lab manual instrumental analysis laboratory 2013 spring...

CHEM 324 Lab Manual, 2012, Middle East Technical University, Ankara

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Lab Manual

Instrumental Analysis Laboratory

2013 Spring Semester

Middle East Technical University

Department of Chemistry


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2013 Spring Semester Analytical Chemistry Laboratory II Groups

GROUP NUMBERS

WEEK 1 2 3 4 5 6 7 8 9 10

1 GLC CV REF FS AAS IR UV IE TLC HPLC

2 HPLC GLC CV REF FS AAS IR UV IE TLC

3 TLC HPLC GLC CV REF FS AAS IR UV IE

4 IE TLC HPLC GLC CV REF FS AAS IR UV

5 UV IE TLC HPLC GLC CV REF FS AAS IR

6 IR UV IE TLC HPLC GLC CV REF FS AAS

7 AAS IR UV IE TLC HPLC GLC CV REF FS

8 FS AAS IR UV IE TLC HPLC GLC CV REF

9 REF FS AAS IR UV IE TLC HPLC GLC CV

10 CV REF FS AAS IR UV IE TLC HPLC GLC

UV : ULTRAVIOLET AND VISIBLE SPECTROMETRY

HPLC : HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

GLC : GAS-LIQUID CHROMATOGRAPHY

CV : CYCLIC VOLTAMMETRY

AAS : ATOMIC ABSORPTION AND EMISSION SPECTROMETRY

FS : FLUORESCENCE SPECTROMETRY

IE : ION EXCHANGE CHROMATOGRAPHY

TLC : THIN LAYER AND PAPER CHROMATOGRAPHY AND

- ELECTROPHORESIS

REF : REFRACTOMETRY AND POLARIMETRY

IR : INFRARED SPECTROMETRY


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GENERAL INFORMATION

1. Total grade for your laboratory work will be evaluated based on 1000 points. Distribution of these points

are;

150 Personal Opinion of lab assistants

200 Reports

200 Quizzes

150 Midterm

300 Final Exam

1000 Total

2. Attendance is compulsory. A medical report is required to have a make-up. Student is not allowed to enter

to the laboratory when she/he is late. Make-up will not be given for being late.

4. Each student may take maximum two make-up experiments with submission of a medical report.

5. Make-up experiments will be performed during the semester.

6. If you miss three experiments, the grade will be FF

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

8. The students must behave in 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.

9. 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.

10. Each student is responsible for his/her own report. Reports will be submitted to Turnitin database in two

parts, first part will be uploaded on the first day of the experiment till 13:30 and consist of the purpose,

theory and the references. Second part consisting of instrumentation, calculation and discussion will

be uploaded on the second day of the following week till 13:30.

11. 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 ‘FD’ and will not be able to continue this course.


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LABORATORY PROGRAM

Each student has two lab periods 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 at home and be ready for the first half day.

1. Please bring the following items with you.

Report consisting of the purpose, theory and the references. (Must be submitted at the

beginning of the experiment)

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

Calculator, water proof pen,

Textbook, (MUST be brought both on FIRST and SECOND LAB PERIODS for ALL

EXPERIMENT. (D. A. Skoog, F. J. Holler, S. R. Crouch, (2007) 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 by entrance quiz. Following the quiz, you will be studying the part of the text book or

documents related to your experiment.

3. A discussion will take place with your assistant. Performance of students will be evaluated.

4. When and if your assistant is convinced that you are ready, a quiz will be given.

5. A grade of less than 6/10 is failure and student’s report will be evaluated out of 100 instead of 200.

SECOND LAB PERIOD

1. Please bring the following items with you.

Report of the previous experiment consisting of instrumentation, data sheet, calculation,

discussion and references. (Must be submitted at the beginning of the experiment)

Instrumentation must present in the second report, not the first part!

Materials listed above.

2. 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.

3. Perform the experiment as a group. Each member of the group should participate in experiment as much

as possible.

4. Complete data sheet of your report. Data sheets without the signature of assistant will not be

evaluated.

5. 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.


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Reading Assignments

UV-VIS

Chapter 13

- An introduction to the Ultraviolet/Visible Molecular Absorption Spectrometry

- Measurement of Transmittance and Absorbance

- Beer’s Law

o Application of Beer’s Law to Mixtures

o Limitations to Beer’s Law

o Real Limitations to Beer’s Law

o Apparent Chemical Deviations

o Apparent Instrumental Deviations with Polychromatic Radiation

o Instrumental Deviations in the Presence of Stray Radiation

- Effect of Slit Width on Absorbance Measurements

- Instrumentation

o Instrument Components

o Sources: Deuterium and Hydrogen Lamps, Tungsten Filament Lamps

o Sample Containers

o Types of Instruments: Single-Beam and Double Beam Instruments

Chapter 14

- Photometric and spectrophotometric titrations

FS

Chapter 15

- Molecular Luminescence Spectrometry

- Theory of Fluorescence and Phosphorescence

- Figure 15-2. Partial energy diagram for a photoluminescent system

- Rates of Absorption and Emission

- Deactivation Processes

o Vibrational Relaxation

o Internal conversion

o External Conversion

o Intersystem Crossing

o Phosphorescence

- Variables affecting fluorescence and phosphorescence

o Quantum Yield

o Transition types in fluorescence

o Quantum efficiency and transiton type

o Fluorescence and structure

- Emission and Excitation Spectra

- Instruments for Measuring Fluorescence and Phosphorescence

- Components of Fluorometers and Spectrofluorometers

o Sources: Lamps

o Filters and Monochromators

o Transducers

o Cells and cell compartments

- Instrument designs

o Fluorometer

o Spectrofluorometer

- Chemiluminescence

- The Chemiluminescence Phenomenon

IR

Chapter 16

- An introduction to Infrared Spectrometry

- Dipole Changes During Vibrations and Rotations


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- Types of Molecular Vibrations

- Vibrational Modes

- Sources

- Thermal Transducers

- Fourier Transform Spectrometers

- Dispersive Instruments

-

Chapter 17

- Applications of Infrared Spectrometry

- Sample Handling : Gases, liquids, solids

- ATR spectrometry

CV

Chapter 22

- An Introduction to Electroanalytical Chemistry

- Electrochemical Cells

o Conduction in a Cell

o Galvanic and Electrolytic Cells

o Anodes and Cathodes

o Cells without Liquid-Junctions

o Schematic Representations of Cells

- Potentials in Electroanalytical Cells

o Liquid-Junction Potentials

Chapter 22

o 22E-2 Polarization

o 22E-3 Mechanism of Mass Transport

o 22E-4 Charge Transfer Polarization

o Voltammetry

Chapter 23

- Potentiometry

- Reference Electrodes

o Calomel Electrodes

o Silver-Silver Chloride Electrodes

Chapter 25

- 25A Excitation Signals In Voltammetry

- 25B Voltammetric Instrumentation

- 25B-3 Voltammograms

- 25D Cyclic Voltammetry

GLC

Chapter 26

- An Introduction to Chromatographic Separations

o A General Description of Chromatography

o Classification of Chromatographic Methods

o Elution Chromatography on Columns

o Chromatograms

- Migration Rates of Solutes

o Distribution Constants

o Retention Time

o Relative Migration Rates The Selectivity Factor

- The Rate Theory of Chromatography

- A Quantitative Description of Column Efficiency

- Column Resolution

- Applications of Chromatography


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Chapter 27

- Gas Chromatography

- Instruments for Gas-Liquid Chromatography

o Carrier Gas Supply

o Sample Injection System

o Column Configurations and Column Ovens

o Flame Ionization Detector

- Gas Chromatographic Columns and Stationary Phases

o The Stationary Phase

- Qualitative Analysis

- Quantitative Analysis

HPLC

Chapter 26

- An Introduction to Chromatographic Separations

o A General Description of Chromatography

o Classification of Chromatographic Methods

o Elution Chromatography on Columns

o Chromatograms

- Migration Rates of Solutes

o Distribution Constants

o Retention Time

o Relative Migration Rates The Selectivity Factor

- The Rate Theory of Chromatography

- A Quantitative Description of Column Efficiency

- Column Resolution

- Applications of Chromatography

Chapter 28

- High-Performance Liquid Chromatography

- Scope of HPLC

- Pumping Systems

- Reciprocating Pumps

- Types of Detectors

- Ultraviolet Absorbance Detectors with Filters

- Partition Chromatography

o Columns for Bonded-Phase Chromatography

o Reversed-Phase and Normal-Phase Packings

- Column Selection in Partition Chromatographic Separations

- Mobile-Phase Selection in Partition Chromatography

Lab Texts

- IE

- TLC

- AAS

- REF-POL

Write any literature 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.

http://www.awc.metu.edu.tr/handouts/APA_Documentation.pdf

http://www.awc.metu.edu.tr/handouts/APA_Documentation.pdf


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CHEM 324

ANALYTICAL CHEMISTRY

LABORATORY II

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 mL, 1.00x10-3 M) from K2Cr2O7: Prepared in 0.03 M H2SO4

solution. b. 500 mL, 0.03 M H2SO4

c. Stock MnO4- solution (100 mL, 3.00x10-3 M) from KMnO4

d. Working solutions: Cr2O72- (50 mL), MnO4

- (50 mL) prepared from their stock solutions in following concentrations:

a) Blank b) 1.00x10-4 M c) 2.00x10-4 M

d) 3.00x10-4 M e) 4.00x10-4 M Procedure: 1. Turn on the instrument before any measurements for 10 min. 2. Correct the baseline between the working wavelength range with distilled water in sample and reference cells (Baseline correction). 3. Take the spectra of MnO4

- and Cr2O72- separately by scanning from 250 nm to 750 nm in

overlay mode and find two wavelengths which can be used for quantification of both ions. 4. Measure the absorbance of MnO4

- and Cr2O72- working solutions at the selected

wavelengths found in Step 3. 5. Ask for unknown solution containing MnO4

- and Cr2O72- ions. Measure the absorbance of

the solution at the two wavelengths selected. Calculations: 1. Plot the calibration curve (Absorbance vs. Molar 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.


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Experiment 2: Photometric Titration of Cu2+ by EDTA Solutions: a. 0.01 M, 100.0 mL Cu2+ prepared from Cu(NO3)2 in ammonia buffer (add 9.00 mL concentrated aqueous NH3 and 1.35 g NH4NO3) b. 0.10 M EDTA solution Procedure:

1. Transfer 60 mL of Cu solution in a 250 mL erlenmeyer flask. 2. Take UV spectrum at the beginning between 400nm and 900 nm. 3. Add EDTA solution dropwise by 1.00 mL increments. 4. Take UV spectrum after addition of each milliliter (Do not discharge the solution

used into waste, put it back to the flask) 5. Continue adding EDTA solution after the endpoint and take at least 5 measurements. 6. Select a suitable wavelength where Cu2+ and Cu-EDTA complex (1:1) have a

maximum ratio in absorbance. 7. Plot the titration curve at the selected wavelength and calculate the amount of Cu2+

in the solution. 8. Compare your result with the theoretical result. Inspect for possible sources of error.


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ATOMIC ABSORPTION AND EMISSION SPECTROMETRY (AAS-AES)

Experiment: Determination of calcium in beverage by AAS and sodium in tap water by AES. Solutions:

a. Stock Calcium Standard Solution (50.0 mL, 1000 mg/L Ca): Prepare from CaCO35H2O.

Note: Dissolve necessary amount of CaCO35H2O in about 20.0 mL Deionized water and

add 1 mL of concentrated HCl. Then dilute to 50.0 mL using deionized water. 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 10.0 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.


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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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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: 2.00 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

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.

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:

Total exchange capacity 1.40 eq/L

Particle size 525-625 μm

Particle density 1.08 g/mL

Water retention capacity 50-60 %

Total swelling 18-20 %


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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 1.00×10-3 M cobalt standard from the stock solution. (Dilutions

will be done with distilled water.)

2. From this solution, prepare 50.0 mL of 1.00×10-4 M , 2.00×10-4 M, 3.00×10-4 M,

5.00×10-4 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.

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,


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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 4.00 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.


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INFRARED SPECTROMETRY (IR)

Experiment: Qualitative and quantitative analysis of film samples by IR spectroscopy, solid

and liquid sample introduction techniques in 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 the mixture of PMMA and PVC - Polyethylene (PE), polystyrene (PS), polytetrafluoroethylene (PTFE) films - Pure KBr for pellet and unknown solid compound for unknown pellet which will be prepared in the laboratory. Procedure: QUANTITATIVE ANALYSIS 1. Make 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 concentrations of 2%, 4%, 6% (w/w) in % transmittance mode 1. Take the IR spectrum of PVC-PMMA unknown film

QUALITATIVE ANALYSIS 1. Take the IR spectrum of PE, PS and PTFE films 2. Take the IR spectrum of unknown solid substance by preparing KBr pellet and using % transmission mode. 3. Take the IR spectrum of solid substance and liquid sample using ATR attachment. Calculations: 1. Draw the calibration plot and calculate the concentration of PMMA in unknown PVC-PMMA film. 2. Make qualitative analysis for the unknown films and for solid compound and liquid sample (Identify the main peaks on each spectrum).


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REFRACTOMETRY AND POLARIMETRY (REF-POL) A) REFRACTOMETRY Experiment: Determination of Refractive Indices of Solvents Solutions: 1. 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 2. Prepare the following standard solutions. (Note that the chosen solvents are miscible with each other; otherwise, true refractive index cannot be obtained).

Concentrations %(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.

dn

nr

1

2

12

2

n : refractive index obtained experimentally d : the density of the solvent

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

d

M

n

nR

2

12

2

M: molecular weight of the analyte


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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, 689nm and 789 nm) - calculate dispersion (nF - nC) using standard dispersion tables

MBAnn CF

- compute Z from the equation:

CF nnZ 5102364.5

- compute Y from the equation:

ZnY D 6108796.2 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:

m

BYn

x

x2

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


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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:

1. Calculate specific rotation [] of each solution using the equation:

cl

netT

net : optical rotation l : length of sample cell (dm) c : concentration in grams per 100 mL. T : temperature, 0C.

2. Plot vs. c

3. Plot net vs. c


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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Experiment: Determination of Ambroksol HCl, Methyl Paraben and Propyl Paraben in a commercial syrup qualitatively by using High Performance Liquid Chromatography (HPLC). Solutions (Already Prepared): Separate Standards: 10 mg/L Ambroksol HCl, 10 mg/L Methyl Paraben, and 10 mg/L Propyl Paraben standards (already prepared). Mobile Phase: Methanol/ 0.5% ammonium acerate buffer (60/40), pH adjusted to 5.5. Column: Dionex 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 10 mg/L Ambroksol HCL solution with syringe. Be careful that the solution inside the syringe must not contain any air bubbles; if there is any it should be removed. 5. Place the syringe into the injection port when it was at the load position and inject the solution from syringe until you see 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 an unknown mixture and identify the peaks. Calculation Calculate number of plate and theoretical plate height for each ion injected alone.


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FLUORESCENCE SPECTROMETRY Experiment: Analysis by spectrofluorimetry. Solutions: Stock Fluorescein Solution (1.00 x 10-3 M): Prepared by technician.

NaOH: 500.0 mL, 0.10 M Working Fluorescein Solutions (50.0 mL): Prepared from stock solution with the

following concentrations. ALL DILUTIONS SHOULD BE DONE WITH 0.10 M NaOH SOLUTION

a) 1.00 x 10-4 M b) 5.00 x 10-5 M c) 1.00 x 10-5 M d) 7.50 x 10-6 M e) 5.00 x 10-6 M f) 2.50 x 10-6 M g) 1.00 x 10-6 M h) 7.50 x 10-7 M i ) 5.00 x 10-7 M

a) Emission and Excitation Spectra: 1. Set up instrument with the help of your assistant. DO NOT TURN ON ANY POWER SUPPLIES YOURSELF. 2. Put 1.00 x 10-5 M fluorescein standard solution into the cell and carefully place in the sample compartment. Set the slit widths of excitation and emission monochromator at minimum values. 3. Take the excitation spectrum between 450 and 530 nm by 5 nm increments on excitation monochromator while keeping the emission monochromator at 520 nm. 4. Take the emission spectrum between 470 and 550 nm by 5 nm increments on emission monochromator while keeping the excitation monochromator at wavelength which gives maximum excitation and determined at Step 3. b) Effect of Slit Width on Excitation and Emission Spectra: 1. Put 1.00 x 10-5 M fluorescein standard solution into the cell and carefully place in the sample compartment. Take the emission spectrum between 470 and 550 nm at maximum slit width. Then, decreasing the slit width to 2.50 mm make the same measurement. Apply this procedure to both excitation and emission monochromators. c) Calibration Plot:

1. Measure fluorescence intensities for each standard solution including blank. 2. Draw a calibration curve (relative fluorescence intensity vs. concentration), from this graph, try to find dynamic range that you can work accurately. 3. Draw a new calibration plot using only the standard solutions in the dynamic ran d) Calculating Limit of Detection:

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

m

sLOD

3 m: Slope of the calibration curve

s: Standard deviation of ten measurements


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Cyclic Voltammetry (CV)

1. Prepare 1, 2, 5, 8 and 10 mM ferricyanide in 0.1 M KNO3 in 25 ml volumetric flasks and

0.1 M KNO3.

2. To run background scan, insert the electrodes (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 is adhering to the bottom of the electrodes, lightly tap the electrode to dislodge

them. Wait ~ 1 minutes before each scan to allow equilibration of the bulk concentration

with that at the electrode surface.

3. Run a CV scan from an initial potential (Ei) of + 600 mV to 0.0 mV and then back to + 600

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 to check the system to the assistant.

2. Run duplicate CVs on each ferricyanide solution (1, 2, 5, 8 and 10 mM) at the scan rate

of 100 mV/s.

5. Run duplicate CVs on the 2 mM ferricyanide solution at scan rates of 20, 50, 200 and 500

mV/s.

6. Label and save each CV with the “save function” if using a computerized potentiostat or

print out a hardcopy of what you want to save.

7. If any dirt on Pt electrode first burn the Pt electrode till a red shiny glow then wait to cool

down and rinse with distilled water. Rub the Ag electrode with sandpaper and rinse with

distilled water.


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THIN LAYER AND PAPER CHROMATOGRAPHY ELECTROPHORESIS (TLC-PC-EF) a) THIN LAYER CHROMATOGRAPHY Experiment: Separation of pH indicator mixtures Silica gel plate: Prepared in the first lab period by coating the glass layer with a mixture of 35 g silica gel and 80 mL distilled water and then drying. Chemicals: Congo Red Phenol Red Bromophenol Blue Fluorescein Methyl Red Unknown Mixture Mobile Phase: a) MP 1 50 mL (30 mL of n-butanol + 10 mL of Ethanol + 10 mL of 2.0 M ammonia) b) MP 2 50 mL (30 mL of n-butanol + 10 mL of Glacial Acetic Acid + 10 mL of Water) Procedure: 1. Pour 50 mL of the solvents into the bottom of the chromatography tank and replace the lids. 2. Take the prepared silica gel plates and apply a different indicator to each of five origins. On the sixth origin apply the mixture using a capillary tube with the instructions of your assistant. 3. Put the plates into the tanks by taking care not to let the solvent touch the spots directly. 4. Close the tanks and run the chromatogram for at least 1.5 hour. Watch the initial flow of solvent across the origin and note how the indicators immediately begin to separate. 5. Remove the plates and mark the solvent fronts with a pencil or spatula along the plate. 6. Dry the chromatograms, calculate the Rf values and identify the components of the

mixture.


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b) PAPER CHROMATOGRAPHY Experiment: Separation of Metal ions (Al+3, Zn+2, Mn+2)

by Ascending chromatography.

Locating Reagents: Rubeanic acid/ethanol (0.1 g/100 mL ethanol) Oxine (8-hydroxyquinoline) 0.5 g/(80 mL ethanol + 20 mL Water) Both are prepared by the technician. Mobile Phase: Mixture composed of 86 mL of Acetone, 6 mL concentrated HCl and 8 mL of water Procedure: 1. Place 50 mL of solvent in the bottom of the cylinder. Replace the lid. 2. Prepare two sheets of the paper of 23 x 23 cm for ascending chromatography and place three spots of the mixture, evenly spaced, along the base line taking care of the flow direction of paper if there is any. 3. Fold one of the papers into a cylinder and secure with the tongued clips. Place the paper, with the spotted end down in the glass tank taking care not to let the paper touch the glass walls and close the tank with the lid. 4. If the chromatogram is observed closely, a yellow band due to Cu may be moving behind the solvent front will be observed. Prepare the locating agents in spray bottle. 5. After the solvent front has traveled about half of the paper, remove the chromatogram from the tank, mark the solvent front with a pencil, open out and dry by using hair dryer. Then; a) Spray rubeanic acid, and expose the chromatogram to vapors of 0.88 M NH3

solution. You should observe nickel as blue, Cobalt as yellow brown, Copper as olive green spots. b) Spray Oxine reagent, and expose the chromatogram to vapors of 0.88 M NH3,

View under UV lamp. This shows aluminum as yellow green fluorescence near starting line and zinc as yellow fluorescence near solvent front. In visible light manganese and cobalt gives light-brown followed by a yellow copper spot. 6. Compute and compare the Rf values of each metal obtained from the ascending and

descending chromatography techniques.


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c) ELECTROPHORESIS Experiment: Separation of dyes Agar gel preparation: Prepared in the first lab period. Place about 4,5 g of agar in a 500 ml beaker. Add 150 ml distilled water. Heat to the boiling until all agar is dissolved. Pour the solution into the tray, place the comb and allow gelation. Chemicals:

Congo Red Cresyl Violet

Phenol Red Crystal Violet

Bromophenol Blue Histidine

Methyl Red Histidine-copper complex

Methyl Orange CuCl2 soluion

Brillant Cresyl Blue Unknown Mixture

Mobile Phase (Phosphate buffer, already prepared. Your assistant will tell you the pH value of the buffer.) Procedure: 1. Similar to TLC, apply a different dye into each origin on the gel. On the last origin, apply the unknown mixture given by the assistant. 2. Place the gel in the tank. 3. Pour the buffer to a level allowing gel to be immersed in the buffer. 4. Close the tank. 5. Set the potential difference to 100 V, period to 20 min and start separation. 6. When the separation is over, stop the process. 7. Note the distance travelled by each indicator and the unknown. 8. Note the pole each indicator moved towards. 9. Remove the tray out of the tank and pour CuCl2 solution on the line where histidine has travelled. Try to observe the formation of histidine-copper complex and again, note the travelled distance. 10. Pour the waste buffer in the tank to the acidic waste container. 11. Clean the tank with distilled water. Questions: 1. Compare the distances travelled by each indicator and find the composition of the unknown mixture. 2. Write your expectations about the travelled distances and the poles preferred by the indicators before the experiment (Consider the molecular weights and charges.). Compare with the experimental results and discuss.


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GAS-LIQUID CHROMATOGRAPHY (GLC)

Experiment 1: Analysis of Mixtures Procedure: 1. Operate the instrument under the surveillance of your assistant a) Set gas chromatograph values as follows during all experiment. i) detector temperature = 260 °C ii) injector temperature = 200 °C b) Set the column temperature as 40 °C and carrier gas flow as 30.0 mL/min.

2. Inject 1.00 L from pure organic compounds (n-butanol, hexane, isopropanol, dichloromethane)

3. Inject 1.00 L of given unknown mixtures, record the peaks and their retention times. 4. Identify the components in the unknown mixture by comparing the retention times of present peaks obtained with that of pure organic compounds. 5. At the end of the experiment, do not shut off the instrument and inform your responsible assistant. Experiment 2: Effect of Carrier Gas Flow Rate Procedure NOTE: Before the injection discard the solvent a few times from the syringe to make sure that the solvent is taken into the syringe.

1. Inject 1.00 L of n-Butanol, record the peak and its retention time. 2. Increase the flow rate by 20.0 mL/min. increments up to 70.0 mL/min., and repeat the injection procedure at each flow rate (30 mL/min, 50 mL/min, 70 ml/min). WAIT FOR AT LEAST TWO MINUTES after each increment of flow rate for stabilization. 3. Calculate the number of the theoretical plates and Height Equivalent of Theoretical Plates (HETP) for each flow rate. 4. Plot the values of HETP versus carrier gas flow rate. 5. Determine the optimum carrier gas flow rate and corresponding HETP (H). Experiment 3: Effects of Column Temperature on Retention Time Procedure: 1. Set the carrier gas flow rate to 30.0 mL/min.

2. Allow the column to stabilize. Then, inject 1.00 L of n-Butanol. Record the peak and its retention time (column temperature=400C). 3. Increase the column temperature to 60 0C and 800C. At each temperature repeat the injection procedure. 4. Plot the retention time versus temperature and discuss the effect of temperature on retention.

lab manual instrumental analysis laboratory 2013 spring...

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