LAB MANUAL

CHEM 324 INSTRUMENTAL ANALYSIS

LABORATORY

Middle East Technical University

Department of Chemistry
2025 Spring

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 GROUP NUMBERS
WEEK 1 2 3 4 5 6 7 8 9 10
1 VIS GC TLC CV FL HPLC REF IR IE AAS
2 AAS VIS GC TLC CV FL HPLC REF IR IE
3 IE AAS VIS GC TLC CV FL HPLC REF IR
4 IR IE AAS VIS GC TLC CV FL HPLC REF
5 REF IR IE AAS VIS GC TLC CV FL HPLC
6 HPLC REF IR IE AAS VIS GC TLC CV FL
7 FL HPLC REF IR IE AAS VIS GC TLC CV
8 CV FL HPLC REF IR IE AAS VIS GC TLC
9 TLC CV FL HPLC REF IR IE AAS VIS GC
10 GC TLC CV FL HPLC REF IR IE AAS VIS

VIS : ULTRAVIOLET AND VISIBLE SPECTROMETRY

HPLC : HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
GC : GAS CHROMATOGRAPHY
CV : CYCLIC VOLTAMMETRY
AAS : ATOMIC ABSORPTION AND EMISSION SPECTROMETRY
FL : FLUORIMETRY
TLC : THIN LAYER AND PAPER CHROMATOGRAPHY, ELECTROPHORESIS
IR : INFRARED SPECTROMETRY
REF : REFRACTOMETRY AND POLARIMETRY
IE : ION EXCHANGE CHROMATOGRAPHY

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 Textbook : Instrumental Analysis Laboratory
D. A. Skoog, F. J. Holler, S. R. Crouch, "Principles of Instrumental Analysis”, 7 th Ed.,
Thomson, Brook/Cole, Canada, 2007.

Lab Hours : Section 1: Monday (13:40-17:30), Tuesday (13:40-17:30)

Section 2: Wednesday (13:40-17:30), Thursday (13:40-17:30)

Instructor :Section 1: Süreyya Özcan Kabasakal Office No: O-212, [email protected]

Section 2: Gülay Ertaş Office No: O-122, [email protected]

Lab Assistants : Dilara Gündoğdu (Coordinator), Chemistry Bld., B38, [email protected]

Deniz Öztürker, Chemistry Bld., B19, [email protected]

Ekinsu Gürsöz, Chemistry Bld., D-158, [email protected]

Mehmet Enes Uzun, Chemistry Bld., C204-C, [email protected]

Esra Korkmaz, Chemistry Bld., D-158, [email protected]

Buse Ayşen Dündar, Chemistry Bld., D248, [email protected]

Selin İlter, METU Research center,1st floor OPV Lab (Günam), [email protected]

Zeynep Nas, Chemistry Bld., D154, [email protected]

Özge Özdemir, Chemistry Bld., D253, [email protected]

Instrumental Analysis Laboratory is performed in the following laboratory: C-57

EXPERIMENTS

1. Ion Exchange Chromatography

- Separation of Iron and Cobalt ions by Ion Exchange Chromatography

2. Atomic Absorption Spectrometry

- Determination of calcium in beverage by AAS and sodium in tap water by AES

3. Infrared Spectrometry
- Qualitative and quantitative analysis of film, solid and liquid sample by IR spectroscopy

4. Thin Layer and Paper Chromatography, Electrophoresis

- Separation of pH indicator mixtures by TLC.
- Separation of Metal ions
- Separation of dyes by electrophoresis.

5. Ultraviolet and Visible Spectrometry

- Determination of 𝑀𝑛𝑂4− and 𝐶𝑟2 𝑂72− in their mixture
- Photometric Titration of Cu2+ by EDTA

6. Gas Chromatography
-Effects of Column Temperature on Retention Time
-Analysis of Mixtures
-Temperature Programming for Mixtures
- Mass Spectral Identification of Toluene and Benzene Using GC-MS
- Quantitative Analysis of Benzene Using External Calibration and Internal Standard Calibration Methods

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7. Refractometry and Polarımetry (REF-POL)
- Determination of Refractive Indices of Solvents
- Determination of Optical Rotation of Rochelle Salt

8. High Performance Liquid Chromatography (HPLC)

- Determination of Ambroxol HCl, Methyl Paraben and Propyl Paraben in a commercial syrup qualitatively by
using High Performance Liquid Chromatography (HPLC).

9. Fluorescence Spectrometry (FS)

- Determination of fluorescein concentration (mol/L) in a given sample by using fluorimetry.

10. Cyclic Voltammetry (CV)

- Determination of diffusion coefficient of redox species using Randles-Sevcik equation by both cyclic
voltammetry and chronoamporometry method, determination of concentration of ascorbic acid in unknown by
cyclic voltammetry & standard addition method.
GENERAL INFORMATION

1. Total grade for your laboratory work will be evaluated based on 100 points. Distribution of these points are;

1. 40 Reports
2. 35 Quizzes
3. 25 Final Exam
4. 100 Total

2. Attendance is compulsory. A medical report for at least 2 days is required to have a make-up. Student is not
allowed to enter the laboratory when she/he is late. Make-up will not be given for being late.

3. Only one lab make-up will be given to a student with a medical report and it must be for two days of lab.
Make-up experiments will be performed during the semester.

4. If a student gets zero from 2 experiments, he/she will fail the course and the course grade will be
NA.

5. No food, drink or smoking is allowed in the laboratories.

6. The students must behave within the limits of common sense in the labs. Any student who does not obey
the rules will be asked to leave the lab and no credits will be given for that experiment.

7. The students who will be working in this lab are expected to be experienced in basics of analytical chemistry.
However, the use and care of the following equipment will be illustrated by your laboratory instructors and
you are expected to strictly follow these instructions.
˗ Analytical balances
˗ Glassware, Pipettes, Burettes, etc.
˗ Keeping the instruments, sink environment clean.
˗ Transfer and care of solutions.

8. In the case of significant overlapping with the literature, you will receive a grade of ‘0’ from the reports for
the first time. Students, who continue to copy, will get ‘FF’ and will not be able to continue this course.

LABORATORY PROGRAM

Each student has two lab periods in a week to complete an experiment. Groups cannot be changed during the
semester.

FIRST LAB PERIOD:

Each student should read the necessary parts from the textbook before the first day of the lab and be ready for the
first half day.

1. Please bring the following items with you.

● A white lab coat, must be kept CLEAN and BUTTONED in the lab,

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 ● Calculator,
● Water proof pen,
● Textbook, (MUST be brought both on FIRST and SECOND LAB PERIODS for ALL
EXPERIMENTS. (D. A. Skoog, F. J. Holler, S. R. Crouch, Principles of Instrumental Analysis.
Thomson Brooks/Cole: Belmont, CA, USA.)
● A small notebook of about 50 pages, to take notes on procedure, discussion etc.

2. Lab session will start with an entrance quiz. If your entrance quiz grade is 25 or lower over 100, your
report for the experiment will be evaluated over 50. Following the quiz, you will discuss the part of the
text book or documents related to your experiment with your assistant.

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SECOND LAB PERIOD:

1. Ask your assistants to give you the necessary valuable accessories. You must return all of these parts in good
conditions when you are finished the experiment.
2. Perform the experiment as a group. Each member of the group should participate in experiment as much as
possible.
3. Complete data sheet of your report. Data sheets without the signature of assistant will not be evaluated.
4. DO NOT turn your instrument off; leave the instrument and glassware as well as the working medium as
clean as possible. Otherwise, your data sheet will not be signed and you will get zero from the
experiment.
5. Lab session will end with an exit quiz.
6. Your laboratory reports must be completed within two half-days and submitted to your assistant by the end of
the second day. Late reports will not be accepted, and the report will be marked as zero in your report average.

REPORT FORMAT (Total: 100 points)

1. Title Page: The title page should include [experiment title, name, lab partners, dates of experimental work,
course title, date submitted and course section] (- 5 pts)
2. Instrumental and Experimental Part: This is a description of the particular instrument and procedure used
for an experiment. Draw the schematic diagram of experimental set up or block diagram of the instrument
used (Do not copy the diagram from the web!) and write the name(s) and function(s) of each component
and lastly brand name of the instrument. Explain briefly how data was collected. (20 pts or 30 pts) (max.
1 page)
3. Data sheet (signed) If data sheet is not provided report will not be evaluated!
4. Graphs and Calculations (Note: Do not write the solution preparation calculations) (40 pts or 30 pts)
5. Post-Lab Questions: In this part you must answer the post-lab questions properly. (40 pts)
6. Literature References (at least three refs): You must correctly list all references used throughout the
report. The literature references should be presented in proper format given by METU Academic Writing
Center. You are expected to use combination of different sources, i.e. books, web pages, articles etc.
7. http://www.awc.metu.edu.tr/handouts/APA_Documentation.pdf

Reports:
● Contain clearly labeled sections
● Include tables, graphs, and charts integrated within the report (Graphs and data table should be clearly
labeled with numbers and titles, use Excel or Origin)
● Contain proper grammar (i.e. write in 3rd person, have agreement in tense, agreement between subject and
verb etc.)

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Reading Assignments
Instrumental Analysis Laboratory
D. A. Skoog, F. J. Holler, S. R. Crouch, "Principles of Instrumental Analysis”, 7 th Ed.,
Thomson, Brook/Cole, Canada, 2007.

UV-VIS

Chapter 6A, 6B, 6B-1, 6B-2, 6C-1

Chapter 7A, 7B-1, 7B-2, 7C, 7E-1
Chapter 13
- An introduction to the Ultraviolet/Visible Molecular Absorption Spectrometry
- Measurement of Transmittance and Absorbance
- Beer’s Law
- Application of Beer’s Law to Mixtures
- Limitations to Beer’s Law
- Real Limitations to Beer’s Law
- Apparent Chemical Deviations
- Apparent Instrumental Deviations with Polychromatic Radiation
- Instrumentation
- Instrument Components
- Sources: Deuterium and Hydrogen Lamps, Tungsten Filament Lamps
- Sample Containers
- Types of Instruments: Single-Beam and Double Beam Instruments
Chapter 14
- Photometric and spectrophotometric titrations

FS

Chapter 6A, 6B, 6B-1, 6B-2, 6C-1

Chapter 7A, 7B-1, 7B-2, 7C, 7E-1
Chapter 15
- Molecular Luminescence Spectrometry
15A - Theory of Fluorescence and Phosphorescence
15A-1 – Excited States Producing Fluorescence and Phosphorescence
o Electron Spin
o Singlet and Triplet Excited States
o Energy-Level Diagrams for Photoluminescent Molecules
15A-2 - Rates of Absorption and Emission
15A-3 - Deactivation Processes
o Vibrational Relaxation
o Internal conversion
o External Conversion
o Intersystem Crossing
o Phosphorescence
15A-4 - Variables Affecting Fluorescence and Phosphorescence
o Quantum Yield
o Transition Types in Fluorescence
o Effect of pH on Fluorescence
o Effect of on Concentration on Fluorescence Intensity
15B - Instruments for Measuring Fluorescence and Phosphorescence
15B-1- Components of Fluorometers and Spectrofluorometers
o Sources: Lamps
o Filters and Monochromators
o Transducers
o Cells and Cell Compartments

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IR

Chapter 6A, 6B, 6B-1, 6B-2, 6C-1

Chapter 7A, 7B-1, 7B-2, 7C, 7E-1
Chapter 16
- An introduction to Infrared Spectrometry
- 16A-1 Introduction
- Dipole Changes During Vibrations and Rotations
- Types of Molecular Vibrations
- 16A-2 Mechanical Model of a Stretching Vibration in a Diatomic Molecule
- 16A-4 Vibrational Modes
- 16A-5 Vibrational Coupling
- 7I-1, 7I-2, 7I-3
- 16A IR INSTRUMENTATION
- 16B-1 Fourier Transform Spectrometers
- Advantages of FT Spectrometers
- 16B-2 Dispersive Instruments
- 16B-3 Nondispersive Instruments
- 16C-1 Sources
- All sources
- 16C-2 IR Transducers
- Thermal Transducers

Chapter 17
- Applications of Infrared Spectrometry
- 17A-1 Sample Handling
- Gases, liquids, solids
- 17A-2 Qualitative Analysis
- Group Frequencies
- The Fingerprint Region
- 17A-3 Quantitative Analysis
- Absorbance Measurement
- Typical Applications
- 17B-3 ATR Spectrometry
- Principles
- Instrumentation
- ATR Spectra

CV

Chapter 22
- 22F
- 22A-7 Mass transfer in Cells with the Passage of Current
22E-3 Mechanism of Mass Transport

Chapter 25
- 25A Excitation Signals In Voltammetry
- 25B Voltammetric Instrumentation
- 25B-1 Working Electrode
- 25B-3 Voltammograms
Profiles for Planar Electrodes in Unstirred Solutions
Profiles for Planar Electrodes in Stirred Solutions
- 25D Cyclic Voltammetry
- Lab text

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AAS-AES

Chapter 6A, 6B, 6B-1, 6B-2, 6C-1

Chapter 7A, 7B-1, 7B-2, 7C, 7E-1
- Lab text.

GC

Chapter 26
- An Introduction to Chromatographic Separations
o A General Description of Chromatography
o Classification of Chromatographic Methods
o Elution in Column Chromatography
o Analyte Dilution
o Chromatograms
o Improving Column Performance
- Migration Rates of Solutes
o Distribution Constants
o Retention Time
o The Rate of Solute Migration: The Retention Factor
o Relative Migration Rates: The Selectivity Factor
- Band Broadening and Column Efficiency
o The Rate Theory of Chromatography
o A Quantative Description of Column Efficiency
o The Definition of Plate Height
o The Experimental Evaluation of H and N
- Kinetic Variables Affecting Column Efficiency
o The Effect of Mobile-Phase Flow Rate
o Theory of Band Broadening
- Optimization of Column Performance
● Column Resolution
● The General Elution Problem

- Applications of Chromatography
o Qualitative Analysis
o Quantitave Analysis

Chapter 27
- Gas Chromatography
- Instruments for Gas-Liquid Chromatography
o Carrier Gas System
o Sample Injection System
o Column Configurations and Column Ovens
o Detection Systems
o Characteristics of the Ideal Detector
o Flame Ionization Detector
- Gas Chromatographic Columns and Stationary Phases
o Open Tubular Columns
o Packed Columns
o Adsorption on Column Packings or Capillary Walls
o The Stationary Phase
- Applications of GC
● Qualitative Analysis
● Quantitative Analysis

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HPLC

Chapter 26
- An Introduction to Chromatographic Separations
o A General Description of Chromatography
o Classification of Chromatographic Methods
o Elution in Column Chromatography
o Analyte Dilution
o Chromatograms
o Improving Column Performance
- Migration Rates of Solutes
o Distribution Constants
o Retention Time
o The Rate of Solute Migration: The Retention Factor
o Relative Migration Rates: The Selectivity Factor
- Band Broadening and Column Efficiency
o The Rate Theory of Chromatography
o A Quantative Description of Column Efficiency
o The Definition of Plate Height
o The Experimental Evaluation of H and N
- Kinetic Variables Affecting Column Efficiency
o The Effect of Mobile-Phase Flow Rate
o Theory of Band Broadening
- Optimization of Column Performance
● Column Resolution
● The General Elution Problem
- Applications of Chromatography
o Qualitative Analysis
o Quantitave Analysis
Chapter 28
- High-Performance Liquid Chromatography
- Scope of HPLC
- LC Instrumentation
- Mobile Phase Reservoirs and Solvent Treatment Systems
- Pumping Systems
o Reciprocating Pumps
- Sample-Injection Systems
- Columns for HPLC
o Analytical Columns
o Guard Columns
o Column Temperature Control
- Detectors
o Characteristics of the Ideal Detectors
o Types of Detectors
o UV Absorption Detectors with Filters
o Absorption Detectors with Scanning Capabilities
- Partition Chromatography
o Columns for Bonded-Phase Chromatography
o Normal- and Reversed-Phase Packings
o Method Development in Partition Chromatography
Column Selection in Partition Chromatographic Separations

TLC, Paper Chromatography and Electrophoresis

- 26A General description of chromatography

- 26A-1 Classification of Chromatographic Methods

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- 26A-2 Elution in column chromatography
- 26B Migration Rates of Solutes
- 26B-1 Distribution Constants
- 26B-2 Retention time
- 26B-5 The Rate of Solute Migration: The Retention Factor
- 26B-6 Relative Migration Rates: The Selectivity Factor
- 28I Thin-layer chromatography
- 28I-1 The scope of thin-layer chromatography
- 28I-2 Principles of thin-layer chromatography
- 28I-3 Performance Characteristics of Thin-Layer Plates
- 28I-4 Applications of thin-layer chromatography
- 30A An overview of electrophoresis
- 30A-1 Types of electrophoresis
- 30A-2 The basis of electrophoretic separations
- 30B Capillary Electrophoresis
- 30B-1 Migration Rates in CE
- 30B-2 Plate Heights in CE
- 30B-3 Electroosmotic Flow
- Lab text

Lab Texts Available in METUCLASS

- TLC-PC-EF
- AAS-AES
- CV
- IE
- REF-POL

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CHEM324 INSTRUMENTAL ANALYSIS
I LABORATORY

PROCEDURES

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 ULTRAVIOLET-VISIBLE SPECTROMETRY (UV-VIS)
Experiment 1: Determination of MnO4- and Cr2O72- in their mixture solution

Solutions:
a. Stock Cr2O72- solution (100.0 mL, 1.0x10-3 M) from K2Cr2O7: Prepared in 0.03 M H2SO4 solution.
b. 500.0 mL, 0.03 M H2SO4
c. Stock MnO4- solution (100.0 mL, 5.0x10-3 M) from KMnO4
d. Intermediate solution: (100.0 mL, 1.0x10 -3 M) from stock solution of MnO4-
e. Working solutions: Cr2O72- (50.0 mL), MnO4- (50.0 mL) prepared from stock solution of Cr2O72- and
intermediate solution of MnO4- in following concentrations:
a) Blank b) 1.0x10-4 M c) 2.0x10-4 M d) 3.0x10-4 M e) 4.0x10-4 M

Procedure:
1. Preliminary Instrument Setup: Before conducting any measurements, ensure that the instrument is powered
on and allowed to warm up for a minimum of 10 minutes to ensure stable readings.
2. Baseline Correction: Correct the baseline within the working wavelength range by using distilled water for
MnO4- and 0.03 M H2SO4 for Cr2O72- measurements both the sample and reference cells.
3. Spectral Scanning and Wavelength Selection: Scan the spectra of MnO4- and Cr2O72- separately, covering
the wavelength range from 250 nm to 750 nm, in overlay mode. Identify and select two distinct wavelengths
that can be reliably used for the quantification of both ions.
4. Absorbance Measurement of Standard Solutions: At the wavelengths identified in Step 3, measure the
absorbance of the prepared working solutions of MnO 4- and Cr2O72-.
5. Analysis of Unknown Solution: Obtain a sample of the unknown solution containing MnO4- and Cr2O72 ions
from your assistant. Measure the absorbance of this solution at the two wavelengths previously selected.
Calculations:
1. Plot the calibration curve (Absorbance vs. concentration) at each wavelength for each ion.
2. Calculate the molar absorptivity constants of ions at the selected wavelengths.
3. Calculate MnO4- and Cr2O72- molar concentrations in unknown solution.

Experiment 2: Photometric Titration of Cu2+ by EDTA

Solutions:
a. 100.0 mL of 0.01 M Cu2+ solution prepared from Cu(NO3)2 in ammonia buffer (to prepare Cu2+ solution dissolve
a certain amount of Cu(NO3)2.3H2O in some water, then add 9.0 mL concentrated aqueous NH 3 and 1.35 g
NH4NO3 and complete the volume to 100.0 mL with distilled water).
b. CuY2- complex solution (1:1 by volume Cu2+:EDTA)
c. 0.10 M EDTA solution

Procedure:
1. Preparation of the Sample: Accurately transfer 60.0 mL of the Cu(NH₄)₃ solution into a clean, dry 250 mL
Erlenmeyer flask.
2. Initial Absorption Spectrum Measurement: Using UV-Vis spectrometer, record the absorption spectrum
of the prepared Cu(NH₄)₃ solution and CuY2- complex solution within the wavelength range of 400 nm to
900 nm.
3. Wavelength Selection: Analyze the collected spectra to determine a suitable wavelength at which the
absorbance ratio between free Cu²⁺ ions and the Cu-EDTA complex (1:1 stoichiometry) is at its maximum.
4. Titration with EDTA: Gradually add 0.1 M EDTA solution to the flask in 1.0 mL increments up to 5.0 mL,
between 5.0 to 7.0 mL 0.5 mL increments, after 7.0 mL use 1.0 mL increments using a calibrated burette or
micropipette. Subsequently, record the absorption spectrum after each increment. Important: Do not
dispose of the solution after measurement. Carefully return it to the Erlenmeyer flask to maintain the
cumulative volume and concentration consistency throughout the titration.
5. Post-Endpoint Measurements: Continue adding 0.1 M EDTA solution beyond the equivalence point to
ensure excess EDTA is present. Record at least five additional absorbance measurements after surpassing
the endpoint. This data will help in identifying the plateau phase of the titration curve.

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6. Titration Curve Construction and Calculation: Plot the titration curve using Excel (or Origin) by
drawing absorbance vs volume of EDTA added at the selected wavelength. Using the titration curve,
identify the equivalence point and calculate the concentration of Cu²⁺ ions in the original solution based on
the stoichiometry of the reaction with EDTA.
7. Data Analysis and Error Evaluation: Compare the experimentally determined Cu²⁺ concentration with the
theoretical value. Conduct a thorough analysis to identify any discrepancies, considering potential sources of
error such as instrumental inaccuracies, pipetting errors, incomplete reactions, or contamination.
Pre-lab Questions:

1. Draw the regions of the electromagnetic spectrum and write the types of interactions between analyte with
electromagnetic radiation in each region.
2. What is the relationship between
a. absorbance and transmitance
b. absorptivity a and molar absoptivity ε
3. How is Beer’s Law applied to solutions containing more than one kind of absorbing substance?
4. Identify factors that cause deviations from the linearity of Beer’s Law.
5. Describe the difference between “real” deviations from Beer’s law and those caused by instrumental or
chemical factors.
6. What is the difference between line source and continuous source?
7. List the types of continuum sources used in optical spectroscopy.
8. What is the function of wavelength selectors?
9. What are the differences between filters and monochromators?
10. How do absorption filters differ from interference filters?
11. List common detectors used in absorption spectroscopy.
12. Briefly explain the working principle of photomultiplier tubes (PMTs)?
13. Draw block diagrams of single and double-beam spectrometers for UV-VIS region.
14. How does photometric titration differ from traditional titration methods? How can photometric titration curves
can be interpreted?
15. Explain the differences between single beam and double beam instruments.

Post-Lab Questions:

1. Why is baseline correction necessary in UV-Vis spectrometry? Explain briefly.

2. Discuss the applicability of Beer’s law to solutions containing more than one kind of absorbing substance.
3. In Experiment 1, two wavelengths will be selected for quantification of each ion in the solution. Calibration
curves for each ion will be plotted at each wavelength to determine molar absorptivities. Which wavelength
is more suitable for quantitative analysis of MnO4- and Cr2O72- ions? Briefly discuss your answer.
4. What is the purpose of diluting the stock K2Cr2O7 solution with H2SO4?
5. Write the reaction between Cu2+ and EDTA in ammonia buffer.
6. How is the end point determined in photometric titration?
7. Why do absorbance values decrease after the endpoint in photometric titration?

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 Data Sheet
Ultraviolet-Visible Spectrometry

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

Part A: Determination of MnO4- and Cr2O72-

Conc. Absorbance Absorbance Conc. Absorbance Absorbance
(Cr2O72-) (…...nm) (…...nm) (MnO4-) (…...nm) (…...nm)
1.0x10-4 M 1.0x10-4 M

2.0x10-4 M 2.0x10-4 M

3.0x10-4 M 3.0x10-4 M

4.0x10-4 M 4.0x10-4 M

Unknown

Slope

Intercept

R2

Part B: Photometric Titration of Cu2+ by EDTA

mL of EDTA added Absorbance Absorbance

(…...nm) (…...nm)
…….. mL

…….. mL

…….. mL

…….. mL

…….. mL

…….. mL

…….. mL

…….. mL

…….. mL
…….. mL

…….. mL

…….. mL

…….. mL

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16
 ATOMIC ABSORPTION AND EMISSION SPECTROMETRY (AAS-AES)
Experiment: Determination of calcium in beverage by AAS and sodium in tap water by AES.

Solutions: Prepare all solutions and make all dilutions with 1% HNO 3 prepared with deionized water!
a. Stock Calcium Standard Solution (50.0 mL, 1000 mg/L Ca): Prepare from CaCO3 .5H2O.
Intermediate Ca Standard Solution (100.0 mL, 100.0 mg/L Ca): Prepare from the stock solution.
Working Solutions:
1. For direct method: Prepare 100.0 mL of 0.50, 1.00, 2.00, 3.00, 4.00 and 5.00 mg/L solutions from
intermediate standard solution.
2. For standard addition method: Place 2.0 mL of sugary beverage in each of three 100 mL volumetric
flasks. Dilute one of them to 100.0 mL with deionized water, that will be the unknown solution for direct
method and “+0” solution for the standard addition method. Then add necessary amount of intermediate
Ca standard solution to other two flasks so that after dilution the added concentrations will be 2.00 mg/L
and 4.00 mg/L. Dilute to the mark with deionized water and label “+2” and “+4”, respectively.

b. Stock Sodium Standard Solution (50.0 mL, 1000 mg/L Na): Prepare from solid NaCl
Intermediate Na standard solution (100.0 mL, 10.0 mg/L Na): Prepare from stock Na solution.
Working solutions: 0.20, 0.40, 0.60, 0.80, 1.00 mg/L, each 100.0 mL from intermediate Na standard solution.

Procedure:

A. Atomic Absorption Spectrometry for the Determination of Ca

1. Turn on the instrument and set up the experimental parameters on the instrument.
2. Set the monochromator to 422.7 nm, the most sensitive line of Ca, and adjust the lamp position to have
maximum intensity.
3. Ignite the flame and investigate the effects of burner position and fuel/oxidant ratio on the absorbance signal.
4. Aspirate the blank and standard solutions, and record their signals.
5. Make 10 replicate measurements of 0.50 mg/L standard solution to calculate the limit of detection (LOD) and
reciprocal sensitivity.
6. Draw calibration line for direct method and find the concentration of Ca in sample (mg/L) (do not forget to
make the blank corrections).
7. Draw calibration line for standard addition method and find the concentration of Ca in sample (mg/L).
8. Calculate LOD and reciprocal sensitivity.
9. Calculate S/N ratio.

B. Atomic Emission Spectrometry for the Determination of Na

1. Set the instrument to the FLAME EMISSION mode and select Na as analyte.
2. Adjust the fuel/oxidant ratio and burner position by aspirating 1.00 mg/L standard Na solution.
3. Set the emission intensity with 1.00 mg/L Na solution.
4. Measure the emission intensities of all standard Na solutions (including the blank solution) and sample
solution after necessary dilutions.
5. Make 10 replicate measurements of 0.20 mg/L standard solution to calculate LOD.
6. Draw the calibration line and find the concentration of Na in sample (mg/L).
7. Calculate S/N ratio.

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Pre- Lab Questions:

1. Write the differences between double-beam UV-VIS spectrometer and double beam AAS.
2. Why is source modulation employed in atomic absorption spectroscopy?
3. Explain the possible interferences occurs in AAS?
4. What are the differences between line and continuous sources?
5. Draw block diagram of a double beam AAS and write the name of each component.

Post -Lab Questions:

1. Compare the concentration values of Ca in the sample calculated by direct and standard addition methods.
2. What is the reason of using standard addition method? Is there any matrix interference? Explain.
3. Why emission mode was used for determination of Na while absorption mode was used for Ca?
4. Which instrumental parameters optimized at the begining of the instrument? What is the purpose of the
optimization?
5. Compare detection limit and reciprocal sensitivity calculated in this experiment with the other methods like
ICP-MS, ICP-OES.

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 Data Sheet
Atomic Absorption-Emission Spectrometry (AAS-AES)

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:

Part A. Determination of Calcium by AAS

Conc. (ppm) Absorbance Conc. (ppm) Absorbance

…….nm ……nm
0.50 0.50

1.00 0.50

2.00 0.50

3.00 0.50

4.00 0.50

5.00 0.50

cherry +0 0.50

cherry +2 0.50

cherry +4 0.50

0.50

Part B. Determination of Sodium by AES

Conc. (ppm) Intensity Blank Conc. (ppm) Intensity Blank

Corrected Corrected
Signal Signal
blank 0.20

0.20 0.20

0.40 0.20

0.60 0.20

0.80 0.20

1.00 0.20

1% Tap Water 0.20

0.20

0.20

0.20

19
20
 INFRARED SPECTROMETRY (IR)
Experiment: Qualitative and quantitative analysis of film samples, solid and liquid samples by IR spectroscopy,
Materials:
- Polyvinyl chloride (PVC) film
- Mixture of polymethyl methacrylate (PMMA) with PVC films which have already been prepared in different
concentrations
- Unknown film which is a mixture of PMMA and PVC
- Polypropylene (PP), polystyrene (PS), polytetrafluoroethylene (PTFE), and polyethylene (PE) films.
- Pure KBr for pellet and unknown solid sample for unknown pellet which will be prepared in the laboratory.

Procedure:
QUANTITATIVE ANALYSIS
1. Perform a background scan and take the IR spectrum of pure PVC in % transmittance mode.
2. Take the IR spectra of standard PMMA-PVC films at 2%, 4%, 6%, and 8% (w/w) concentrations in %
transmittance mode.
3. Take the IR spectrum of PVC-PMMA unknown film.
QUALITATIVE ANALYSIS
1. Take the IR spectra of PP, PE, PS, and PTFE films.
2. Take the IR spectrum of the unknown solid substance by preparing the KBr pellet and using % transmission
mode.
3. Take the IR spectrum of liquid samples of n-butanol, acetone, and acetonitrile.

Calculations:
1. Draw the calibration plot and calculate the % (w/w) concentration of PMMA in unknown PVC-PMMA film.
2. Perform qualitative analysis for the unknown films, unknown solid compound, and liquid samples (Identify the
main peaks on each spectrum).
3. Calculate film thicknesses of PP and PS films.

Pre-lab Questions

1. What type of molecules can absorb IR radiation?

2. Why is wavenumber usually used in the abscissa for IR spectra?
3. Why does the C-O bond in methanol absorb at a different wavelength compared to the C-O bond in ethanol
or butanol?
4. Write the advantages of FTIR instruments over dispersive instruments.
5. What is the advantage of putting a monochromator after the sample compartment in dispersive instruments?
6. What is the reason for using shorter path lengths in liquid cells?
7. Why aren’t photomultiplier tubes used in IR spectrometers?
8. What are the differences between a photon detector and a heat detector?

Post Lab Questions:

1. What types of peaks originate from air could show up on the spectra? Write also their wavenumbers.
2. Discuss the effect of the number of scans on S/N for a specific region of the polystyrene spectrum.
3. Explain why we use KBr in the preparation of pellets.
4. Is it possible to take the IR spectrum without closing the lid of the spectrometer? Explain briefly.
5. Humidity can damage the components of the IR spectrometers. What are these components? Explain the
reason(s)
6. Consider the reaction between acetonitrile as the reactant and acetone as the product. Is it possible to
monitor this reaction with IR spectroscopy? Explain by writing the reaction.
7. Draw the structures of PVC, PMMA, PP, PS, PTFE, acetone, acetonitrile, and n-butanol to show the
functional groups and their possible vibrational modes observed in the IR spectrum.

21
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Experiment: Determination of Ambroxol HCl, Methyl Paraben, and Propyl Paraben in a commercial syrup
qualitatively and in an unknown mixture quantitatively by using High Performance Liquid Chromatography
(HPLC).
Solutions:
Dilute all solutions with Deionized water.
Separate Standards for Qualitative Analysis: 100 mg/L Ambroxol HCl, 100 mg/L Methyl Paraben, and 100
mg/L Propyl Paraben standards, 100 mg/L commercial syrup, and 100 mg/L mixture of Ambroxol HCl, Methyl
Paraben, and Propyl Paraben.
Working solutions for Quantitative Analysis: 5.0, 10.0, 15.0, 20.0 and 25.0 mg/L mixture, an unknown solution
(from assistant)
Mobile Phase: Methanol / 0.5% (w/v) ammonium acetate buffer (60/40), pH adjusted to 5.5 with acetic acid.
Column:
Thermo Scientific Acclaim, C18
Procedure:
1. Turn on the pump and UV absorbance detector.
2. Adjust the flow rate to 1.0 mL/min.
3. Wait until the detector reads a constant absorbance value, if it is different than 0.000 press the autozero button.
4. Take a few milliliters of 100 mg/L Ambroxol HCL solution with the syringe. Be careful that the solution
inside the syringe does not contain any air bubbles; if there are any they should be removed.
5. Place the syringe into the injection port when it is at the load position and inject the solution from the syringe
until you see a few droplets of the solution coming out of the stainless steel pipe.
6. Then, turn the knob to the inject position and at the same time initiate data acquisition. First, the system peak
will appear on the chromatogram. Then, the analyte peak will be observed. From the chromatogram find the
maximum point of the peak and read the retention time of the analyte.
7. Repeat the same procedure for the Methyl Paraben and Propyl Paraben solutions to find their retention times.
8. Inject the unknown mixture (commercial syrup) and identify the peaks.
9. Calculate the number of plates and theoretical plate height for each ion injected alone.
10. For the quantitative analysis of the unknown mixture, inject 5.0, 10.0, 15.0, 20.0, 25.0 mg/L working solutions,
and the unknown solution. Record the peak areas of working solutions and the unknown solution.
11. Plot a calibration curve and determine the unknown concentration.

Pre-Lab Questions:
1. Define chromatogram, tM, tS, tR.
2. Draw the block diagram of HPLC. Explain the function of each component.
3. What kind of samples can be analyzed using HPLC?
4. What are the requirements for an HPLC pump?
5. Explain working principle of a reciprocating pump and what are the advantages of this pump compared to
the other pumps used in HPLC?
6. Write the types of HPLC detectors.
7. Explain normal- and reversed-phase chromatography.

Post-Lab Questions:
1. Why were four wavelengths used during the analysis? Explain considering the molecular structures of
analytes.
2. Discuss the effect of polarity of methyl paraben, propyl paraben and ambroksol HCl and stationary phase
used in the experiment on the retention times.
3. If you increase the polarity of the mobile phase, how will the retention times of methyl paraben, propyl
paraben and ambroxol HCl be effected? Explain briefly.
4. Some HPLC devices are equipped with online degasser system. What will be the advantage(s) of such an
addition to the instrument? If there is no online degasser in the instrument, what kind of precautions should
you take?
5. Why was propyl paraben peak broader than other peaks in the experiment?
6. Explain the possible reason(s) observing tm peak in HPLC chromatograms.
7. What is pulse-free output? How did we achieve it?
8. Explain the importance of filtering every solution used in HPLC experiments.
9. Define isocratic and gradient elution. Which one did we use in the experiment?

22
 Data Sheet
High Performance Liquid Chromatography (HPLC)

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

Part I: Qualitative Analysis. Calculate N and H values for each peak.

Selected
Sample Name Retention Time Peak Width
Wavelength

Peak I:

Unknown Peak II:

Peak III:

Part II: Quantitative Analysis. Calculate the concentration of each component in the unknown.

Sample Peak I Peak II Peak III

Selected Retentio Peak Selected Retentio Peak Selected Retentio Peak
Wavele n Time Area Wavele n Time Area Wavele n Time Area
ngth ngth ngth
5 ppm
10 ppm
15 ppm
20 ppm
25 ppm
Unknow
n

23
24
 FLUORESCENCE SPECTROMETRY
Experiment: Determination of fluorescein concentration (mol/L) in an unknown sample by using fluorimetry.

Solutions: Stock Fluorescein Solution (1.0 x 10-3 M): Prepared by technician.

NaOH: 500 mL, 0.10 M
Working Fluorescein Solutions (50 mL): Prepared from stock solution with the following concentrations.
Remember: ALL STANDARD AND SAMPLE SOLUTIONS SHOULD BE DILUTED WITH 0.10 M NaOH
SOLUTION.
a) 1.0 x 10-4 M b) 5.0 x 10-5 M c) 2.5 x 10-5 M
-5 -6
d) 1.0 x 10 M e) 5.0 x 10 M f) 1.0 x 10-6 M
g) 5.0 x 10-7 M h) 1.0 x 10 -7 M

a) Emission and Excitation Spectra:

1. Set up the instrument with the help of your assistant. DO NOT TURN ON ANY POWER SUPPLIES
YOURSELF.
a. Fill 1.00 x 10-5 M fluorescein solution into the cell and carefully put into the sample compartment.
Take the excitation spectrum between 430 and 530 nm and set emission MC to 515 nm. Set
bandwidth of both MC to 2.5 nm.
b. Take the emission spectrum between 470 and 550 nm and set excitation MC to 490 nm. Set
bandwidth of both MC to 2.5 nm.
c. Draw excitation and emission spectra on the same graph and compare fluoresence intensity of a and
b.
2. Take the emission spectrum of fluorescein between 470 and 550 nm and set excitation MC to 440 nm and
then to 450 nm. Compare these spectra with 1b.
3. Take the emission spectrum between 470 and 550 nm and set excitation MC to 490 nm. Set slit width of both
MC 2.5 nm. and then fix excitation spectrum to 2.5 nm slit width and change emission MC bandpass to 5.0,
10 and 20 nm.

b) Calibration Curve:
1. Measure fluorescence intensities for each standard solution including blank and then for the unknown solution.
2. Draw a calibration curve (relative fluorescence intensity vs. concentration), from the data and find dynamic
range.
3. Calculate the concentration of fluorescein in the unknown solution using calibration curve.

c) Calculating Limit of Detection and Limit of Quantitation:

1. Do ten replicate measurements for the most diluted standard solution in order to calculate the limit of detection
(LOD) according to the equation below:

LOD = (3sC) / Iavg

Iavg: Average fluorescence intensities of 10 measurements

s: Standard deviation of ten measurements
C: Minimum standard concentration that is detected

2. Calculate the limit of quantitation (LOQ) according to the equation below:

LOQ = (10sC) / Iavg

d) Effect of pH on the fluorescence of a material:
1. Take 5.0 mL samples of standard solutions c and d into separate containers and add 2 drops of concentrated
HCl to each.
2. Take excitation and emission spectra for c as you have done in Part a.
3. Decide which wavelengths should be used for excitation and emission.
4. Take the emission spectrum for d.

25
Pre-lab Questions

1. Draw the block diagram of fluorometry instrument (double and single beam). Please compare them with the
other spectrometric analysis instruments.
2. Explain the difference between emission spectrum and excitation spectrum.
3. Briefly explain singlet-singlet transition state and singlet-triplet transition state.
4. Please find at least three molecules have fluorescence.
5. Write three application area of fluorometry.
6. Why does structural rigidity enhance the quantum yield? Discuss in detail.
7. Why do some absorbing compounds fluoresce but others do not?
8. Discuss the major reasons why molecular phosphorescence spectrometry has not been as widely used as
molecular fluorescence spectrometry.

Post-lab Questions

1. What is the difference between a fluorescence emission spectrum and a fluorescence excitation spectrum?
Which more closely resembles an absorption spectrum? Explain differences with using your experimental
data in part b.
2. Why is spectrofluorometry potentially more sensitive than spectrophotometry?
3. Considering the structure of fluorescein discuss what affects the fluorescence signal of fluorescein? What is
the reason for using NaOH for the dilution of solutions?
4. Discuss the effect of source intensity on absorbance and fluorescence intensity.
5. Discuss the effect of slit width on fluorescence intensity using data in part A.
6. Discuss the effect of pH on fluorescence intensity using data in part e and part b.
7. For the quantitative analysis of your unknown, explain the behavior of your calibration curve. What happens
at high concentrations (a and b solution in part b)? Why did you see this effect on these solutions? Which
portion of the curve did you use? Why did you do so? Please explain using your experimental data.

26
 Data Sheet
Fluorescence Spectrometry (FS)

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

B. Calibration Curve

Sample Intensity

Blank

1.0 x 10-7 M (h)

5.0 x 10-7 M (g)

1.0 x 10-6 M (f)

5.0 x 10-6 M (e)

1.0 x 10-5 M (d)

2.5 x 10-5 M (c)

5.0 x 10-5 M (b)

1.0 x 10-4 M (a)

Unknown

C. Detection Limit
Sample:

Number of measurement Intensity

1
2
3
4
5
6
7
8
9
10

Theoretical concentration of unknown:

27
28
 CYCLIC VOLTAMMETRY
Experiment: Determination of diffusion coefficient of redox species using Randles-Sevcik equation by both
cyclic voltammetry and chronoamperometry method, determination of concentration of ascorbic acid in unknown
by cyclic voltammetry & standard addition method.

Determination of Diffusion Coefficient by CV and CA

1. Prepare 0.1 M KNO3 in a 25 mL volumetric flask and a mixture of 2 mM ferricyanide and 2 Mm ferrocyanide
in 0.1 M KNO3 in 10 ml volumetric flasks by using deionized water.
2. To run a background scan, insert the electrodes (working glassy carbon Pt, reference Ag/AgCl and Pt
auxiliary) and fill the cell with 0.1 M KNO3 until the ends of the electrodes are immersed. If any tiny gas
bubbles are adhering to the bottom of the electrodes, lightly tap the electrode to dislodge them. Wait ~ 1
minute before each scan to allow equilibration of the bulk concentration with that at the electrode surface.
3. If any dirt on the counter Pt electrode first burns the Pt electrode till a red shiny glow, then wait to cool down
and rinse with distilled water. Rub the glassy carbon working Ag electrode with sandpaper and rinse with
distilled water.
4. Run a CV scan from an initial potential (Ei) of + 0.6 mV to 0.0 mV and then back to + 0.6 mV at a scan rate
of 100 mV/s. Repeat the scan after stirring the solution for 10-15 seconds and allowing the solution to quiet.
The two voltammograms should match each other within 2-3 %. If not, re-clean the Pt electrode and rerun.
Ask the assistant to check the system.
5. Run duplicate CVs on the 2 mM ferricyanide-ferrocyanide mixture solution at scan rates of 20, 40, 60, 80,
100, 120 and 150 mV/s.
6. Use Gamry software to read the peak heights of anodic scan and cathodic scan separately and construct a
calibration plot for both anodic and cathodic peak currents versus square root of scan rate (v 1/2).
7. If the plot is linear, determine the value of diffusion coefficient from slope by using Randles-Sevcik equation:
i p = (2.69×105 )n3 / 2 AD 1/ 2 v1/ 2 C*, where the slope is equal to (2.69×105 )n3 / 2 AD 1/ 2 C*. Thus, the value of
D can be obtained if the electrode area is known.
8. After finishing CV measurements, repeat the experiment with chronoamperometry.
9. Firstly, determine the peak potential of anodic and cathodic scans from CV.
10. Run Chronoamperometry measurements at these potentials for 5 and 10 seconds.
11. Choose second chart option from software to obtain a plot of I versus t -1/2
12. Calculate diffusion coefficient from Cottrell equation.
13. Compare the results in the calculation part.

B. Determination of Ascorbic Acid in Tablet by CV using Standard Addition Method

1. Prepare 0.1 M KCl solution in 25 ml volumetric flask and 0.1 M ascorbic acid solution in a 25 10
mLvolumetric flask with deionized water.
2. Prepare Vitamin C tablet solution by taking 40 mg of tablet and 75 mg of KCl into a 10 mL volumetric flask
and diluting to 10 mL with deionized water.
3. To run a background scan, take 2 mL of 0.1 M KCl solution into a cell and insert the electrodes (working Pt,
reference Ag/AgCl and Pt auxiliary) until the ends of the electrodes are immersed. If any tiny gas bubbles are
adhering to the bottom of the electrodes, lightly tap the electrode to dislodge them. Wait ~ 1 minute before
each scan to allow equilibration of the bulk concentration with that at the electrode surface.
4. Run a CV scan from an initial potential (Ei) of - 0.1 V to 1.0 V and then back to – 0.1 V at a scan rate of 100
mV/s. Repeat the scan after stirring the solution for 10-15 seconds and allowing the solution to quiet. The two
voltammograms should match each other within 2-3 %. If not, re-clean the Pt electrode and rerun. Ask the
assistant to check the system.
5. Then, add 1 mL of tablet solution into the cell containing 2 mL of KCl solution to obtain a 3 mL sample
solution totally and stir the sample solution and run a duplicate CV on this solution. Take at least 5 cycles for
each measurement.
6. Then, add the necessary amount of 0.1 M ascorbic acid solution to this sample solution so that after addition
the concentration of ascorbic acid will be 1 mM. After addition, stir the solution and run a duplicate CV on
this solution. Take at least 5 cycles.

29
7. Repeat the additions of 0.1 M ascorbic acid solution into the sample solution until the concentration of ascorbic
acid will be 2.5, 5, 7.5 and 10 mM. DON’T FORGET to run CV after each addition.
8. After each run, read the peak maximum of cathodic anodic scan for the same cycle and construct a calibration
plot in the ascorbic acid concentration range (1 mM – 10 mM).
9. The extrapolation of the resulting line to zero current gives the concentration of ascorbic acid in sample
solution. Compare the result with the theoretical value in the calculation part. The 4500 mg tablet contains
1000 mg vitamin C.

Pre Lab Questions:

1. What are the advantages of electrochemical methods?

2. What are the differences between potentiometry, coulometry and voltammetry?
3. What kind of experiments are studied by using potentiometry?
4. What are the differences between three electrode systems and two electrode systems?
5. What is the basic working principle of voltammetry?
6. What are the mass transport mechanisms in voltammetry? Explain.
7. What are the differences between (a) linear scan voltammetry and pulse voltammetry, (b) differential pulse
voltammetry and squarewave voltammetry?
8. What is the basic working principle of cyclic voltammetry?
9. What are the components of an electrochemical cell? Explain each component.
10. What are the differences between electrochemical and galvanic cells?
11. What is the electrode potential for a half-cell consisting of a cadmium electrode immersed in a solution that
is 0.0150 M in Cd2+? (See example 22-2)
12. Calculate potentials for the following cell using (a) concentrations and (b) activities:
Zn I ZnSO4,(c ZnSO4) , PbSO4,(sat'd) I Pb
where c ZnSO4 = 5.00 x 10-4 , 2.00 x 10-3 , 1.00 x 10-2, 2.00 x 10-2 and , 5.00 x 10-2
(See example 22-2)
13. How does conduction take place in a cell?
14. What is the electrical double layer?
15. What are the properties of the cells without liquid junctions?

Post Lab Questions:

1. Why is a high supporting electrolyte concentration used in most electroanalytical procedures?

2. Why is the reference electrode placed near the working electrode in a three electrode cell?
3. What is the role of auxiliary/counter electrode in cyclic voltammetry?
4. Why is a three electrode system used in voltammetry instead of a two-electrode system?
5. How does peak change with scan rate in cyclic voltammetry? Why?
6. How can it be understood that a redox process is diffusion controlled or not?

30
 Data Sheet
Cyclic Voltammetry (CV)

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

A. Determination of Diffusion Coefficient by CV and CA

DA (in cm2/s): D C (in cm2/s):
Scan Rate (mV/s) ip,a (µA) ip,c (µA)

20

40

60

80

100

120

150

Experimental Value of Diffusion Coefficient (in cm 2/s):

Diffusion Coefficients Calculated From Cottrell Equation:
For 5 Seconds (in cm2/s): For 10 Seconds (in cm 2/s):
Theoretical Value of Diffusion Coefficient (in cm 2/s):
% Error:

B. Determination of Ascorbic Acid in Tablet by CV using Standard Addition Method:

Addition (µL) Concentration (mM) ip,a (µA)

2.5

7.5

10

Amount of ascorbic acid in 40 mg vitamin C Tablet (in mg): % Error:

31
32
 THIN LAYER AND PAPER CHROMATOGRAPHY, ELECTROPHORESIS
(TLC-PC-EF)

A) THIN LAYER CHROMATOGRAPHY (TLC)

Experiment: Separation of pH indicator mixtures

Silica gel plate: Use silica coated plates in the cabinet.

Chemicals:
Congo Red, Phenol Red, Bromophenol Blue, Cresyl Violet, Brilliant Cresyl Blue, Unknown Mixture

Mobile Phases:
a) MP1: 5.0 mL (3.0 mL of n-Butanol + 1.0 mL of Ethanol + 1.0 mL of 2.0 M Ammonia)
b) MP2: 5.0 mL (3.0 mL of n-Butanol + 1.0 mL of Glacial Acetic Acid + 1.0 mL of Water)

Procedure:
1. Pour 5.0 mL of the chosen solvent into the chromatography tank and close the lid.
2. Take the prepared silica gel plates and prepare the following TLC plate by using pencil
and scissors. (Height of the plate: 6 cm, Length: 6 cm, Marking line 1 cm above the
bottom)
3. Apply one drop of each indicator to the labeled spots using a capillary tube. On the
sixth origin apply one drop of mixture.
4. Put the plate into the tank by taking care not to let the solvent touch the spots directly.
5. Close the tank and run the chromatogram.
6. Remove the plate and mark the solvent front with a pencil or spatula along the plate.
7. Dry the chromatogram and calculate Rf values and identify the components of the mixture.

B) PAPER CHROMATOGRAPHY (PC)

Experiment: Separation of Metal ions (Ni2+, Cu2+, Co2+) by ascending chromatography.

Chemicals:
Ni2+, Cu2+, Co2+ complex solutions
Rubeanic acid/ethanol (0.1 g in 100 mL of ethanol) (Prepared by technician)

Mobile Phase: Mixture composed of 43.0 mL of Acetone, 3.0 mL concentrated HCl and 4.0 mL of water.

Procedure:
1. Place 50 mL of mobile phase into the cylinder tank. Close the lid.
2. Take a sheet of “23 x 23 cm ascending chromatography” paper and mark the line 2.5 cm above the bottom
taking care of the flow direction of paper if there is any.
3. Place the spots of the metal ions and mixture, evenly spaced, along the baseline.
4. Fold the paper and secure it with paper clips. Then place the paper with the spotted end down in the glass tank
not to let the paper touch the glass wall. Close the tank with the lid.
5. If the chromatogram is observed closely, a yellow band due to Cu may be moving on the solvent front.
6. After the solvent front has traveled about half of the paper, remove the chromatogram from the tank, open
out, mark the solvent front with a pencil, and dry by using a hair dryer. Then; spray rubeanic acid, and expose
the chromatogram to vapors of conc. NH3 solution. Record your observations.
7. Calculate Rf values and identify the components of the mixture, compare the Rf values of each metal obtained
from the ascending chromatography.

Pre-Lab Questions:

1. Explain how quantitative analysis is done with TLC and PC.

2. Define two dimensional thin layer chromatography.
3. What is the driving force for the sample to be carried through the stationary phase on TLC and PC?
4. What is the basic principle(s) of that some samples can be carried faster than other samples through the
stationary phase by mobile phase on TLC?
5. Write one of the ways to locate analytes on the plate used by TLC?
6. Explain the Rf value. How can we calculate and interpret it?

33
7. Draw the structure of silica gel and explain its properties.

Post Lab Questions (TLC)

1. Explain the reason(s) of closing the solvent tank with a lid.

2. Compare mobile phases. Which solvent is more suitable for the separation of these dyes? Explain briefly.
3. Which one of the indicators used in the experiment changed its color in the mobile phases? Explain the
reason(s) in the color change of the indicator(s).
4. According to your calculations, order the polarities of these dyes from the most polar to the least polar. Explain
considering their structures.

Post Lab Question (PC)

1. Why do you use Rubeanic acid? What is the reason for the change in the color of the spot when you sprayed
it to the paper surface?

C) ELECTROPHORESIS

Experiment: Separation of dyes by electrophoresis.

Materials:
Congo Red, Phenol Red, Bromophenol Blue, Methyl Red, Cresyl Violet, Brilliant Cresyl Blue
Buffer solution (500 mL of buffer with a of pH 7.4 using KH 2PO4 and K2H2PO4)
Agarose gel (1.0 % (w/v))

Procedure:
1. To prepare gel; 0.5 g agarose powder is mixed with a 50 mL phosphate buffer and heated in a microwave
oven until it completely dissolves. Then it is poured into a casting tray containing a sample comb and allowed
to solidify at room temperature. After the gel is solidified, the comb is removed, taking care not to rip the
bottom of the wells. The gel, still in a plastic tray, is inserted horizontally into the electrophoresis chamber
and is covered with the buffer.
2. Apply each dye into the wells of the gel.
3. Place the tray in the tank.
4. Pour the buffer into the tank until it covers the gel.
5. Put the lid on the tank.
6. Select constant mode and set the potential difference to 60 V for 20 min.
7. After 20 minutes, note your observations about the position of the dyes on the gel (charge, distance, shape of
the stain, etc.)
8. Start another 20 minutes separation at 60 V if necessary after it cooled for a while.
9. After 20 minutes, note your final observations.

Pre-lab Questions

1. What is the function of agarose gel?

2. Which equipment(s) are required for preparing and running agarose gel?
3. Describe preparation of 1.0% (w/v) agarose gel in a phosphate buffer solution.
4. Write the name(s) of parameters affects the mobility of analytes in the gel?

Post-Lab Questions

1. Draw the chemical structures of each indicator used in the EP part. Depending on pH of the medium, determine
the direction of these charged species. Is the chromatogram obtained from the experiment consistent with the
theory? Explain briefly.
2. Compare the size of the dyes and discuss the travel distance of each dye regardless of the charge?

34
 Data Sheet
Thin Layer and Paper Chromatography, Electrophoresis

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

A.Thin Layer Chromatography

Rf Values of Dyes in MP1 Rf Values of Dyes in MP2

Congo Red: Congo Red:

Phenol Red: Phenol Red:

Bromophenol Blue: Bromophenol Blue:

Cresyl Violet: Cresyl Violet:

Brilliant Cresyl Blue: Brilliant Cresyl Blue:

Unknown: Unknown:

Identity of the Unknown:

B.Paper Chromatography:

Rf Values

Ni2+:

Cu2+:

Co2+:

Unknown:

Identity of the Unknown:

35
36
 GAS-LIQUID CHROMATOGRAPHY (GLC)
GC-FID

Experiment 1: Qualitative Analysis

1. Set Gas Chromatography values as follows;

i) column temperature = 40 °C
ii) flow rate = 30 mL/min
2. Inject 1.00 µL from organic compounds diluted with methanol 1+5 (v/v) (n-butanol, n-hexane, isopropanol
(IPA), dichloromethane(DCM))
3. Prepare the following mixtures:
Mixture: 0.5 mL DCM, 0.5 mL n-Hexane, 0.5 mL n-Butanol and 0.5 mL IPA.
4. Inject 1.00 µL of the mixture, record the peaks and their retention times.
5. Identify the components in the mixture by comparing the retention times of present peaks obtained with that
of organic compounds.

Experiment 2: Effects of Column Temperature on Retention Time

1. Set Gas Chromatography values as follows;

i) column temperature = 40 °C
ii) flow rate = 30 mL/min
Allow the column to stabilize. Then, inject 1.00 μL of mixture 2. Record the peaks and their retention
time.
2. Increase the column temperature to 60 °C, 80 °C, 100 °C and 120 °C. At each temperature repeat the injection
procedure after allowing the column to stabilize.

Experiment 3: Temperature Programming for Mixtures

1. Set Gas Chromatography values as follows for experiment 3:

i. column temp. = 30 °C
ii. flow rate = 30 mL/min
iii. initial time = 1.0 min
iv. ramp = 20 °C /min
v. upper temp. = 120 °C
vi. upper time = 0.5 min
2. Inject 1.00 µL of mixture 2, record the peaks and their retention times.
3. At the end of the experiment, do not shut off the instrument and inform your responsible assistant.

Graphs and Calculations:

● Plot the retention time vs temperature for DCM, IPA, n-butanol and n-hexane graph. (Use data in Experiment
2, all in one graph)
● Find W values from your chromatograms and calculate N & H for n-butanol at all T values (from experiment
2 & 3)

GC-MS

Experiment 1: Mass Spectral Identification of Toluene and Benzene Using GC-MS

Chromatographic method:

i. Solvent delay: 2.5 min

ii. Flow rate: 0.9 mL/min

iii. For SCAN mode: m/z 25-150 amu

For SIM mode: Benzene Group: m/z 51 and 78 amu Toluene Group: m/z 91 and 92 amu

37
 iv. Initial column T: 30°C

v. Hold: 2.5 min vi. Ramp: 60 °C /min

vi. Upper T: 150 °C

vii. Hold: 1 min

viii. Total runtime: 5.5 min.

1. Preparation of Solutions: Prepare solutions of 10.0 mL of 10 mg/L toluene and benzene in methanol
using 1000 mg/L methanolic solutions of benzene and toluene. Prepare a series of calibration solutions
of benzene in methanol by taking appropriate volumes from 10.0 mg/L stock solution to have 1.0 mL of
0.0, 0.5, 1.0, 2.0, 3.0, and 5.0 mg/L benzene. Add to each calibration solution an appropriate volume of
toluene stock solution (10.0 mg/L) as an internal standard to have 2.0 mg/L toluene in the final solutions.
Using the chromatographic method described above, inject 1.0 µL of the 5.0 mg/L benzene solution
containing 2.0 mg/L toluene as the internal standard in full scan mode. For a full scan, use m/z range from
25 to 150. 5.
2. Take the mass spectra of each peak at its retention time and determine the identity of each peak using
fragmentation patterns and the NIST library.
3. Determine the m/z values for toluene and benzene from the full scan spectra that can be used for
quantitative analysis in single ion monitoring (SIM) mode.

Experiment 2: Quantitative Analysis of Benzene Using External Calibration and Internal Standard
Calibration Methods

1. Using the chromatographic method described above, inject 1.0 µL of each calibration solution in SIM
mode to GC-MS.
2. Sketch the external calibration curve based on peak area versus concentration of benzene in the calibration
solutions. Determine the linear equation.
3. Sketch the internal calibration curve based on the peak area ratio of benzene to toluene versus the
concentration of benzene in the calibration solution. Determine the linear equation.
4. Inject the unknown sample and determine the concentration of benzene in the sample using external and
internal calibration curves.

Pre-Lab Questions:

1. What is a chromatogram?

2. How do gas-liquid and gas-solid chromatography differ?

3. What properties should the stationary and mobile phases have in gas-liquid chromatography?

4. Describe the principle on which each of the following gas chromatography detectors is based and what are the
advantages and principal disadvantages of these detectors: a) thermal conductivity, b) flame ionization, c)
electron capture, d) thermionic, e) photoionization.

5. How can the number of plates in a column be calculated?

6. Draw the block diagram of a gas chromatograph. Explain the function of each component.

38
7. What is the relation between the efficiency of chromatography and the Height Equivalent of Theoretical Plates
(HETP) number of plates?

8. What is the function of a sample splitter and how does that work?

9. What is a mass spectrometer?

10. What is SIM mode in mass spectrometry?

11. What is full scan mode in mass spectrometry? 24

12. What is the difference between soft and hard ionization?

13. How does an electron impact ionization work?

14. What is an internal standard?

15. What is the purpose of using internal calibration?

Post-Lab Questions:

1. Discuss the effect of boiling point and polarity of the analytes used in this experiment on the retention times.
Are your results consistent with the literature values? Explain briefly.

2. Discuss the effect of column temperature on retention time in Exp 2.

3. Discuss the effect of temperature programming on an unknown chromatogram (Compare the retention times
and resolution with that obtained in Exp 2&3)

4. Even if the concentration of analytes is the same, the areas under the peaks may be different from each other.
Explain briefly.

5. Discuss which one of the calibration methods provides more reliable results in GC-MS.

6. Discuss the fragmentation pattern of benzene and toluene in GC-MS.

7. Compare the peak areas of 5.0 mg/L benzene solution containing 2.0 mg/L toluene as the internal standard
obtained by full scan mode and SIM mode. Discuss the advantages of each mode.

39
40
 Data Sheet
Gas Chromatography (GC)

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

GC-FID

Experiment 1
Mixture:
Retention
Time (tr), min
Methanol
n-Buthanol
n-Hexane
Isopropanol (IPA)
Dichloromethane (DCM)
Retention Time (tr), min Component

Experiment 2

Width of
tr of n- tr of n- Plate
Temperature tr of DCM, tr of IPA, n- Number of
Buthanol, Hexane, Height
(° C) min min Buthanol Plates (N)
min min (H), m
(W), min
40
80
100

Experiment 3

tr of tr of tr of n- tr of n- Width of n- Plate
Number of
Temperature DCM, IPA, Buthanol, Hexane, Buthanol Height (H),
Plates (N)
Programming min min min min (W), min m

GC-MS

Concentration (ppm) Peak Area of Benzene Peak Area of Toluene Ratio of Peak Areas
0.5
1.0
2.0
3.0
5.0
Unknown

True Value of Unknown Concentration= % Relative Error=

41
42
 REFRACTOMETRY AND POLARIMETRY (REF-POL)
A . REFRACTOMETRY

Experiment:
Determination of Refractive Indices of Solvents

Solutions:
Place the following solvents about 2-3 mL in tubes (do not inhale and avoid skin contact with solvents)
a) Distilled water b) Carbon tetrachloride
c) Chloroform d) Diethyl ether
e) Ethanol

Prepare the following standard solutions. (Note that the chosen solvents must be miscible with each other;
otherwise, refractive index cannot be measured).

Concentration %(v/v)
Solvent A 0 20 40 60 80 100
Solvent B 100 80 60 40 20 0
A: Ethanol B: Water

Procedure:
1. Clean the prisms with ethanol.
2. Place two or three drops of distilled water over prism with a droplet.
3. Turn the dispersion knob until a sharp line appears between the light and dark halves in the field.
4. Record the refractive index of distilled water.
5. Repeat the refractive index measurement for other solvents, standard solutions and an unknown solution given
by your assistant.
6. Determine the composition of unknown solution by using the calibration plot drawn with the standards.
7. Clean the prism with ethanol and all glassware with distilled water that were used in the experiment.
8. Write down all data on Data Sheet using a pen, not a pencil.

Calculations:

Questions 1, 2 and 3 will be answered for pure solvents, not for mixtures!

1. Calculate specific refractivity r by using following equation.

n2 −1 1
r= 2 
n +2 d
n : refractive index obtained experimentally
d : the density of the solvent

2. Calculate molar refractivity (R) experimentally using Lorentz-Lorenz formula

n2 −1 M
R= 
n2 + 2 d M: molecular weight of the analyte

43
3. Calculate the molar refractivity (R) theoretically by summing up the atomic refractivities for each molecule.

Atoms Atomic Refractivities

C 2.42
H 1.40
O 1.52
Cl 5.9

4. From the dispersion measurements, it is possible to estimate the refractive index at other wavelengths. To do
this, proceed in the following manner:
(The following calculations will be made ONLY for H2O at 489 nm, 689 nm and 789 nm)

- calculate dispersion (nF - nC) using standard dispersion tables

n F − nC = A + ( B  M )

- compute Z from the equation:

Z = 5.2364  10 5  (n F − nC )

- compute Y from the equation:

Y = n D − 2.8796  10 −6  Z nD: refractive index at sodium D line

Using the following equation, calculate the refractive indices of pure water at 489 nm, 689 nm and 789 nm:

B
nx = Y +
 (m)
2
x

Dispersion Table
nD A B M
1.30 0.02446 0.03829 1.000
1.31 0.02441 0.03811 0.999
1.32 0.02436 0.03791 0.995
1.33 0.02431 0.03769 0.988
1.34 0.02426 0.03746 0.978
1.35 0.02422 0.03721 0.966
1.36 0.02418 0.03694 0.951
1.37 0.02414 0.03665 0.934
1.38 0.02410 0.03634 0.914
1.39 0.02406 0.03601 0.891
1.40 0.02402 0.03567 0.866

44
B . POLARIMETRY

Experiment: Determination of Optical Rotation of Rochelle Salt

Solutions:
Standard Rochelle Salt (Potassium Sodium Tartarate) Solution: 100.0 mL, 0.75 M.
Working Rochelle Salt Solutions (25.0 mL): prepared from Standard Rochelle Salt Solution
a) Blank b) 20 % (v/v)
c) 40 % (v/v) d) 60 % (v/v)
e) 80 % (v/v) f) 100 % (v/v)

Procedure:
1. Place first blank solution then other working solutions in the polarimeter tube and measure the angle of
rotations.
2. Ask for unknown solutions to assistant.
3. Clean all glassware with distilled water that was used in the experiment.
4. Write down all data on Data Sheet with pen.

Calculations:
Calculate specific rotation [] of each solution using the equation:
 net
 T =
lc
net : optical rotation
l : length of sample cell (dm)
c : concentration in grams per 100 mL.
T : temperature, 0C.

1. Plot   vs. c
2. Plot net vs. c

Pre Lab. Questions:

1. Define followings; refraction, refractive index, Snell’s Law, Specific and Molar Refraction, Critical angle,
unpolarized light, polarization, nicol prism

2. Explain the working principle of Amici Prism

3. What are variables that affect refractive index measurements?
4. Draw the block diagram of refractometer. Explain briefly the function of each component.
5. Describe the preparation of 50.0 mL of 30% ethanol (v/v) solution.
6. Please draw the block diagram of polarimeter. Explain briefly the function of each component.
7. Please explain the working principle of Nicol Prism.

Post Lab. Questions:

1. Why do we use Sodium lamp for refractometry? Can we use another type of lamp? Please explain.
2. Why is the position of the analyzer different from the position of the polarizer in the instrument?
3. Calculate percent relative error for molar and specific refractivity in part A? Explain possible sources of the
errors?
4. Explain briefly the reason(s) of deviation from the linearity in the calibration curve of water-ethanol
solutions in refractometry. (Hint: intermolecular forces)
5. What is the effect of bubbles in test tube in the measurement of Rochelle Salt solutions? Explain briefly.
6. Please discuss your graph of specific rotation-concentration in polarimetry.

45
46
 Data Sheet
Refractometry-Polarimetry (Ref-Pol)

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

A.Refractometry

Sample Refractive Index (nD)

Distilled water
Chloroform
Diethyl ether
Ethanol
0% Ethanol + Water
20% Ethanol + Water
40% Ethanol + Water
60% Ethanol + Water
80% Ethanol + Water
100% Ethanol + Water
Unknown

Theoretical concentration of unknown:

B.Polarimetry

Sample αnet

Blank

20%

40%

60%

80%

100%

Unknown

Theoretical concentration of unknown:

47
48
 ION EXCHANGE CHROMATOGRAPHY (IE)
Experiment: Separation of Iron and Cobalt Ions by Ion Exchange Chromatography
Solutions and materials:
▪ Hydroxylamine hydrochloride, 5%: prepared by technician
▪ o-phenantroline, 0.1%: prepared by technician
▪ Ammonium acetate: 2.0 M, 100.0 mL
▪ Ammonium thiocyanate: 10.0 M, 50.0 mL
▪ Acetone-ammonium thiocyanate mixture: Prepared by mixing all of ammonium thiocyanate solution with
240.0 mL of acetone (put into volumetric flask and close tightly)
▪ HCl: 500.0 mL, 4.50 M
▪ Dowex 21K; Strongly basic anion exchange resin

The resin is made up of styrene-DVB gel which contains quaternary amine functional
groups which provide the resin ability to exchange anions. When in the OH - ionic
form, the resin acts as a strong insoluble base in solution. These OH - groups are
replaced by anions during the ion-exchange process.
The properties are given below:

▪ Congo red paper

▪ Iron stock solution : 50.0 mL, 0.02 M from FeCl3.6H2O
▪ Cobalt stock solution: 50.0 mL, 0.05 M from CoCl2.6H2O
▪
NOTE: PREPARE THE STOCK SOLUTIONS IN 4.50 M HCl USING VOLUMETRIC FLASKS
Separation Procedure
Cobalt Recovery:
1. Elute the column 2 times with approximately 20.0 mL portions of 4.50 M HCl
2. Inject 2.00 mL of sample solution which is prepared by your assistant. by mixing iron and cobalt stock
solutions in unknown proportions.
3. Allow the sample to be drained on by the column, NEVER ALLOW THE COLUMN TO DRY.
4. Add approximately 1mL of 4.50 M HCl to wash the solution down onto the resin and again allow this solution
to be drained (repeat once more).
5. Fill the column with 4.50 M HCl.
6. Elute the column with 4.50 M HCl and start to collect the effluent into 100.0 mL beaker when cobalt starts to
come out of the column until no indication of cobalt with thiocyanate test. (Treat the effluent with the mixture
of acetone-ammonium thiocyanate solution. The green color confirms the presence of cobalt.)
7. Transfer the effluent into a 100.0 mL volumetric flask and treat same as cobalt standard solutions.

Preparation of Standard Cobalt Solutions and Color Development:

1. Prepare 100.0 mL of 1.0 ×10-3 M cobalt standard from the stock solution. (Dilutions will be done with
distilled water.)
2. From this solution, prepare 50.0 mL of 1.0 ×10-4 M, 2.0 ×10-4 M, 3.0 ×10-4 M, 5.0 ×10-4 M standard
solutions. (Dilutions must be done with acetone-ammonium thiocyanate mixture JUST BEFORE
ABSORBANCE MEASUREMENTS.)
3. After preparing a blank solution measure absorbance at 620 nm as soon as possible because the color is not
stable.
4. Determine percent cobalt recovery by dividing the amount of cobalt taken from the column to the amount of
cobalt injected to the column.

49
Iron Recovery
1. After all cobalt ions are collected from the column use distilled water as eluent for the recovery of iron.
2. Elute the column with distilled water and start to collect the effluent into 50.0 mL beaker when iron starts to
come out of the column until no indication of iron with thiocyanate test. (Treat a few drops of effluent with
the mixture of acetone-ammonium thiocyanate solution on a watch glass. The dark red color confirms the
presence of iron.)
3. Transfer all the collected effluent to 100.0 mL volumetric flask and apply color development method together
with iron standard solutions.

Preparation of Standard Iron Solutions

1. Prepare 100.0 mL of 4.00×10-4 M intermediate standard iron solution from the stock solution (use distilled
water for dilution).
2. Put necessary amount of intermediate standard solution into each 100.0 mL volumetric flask for the
preparation of 2.00×10-5 M, 4.00×10-5 M, 6.00×10-5 M, 8.00×10-5 M iron solutions and dilute to the mark by
using color development method.
3. Prepare a blank solution and apply the same procedure to the blank.

Color Development of Iron Solutions

The following steps are also valid for effluent and blank solutions.
1. Add 4.0 mL of 5 % hydroxyl amine hydrochloride into each flask
2. Place a small piece of Congo-red paper into each volumetric flask, its color will turn blue.
3. Then add ammonium acetate solution dropwise until its color just turns to red again.
4. Add 4.00 mL of o-phenanthroline solution and dilute to the mark with distilled water.
5. After preparing a blank solution, measure the absorbance values at 500 nm.
6. Draw the calibration plot. Find the amount of iron collected from the column and determine the percent iron
recovery.

Pre Lab. Questions

1. How do strong and weak acid synthetic ion-exchange resins differ in structure?
2. Write equilibrium reaction of a strong cation exchanger and a strong anion exchanger?
3. Draw structure of a cross linked polystyrene ion-exchange resin.
4. Give examples to applications of ion-exchange chromatography.
5. Please define followings;
- Anion/cation exchanger
- Stationary/Mobile phase
- Capacity
- Selectivity

6. Describe the preparation of 500 mL of 4.5 M HCl from a concentrated HCl solution which has a density of
1.19 g/mL and 37% (w/w). (HCl: 36.5 g/mol)
7. Describe the preparation of 50.0 mL, 0.02 M FeCl3.6H2O (FeCl3.6H2O=270.2956 g/mol)
8. Please write properties of ion-exchange resins.

Discussion Questions:
1. What is the type of resin used in this experiment?
2. Write all reactions taking place in the ion Exchange column.
3. Eluent concentration is critical for recovery of the ions in the unknown mixture. Why?
4. Why the column resin should not be allowed to dry out? Explain briefly.
5. Discuss the functions of each reagent used for the color development of iron and cobalt ions.
6. Discuss the reasons of low recovery for each ion.

50
 Data Sheet
Ion Exchange Chromatography (IE)

Student’s Name: Group Number:

Group Members:
Signature of the Assistant:
Date:

Cobalt Solution:

Concentration Absorbance

1.0×10-4 M

2.0×10-4 M

3.0×10-4 M

5.0×10-4 M

Unknown:
Dilution Factor (if applicable) :

Iron Solution:

Concentration Absorbance

2.0×10-5 M

4.0×10-5 M

6.0×10-5 M

8.0×10-5 M

Unknown:
Dilution Factor (if applicable) :

51