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BIC150X PRACTICAL SCHEDULE 202 5
Experiment Title Date 1 Acid Determination in Apple Juice 21 /07/2 5 2 ANALYSIS OF ASPIRIN TABLETS ("BACK TITRATION") 28 /0 7 /2 5 3 DETERMINE THE CONCENTRATION (in% m/m) OF SODIUM CHLORIDE IN FETA CHEESE
4 DETERMINATION OF THE PERCENTAGE NITRITE IN
PRAGUE SALT
5 DETERMINATION OF THE% m/m CALCIUM IN DRIED MILK POWDER WITH EDTA
6 pH MEASUREMENT TECHNIQUE 25 /0 8 /2 5 7 A POLARIMETRY 25 /0 9 /2 5 7 B REFRACTOMETRY 25 /0 9 /2 5 8 A SPECTROSCOPY 01 /09/2 5 8 B THIN LAYER CHROMATOGRAPHY (TLC) 01 /09/2 5 11 Practical Test 15 /09/2 5
FACULTY OF APPLIED SCIENCES
Diploma in Biotechnology
SUBJECT NAME: BIOANALYTICAL CHEMISTRY 1
P SUBJECT CODE: BIC150X
RACTICAL
MANUAL
REPORT WRITING RUBRIC
Date Date on which the experiment was performed and not the date of the report submission
2 Title A short line summarizing the experiment undertaken 1 3 Aim A sentence or two describing the objective of the experiment
4 Introduction Brief theory describing chemical reactions, and principles of the experiment completed. This includes the necessary balanced equation. Precautions and the application of the technique
5 Procedure Past tense description of the experimental method. This includes mass, volume, and procedure.
6 Results a) Data obtained; Tabulated, presented using correct significant figures, Triplicated where necessary with relevant statistical analysis( mean, standard deviation) b) Mass and volumes necessary to complete the calculations using the data c) Calculations: Representative example calculation where replicates are used and the rest of the calculations can be tabulated in data. Clear balanced equations used in the calculations
7 Discussion Discuss the data obtained by Commenting on the accuracy and precision of the results as well as the possible errors that can be obtained. Compare your results with theoretical values usually search from the internet
8 Answer questions Answer questions from the practical if present 3 9 References^ List of^ literature consulted minimum 3^2 10 Pre-practical^ Schematic representation of the experimental method.
[Total 25]
EXPERIMENT 2
ANALYSIS OF ASPIRIN TABLETS ("BACK TITRATION")
Theory Aspirin is a compound of two acids - acetic acid and salicylic acid. It can be hydrolysed by alkali and the two acids neutralised simultaneously: CH 3 CO 2 C 6 H 4 CO 2 H + 2NaOH → CH 3 CO 2 Na+ HOC 6 H 4 CO 2 Na +H 2 O The presence of the weak acid salts means that the excess alkali is best titrated using phenol red indicator (pH range 6,8 to 8,4 ), although phenolphthalein is satisfactory. Phenol red changes colour from yellow below pH 6.8 to bright pink above pH 8.2. Chemical structures of acetic acid, salicylic acid, and acetylsalicylic acid Method Weigh out accurately a definite number of aspirin tablets (roughly 1,5 g) into a conical flask. Add 20 mL aliquot of standard sodium hydroxide (approx. 1 mol/L) and about the same volume of de-ionized water to the tablets. Simmer gently for ten minutes to hydrolyse the aspirin. Cool the reaction mixture and transfer it with washings to a 250
mL volumetric flask. Dilute to the calibration mark with pure water, then mix well by inverting repeatedly. Pipette 25 mL of the solution into 3 conical flasks, add 3 drops of phenol red indicator, and titrate with standard 0,05 mol/L sulphuric acid. Calculations Calculate the mass of acetylsalicylic acid in each tablet and compare your result with the specification.
SCN-^ + Ag+^ → AgSCN Iron (III) ions are used as an indicator Fe3+^ + SCN-^ → Fe(CNS)2+^ (red) Calculate the molar concentration of NH 4 CNS
3. Determination of the %NaCl in feta cheese by the volhard method Accurately weigh three portions of cheese of about 0,5 g each into three conical flasks. Add about 10 mL of water, 10 mL of concentrated Nitric acid and exactly 50 mL of ± 0,01 mol/L AgNO 3 (std.). Digest on a hot plate / water bath by boiling gently for 10 mins. The digestion is complete when the liquid is a clear lemon colour and the fat layer is free of solids, except for granular AgCI. Cool the mixture. Add exactly 2. 0 mL ferric alum indicator and 1 mL nitrobenzene. Titrate against NH 4 CNS (std.) shaking thoroughly until the first permanent reddish tinge appears. Calculate the %NaCl in the feta cheese.
EXPERIMENT 4
DETERMINATION OF THE PERCENTAGE NITRITE IN
PRAGUE SALT
Background Curing salts are used in food preservation to prevent or slow spoilage by bacteria or fungus. Generally, they are used for pickling meats as part of the process to make sausage or cured meat. Prague salt is generally a mixture of table salt with 6, 25 % sodium nitrite added, which inhibits the growth of bacteria, specifically Clostridium botulinum , to prevent botulism. It also helps preserve the colour of cured meat. Many curing salts also contain red dye that makes them pink to prevent them from being confused with common table salt. Thus curing salt is sometimes referred to as "pink salt". (Curing salts are not to be confused with Himalayan pink salt, which is pure salt with trace elements that give it a pink colour). Figure 1: Prague salt
1. Standardization of iron (II) solution with standard KMnO 4 Pipette 25,00 mL of the iron (II) solution into a conical flask, add 12 mL of (~1,0 mol/L) sulphuric acid and titrate with standard 0,02 mol/L potassium permanganate till a faint permanent pink colour is produced. Repeat for triplicate results. MNO 4 -^ + 5Fe2+^ + 8H+^ → Mn2+^ + 5Fe3+^ + 4H 2 O
EXPERIMENT 5
DETERMINATION OF THE % m/m CALCIUM IN DRIED MILK
POWDER WITH EDTA
Background EDTA forms 1:1 complexes with most metals. A metal ion indicator (e.g. Eriochrome Black T) is used in the detection of the endpoints. In the pH range 7-11 the addition of metal salts changes the colour of the indicator from blue to red. M-In + EDTA → M-EDTA + In Wine red Blue However, when calcium ions alone are titrated with EDTA no sharp endpoint can be obtained with Eriochrome Black T indicator and the transition from red to pure blue is not observed. This is because a relatively stable calcium complex is formed, with a relatively less stable calcium-indicator complex. However, with magnesium ions, a somewhat less stable complex is formed, while the magnesium-indicator complex is more stable than the calcium-indicator complex. So, if magnesium ions are not present in the solution containing calcium ions they must be added, since they are required for the colour change of the indicator. During the titration of a solution containing magnesium and calcium ions with EDTA in the presence of Eriochrome Black T, the EDTA reacts first with the free calcium ions, then with the free magnesium ions, and finally with the magnesium-indicator complex. Since the magnesium-indicator complex is wine red in colour and the free indicator is blue between pH 7 and 11, the colour of the solution changes from red to blue at the endpoint. METHOD Standardization of EDTA with MgSO 4 Prepare 250 mL of a 0,05 mol/L MgSO 4 standard solution using the AR salt. Pipette 25 mL of this solution into 3 conical flasks. Add ~ 1 0 mL of pH 1 0 Buffer and 3- 4 drops of Eriochrome Black T indicator solution. Titrate slowly with EDTA solution until the colour changes from red to full blue.
Determination of % m/m Calcium in dried milk powder Make up the milk powder according to the manufacturer's instructions using 10 g in 100 mL warm water. Pipette 25 mL of this solution into 3 conical flasks. Add exactly 1 0 mL of a ~0,05 mol/L MgSO 4 standard solution. Add ~ 10 mL of pH 10 buffer and 3
- 4 drops of Eriochrome Black T indicator solution. Titrate slowly with EDTA solution until the colour changes from pink to true blue. Calculations
- Calculate the mmoles of EDTA that reacted with the 10 mL MgSO 4 standard.
- calculate the mmoles of EDTA that reacted with the Ca2+.
- Finally, calculate the mass of Ca2+^ per 100 mL and subsequently the % m/m calcium in the milk powder.
➢ Keep the reference hole covered during measurement; Store the electrode dry or in de-ionized water. ➢ Stir the sample or buffers with the electrode. ➢ Re-use buffers. ➢ Wipe the bulb. ➢ Assume pH measurement is simple. Standardization pH meters should not be used without appropriate standardization i.e. measuring the pH of a solution of accurately known pH and adjusting the meter so that the reading corresponds to the known value of the standard buffer solution. In fact, for accurate work, the pH meter is calibrated at two pH values that bracket the expected readings i.e. pH 4.00 and 7.00 for acids; pH 7.00 and 10.00 for alkalis; and pH 4.00 and 10. 00 for the whole range. The buffer solution used for calibration must be stable and must be repeatable from day to day. If a buffer solution is used from a container it should not be returned to the container after use. Try to calibrate with a pH close to the measurement pH. Methods
1. Calibration ➢ Pour sufficient pH 7.00 buffer into a 100 mL beaker so that the electrode can be immersed past the reference liquid junction. ➢ Rinse the electrode with distilled water and blot dry with a tissue. ➢ Place the electrode in the buffer solution and stir the solution with a stirring rod. ➢ Allow 1 minute for electrodes to equilibrate. ➢ Adjust "OFFSET" control to the buffer pH value. ➢ Remove the electrode and rinse with distilled water.· ➢ Repeat the above using pH 4. 00 and pH 10.00 buffers, using the "SLOPE" button to adjust the reading to the correct pH. 2. pH measurement ➢ Rinse the electrode with a sample aliquot. ➢ Place the electrode in the sample and activate the meter. Stir with a stirring rod. ➢ Allow electrodes to equilibrate and reading to stabilize. Record the pH.
➢ Remove the electrode and rinse with distilled water. ➢ Measure and record the pH of the various products provided. Comment on their relative acidity or alkalinity. ➢ When finished store the electrode in a storage solution (usually KOH).
3. Effect of buffers ➢ Pour out 2 separate aliquots (50 mL) of the buffer provided, measure and record the pH. ➢ To one solution add 1 mL 0 .1 mol/L NaOH, stir well, measure, and record the pH. ➢ To the other solution add 1 mL 0 .1 mol/L HCI, measure and record the pH after stirring well. ➢ Comment on your findings ➢ Dilute some fresh buffer 1 0 times. ➢ Measure the pH before and after adding 1 mL 0 .1 mol/L NaOH to one aliquot. ➢ Measure the pH before and after adding 1 mL 0 .1 mol/L HCI to the other aliquot. ➢ Repeat using water instead of buffer. ➢ Record all results and comment.
Experiment 7 B
REFRACTOMETRY
Introduction Refraction is the changing of direction of a beam of light passing into a medium of a different optical density. The refractive index varies with temperature, pressure and the wavelength of light used. It is often expressed as specific refraction RD where p is the density of the solution. RD is independent of temperature and pressure. The instrument used for refractive index is called a refractometer. μ is characteristic for a substance and is thus used to identify compounds and mixtures of compounds. We will use the Abbe refractometer as well as an immersion refractometer to determine the concentration of an ethanol/water mixture. Method ➢ You are provided with an unknown mixture of ethanol and water. Make up the following ethanol/water mixtures: ➢ 0, 10, 20, 30, 40, 50, 60, 70, 80 % ethanol (volume per volume). ➢ Take readings on both refractometers for each of these and for the unknown solution ( at 20 °C ). Also, take a reading for pure ethanol. ➢ Plot a calibration curve for the known ethanol concentrations as %( v/v ) against your readings.
➢ Determine the % ethanol in the unknown solution. ➢ Calculate the specific refraction of pure H 2 O and pure CH 3 CH 2 OH. ➢ There will be one unknown organic solvent that you need to identify using the refractometer reading and a table of R. I. readings for these solvents Relative Density at 20 °C: Water 0. 99823 Ethanol 0. 78945
Experiment 9
THIN LAYER CHROMATOGRAPHY
Introduction Extraction of caffeine from Coke or Coffee Caffeine containing beverages have been used as a stimulant for over a thousand years. Caffeine is a heterocyclic amine found in many ,plant materials and is classified as an alkaloid. Many alkaloids have pharmacological effects, including morphine, codeine, cocaine and nicotine. TLC is a standard technique for the qualitative analysis of mixtures and it is used in organic chemistry to monitor the progress of a reaction or to identify the components of a mixture. The caffeine present in the beverage must first be separated out using an extraction procedure. This involves treating the substance with an organic solvent that will dissolve out the caffeine. Caffeine is more soluble in chloroform than in water, so on shaking the sample with chloroform, any caffeine present in the beverage will go into the chloroform layer. A TLC plate is prepared by spotting a standard and the extracts at the bottom edge of a silica gel coated plastic sheet. The silica gel contains fluorescein F 254 which aids in visualization under UV light. The plate is developed by placing it in a beaker filled with a small amount of developing solvent. As this mobile phase flows up the plate by capillary action, it carries the caffeine along with it. The rate at which a particular component moves depends on whether it tends to dissolve in the developing solvent or to remain adsorbed on the surface of the silica gel stationary phase. More polar compounds will be absorbed more strongly onto the silica gel and will thus move more slowly up the TLC plate. The less polar compounds will move faster. The polarity of the mobile phase can be adjusted by varying the proportions of the solvents.
Figure 1: Procedure for thin layer chromatography (TLC) The results of a chromatographic separation are expressed in terms of Rf values. The Rf is the distance a component has moved relative to the distance moved by the developing solvent. Figure 2: Reading your TLC plate and calculating the Retention Factor (Rf)
