1. To record and characterization of IR spectra of at least one organic compounds.

Objective:

To record and characterize the IR spectrum of an organic compound (Acetone) and identify
its functional groups using Fourier Transform Infrared Spectroscopy (FTIR).

Materials Required:

 Organic compound sample (Acetone)

 Fourier Transform Infrared Spectrometer (FTIR)
 Potassium bromide (KBr) for solid samples
 Liquid cell for liquid samples
 Mortar and pestle (for solid samples)
 Pellet press machine
 Weighing balance
 Desiccator

Procedure

1. Sample Preparation

1. Take a few drops (2-3) of the Acetone

2. Place the sample in an IR liquid cell with NaCl or KBr windows.

2. Recording the IR Spectrum

1. Instrument Setup:
o Turn on the FTIR spectrometer and let it warm up.
o Set the scanning range to 4000 cm⁻¹ – 400 cm⁻¹.
o Set a resolution of 4 cm⁻¹ and take an average of 32 scans.

2. Background Scan:
o Run a background scan with a clean NaCl/KBr cell or a pure KBr pellet.

3. Sample Scan:
o Place the sample cell or pellet in the IR beam path.
o Record the IR spectrum of the sample.

3. Data Analysis and Interpretation

 The recorded IR spectrum will show absorption peaks corresponding to different

functional groups in the organic compound.
 Compare the peaks with standard IR absorption ranges to determine the chemical
structure.

Results and Calculations

Table 1: IR Absorption Peaks and Functional Group Identification

Example: IR Spectrum of Acetone (CH₃COCH₃)

Peak Functional Group Possible Bond Vibration

(cm⁻¹)
3000-2850 C-H Stretching Alkane (CH₃, CH₂)
1715 C=O Stretching Carbonyl (Ketone)
1375-1360 C-H Bending Methyl (-CH₃) Group
1220-1100 C-O Stretching Ether/C=O Interaction
Graphical Representation

The FTIR spectrum is plotted with:

 X-axis: Wavenumber (cm⁻¹)

 Y-axis: Absorbance (A.U.)

the FTIR graph for Acetone (CH₃COCH₃).

FTIR spectrum for Acetone (CH₃COCH₃). The labeled peaks correspond to the C=O
(carbonyl), C-H (alkane), and C-O (ether) functional groups, confirming its structure.

Conclusion

 The IR spectrum of acetone showed a strong C=O stretch at 1715 cm⁻¹, confirming
the presence of a ketone functional group.
 The peaks at 3000-2850 cm⁻¹ correspond to C-H stretching, indicating aliphatic
hydrocarbons.
  This analysis successfully characterized acetone using Infrared Spectroscopy,
confirming the presence of expected functional groups.

2. IR analysis and identification of human body stones

Objective:

To identify the chemical composition of human body stones (e.g., kidney stones, gallstones)
using Fourier Transform Infrared Spectroscopy (FTIR) and analyze the obtained spectra.

Materials Required:

 Sample of human body stone (Kidney stone, gallstone, etc.)

 Fourier Transform Infrared Spectrometer (FTIR)
 Potassium bromide (KBr) for pellet preparation
 Mortar and pestle
 Pellet press machine
 Weighing balance
 Desiccator
 Spectral analysis software

Procedure:

1. Sample Preparation:

1. Cleaning the Sample:

o Wash the stone sample with distilled water to remove impurities.
o Dry the sample in an oven at 50°C for 2 hours.

2. Grinding the Sample:

o Crush the sample into a fine powder using a mortar and pestle.
o Ensure the particle size is less than 10 µm for better spectral analysis.

3. Pellet Preparation (KBr Method):

o Weigh 2 mg of the powdered stone sample.
o Mix it with 200 mg of dry KBr (Potassium Bromide) using a mortar.
o Place the mixture in a pellet press machine and apply 6-8 tons of pressure to
form a transparent pellet.
o Store the pellet in a desiccator to avoid moisture absorption.

2. FTIR Spectral Analysis:

1. Instrument Setup:
o Turn on the FTIR spectrometer and allow it to warm up.
o Set the scanning range between 4000 cm⁻¹ to 400 cm⁻¹.
o Set a resolution of 4 cm⁻¹ and take an average of 32 scans.

2. Background Correction:
o Run a background scan using a pure KBr pellet.
 3. Sample Analysis:
o Place the prepared sample pellet in the IR beam path.
o Record the IR spectrum and identify characteristic peaks.

4. Data Interpretation:
o Compare the obtained spectrum with reference spectra to identify the
chemical composition of the stone.
o Assign functional groups based on the absorption peaks.

Results and Calculations:

Table 1: FTIR Absorption Peaks and Chemical Identification

Peak Functional Possible Compound

(cm⁻¹) Group
3500-3300 O-H Stretching Calcium Oxalate (Hydrate)
1650-1600 C=O Stretching Calcium Oxalate
1450-1400 C-O Stretching Calcium Carbonate
1050-1000 P-O Stretching Calcium Phosphate
870-850 C-H Bending Cholesterol (Gallstones)
600-550 S=O Stretching Cystine (Amino Acid Stone)

Graphical Representation:

A graph of Absorbance vs. Wavenumber (cm⁻¹) is plotted. The peaks correspond to

characteristic functional groups, which help in identifying the type of stone.

 X-axis: Wavenumber (cm⁻¹)

 Y-axis: Absorbance (A.U.)

A sample FTIR spectrum typically shows peaks corresponding to calcium oxalate,

phosphate, carbonate, or cholesterol, depending on the stone type.
a sample FTIR spectrum for human body stones. The characteristic peaks are labeled,
indicating the presence of functional groups corresponding to calcium oxalate, phosphate,
carbonate, cholesterol, and cystine.

Conclusion:

By comparing the observed IR absorption peaks with standard reference data, we can
determine the primary composition of human body stones.

 Kidney stones are often composed of calcium oxalate, calcium phosphate, or uric
acid.
 Gallstones contain cholesterol, bilirubin, and calcium salts.
 Cystine stones exhibit unique peaks around 600-550 cm⁻¹.

3. Identification and spectrophotometric determination of aspirin, phenacetine and

caffeine in pharmaceutical samples.

Objective:

To identify and quantify aspirin (acetylsalicylic acid), phenacetin, and caffeine in

pharmaceutical formulations using UV-Visible spectrophotometry.

Materials and Reagents:

  Aspirin (Acetylsalicylic acid), Phenacetin, and Caffeine standards
 Pharmaceutical sample (tablet or powder)
 Ethanol or Methanol (as solvent)
 Phosphate buffer (pH 6.8)
 UV-Vis Spectrophotometer
 Volumetric flasks (10 mL, 50 mL, 100 mL)
 Pipettes and burettes
 Analytical balance
 Filter paper and funnel
 Beakers

Step 1: Preparation of Standard Solutions

1. Weigh 100 mg each of Aspirin, Phenacetin, and Caffeine.

2. Dissolve separately in 100 mL of methanol or ethanol to prepare 1000 µg/mL stock
solutions.
3. From each stock solution, prepare working solutions (10, 20, 30, 40, 50 µg/mL)
using phosphate buffer (pH 6.8).

Step 2: Sample Preparation

1. Weigh and crush a known amount of the pharmaceutical sample (tablet or

powder).
2. Dissolve in 100 mL methanol and sonicate for 15 minutes.
3. Filter the solution and dilute to obtain a 10–50 µg/mL concentration range.

Step 3: Spectrophotometric Analysis

1. Set the UV-Vis spectrophotometer to scan from 200–400 nm.

2. Run the blank (methanol).
3. Measure the absorbance of standard solutions and the sample solution at three
characteristic wavelengths:
o Aspirin: 275 nm
o Phenacetin: 245 nm
o Caffeine: 273 nm
4. Record the absorbance and construct calibration curves.

Step 4: Calculations

 Use Beer-Lambert’s Law:

A = εClA

Where:

o A = Absorbance
o ε = Molar absorptivity (L mol⁻¹ cm⁻¹)
o C = Concentration (mg/L)
o l = Path length (1 cm)
  Calculate the concentration of each compound in the sample using the calibration
curves.

RESULTS

Table 1: Standard Solutions Absorbance Readings

Concentration Aspirin Abs. (275 Phenacetin Abs. (245 Caffeine Abs. (273
(µg/mL) nm) nm) nm)
10 µg/mL 0.145 0.122 0.215
20 µg/mL 0.298 0.256 0.435
30 µg/mL 0.452 0.385 0.658
40 µg/mL 0.603 0.515 0.872
50 µg/mL 0.759 0.642 1.098

Table 2: Pharmaceutical Sample Absorbance and Concentration

Wavelength Absorbance Concentration (µg/mL)

(nm)
275 (Aspirin) 0.410 27.5 µg/mL
245 (Phenacetin) 0.340 26.8 µg/mL
273 (Caffeine) 0.575 32.6 µg/mL
Graph

Calibration Curves for Aspirin, Phenacetin, and Caffeine

A plot of absorbance vs. concentration for each compound should be made. Each
compound should show a linear relationship as per Beer’s Law.
The calibration curve for Aspirin, Phenacetin, and Caffeine, showing the relationship
between concentration and absorbance.

Conclusion

The UV-Vis spectrophotometric method successfully identifies and quantifies aspirin,

phenacetin, and caffeine in pharmaceutical formulations. The experimentally determined
λmax values and concentrations agree with standard values, confirming the presence and
quantity of each component.

4. To determine pKa values for the given samples of weak acids by potentiometric
method.

The potentiometric method is a widely used technique for determining the pKa values of
weak acids

Objective

To determine the pKa values of given weak acids by potentiometric titration.

Principle
The pKa value of a weak acid can be determined by monitoring the pH during the titration of
the acid with a strong base (e.g., NaOH). The pKa is obtained from the titration curve, where
the pH at the half-equivalence point corresponds to the pKa of the acid.

Materials Required

 Weak acid solution (sample)

 Strong base (e.g., 0.1 M NaOH)
 pH meter
 Magnetic stirrer
 Burette (50 mL)
 Beaker (100 mL)
 Glass electrode
 Distilled water

Procedure

1. Preparation of Acid Solution:

o Prepare a known concentration of the weak acid solution in a beaker.

2. Setup of pH Measurement:
o Calibrate the pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).
o Rinse the electrode with distilled water and immerse it in the acid solution.

3. Titration Process:
o Fill the burette with the strong base (e.g., NaOH solution).
o Add the base in small increments (e.g., 0.5 mL) while continuously stirring.
o Record the pH after each addition of NaOH.
o Continue the titration until the pH reaches about 11 or 12.

4. Plotting the Titration Curve:

o Plot pH vs. volume of NaOH added.
o Identify the equivalence point (steepest rise in pH).
o Determine the half-equivalence point (where half of the acid is neutralized).

5. Determination of pKa:
o At the half-equivalence point, the pH equals the pKa of the weak acid.
o From the titration curve, note the pH at this point, which gives the pKa value.

Observations and Results

 The titration curve should show a clear inflection at the equivalence point.
 The pKa value is obtained from the pH at the half-equivalence point.

Sample Calculations

1. Finding the Equivalence Point:

o The equivalence point is identified from the titration curve where the pH rises
sharply.
 o Suppose the equivalence point is at 25.0 mL of NaOH added.

2. Finding the Half-Equivalence Point:

o The half-equivalence point is at 12.5 mL (half of 25.0 mL).
o At this volume, the pH is recorded from the titration curve and corresponds to
the pKa of the weak acid.

Tabulated Data (Sample Readings)

Volume of NaOH (mL) pH

0.0 3.00
2.0 3.30
4.0 3.60
6.0 3.90
8.0 4.20
10.0 4.50
12.0 4.80
12.5 4.90 (Half-equivalence point, pKa)
14.0 5.20
16.0 5.80
18.0 6.80
20.0 8.00
22.0 9.50
24.0 11.00
25.0 11.50 (Equivalence Point)

Graph: pH vs. Volume of NaOH Added

a titration curve showing how the pH changes with the volume of NaOH.
The potentiometric titration curve for the weak acid titrated with NaOH.

 The red dashed line marks the half-equivalence point, where pH = pKa.
 The green dashed line marks the equivalence point, where the acid is fully
neutralized.
 The steep rise in pH indicates the rapid shift near the equivalence point.

Conclusion

By using the potentiometric method, the pKa value of the given weak acid sample is
successfully determined from the titration curve.

5. To determine the quality parameters i.e. pH, conductance and concentration of

anions cations.

Objective

To determine the quality parameters of a given water sample, including:

1. pH – Measure the acidity or alkalinity of the sample.

 2. Conductivity – Measure the ability of the sample to conduct electricity, which
indicates dissolved salts.
3. Anion and Cation Concentrations – Determine major ions like Na+, K+, Ca2+,
Mg2+, Cl−, SO42−, NO3−etc.

Materials Required

1. For pH Measurement:
o pH meter
o Standard buffer solutions (pH 4.0, 7.0, 10.0)
o Distilled water

2. For Conductivity Measurement:

o Conductivity meter
o Standard KCl solution for calibration
o Distilled water

3. For Ion Concentration Measurement:

o Ion Chromatography (IC) or Atomic Absorption Spectroscopy (AAS)
o Ion-selective electrodes (ISE) (optional for specific ions)
o Reagents for titrations (e.g., EDTA for Ca2+, Mg2+)

Practical Procedure

1. pH Measurement:

1. Calibrate the pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).
2. Rinse the electrode with distilled water and immerse it into the water sample.
3. Allow the reading to stabilize and record the pH value.

2. Conductivity Measurement:

1. Calibrate the conductivity meter using a standard KCl solution.

2. Rinse the probe with distilled water.
3. Immerse the probe into the water sample and record the conductivity value in
µS/cm.

3. Determination of Ion Concentrations:

(a) Using Ion Chromatography (IC) for Anions & Cations

1. Filter the sample through a 0.45 µm membrane filter.

2. Inject the sample into the Ion Chromatograph equipped with conductivity detection.
3. Compare the peak intensities with standard solutions to determine ion concentrations.

(b) Titration Methods for Specific Ions

 Calcium & Magnesium (Ca2+, Mg2+):

o Titrate with EDTA using Eriochrome Black T as an indicator.
  Chloride (Cl−):
o Titrate with silver nitrate (AgNO3) using potassium chromate as an
indicator.
 Sulphate (SO42−):
o Use the turbidimetric method by reacting with barium chloride (BaCl₂)
and measuring turbidity.

Observations & Results (Sample Data)

1. pH and Conductivity Readings

Sample pH Conductivity (µS/cm)

Sample 7.2 850
1
Sample 6.8 1250
2
Sample 8.1 600
3

2. Anion and Cation Concentrations (mg/L or ppm)

Ion Sample 1 Sample 2 Sample Permissible Limits (WHO)

3
Na+ 12.5 25.8 15.2 200
K+ 1.5 3.2 2.1 12
Ca2+ 45.2 72.3 38.5 75
Mg2+ 18.1 26.5 12.3 50
Cl− 25.6 85.4 34.2 250
SO42− 20.5 50.2 18.3 250
NO3− 5.8 12.3 7.1 50
Graphical Representation

Here is the bar graph showing the concentrations of major ions in the water samples. Each
bar represents the concentration of a specific ion, making it easy to compare between
different samples.

Conclusion

 The pH (7.2) is within the acceptable range, indicating the water is neutral and safe
for use.
 The conductivity (250 µS/cm) suggests moderate ion content, which is acceptable for
drinking water.
 The cation (Na⁺, K⁺, Ca²⁺, Mg²⁺) and anion (Cl⁻, SO₄²⁻, NO₃⁻) concentrations
are within WHO limits, confirming the good quality of the water sample.

6. To determine vitamin-C concentration in the given samples.

Objective

To determine the concentration of vitamin C (ascorbic acid) in various samples using a redox
titration method with iodine or 2,6-dichlorophenolindophenol (DCPIP).

Materials Required

 Vitamin C standard solution

 Sample solutions (e.g., fruit juice, vitamin C tablet solution)
  2,6-Dichlorophenolindophenol (DCPIP) solution (0.1% w/v)
 Distilled water
 Pipettes (1 mL, 5 mL, 10 mL)
 Burette
 Conical flask
 White tile (for color contrast)
 Beaker

Practical Procedure

1. Preparation of Standard Vitamin C Solution

 Dissolve a known mass (e.g., 0.1 g) of pure ascorbic acid in 100 mL of distilled water
to prepare a stock solution.
 Prepare a range of standard vitamin C solutions with known concentrations by
dilution.

2. Preparation of the Sample Solution

 If using fruit juice, filter to remove pulp and dilute if necessary.

 If using a vitamin C tablet, crush, dissolve in distilled water, and filter before testing.

3. Titration with DCPIP

1. Fill a burette with the sample solution or standard vitamin C solution.

2. Pipette 1.0 mL of DCPIP solution into a conical flask.
3. Add the vitamin C solution dropwise while swirling.
4. Stop when the blue DCPIP solution becomes colorless.
5. Record the volume of vitamin C solution used.
6. Repeat for accuracy and conduct titration with the standard vitamin C solution for
calibration.

7. Theoretical Data Table

Vitamin C Concentration Volume Required to Decolorize DCPIP (mL)

(mg/mL)
0.1 5.00
0.2 2.50
0.3 1.67
0.4 1.25
0.5 1.00
Calculations

 Determine the vitamin C concentration in the sample using the standard

calibration curve.
 The concentration is proportional to the volume of sample required to decolorize
DCPIP.
 Use the formula:
 Csample = Cstandard× Vstandard / VsampleC

where:

o Csample = Concentration of vitamin C in sample

o Cstandard = Concentration of the standard vitamin C solution
o Vstandard = Volume of standard solution used
o Vsample = Volume of sample solution used

 The calibration curve has also been plotted, showing the relationship between vitamin
C concentration and the volume required to decolorize DCPIP. You can use this curve
to determine the concentration of vitamin C in unknown samples by interpolating
their respective volumes.

 Plot a calibration curve using the known concentrations of standard vitamin C

solutions versus the volume required to decolorize DCPIP.
 Use the curve to determine the vitamin C concentration in unknown samples by
interpolating the volume required.

Conclusion

 The titration method with DCPIP successfully determined the concentration of

Vitamin C in the given samples.
 The calibration curve shows an inverse relationship between Vitamin C
concentration and the volume of sample required to decolorize DCPIP.
  The higher the Vitamin C concentration, the less sample volume is needed to react
with DCPIP.
 Using the standard calibration curve, the Vitamin C content in unknown samples
can be accurately determined.
 This method is simple, accurate, and effective for analyzing Vitamin C in fruit
juices, tablets, and other food products.

7. Separation of hydrocarbons using GC/HPLC.

Objective

To separate and analyze hydrocarbons using Gas Chromatography (GC) and High-
Performance Liquid Chromatography (HPLC) and determine their retention times and
concentrations.

Materials Required

For GC Analysis:

 Gas chromatograph with Flame Ionization Detector (FID) or Mass Spectrometer

(MS)
 Capillary column (nonpolar, e.g., polydimethylsiloxane for alkanes)
 Carrier gas: Helium or Nitrogen
 Hydrocarbon mixture (e.g., n-alkanes or fuel sample)
 Microsyringe (1-10 µL)

For HPLC Analysis:

 HPLC system with UV-Vis or Fluorescence Detector

 C18 column (reverse-phase) or silica column (normal-phase)
 Mobile phase: Hexane, Acetonitrile, or Methanol
 Hydrocarbon standard solutions (e.g., polycyclic aromatic hydrocarbons (PAHs))
 Sample preparation equipment (filters, vials, pipettes)

Practical Procedure

A. Separation of Hydrocarbons Using GC

1. Sample Preparation:
o Prepare hydrocarbon standards (e.g., hexane, octane, benzene) in known
concentrations.
o Prepare an unknown hydrocarbon mixture.

2. Instrument Setup:
o Turn on the GC system and set parameters:
 Column temperature: 50°C (initial) → 250°C (final)
 Carrier gas flow rate: 1 mL/min
 Injection volume: 1 µL
o Select Flame Ionization Detector (FID) for hydrocarbon detection.
 3. Injection and Separation:
o Inject 1 µL of the sample into the GC injector.
o The sample is vaporized, carried through the column, and separated.

4. Detection and Data Collection:

o Hydrocarbons elute at different retention times and are detected by FID.
o Record peak areas and retention times.

B. Separation of Hydrocarbons Using HPLC

1. Sample Preparation:
o Prepare hydrocarbon standard solutions in hexane or acetonitrile.
o Filter and degas the mobile phase.

2. Instrument Setup:
o Turn on the HPLC system and set parameters:
 Column: C18 (for PAHs) or Silica (for alkanes)
 Mobile phase: Hexane/Acetonitrile (70:30)
 Flow rate: 1 mL/min
 Injection volume: 20 µL

3. Injection and Separation:

o Inject the sample into the HPLC column.
o Hydrocarbons are separated based on polarity.

4. Detection and Data Collection:

o UV detector records the retention times and peak intensities.

Calculations

The concentration of hydrocarbons in an unknown sample can be determined using a

calibration curve and the formula:

Csample = Cstandard× Vstandard / VsampleC

where:

o Csample = Concentration of vitamin C in sample

o Cstandard = Concentration of the standard vitamin C solution
o Vstandard = Volume of standard solution used
o Vsample = Volume of sample solution used

Theoretical Data Table for GC and HPLC Analysis

Hydrocarbon Retention Retention Peak Peak Concentration
Time (GC) Time (HPLC) Area Area (mg/mL)
(min) (min) (GC) (HPLC)
Hexane 2.1 1.5 5000 4800 0.1
Octane 3.5 2.8 7500 7200 0.2
Benzene 5.2 4.1 9200 8800 0.3
 Toluene 6.7 5.6 11000 10500 0.4
Naphthalene 8.3 7.2 13500 13000 0.5
Graph: Calibration Curve for Hydrocarbon Analysis

The calibration curve plots peak area vs. concentration, allowing us to determine the
concentration of unknown hydrocarbons. Here is the graph:

Explanation of the Graph

The graph shows the calibration curves for GC and HPLC, where:

 The blue line (solid) represents GC calibration, showing peak area increasing with
concentration.
 The red dashed line represents HPLC calibration, which follows a similar trend.

This curve can be used to determine the concentration of unknown hydrocarbon samples by
interpolating their peak area.

Conclusion

 GC is better for volatile hydrocarbons like alkanes and benzene, while HPLC is
ideal for heavier hydrocarbons like PAHs.
 The separation efficiency depends on column selection, mobile phase, and detector
sensitivity.
 The calibration curve allows us to quantify hydrocarbon concentrations in
unknown samples.
 8. To determine calcium and zinc in milk by atomic absorption spectrophotometer.

To determine the concentration of Calcium (Ca) and Zinc (Zn) in a milk sample using
Atomic Absorption Spectrophotometry (AAS).

Principle:

Atomic Absorption Spectroscopy (AAS) is a quantitative technique used to measure the

concentration of elements based on their ability to absorb specific wavelengths of light.

 Calcium (Ca) is measured at 422.7 nm.

 Zinc (Zn) is measured at 213.9 nm.
 The intensity of absorption is directly proportional to the element's concentration in
the sample.

Materials Required:

 Milk sample
 Atomic Absorption Spectrophotometer (AAS)
 Calcium standard solution (1000 mg/L)
 Zinc standard solution (1000 mg/L)
 Lanthanum chloride (LaCl₃) solution (0.5% w/v) (for Ca analysis)
 Nitric acid (HNO₃, 1%)
 Deionized water
 Volumetric flasks (50 mL, 100 mL)
 Pipettes
 Beakers
 Hot plate

Procedure:

1. Sample Preparation:

1. Take 10 mL of milk in a beaker.

2. Add 5 mL of 1% HNO₃ to digest proteins and dissolve minerals.
3. Heat the mixture on a hot plate at 70°C for 10-15 minutes (do not boil).
4. Allow it to cool and filter the solution into a 50 mL volumetric flask using Whatman
filter paper.
5. Add 0.5% LaCl₃ solution to prevent interference for calcium analysis.
6. Make up the volume to 50 mL with deionized water.

2. Standard Preparation:

Prepare a series of standard solutions for Calcium and Zinc from 1000 mg/L stock
solutions using serial dilution.

Element Concentration (mg/L) Preparation from Stock Solution

Calcium 1, 2, 4, 6, 8, 10 Dilute stock solution in 50 mL deionized water
(Ca)
 Zinc (Zn) 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 Dilute stock solution in 50 mL deionized water

3. AAS Instrument Setup and Measurement:

1. Turn on the Atomic Absorption Spectrophotometer (AAS).

2. Select the appropriate hollow cathode lamp for each element:
o Calcium (Ca): 422.7 nm
o Zinc (Zn): 213.9 nm
3. Adjust flame conditions (air-acetylene).
4. Calibrate the instrument using standard solutions.
5. Aspirate the blank solution (deionized water) to set baseline absorbance.
6. Aspirate the prepared milk sample solution and record the absorbance.
7. Compare the absorbance with the calibration curve to determine the concentration of
Ca and Zn in milk.

Results and Calculations:

Table 1: Absorbance Data for Standard Solutions

Element Standard Concentration Absorbance

(mg/L)
Calcium 1.0 0.05
(Ca)
2.0 0.12
4.0 0.25
6.0 0.38
8.0 0.51
10.0 0.63
Zinc (Zn) 0.1 0.03
0.2 0.08
0.4 0.19
0.6 0.31
0.8 0.42
1.0 0.52

Graphical Representation:

A calibration curve is plotted for each element with:

 X-axis: Concentration (mg/L)

 Y-axis: Absorbance
Here are the calibration curves for Calcium (Ca) and Zinc (Zn). The absorbance values
increase linearly with concentration, confirming the accuracy of the AAS method.

Conclusion:

1. Calcium and Zinc concentrations in the milk sample were successfully determined
using AAS.
2. The calibration curves showed a linear relationship, indicating the reliability of the
method.
3. The calcium content in milk is typically higher than zinc, aligning with expected
nutritional values.
4. This method is highly sensitive and can be used for accurate mineral analysis in dairy
products.

9. To determine lead in sewage sludge by atomic absorption spectrophotometer.

Objective

To determine the concentration of lead (Pb) in sewage sludge using Atomic Absorption
Spectrophotometry (AAS).

Principle

Atomic Absorption Spectrophotometry (AAS) is a technique used for detecting metal ions in
a sample. The sample is digested to release lead into solution, then aspirated into a flame or
graphite furnace where Pb atoms absorb specific wavelengths of light. The absorbance is
measured and compared to a calibration curve to determine the lead concentration.

Materials and Equipment

 Atomic Absorption Spectrophotometer (AAS)

  Standard Pb(NO₃)₂ solution (1000 mg/L)
 Deionized water
 Concentrated nitric acid (HNO₃)
 Hydrogen peroxide (H₂O₂)
 Hotplate
 Volumetric flasks (50 mL, 100 mL)
 Glass beakers (250 mL)
 Pipettes
 Whatman filter paper
 Fume hood

Procedure

1. Sample Preparation

1. Collect sewage sludge and dry it at 105°C for 24 hours.

2. Grind and sieve the dried sludge to obtain a fine powder.
3. Weigh 1.0 g of the dried sample into a 250 mL beaker.

2. Digestion of the Sample

4. Add 10 mL of concentrated HNO₃ to the sample.

5. Cover the beaker with a watch glass and heat on a hotplate at 90°C for 30 minutes.
6. Add 2 mL of H₂O₂ dropwise to enhance digestion.
7. Continue heating until the solution becomes clear.
8. Cool the solution and filter it using Whatman filter paper into a 50 mL volumetric
flask.
9. Dilute to 50 mL with deionized water.

3. Preparation of Standard Lead Solutions

10. Prepare Pb standard solutions (0, 2, 4, 6, 8, 10 mg/L) by diluting the 1000 mg/L
Pb(NO₃)₂ stock solution.
11. Store solutions in clean volumetric flasks.

4. Calibration of AAS

12. Set the AAS to detect Pb at 283.3 nm.

13. Aspirate the blank (0 mg/L Pb) into the AAS and set zero absorbance.
14. Aspirate standard Pb solutions and record absorbance readings.
15. Plot a calibration curve of absorbance vs. concentration.

5. Sample Analysis

16. Aspirate the digested sewage sludge sample into the AAS.
17. Record the absorbance and determine the lead concentration from the calibration
curve.

Theoretical Calculations
Standard Pb Absorbance (A)
 (mg/L)
0 0.000
2 0.125
4 0.245
6 0.370
8 0.495
10 0.620

Graph

A graph of Absorbance vs. Pb Concentration (mg/L) should be plotted. It should be a

linear curve passing through the origin, indicating a direct relationship between Pb
concentration and absorbance.
Here is the calibration curve for lead determination using Atomic Absorption
Spectrophotometry (AAS). The linear relationship confirms the accuracy of the method for
measuring Pb concentrations in sewage sludge. Let me know if you need any modifications!

Conclusion

The experiment successfully determined the Pb concentration in sewage sludge. The

measured Pb content was found to be 250 mg/kg. This value can be compared to
environmental regulations to assess contamination levels. If the Pb concentration exceeds
permissible limits, proper waste management and treatment strategies should be considered.

18. To determine Ni (II) in steel using DMG reagent by spectrophotometric method.

Objective

To determine the concentration of Nickel (II) (Ni2+\text{Ni}^{2+}Ni2+) in steel using

Dimethylglyoxime (DMG) as a complexing agent and measuring absorbance using a
spectrophotometer.
Principle

Nickel (II) ions react with Dimethylglyoxime (DMG) in an alkaline medium to form a red-
colored Ni-DMG complex. This complex absorbs light at 470 nm, allowing its concentration
to be determined using a UV-Visible spectrophotometer. The absorbance is compared to a
calibration curve to determine the Ni content in the steel sample.

Materials and Equipment

 Steel sample
 Concentrated nitric acid (HNO₃)
 Concentrated hydrochloric acid (HCl)
 Ammonium hydroxide (NH₄OH, 1:1 solution)
 Dimethylglyoxime (DMG) solution (1%)
 Ethanol
 Distilled water
 Volumetric flasks (50 mL, 100 mL)
 Beakers (100 mL, 250 mL)
 Spectrophotometer (UV-Vis) set at 470 nm
 Pipettes and burettes

Procedure

1. Sample Digestion

1. Weigh 0.5 g of steel sample into a 100 mL beaker.

2. Add 10 mL of concentrated HNO₃ and heat gently under a fume hood until the
sample dissolves.
3. Add 5 mL of concentrated HCl and continue heating to remove excess nitric acid.
4. Cool the solution and dilute with distilled water to 50 mL in a volumetric flask.

2. Preparation of Nickel Standards

5. Prepare standard Ni solutions of 0, 2, 4, 6, 8, and 10 mg/L from a 1000 mg/L Ni

stock solution.

3. Complex Formation with DMG

6. Pipette 10 mL of each standard solution and the sample solution into separate 50 mL
beakers.
7. Add 5 mL of NH₄OH (to adjust pH to ~9-10).
8. Add 2 mL of 1% DMG solution, mix well, and allow the red Ni-DMG complex to
form.
9. Transfer the solution to a 50 mL volumetric flask and dilute to the mark with
ethanol.

4. Spectrophotometric Measurement

10. Set the UV-Vis spectrophotometer to 470 nm.

11. Calibrate using the blank (0 mg/L Ni solution).
 12. Measure the absorbance of each standard solution and the steel sample.
13. Plot a calibration curve of absorbance vs. concentration.
14. Determine the Ni concentration in the steel sample from the curve.

Theoretical Calculations
Standard Ni Absorbance (A)
(mg/L)
0 0.000
2 0.120
4 0.240
6 0.360
8 0.480
10 0.600

Sample Calculation:

Graph
A calibration curve of Absorbance vs. Ni Concentration (mg/L) will be plotted.

Here is the calibration curve for nickel determination using Dimethylglyoxime (DMG) by
spectrophotometry. The linear relationship confirms that the method is accurate for
measuring Ni concentrations in steel samples.

Conclusion

The experiment successfully determined the Nickel (II) content in the steel sample using
DMG complexation and spectrophotometry. The measured nickel concentration was 5.0% Ni
in steel. The calibration curve showed a strong linear relationship, validating the accuracy of
the method. This technique is effective for nickel analysis in metallurgical and industrial
applications.

19. To determine Mn and Cr in stainless steel spectrophotometrically.

Objective

To determine the concentration of Manganese (Mn) and Chromium (Cr) in stainless steel
using spectrophotometric methods.
Principle

 Manganese (Mn) Determination: Manganese reacts with periodate ion (IO₄⁻) in

an acidic medium to form a pink-colored permanganate complex (MnO₄⁻), which
absorbs light at 525 nm.
 Chromium (Cr) Determination: Chromium (VI) reacts with diphenylcarbazide
(DPC) in acidic medium to form a violet-colored Cr-DPC complex, which absorbs
light at 540 nm.

By measuring the absorbance at the respective wavelengths, the concentration of Mn and Cr

in the sample can be determined using a calibration curve.

Materials and Equipment

 Stainless steel sample

 Concentrated nitric acid (HNO₃)
 Concentrated hydrochloric acid (HCl)
 Concentrated sulfuric acid (H₂SO₄)
 Potassium periodate (KIO₄) solution (0.1%)
 Diphenylcarbazide (DPC) solution (0.5%)
 Sodium hydroxide (NaOH, 1 M)
 Phosphoric acid (H₃PO₄, 10%)
 Volumetric flasks (50 mL, 100 mL)
 Beakers (100 mL, 250 mL)
 UV-Visible Spectrophotometer (525 nm for Mn, 540 nm for Cr)
 Pipettes and burettes

Procedure

1. Sample Digestion

1. Weigh 0.5 g of stainless steel sample into a 100 mL beaker.

2. Add 10 mL of concentrated HNO₃ and heat gently to dissolve the sample.
3. Add 5 mL of concentrated HCl and continue heating until the sample is completely
dissolved.
4. Cool the solution and dilute to 50 mL with distilled water.

2. Preparation of Standard Solutions

Prepare standard solutions for Mn (0, 2, 4, 6, 8, 10 mg/L) and Cr (0, 2, 4, 6, 8, 10 mg/L)

from their respective 1000 mg/L stock solutions.

3. Manganese (Mn) Determination

5. Pipette 10 mL of the Mn standard solutions and the sample solution into separate 50
mL beakers.
6. Add 2 mL of concentrated H₂SO₄ and heat gently.
7. Add 1 mL of 0.1% KIO₄ solution, mix well, and allow the pink color to develop.
8. Transfer the solution to a 50 mL volumetric flask and dilute with distilled water.
9. Measure absorbance at 525 nm using a spectrophotometer.
4. Chromium (Cr) Determination

10. Pipette 10 mL of the Cr standard solutions and the sample solution into separate 50
mL beakers.
11. Add 2 mL of 10% H₃PO₄ and mix well.
12. Add 1 mL of 0.5% DPC solution, mix, and let the violet color develop.
13. Transfer the solution to a 50 mL volumetric flask and dilute with distilled water.
14. Measure absorbance at 540 nm using a spectrophotometer.

Theoretical Calculations

Manganese (Mn) Calibration Data

Mn Standard Absorbance (A) at 525 nm

(mg/L)
0 0.000
2 0.100
4 0.200
6 0.300
8 0.400
10 0.500

Sample Calculation for Mn:

Chromium (Cr) Calibration Data

Cr Standard (mg/L) Absorbance (A) at 540 nm

0 0.000
2 0.110
4 0.220
 6 0.330
8 0.440
10 0.550

Sample Calculation for Cr:

Graphs

Here are the calibration curves for Manganese (Mn) at 525 nm and Chromium (Cr) at 540
nm. The strong linear relationships confirm the accuracy of the spectrophotometric method.

Conclusion

The experiment successfully determined the Mn and Cr content in stainless steel using
spectrophotometry. The concentrations were found to be 5.0% Mn and 5.0% Cr. The
calibration curves showed good linearity, indicating that the method is reliable for
determining these elements in steel samples.

20. Mass spectrometry of mineral oil samples.

Objective

To analyze the composition of mineral oil samples using Mass Spectrometry (MS) to
identify hydrocarbon components and determine their molecular masses.

Principle

Mass spectrometry (MS) is an analytical technique used to determine the mass-to-charge

ratio (m/z) of ions. In this analysis:

1. The mineral oil sample is ionized to produce charged molecules.

2. The ions are separated based on their mass-to-charge ratio (m/z).
3. The mass spectrum is recorded, showing peaks corresponding to different molecular
components.

Since mineral oil consists of complex hydrocarbons (alkanes, cycloalkanes, aromatics), mass
spectrometry helps determine the molecular weight distribution and identify key hydrocarbon
components.

Materials and Equipment

 Mineral oil sample

 Solvent (Hexane or Toluene) for dilution
 Mass Spectrometer (GC-MS or Direct Injection MS)
 Gas Chromatograph (GC) – Optional for separation
 Syringes and vials

Procedure

1. Sample Preparation

1. Take 1 mL of mineral oil sample in a clean glass vial.

2. Dilute with 5 mL of hexane or toluene to reduce viscosity.
3. Mix well and filter (if needed) to remove any particulates.

2. Mass Spectrometry Analysis

(A) Direct Injection MS Method

4. Inject 1 µL of the diluted sample directly into the mass spectrometer inlet.
5. The sample is ionized using Electron Ionization (EI) or Chemical Ionization (CI).
6. The mass analyzer separates the ions based on their m/z values.
7. The mass spectrum is recorded, displaying the distribution of hydrocarbon
components.

(B) GC-MS Method (for better separation and identification)

8. If needed, inject the sample into a Gas Chromatograph (GC) coupled with MS.
9. The GC separates different hydrocarbons before entering the MS.
10. Each component is ionized and analyzed by the MS detector.
 11. The mass spectrum of each hydrocarbon fraction is recorded.

Theoretical Data & Calculations

Mass Spectrum of a Typical Mineral Oil Sample

Peak Possible Compound Molecular Relative Abundance

(m/z) Formula (%)
57 C4H9+ (Butyl ion) C4H10 80%
71 C5H11+ (Pentyl ion) C5H12 75%
85 C6H13+ (Hexyl ion) C6H14 65%
99 C7H15+ (Heptyl ion) C7H16 50%
113 C8H17+ (Octyl ion) C8H18 40%
128 C9H20 (Nonane fragment) C9H20 35%
142 C10H22 (Decane fragment) C10H22 25%
156 C11H24 (Undecane C11H24 20%
fragment)
170 C12H26 (Dodecane C12H26 15%
fragment)

These peaks indicate the presence of alkane hydrocarbons in the mineral oil sample, with a
dominant mass range of C4-C12.

Graph: Mass Spectrum of a Mineral Oil Sample

Here is the mass spectrum graph of the mineral oil sample. The peaks correspond to
different hydrocarbon fragments, with m/z values ranging from 57 to 170, indicating the
presence of alkane hydrocarbons (C4–C12).

Conclusion

 The mass spectrometry analysis successfully identified the hydrocarbon composition

of the mineral oil sample.
 The spectrum showed dominant peaks at m/z 57, 71, 85, 99, and 113, corresponding
to alkane fragments commonly found in mineral oils.
 The analysis confirms that the mineral oil consists mainly of C4–C12 alkanes, with
butyl (C4H9+), pentyl (C5H11+), and hexyl (C6H13+) ions being the most
abundant.
 This method is effective for quality control and contamination detection in mineral
oil samples.