Organic Chemistry II

CHEM 202

Instructor: Prof. Khaled S. Abdel Halim

k.abdulhalem@uoh.edu.sa 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.