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Organic Chemistry II
CHEM 202
Instructor: Prof. Khaled S. Abdel Halim
[email protected] http://faculty.uoh.edu.sa/k.abdulhalem/index.html
UOH Blackboard at www.uoh.edu.sa Lab Sessions/Syllabus
Objective:
To provide students to the basics of spectroscopic techniques, laboratory synthesis of
organic chemicals and multistep synthesis. To practice the fundamentals of organic
spectroscopic equipments such as FTIR and UV-vis. To discuss how HPLC (High
performance liquid chromatography) is a powerful tool in analysis and how to carry
experiments on HPLC.
Outcomes:
Upon successful completion of this course, students will be able to:
Understand the principals of organic spectroscopy.
Practice the identification of organic compounds by FTIR & UV measurements.
Practice the identification of organic compounds by instrumental analysis.
Know the fundamentals of HPLC.
Analyze organic chemistry problems & prepare information and data.
Demonstrate teamwork skills through assignments tasks.
Demonstrate communications effectively in oral and written form.
3
Lab regulations
Total points: 20 % of the CHEM 202 grade (15 % for lab assignments,
reports, homework & attendance and 5 % for final lab sheet exam) .
Safety measures are strictly followed in the lab including: proper clothing
(pant, shirt and shoes), wearing safety goggles, and shoes.
Homework problems can be found in the provided manual.
Homework will be collected on the due date for grading.
No late homework will be accepted, unless prior arrangements have been
made with the instructor.
Comments will be made on solution techniques, and mistakes will be
indicated.
Homework
Assignments
Teams will be formed, with four or five students per group. These groups
will be used for reporting and presenting on assignments.
Students are encouraged to work on homework assignments in groups. At
the end of the semester, each group member may be asked to evaluate (score)
the other members on contributions to team success.
All writing assignments will be evaluated on the basis of the quality of the
communications, as well as on technical content.
Every assignment will be made to relate the information developed in the
lab to the real world of organic chemistry. Students will be asked during
presentations to develop their communications skills.
5
Lab Reports Each student has to write his own report. A copied report will get zero for the first
time, and if repeated other actions will be taken including dismissal.
Lab reports will be graded based on the quality and compliance with the suggested
format.
If a student misses a lab without official excuse, he will not be allowed to submit the
lab report for that lab and he will get zero for that lab.
Hand plotting is not accepted.
Safety measures are strictly followed in the lab including: proper clothing (pant, shirt
and shoes), wearing safety goggles, and shoes.
Every student should submit his lab report on the due date of the report (one week
after performing the experiment)
6
Report Format and Grading:
Each lab report should include the followings:
1) Cover Page (title of the experiment, date, student name, instructor name, and
section number) (1 point)
2) Introduction and Objectives (background related to the experiment and the
experiment objectives). (3 points)
3) Experimental Procedure (brief description of the test procedure and listing the
main equipment used). (3 points)
4) Results and Discussion (raw data, observation, example calculation, and evaluation
of data generated, and good quality graphs). (10 points)
5) Conclusion (main findings or what the experiment added to your understanding).
(3 points)
Manual Outlines
This manual will provide an introduction to basic spectroscopic techniques, laboratory
synthesis of organic chemicals and multistep synthesis. The manual will introduce the
fundamentals of organic spectroscopic equipments such as FTIR and UV-vis.
Furthermore, the manual will discuss how HPLC (High performance liquid
chromatography) is a powerful tool in analysis and how to carry experiments on HPLC.
Finally, a tutorial for synthesis, purification and identification of organic compounds
will be introduced. It is important for chemical engineer to have some background in
organic spectroscopy and organic synthesis tests to understand how dealing with
different types of organic compounds.
Lab. I Fundamental of organic Spectroscopy ……..(Assignment I)
Lab. II Organic Spectroscopic Problems -(Assignment I Discussions)
Lab.III FTIR tutorial & polystyrene test …………..............(Report 1)
Lab.IV UV-vis - Demo & UV – spectrum test…………….(Report 2)
Lab.V Organic Chemistry problems I
(Aromatic compounds- Aldehydes& Ketones) ……… HW 1
Lab. VI HPLC- tutorial………………………………(Assignment II)
Lab.VII Assignment II Discussions
Lab.VIII Synthesis of organic compounds- tutorial ......(Assignment III)
Lab. IX Assignment III Discussions
Lab.X Organic Chemistry Problems II
(Carboxylic acids & derivatives- Amines) ….………..HW 2
Lab XI Organic Chemistry Problems III
(Biochemistry Problems)……………………………..HW3
Lab I: Organic Spectroscopy
In this part, we shall study the following topics:
Spectroscopic methods.
Infrared Spectroscopy.
Ultraviolet and visible spectra.
* Modern spectroscopic methods have largely replaced chemical tests as the standard means of identifying
chemical structures (particularly in organic chemistry). Spectroscopic techniques are fundamentally based on
the molecules absorbing energy then monitoring the affect this has on the molecule.
Overview
* Spectroscopic methods:
1. Infrared technique (IR)
2. Ultraviolet technique (UV)
3. Mass Spectroscopy
4. Nuclear Magnetic Resonance
Electromagnetic Radiation (EMR) : “Visible light is the common type of EMR but this is just a
small portion of all possible types. Electromagnetic radiation has both particle and wave
properties
A particle of energy is called a photon. Each photon has a discrete amount of energy a quantum, ∆E = h ν= h c / λ (h = Planck's constant)
Electromagnetic radiation
Assignment I
Design a presentation discussing how organic spectroscopy
is used in identification of organic compounds?!
Lab II: Organic spectroscopy problems
Suggested approach to solving spectroscopy problems:
Step 1: Calculate the degree of unsaturation to limit the number of possible structures. The degree of unsaturation indicates
whether or not the compound has one or more double bonds.
Step 2: Look at the IR spectrum, especially the region of wavenumbers greater than 1500. Look for likely functional groups,
working in conjunction with the degree of unsaturation result from Step 1 and with the molecular formula. For instance, if there
is one double bond and an oxygen, it could be a carbonyl; if there is no double bond and an oxygen, it will be either an ether or
an alcohol; if there are 4 double bonds, there might be an aromatic ring (and so on).
Step 3: Study the NMR to determine the connectivity of the compound. Draw the likely functional groups proposed in step 2 and
see how they might fit together to be consistent with the given NMR spectrum.
Step 4: Draw a structure consistent with the unsaturation, IR, and NMR data. Check that you have the proper total number of
carbons, oxygens, hydrogens (and etc.) as consistent with the molecular formula. Check that you have 4 bonds to each carbon.
Check that the structure you have drawn "fits" with the NMR. If it doesn't, draw another possible structure and check again.
Degree of Unsaturation Rule 1: Replace all halogens in the molecular formula by hydrogens. Rule 2: Omit oxygens and sulfurs. Rule 3: For each nitrogen, omit the nitrogen and one hydrogen.
for the formula CnHm
Applying these rules to the molecular formula C8H
8NOBr
•Rule 1, replace halogens with hydrogens: C8H9NO
•Rule 2, omit oxygens: C8H9N
•Rule 3, omit the nitrogen and one hydrogen: C8H8
Therefore, Dou = 5
Try with the following compounds: Benzene acetylene ethylene propane
Problem 10:
Lab III: FTIR spectrometer
(Polystyrene test)
Lab IV: UV spectrum test
Report 1:
Spectrophotometer test of CoCl2
Lab V: Organic Chemistry Problems on:
Aromatic compounds, Aldehydes & Ketones
Nomenclature of substituted benzenes
Problem 1:
Problem 2:
IR spectra of benzene substituted
Aromaticity
Electrophilic substitution reactions
Homework 1
HPLC Basics
Page 1
HPLC Basics Fundamentals of Liquid
Chromatography (HPLC)
Courtesy of Agilent Technologies, Inc.
Pump Injector
Column and
column oven
Detector
Control and
data processing
min0 2 4 6 8 10 12 14
Compound A
Compound B
Compound C
Chromatogram
Page 2
Fundamentals of High Performance Liquid
Chromatography (HPLC)
This course will enable you to:
• Explain the general principles of HPLC analyses
• Know the major application areas of HPLC
• Identify the major components of an HPLC system
and explain their principles of operation
HPLC Basics
Chromatographic Separation TechniquesWhich separation technique for which compound?
Page 3
HPLC Basics
Page 4
First, What is Liquid Chromatography?
• Liquid chromatography is a separation technique that involves:
• the placement (injection) of a small volume of liquid sample
• into a tube packed with porous particles (stationary phase)
• where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.
Note: The modern form of liquid chromatography is now referred to as “flash chromatography”
• The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.
• The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount.
Note: Look for the comparison with HPLC on page 7
HPLC Basics
Page 5
Principles of Liquid Chromatography
Column
containing
stationary
phase
Load
sampleAdd
solvent
Collect
components
Time
HPLC Basics
• HPLC is an abbreviation for
High Performance Liquid Chromatography
(It has also been referred to as High Pressure LC)
• HPLC has been around for about 35 years and is the largest
separations technique used
• The history of HPLC:
• Beginning of the 60’s: start of HPLC as High Pressure
Liquid Chromatography
• End of the 70’s improvements of column material and
instrumentation – High Performance Liquid
Chromatography
• Since beginning of the 80’s: “boom” in HPLC started
• Since 2006 new terms popped up like UPLC, RRLC,
UFLC, RSLC, …..
Page 6
Then, What is HPLC?
HPLC in 1973
HPLC in 2009
HPLC Basics
Page 7
What is HPLC?
• HPLC is a separation technique that involves:
• the injection of a small volume of liquid sample
• into a tube packed with tiny particles (3 to 5 micron (µm) in diameter called the stationary phase)
• where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.
In principle, LC and HPLC work the same way except the speed,efficiency, sensitivity and ease of operation of HPLC is vastly superior.
• These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.
• These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.
Note: Compare this description to that on page 4 about “Liquid Chromatography”
HPLC Basics
Page 8
Time after injection
This is the chromatogram resulting from the injection of a small volume of liquid
extracted from a vitamin E capsule that was dissolved in an organic solvent. Modern
HPLC separations usually require 10- to 30-minutes each.
min0 2 4 6 8 10 12 14
Point of sample injection
into the column
These are called chromatographic
peaks and each one represents a
separated compound
Compound A
Compound B
Compound C
What Does a Liquid Chromatogram Look Like?
HPLC Basics
Page 9
What does a high pressure LC look like?(1) Describing the 5 major HPLC components and their functions …
1. Pump:
• The role of the pump is to force a liquid (called the mobile phase) through the liquid
chromatograph at a specific flow rate, expressed in milliliters per min (mL/min).
• Normal flow rates in HPLC are in the 1- to 2-mL/min range.
• Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-bar).
• During the chromatographic experiment, a pump can deliver a constant mobile phase composition
(isocratic) or an increasing mobile phase composition (gradient).
2. Injector:
• The injector serves to introduce the liquid sample into the flow stream of the mobile phase.
• Typical sample volumes are 5- to 20-microliters (µL).
• The injector must also be able to withstand the high pressures of the liquid system.
• An autosampler is the automatic version for when the user has many samples to analyze or
when manual injection is not practical.
1
2
4
5
Solvent reservoirs
and degassing
3
HPLC Basics
Page 10
(2) Describing the 5 major HPLC components and their functions …
3. Column:
• Considered the “heart of the chromatograph” the column’s stationary phase separates the
sample components of interest using various physical and chemical parameters.
• The small particles inside the column are what cause the high backpressure at normal
flow rates.
• The pump must push hard to move the mobile phase through the column and this
resistance causes a high pressure within the chromatograph.
4. Detector:
• The detector can see (detect) the individual molecules that come out (elute) from the column.
• A detector serves to measure the amount of those molecules so that the chemist can
quantitatively analyze the sample components.
• The detector provides an output to a recorder or computer that results in the liquid
chromatogram (i.e., the graph of the detector response).
5. Computer:
• Frequently called the data system, the computer not only controls all the modules of the
HPLC instrument but it takes the signal from the detector and uses it to determine the time of
elution (retention time) of the sample components (qualitative analysis) and the amount of
sample (quantitative analysis).
1
2
4
5
Solvent reservoirs
and degassing
3
HPLC Basics
Page 11
What is HPLC used for?Separation and analysis of non-volatile or thermally-unstable compounds
HPLC is optimum for the separation of chemical and biological compounds
that are non-volatile
Typical non-volatile compounds are:
Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
Salts like sodium chloride and potassium phosphate
Proteins like egg white or blood protein
Organic chemicals like polymers (e.g. polystyrene, polyethylene)
Heavy hydrocarbons like asphalt or motor oil
Many natural products such as ginseng, herbal medicines, plant extracts
Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline,
etc.), gas chromatography is a better separation technique.
HPLC Basics
Page 12
The identification (ID) of individual compounds in the sample;
• the most common parameter for compound ID is its retention time (the time it takes for that
specific compound to elute from the column after injection);
• depending on the detector used, compound ID is also based on the chemical structure, molecular
weight or some other molecular parameter.
min0 2.5 5 7.5 10 12.5 15
Injection point
(time zero)
Retention time
of compound A
Retention time
of compound B
Time after injection
What is HPLC used for?Qualitative analysis
HPLC Basics
Page 13
What is HPLC used for?Quantitative Analysis
The measurement of the amount of a compound in a sample (concentration); meaning, how much is there?;
There are two main ways to interpret a chromatogram (i.e. perform quantification):
1. determination of the peak height of a chromatographic peak as measured from the baseline;
2. determination of the peak area (see figure below);
In order to make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured. In many cases, there is a linear relationship between the height or area and the amount of sample.
min0 2 4 6 8 10 12 14
AB
C
D
Peak area of Compound A
Peak area of Compound A
Peak area of Compound A
Peak area of Compound A
Peak height
of
Compound A
HPLC Basics
Page 14
What is HPLC used for?Preparation of Pure Compound(s)
- By collecting the chromatographic peaks at the exit of the detector,
- and concentrating the compound (analyte) by removing/evaporating the solvent,
- a pure substance can be prepared for later use (e.g. organic synthesis, clinical
studies, toxicology studies, etc.).
This methodology is called preparative chromatography.
Concentrate by
evaporation
Collect pure peak
from preparative
chromatography
column
Start Stop
Preparative LC Columns
HPLC Basics
Use for animal
testing
Page 15
What is HPLC used for?Trace analysis
A trace compound is a compound that is of interest to the analyst but it’s concentration is
very low, usually less than 1% by weight, often parts per million (ppm) or lower;
• the determination of trace compounds is very important in pharmaceutical, biological,
toxicology, and environmental studies since even a trace substance can be harmful or
poisonous;
• in a chromatogram trace substances can be difficult to separate or detect;
• high resolution separations and very sensitive detectors are required.
min0 2 4 6 8 10 12 14
Time after injection
These are the main
substances in the
sample
These are the trace
substances in the sample
HPLC Basics
Page 16
Modular HPLC System –
basic configuration with
isocratic pump, manual
injector, variable
wavelength detector, and
hand-held controller
Modular HPLC System –
high-end configuration with
quaternary pump,
autosampler, column
thermostat, diode array
detector, and computer with
control and data analysis
SW
Integrated HPLC System
“all parts in one box” –
different configurations
possible, here with
gradient pump,
autosampler, column oven,
VWD, and computer with
control and data analysis
SW (not shown on picture)
Examples of Different Instruments and
Configurations
HPLC Basics
Lab VII
Assignment II Discussions
Lab VIII Synthesis of organic compounds- tutorial
https://www.youtube.com/watch?v=ToSmwYgbvI0
Assignment III Discussions
Lab IX
Lab X: Organic Chemistry Problems on:
Carboxylic acids, Acid derivatives, Amines
Homework II
Lab XI: Organic Chemistry Problems on:
Biochemistry
1. Providing the chemical structure of amino acids in the corresponding table, answer
the following questions: Methionine (met)
Leucine (leu)
Alanine (ala)
Glycine (gly)
Lysine (lys)
(1) Write an equation showing dipolar ion formation for each.
(2) Write flow equations showing two different ways to synthesis of alanine.
(3)Express by chemical equation the acylation reaction of leucine with acetic anhydride.
(4)Show the chemical structure for two different dipeptides from alanine and glycine.
(5) What is the structure of leu-lys-met?
2. Given the structure of the following carbohydrates (A, B, C, D):
(i) Classify each compound according to their names (for example D-aldohexose) ?
(ii) Show by chemical equations the effect of adding Tollen’s reagent to each compound?
(iii) Show by chemical equations the effect of dilute nitric acid on the A & C ?
(iv) Write the equation to show the effect of enzyme on B?
(v) Show by chemical equations the reduction products of each compound?
3. Match each of the following classes of compound with a structure on the right:
4. “Although fructose is a ketone, it is a reducing sugar”. Explain by
equations how and why fructose is a reducing sugar.
5. For each of the following structures identify :
(i) The base
(ii) the carbohydrate
(iii) nucleoside or a nucleotide ?
(iv ) DNA or a RNA system ?
Homework III 1. Predict the product (if any) of bromine oxidation of each of the following compounds:
2. Write equations for cyclization of the following monosaccharides
3. Classify the following molecules into: Aldotetrose, Hemi-acetalpyranose, Hemi-ketalfuranose
4. Predict the products for each of the following reactions:
Methionine (met)
Leucine (leu)
Alanine (ala)
Glycine (gly)
Lysine (lys)
5. Providing the chemical structure of amino acids in the corresponding table, answer the following
questions:
(1) Show the structure of dipolar ion formation for each.
(2) Write chemical equations showing two different ways to synthesis of alanine
(3)Express by chemical equation the acylation reaction of leucine with acetic anhydride.
(4)Show the chemical structure for two different dipeptides from alanine and glycine.
(5) What is the structure of leu-ala?
*Write the selected amino acids in dipolar form.
*Show the effect of adding acetic anhydride to the selected amino acids.
*Write the structure of dipeptide formed from the selected amino acids.
6. Which of the following structures illustrates an amino acid that might be important
biologically, that is a-amino acids, be incorporated into proteins.