between the final and initial
volumes read from the burette.
The mass volume is calculated by weighing the flask after adding each 10ml increment by
means of an electronic balance
The actual volume is calculated by simple proportion using the density of water 23°C as
shown:
Example:
1g of water weighs 0.9975415g
Thus Yg of water weighs ? g
Actual volume of water delivered = (0.9975415 x Y)/1 = Zml
The correction factor is calculated by finding the difference between the actual volume and
the apparent volume in each case.
1.5
Further Data Manipulation
1.5.1
Calculation of Standard Deviation:
σ=
Σ [ x – x]2
n-1
1.5.2
Calculation of Absolute Error
Absolute Error = measured value – actual value
1.5.3
Calculation of Relative Error
Absolute error / actual value * 100
1.5.4
Significance of Observed Results
Comment on the significance of the results obtained.
12
Practical 2 Marking Scheme
Calculation of the Percentage Moisture in Flour Using the Hot Air Oven
Name of Student
Carrying out the Experiment
7 marks
Marks
obtained
Criteria
Maximum
marks
2
handles equipment correctly and with skill
observes/ measures systematically and accurately
2
records the results accurately
3
7
Calculations
3 marks
Marks
Obtained
Criteria
Maximum
marks
3
calculates the percentage humidity in the provided sample accurately
3
Evaluation
10 marks
Mark
obtained
Criteria
comments on results obtained.
Maximum
marks
2
shows scientific knowledge and understanding of the results obtained
3
comments on the quality of the experiment and the results obtained
2
suggests improvements to the experiment
3
10
Total Score
/ 20 marks
Name of Demonstrator: _____________________________
Signature of Demonstrator: __________________________
13
Experiment 2
Calculation of the Percentage Moisture in Flour Using the Hot Air Oven
2.1
Introduction
Flour is derived from wheat after this has been subjected to a process known as milling.
Accurate determination of the moisture content of flour is considered to be a very important
process that is instrumental in the determination of its shelf life. The lower the level of
moisture in flour, the better its storage stability will be. The deterioration of baking quality is
also less at lower moisture content. This may be attributed to the retarded respiration and
activity of microorganisms.
Moisture is an important factor in controlling grain infestation. Insects that live on stored
grains and their products, depend upon the moisture supply. Generally, a moisture content of
9% or lower is considered restrictive to infestation. Moisture is also of great importance for
the safe storage of cereals and their products regarding microorganisms, particularly certain
species of fungi. At lower moisture fungi will not grow but at about 14% or slightly above,
fungal growth takes place.
Higher lipolytic and proteolytic activities are related to higher moisture content, which
further lead to loss in nutrients (protein and fat) and production of more free fatty acids
resulting in inferior characteristics.
Adequate food packaging is consequently very important because of the protection that this
affords the enclosed product from contamination by macro and micro-organisms, prevention
from loss or gain of moisture, shielding the product from oxygen and facilitation of
handling.
With respect to moisture, the establishment of methods which reliably quantify the resident
humidity are important in establishing the quality and shelf life of flour samples destined for
human consumption.
2.2
Methodology
The Hot Air Oven Method
Switch the oven on and set it to 131OC
Label the supplied petri-dishes and their corresponding covers
Place the uncovered petri dishes and their corresponding lids into the previously
heated oven facing upwards
After 10 mins have elapsed, remove the petri dishes and their lids from the oven, and
place them into a dessicator until they reach room temperature
Weigh the covered petri dishes and record their weight
Weigh 10g ± 0.05g of flour for each supplied petri dish
Place this amount into each petri dish
Cover each petri dish
Weigh and record the weight of each covered petri dish
Remove the cover once again
Place the uncovered petri dishes together with their cover into the oven for 20
minutes
14
After 30 minutes have elapsed, cover the petri-dishes with their corresponding lid
while these are still in the oven
Remove them from the oven and place them in the dessicator until they reach room
temperature
Weigh and record the weight of the covered petri dishes.
Calculate the moisture content of each sample.
2.3
Data Manipulation
Present your result in tabulated format
Calculate the percentage humidity in the provided sample
Comment on your results critically
15
Practical 3 Marking Scheme
Water for Injections
Name of Student
Carrying out the Experiment
6 marks
Marks
obtained
Criteria
handles equipment correctly and with skill
Maximum
marks
3
observes/ measures systematically and accurately
3
6
Results
4 marks
Marks
Obtained
Criteria
records the results accurately
Maximum
marks
4
4
Evaluation
10 marks
Mark
obtained
Criteria
comments on the results obtained.
Maximum
marks
2
shows scientific knowledge and understanding of the results obtained
3
comments on the quality of the experiment and the results obtained
3
suggests improvements to the experiment
2
10
Total Mark
/ 20 marks
Name of Demonstrator: _____________________________
Signature of Demonstrator: __________________________
16
Experiment 3
Water for Injections
3.1
Introduction:
Water for Injection (WFI) is water purified by distillation or reverse osmosis. It is used for
the preparation of parenteral medicines. Water is used as a vehicle (water for injections in
bulk) and for dissolving or diluting substances or preparations for parenteral administration
(sterilised water for injections).
Water for injections in bulk is a clear, colourless, odourless, and tasteless liquid. It is
obtained from water that complies with the regulations on water intended for human
consumption laid down by competent authorities or from purified water by distillation in an
apparatus of which the parts in contact with the water are of neutral glass, quartz or suitable
metal and which is fitted with an effective device to prevent the entrainment of droplets. The
correct maintenance of the apparatus is essential.
During production and subsequent storage, appropriate measures are taken to ensure that the
total viable aerobic count is adequately controlled and monitored. Appropriate alert and
action limits are set so as to detect adverse trends. Under normal conditions, an appropriate
limit is a total viable aerobic count of 10 microorganisms per 100ml. For aseptic processing,
stricter alert limits may need to be applied. Water for injections in bulk is stored and
distributed in conditions designed to prevent growth of microorganisms and to avoid any
other contamination.
Sterilised water for injections is water for injections in bulk that has been distributed into
suitable containers, closed and sterilised by heat in conditions which ensure that the product
still complies with the test for bacterial endotoxins. Sterilised water for injections is free
from any added substances. It is clear and colourless.
17
Methodology
3.2
The following parameters will be determined for the supplied sample of water for injections:
Acidity or Alkalinity
•
Oxidisable substances
•
Sulphates
•
Residue on evaporation
•
pH
3.2.1
Acidity or Alkalinity
Measure 20ml of tap water in a measuring cylinder and transfer this to a beaker.
Add 0.05ml of phenol red solution. Note any colour change
Add 0.1ml of 0.01M NaOH. Note any colour change
Add 0.15ml of 0.01M HCl. Note any colour change.
Repeat the above tests on a 20ml sample of distilled water
Comment on your results
3.2.2
Oxidisable Substances
Transfer 100ml of tap water into a beaker.
Add 10ml of dilute H2SO4 and boil the mixture.
Add 0.2ml of 0.02M KMnO4 and allow the mixture to re-boil for 5 minutes
Note any colour change
Comment on your results
3.2.3
Sulphates
Transfer 10ml of tap water to a beaker.
Add 1ml of dilute (0.01M) HCl and 0.1ml of 0.001M BaCl2 solution.
Leave the solution to stand for at least an hour.
Note any changes to the appearance of the solution
Comment on your results
18
3.2.4
Residue on evaporation
Weigh an empty beaker
Add 100ml tap water
Weigh the beaker containing 100ml water
Evaporate the tap water to dryness.
Weigh the beaker after the tap water has been evaporated to dryness
Quantify the concentration of the residue on evaporation
Repeat the entire procedure using 100ml water for injection
Comment on your results
3.2.5
pH
Allow an electrode to stand for a few minutes in a beaker containing unionized water.
Record the reading on the pH meter
Fully immerse the electrode into a beaker containing 100ml of tap water.
Record the reading on the pH meter
Comment on your results
3.3
Data Manipulation
Discuss the results obtained. Mention other tests that you consider vital in the quality control
of water for injections.
19
Practical 4 Marking Scheme
Determination of Sugar Content in Honey
Name of Student
Carrying out the Experiment
6 marks
Marks
obtained
Criteria
Maximum
marks
2
handles equipment correctly and with skill
observes/ measures systematically and accurately
2
records the results accurately
2
6
Calculations
6 marks
Criteria
Standard solution: calculates the total sugar required to reduce the Cu 2+
Marks
Obtained
Maximum
marks
3
ions
calculates the percentage reducing sugars in honey
3
6
Evaluation
8 marks
Mark
obtained
Criteria
comments on the results obtained.
Maximum
marks
2
shows scientific knowledge and understanding of the results obtained
2
comments on the quality of the experiment and the results obtained
2
suggests improvements to the experiment
2
8
Total Mark
/ 20 marks
Name of Demonstrator: _____________________________
Signature of Demonstrator: __________________________
20
Experiment 4
Determination of the Sugar Content in Honey
4.1
Introduction
Honey is a sweet fluid produced by honey bees (genus Apis), and derived from the nectar of
flowers. A label stating pure honey implies that no other additives have been added to the
honey including water or other sweeteners.
Honey derives its sweetness from the monosaccharides fructose and glucose and has
approximately the same relative sweetness as granulated sugar (97% of the sweetness of
sucrose, a disaccharide). Honey has attractive chemical properties for baking, and a
distinctive flavor which leads some people to prefer it over sugar and other sweeteners.
Most micro-organisms do not grow in honey because of its low water activity of 0.6.
However, honey frequently contains dormant endospores of the bacterium Clostridium
botulinum, which can be dangerous to infants as the endospores can transform into toxinproducing bacteria in the infant's immature intestinal tract, leading to illness and even death.
The study of pollens and spores in raw honey (melissopalynology) can determine floral
sources of honey. A main effect of bees collecting nectar to make honey is pollination,
which is crucial for flowering plants.
4.2
Preparing a Sugar Standard Solution – 1%
1. Weigh 4.75g pure sucrose.
2. Add 7.5ml HCl and dilute with water to approximately 50ml.
3. Store several days at room temperature (about 7 days at 12-15oC or 3days at 2025oC).
4. Dilute to 500ml. (acidified 1% invert sugar solution is stable for several months)
5. Neutralise approximately 75ml of the sugar solution with 1M and 0.5M NaOH
accordingly.
6. Dilute to desired concentration immediately before use.
4.3
Standardisation
1. Accurately pipette 12.5ml of Fehling’s solution A and Fehling’s solution B into a
conical flask.
2. Add antibumping granules.
3. Fill the burette with the neutralised sugar solution till the 0.00ml mark.
4. Add 8ml of the sugar solution into the conical flask.
5. Heat the cold mixture to its boiling point on wire gauze over a Bunsen burner and
maintain moderate boiling for 1 minute.
6. Without removing from flame add 1ml of 0.2% aqueous methylene blue solution.
7. Complete titration within total boiling time of approximately 3 minutes by small
additions (2-3 drops) of sugar solution to decolouration of indicator.
8. Maintain continuous evolution of steam to prevent reoxidation of Cu+ or
indicator
9. After complete reduction of Cu2+ methylene blue is reduced to colourless compound
and the solution resumes an orange colour which it had before addition of indicator.
10. Repeat step 1 – 9 to obtain three titre values
21
4.4
Results
Final Reading (ml)
Initial Reading (ml)
Titre value (ml)
Average titre value for standard sugar solution: __________________________
4.5
Calculations
1. Taking the first titre value as a rough value, use the other two titre values to obtain an
average.
2. Multiply the average titre by mg/ml standard solution to obtain the total sugar
required to reduce the Cu2+.
4.6
Determination
1. Accurately pipette 12.5ml of Fehling’s solution A and Fehling’s solution B into a
conical flask.
2. Add antibumping granules.
3. Fill the burette with the honey solution till the 0.00ml mark.
4. Add 5ml of the honey solution into the conical flask.
5. Heat the cold mixture to its boiling point on wire gauze over a Bunsen burner and
maintain moderate boiling for 15 seconds.
6. Without removing from flame add 1ml of 0.2% aqueous methylene blue solution.
7. Complete titration by small additions (2-3 drops) of honey solution to decolouration
of indicator.
8. Maintain continuous evolution of steam to prevent reoxidation of Cu+ or indicator.
9. After complete reduction of Cu2+ methylene blue is reduced to colourless compound
and the solution resumes an orange colour which it had before addition of indicator.
4.7
Results
Final Reading (ml)
Initial Reading (ml)
Titre value (ml)
Average titre value for honey solution: __________________________
4.8
Calculations
1. Taking the first titre value as a rough value, use the other two titre values to obtain an
average.
2. Calculating the % reducing sugar in honey:
Titre value for standard sugar solution
Titre value for honey solution
4.9
Data Manipulation
Comment on the results obtained
22
X 100 =
Experiment 5
Dry Run: Nitrite and Nitrate Analysis in Food
5.1
Introduction
Nitrites and nitrates occur naturally in meats, but are also used as preservatives for hams,
bacon and pickled meats. The brine baths used for this purpose may contain nitrite and
nitrate levels well over a 1,000ppm and needs to be monitored on a regular basis in order to
ensure that the meats do not exceed the statutory level of 200ppm. Apart from their
preservative properties nitrites and nitrates have the effect of enhancing the natural redness
of the meat products.
5.2
Supplied Data
The following is a table of results obtained in a local survey conducted to measure the
residual level of nitrite and nitrate in a variety of meat samples.
25
25
25
25
25
26
B3
B3
B3
B3
B3
B4
26
B4
26
B4
26
B4
26
B4
26
B4
27
27
27
27
27
27
B4
B4
B4
B4
B4
B4
B7
B7
B7
B7
B7
B7
31
31
31
31
31
31
Foodstuff
Sample
No.
Residual Nitrate
Level
(ppm)
Country
2
3
4
5
6
1
Residual Nitrite
Level
(ppm)
5.8
5.6
5.3
6.4
8.2
5.7
Ham
Ham
Ham
Ham
Ham
Mortadella (Sausage
Type)A
Mortadella (Sausage
Type)A
Mortadella (Sausage
Type)A
Mortadella (Sausage
Type)A
Mortadella (Sausage
Type)A
Mortadella (Sausage
Type)A
Mortadella B
Mortadella B
Mortadella B
Mortadella B
Mortadella B
Mortadella B
Sausages (Maltese)
Sausages (Maltese)
Sausages (Maltese)
Sausages (Maltese)
Sausages (Maltese)
Sausages (Maltese)
24.6
28.9
32.4
33.9
41.4
169.4
Malta
Malta
Malta
Malta
Malta
Malta
2
5.5
160.0
Malta
3
5.4
135.1
Malta
4
5.8
155.9
Malta
5
6.5
185.7
Malta
6
6.1
169.8
Malta
1
2
3
4
5
6
1
2
3
4
5
6
5.2
6.2
5.2
7.5
6.4
6.2
0.2
0.1
0.1
0.1
0.0
0.0
129.3
114.4
147.7
132.5
131.4
156.0
16.0
12.5
14.7
17.3
10.1
10.8
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
23
32
32
32
32
32
32
33
33
33
33
33
33
34
34
34
34
34
34
35
35
35
35
35
35
36
36
36
36
36
36
37
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C3
37
C3
37
C3
37
C3
37
C3
37
C3
38
38
38
38
38
38
39
39
39
39
39
39
40
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
Bacon (Back) A
Bacon (Back) A
Bacon (Back) A
Bacon (Back) A
Bacon (Back) A
Bacon (Back) A
Bacon (Collar) B
Bacon (Collar) B
Bacon (Collar) B
Bacon (Collar) B
Bacon (Collar) B
Bacon (Collar) B
Bacon (Streaky)C
Bacon (Streaky)C
Bacon (Streaky)C
Bacon (Streaky)C
Bacon (Streaky)C
Bacon (Streaky)C
Beef Burgers A
Beef Burgers A
Beef Burgers A
Beef Burgers A
Beef Burgers A
Beef Burgers A
Beef Burgers B
Beef Burgers B
Beef Burgers B
Beef Burgers B
Beef Burgers B
Beef Burgers B
Sausages
(BBQ/Grill) A
Sausages
(BBQ/Grill) A
Sausages
(BBQ/Grill) A
Sausages
(BBQ/Grill) A
Sausages
(BBQ/Grill) A
Sausages
(BBQ/Grill) A
Sausages(Beef)B
Sausages(Beef)B
Sausages(Beef)B
Sausages(Beef)B
Sausages(Beef)B
Sausages(Beef)B
Sausages(Beef)C
Sausages(Beef)C
Sausages(Beef)C
Sausages(Beef)C
Sausages(Beef)C
Sausages(Beef)C
Sausages (Chick. &
Turk. Franks.)D
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
58.7
54.1
58.9
54.1
54.0
55.7
4.0
3.6
3.9
3.2
4.2
3.3
0.0
17.9
19.0
20.4
18.3
17.2
0.3
0.4
0.3
0.2
0.3
0.2
0.3
0.2
0.3
0.2
0.3
0.4
0.1
49.9
52.9
53.3
46.7
51.4
44.0
501.7
482.6
491.5
477.4
506.6
464.5
81.0
71.3
75.3
77.5
75.1
79.5
31.1
41.9
31.3
34.6
45.3
31.4
21.1
20.5
27.8
21.5
19.1
25.3
37.5
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
2
0.2
35.7
Malta
3
0.2
34.4
Malta
4
0.2
35.4
Malta
5
0.2
33.8
Malta
6
0.1
34.4
Malta
1
2
3
4
5
6
1
2
3
4
5
6
1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.1
82.6
73.3
75.0
78.7
67.3
70.6
69.1
57.1
52.5
51.2
52.9
51.8
161.2
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
24
40
C3
40
C3
40
C3
40
C3
40
C3
41
C3
41
C3
41
C3
41
C3
41
C3
41
C3
42
C3
42
C3
42
C3
42
C3
42
C3
42
C3
43
C3
43
C3
43
C3
43
C3
43
C3
43
C3
44
C3
44
C3
44
C3
44
C3
44
C3
Sausages (Chick. &
Turk. Franks.)D
Sausages (Chick. &
Turk. Franks.)D
Sausages (Chick. &
Turk. Franks.)D
Sausages (Chick. &
Turk. Franks.)D
Sausages
(Frankfurters) E
Sausages
(Frankfurters) E
Sausages
(Frankfurters) E
Sausages
(Frankfurters) E
Sausages
(Frankfurters) E
Sausages
(Frankfurters) E
Sausages
(Frankfurters) E
Sausages
(Frankfurters) F
Sausages
(Frankfurters) F
Sausages
(Frankfurters) F
Sausages
(Frankfurters) F
Sausages
(Frankfurters) F
Sausages
(Frankfurters) F
Sausages
(Frankfurters) G
Sausages
(Frankfurters) G
Sausages
(Frankfurters) G
Sausages
(Frankfurters) G
Sausages
(Frankfurters) G
Sausages
(Frankfurters) G
Sausages
(Frankfurters) H
Sausages
(Frankfurters) H
Sausages
(Frankfurters) H
Sausages
(Frankfurters) H
Sausages
(Frankfurters) H
2
6.1
162.6
Malta
3
4.9
154.5
Malta
4
5.8
157.9
Malta
5
5.4
162.4
Malta
6
5.8
148.3
Malta
1
2.5
26.9
Malta
2
2.4
24.6
Malta
3
2.3
28.0
Malta
4
2.1
23.5
Malta
5
2.6
31.3
Malta
6
2.6
31.4
Malta
1
40.1
170.9
Malta
2
37.8
160.2
Malta
3
37.5
151.6
Malta
4
39.3
169.8
Malta
5
38.9
165.4
Malta
6
39.0
168.1
Malta
1
8.6
215.5
Malta
2
8.7
210.6
Malta
3
7.3
201.5
Malta
4
9.6
221.2
Malta
5
9.1
218.7
Malta
6
8.2
206.7
Malta
1
8.3
57.9
Malta
2
8.3
60.7
Malta
3
8.3
61.9
Malta
4
8.4
57.1
Malta
5
8.0
59.0
Malta
25
44
C3
45
45
45
45
45
45
46
46
46
46
46
46
47
47
47
47
47
47
48
48
48
48
48
48
49
49
49
49
49
49
50
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
C3
5.3
Sausages
(Frankfurters) H
Sausages (Pork) I
Sausages (Pork) I
Sausages (Pork) I
Sausages (Pork) I
Sausages (Pork) I
Sausages (Pork) I
Sausages (Pork)J
Sausages (Pork)J
Sausages (Pork)J
Sausages (Pork)J
Sausages (Pork)J
Sausages (Pork)J
Sausages (Other) K
Sausages (Other) K
Sausages (Other) K
Sausages (Other) K
Sausages (Other) K
Sausages (Other) K
Sausages (Other) L
Sausages (Other) L
Sausages (Other) L
Sausages (Other) L
Sausages (Other) L
Sausages (Other) L
Sausages (Other) M
Sausages (Other) M
Sausages (Other) M
Sausages (Other) M
Sausages (Other) M
Sausages (Other) M
Sausages (Other) N
6
8.5
62.4
Malta
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.3
7.7
8.9
8.4
7.4
7.6
6.5
6.8
5.5
5.6
6.3
5.8
10.0
10.4
9.9
10.2
9.8
10.8
0.6
57.4
61.1
74.4
60.0
76.9
52.0
68.5
60.2
59.4
51.9
50.9
55.2
58.0
56.4
60.8
60.7
56.2
59.9
35.3
48.1
45.1
48.8
45.2
43.3
56.9
56.6
56.1
56.8
54.9
55.8
26.5
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Malta
Data Manipulation
Describe the methodologies for testing of nitrites and nitrates. Discuss their main use and
importance in preservation. Compare these results to the permitted levels according to local
regulations on the use of preservatives in foodstuffs and discuss.
26
READING LIST
European Pharmacopoeia: 6th Edition. Council of Europe; 31 Jul 2007
Kanare, H. M. Writing the Laboratory Notebook, Washington DC: American
Chemical Society, 1985.
27