1. No category

CHEM 202
2nd Term 2013-2014
Organic Chemistry II
Instructor: Prof. Khaled S. Abdel Halim
k.abdulhalem@uoh.edu.sa
http://faculty.uoh.edu.sa/k.abdulhalem/
CHEM 202 Lab Manual
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
Spectroscopy I: theoretical approach (IR)
Lab. II
Spectroscopy II: UV-vis fundamentals
Lab.III
FTIR tutorial & polystyrene test
Lab.IV
FTIR- Aspirin analysis
Lab.V
UV-vis - Demo
Lab. VI
UV – spectrum test
Lab.VII
HPLC- tutorial
Lab. VIII
HPLC test
Lab. IX
Synthesis of organic compounds- tutorial
Lab. I & II
Organic Spectroscopy
Theoretical approach
Organic Spectroscopy
In this chapter, we shall study the following topics:
•Spectroscopic methods.
•Infrared Spectroscopy.
•Ultraviolet and visible spectra.
Overview
* 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.
Spectroscopic methods
Infra red (IR)
Ultra-violet / visible (UV-VIS)
Nuclear magnetic Resonance (NMR)
Mass Spectrometry (MS)
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)
Infrared Spectroscopy
Electromagnetic radiation is energy that transmitted through space in the form of waves such as radio waves,
ultraviolet, infrared, visible ,…etc. Each type is characterized by wavelength and frequency.
Wavelength is the distance from the crest of one wave to the crest of the next wave while frequency is the
number of complete cycle per second. Wave number is the number of cycles per cm. Wavelength and frequency
are inversely proportional while energy and frequency are directly proportional. .
The amount of energy is determined by the
intensity or brightness of the radiation. The
intensity is proportional with the number of
photons.
Feature of a spectrum
Spectrum is a graph of wavelength (or frequency) versus % of transmission (%T) or absorbance (A). For IR
spectra, wavelength or frequency is usually plotted with %T while ultraviolet spectra is usually represented as
graphs of A versus wavelength.
The absence of absorption by a compound at a specific wavelength is recorded as 100% T (ideal). When a
compound absorb radiation at specific wavelength, the intensity of radiation being transmitted decreases, so
%T will decrease and appear in spectrum as a dip called absorption peak or absorption band. 100 % T is base
line is recorded at the top of IR spectrum.
Atoms form covalent bonds undergo vibrations or
oscillations (like two balls attached by a spring).
When molecules absorb IR, the amplitude of vibration increased (excited
vibration state). The absorbed energy is dissipated as heat when molecule
returns to ground state.
The wavelength of absorption by a given bond depends on the energy of difference ground state and excited state. So, different
types of bonds like C-H or C=O absorb IR at different and characteristics wavelength. Nonpolar bonds do not absorb IR (no change
in bond moment when vibrate). Relatively nonpolar bond as C-C or C-H give weak absorption while O-H or C=O exhibt strong
absorption.
IR spectrum
IR spectrophotometer is the instrument used to measure the absorption of IR radiation. A diagram
obtained from IR spectrophotometer is called IR spectra. Modern IR instrument used fast Fourier
analysis called Fourier transform infrared spectrometer (FTIR).
IR bands can be classified according to intensity
into strong (s), medium (m), weak (w) and
shoulder (sh). Shoulder is given for overlapping
stronger and weaker bands.
Single OH group produces strong absorption
(polar) while single C-H gives weak absorption.
Multiply C-H bonds gives medium or even strong
absorption
Analysis of IR spectra
Typical ranges of wavelengths of absorption for each functional group were determined and
summarized in correlation charts .
IR Spectra of compounds C-C and C-H
IR of Haloalkanes
C-X bond falls in the fingerprint
region of IR spectra 500-1430 cm-1
Alcohols & amines
IR Spectra of ethers
C-O bond absorption is usually strong
because of highly electronegativity of O
atom. C-O stretching band in the
fingerprint region at 1050-1260 cm-1.
Sp3 C-H
C-C
C-O
at 2800-2900 cm-1
at 1380 cm-1
at 1110 cm-1
IR spectra of carbonyl compounds
Providing that you have the following IR data, answer the following questions:
(i) A compound of formula C4H8O2, which structure shown below is consistent with the
following IR spectrum?
(ii) A molecule with formula C6H12O shows a strong peak at 1725 cm-1 and no peak at 3300-3600
cm-1. Circle all possible structures that are consistent with this data.
(iii) The following IR Spectrum best represents which of the following compounds?
(1) Ultraviolet and visible spectra
The visible spectrum spans from about 400 nm (violet) to 750 nm (red), while the ultraviolet spectrum
ranges from 100 to 400 nm. The quantity of energy absorbed by a compound is inversely proportional
to the wavelength of the radiation.
Both UV and visible radiation are of higher energy than infrared radiation. Absorption of UV or visible light
results in electronic transition (from lower to higher energy level). These transition require 40-300 kcal/mole.
The wave length of UV or visible light absorbed depends on electronic transition, molecules require more
energy for transition absorb at shorter wavelength. Compounds that absorb light in the visible region (colored
compounds) have more easily promoted electron than compounds that absorb at shorter UV wavelengths.
Modern UV spectrometer
sample cell
UV Spectrum
The UV Spectrum:
1.
The x-axis of the spectrum is in wavelength; 200-350 nm for UV, 200-700 for UVVIS determinations
2.
Due to the lack of any fine structure, spectra are rarely shown in their raw form,
rather, the peak maxima are simply reported as a numerical list of “lamba max”
values or lmax
lmax =
206 nm
252
317
376
Absorbance in UV spectroscopy
The absorption of energy in UV is recorded as
absorbance (not transmittance as in IR spectra).
The absorbance depends on the electronic structure
of the compound and also on the concentration of
the sample and the length of the sample cell. So ,
the actual absorbance is defined as molar
absorptivity.
Mechanism of electron absorption in UV spectroscopy
There are 3 types of electron transitions give rise
to UV or visible spectra. The most useful region of
UV spectrum at wavelength longer than 200 nm.
Energy
s*
Unoccupied levels
p*
Atomic orbital
n
Atomic orbital
Occupied levels
p
p*
s
Molecular orbitals
s*
Energy
n
p
s
s
s* alkanes
s
p* carbonyls
p
p* unsaturated cmpds.
n
s* O, N, S, halogens
n
p* carbonyls
Absorption arising from transitions of n electrons
Acetone for example contain both pi bond and n electrons exhibits both
http://chemistry.boisestate.edu/people/richardbanks/organic/mc/vol8/mcquestions317h.htm
http://chemistry.boisestate.edu/people/richardbanks/organic/mc/vol5/mcquestions317e.htm
http://www.chem.ucalgary.ca/courses/350/Carey5th/Ch13/ch13-ir-1.html
http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Questions/problems/exam6.htm
Lab.III
FTIR tutorial & polystyrene test
Introduction to
Fourier Transform
Infrared Spectrometry
© 2001 Thermo Nicolet Corporation
All rights reserved, worldwide.
I N T R O D U C T I O N
What is FT-IR?
FT-IR stands for Fourier Transform InfraRed, the preferred method of infrared spectroscopy. In
infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation is
absorbed by the sample and
some of it is passed through
(transmitted). The resulting
spectrum represents the molecular
absorption and transmission,
creating a molecular fingerprint
of the sample. Like a fingerprint
no two unique molecular
structures produce the same
infrared spectrum. This makes
infrared spectroscopy useful for
several types of analysis.
So, what information can FT-IR provide?
• It can identify unknown materials
• It can determine the quality or consistency of a sample
• It can determine the amount of components in a mixture
This booklet is an introduction to the concepts behind FT-IR spectroscopy. It covers both the
basic theory of FT-IR and how it works as well as discussing some the practical aspects of FT-IR use.
We hope that it gives you a good understanding of the importance and usefulness of this
powerful technique.
2
T H E O R Y
O F
F T - I R
Why Infrared Spectroscopy?
Infrared spectroscopy has been a workhorse technique for materials analysis in the laboratory for over
seventy years. An infrared spectrum represents a fingerprint of a sample with absorption peaks which
correspond to the frequencies of vibrations between the bonds of the atoms making up the material.
Because each different material is a unique combination of atoms, no two compounds produce the
exact same infrared spectrum. Therefore, infrared spectroscopy can result in a positive identification
(qualitative analysis) of every different kind of material. In addition, the size of the peaks in the
spectrum is a direct indication of the amount of material present. With modern software algorithms,
infrared is an excellent tool for quantitative analysis.
Older Technology
The original infrared instruments were of the dispersive type. These instruments separated the
individual frequencies of energy emitted from the infrared source. This was accomplished by the use
of a prism or grating. An infrared prism works exactly the same as a visible prism which separates
visible light into its colors (frequencies). A grating is a more modern dispersive element which better
separates the frequencies of infrared energy. The detector measures the amount of energy at each
frequency which has passed through the sample. This results in a spectrum which is a plot of
intensity vs. frequency.
Fourier transform infrared spectroscopy is preferred over dispersive or filter methods of infrared
spectral analysis for several reasons:
• It is a non-destructive technique
• It provides a precise measurement method which requires no external calibration
• It can increase speed, collecting a scan every second
• It can increase sensitivity – one second scans can be co-added together to ratio out random noise
• It has greater optical throughput
• It is mechanically simple with only one moving part
3
Why FT-IR?
Fourier Transform Infrared (FT-IR) spectrometry was developed in order to overcome the limitations
encountered with dispersive instruments. The main difficulty was the slow scanning process. A method
for measuring all of the infrared frequencies simultaneously, rather than individually, was needed.
A solution was developed which employed a very simple optical device called an interferometer.
The interferometer produces a unique type of signal which has all of the infrared frequencies “encoded”
into it. The signal can be measured very quickly, usually on the order of one second or so. Thus,
the time element per sample is reduced to a matter of a few seconds rather than several minutes.
Most interferometers employ a beamsplitter which takes the incoming infrared beam and
divides it into two optical beams. One beam reflects off of a flat mirror which is fixed in place. The
other beam reflects off of a flat mirror which is on a
mechanism which allows this mirror to move a very
short distance (typically a few millimeters) away from
the beamsplitter. The two beams reflect off of their
respective mirrors and are recombined when they meet
back at the beamsplitter. Because the path that one beam
travels is a fixed length and the other is constantly
changing as its mirror moves, the signal which exits
the interferometer is the result of these two beams “interfering” with each other. The resulting signal
is called an interferogram which has the unique property that every data point (a function of the
moving mirror position) which makes up the signal has information about every infrared frequency
which comes from the source.
This means that as the interferogram is measured, all frequencies are being measured
simultaneously. Thus, the use of the interferometer results in extremely fast measurements.
Because the analyst requires a frequency spectrum (a plot of the intensity at each individual
frequency) in order to make an identification, the measured interferogram signal can not be interpreted
directly. A means of “decoding” the individual frequencies is required. This can be accomplished via a
well-known mathematical technique called the Fourier transformation. This transformation is
performed by the computer which then presents the user with the desired spectral information for analysis.
90
Polystyrene run as film
80
70
60
50
FFT
Calculations
% T 40
30
20
10
0
4000
3500
3000
2500
2000
1500
Wavenumbers (cm-1)
Interferograms
4
CPU
Spectrum
1000
500
The Sample Analysis Process
The normal instrumental process is as follows:
1. The Source: Infrared energy is emitted from a glowing black-body source. This beam passes
through an aperture which controls the amount of energy presented to the sample (and, ultimately,
to the detector).
2. The Interferometer: The beam enters the interferometer where the “spectral encoding” takes
place. The resulting interferogram signal then exits the interferometer.
3. The Sample: The beam enters the sample compartment where it is transmitted through or reflected
off of the surface of the sample, depending on the type of analysis being accomplished. This is
where specific frequencies of energy, which are uniquely characteristic of the sample, are absorbed.
4. The Detector: The beam finally passes to the detector for final measurement. The detectors used
are specially designed to measure the special interferogram signal.
5. The Computer: The measured signal is digitized and sent to the computer where the Fourier
transformation takes place. The final infrared spectrum is then presented to the user for
interpretation and any further manipulation.
Spectrometer
1. Source
2. Interferometer
90
Polystyrene run as film
80
70
60
50
% T 40
30
3. Sample
20
10
0
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
Interferogram
FFT
5. Computer
Spectrum
4. Detector
Because there needs to be a relative scale for the absorption intensity, a background spectrum
must also be measured. This is normally a measurement with no sample in the beam. This can be
compared to the measurement with the sample in the beam to determine the “percent transmittance.”
This technique results in a spectrum which has all of the instrumental characteristics removed.
Thus, all spectral features which are present are strictly due to the sample. A single background
measurement can be used for many sample measurements because this spectrum is characteristic of
the instrument itself.
5
A Simple Spectrometer Layout
6
Advantages of FT-IR
Some of the major advantages of FT-IR over the dispersive technique include:
• Speed: Because all of the frequencies are measured simultaneously, most measurements by FT-IR
are made in a matter of seconds rather than several minutes. This is sometimes referred to as the
Felgett Advantage.
• Sensitivity: Sensitivity is dramatically improved with FT-IR for many reasons. The detectors
employed are much more sensitive, the optical throughput is much higher (referred to as the
Jacquinot Advantage) which results in much lower noise levels, and the fast scans enable the
coaddition of several scans in order to reduce the random measurement noise to any desired level
(referred to as signal averaging).
• Mechanical Simplicity: The moving mirror in the interferometer is the only continuously
moving part in the instrument. Thus, there is very little possibility of mechanical breakdown.
• Internally Calibrated: These instruments employ a HeNe laser as an internal wavelength
calibration standard (referred to as the Connes Advantage). These instruments are self-calibrating
and never need to be calibrated by the user.
These advantages, along with several others, make measurements made by FT-IR extremely
accurate and reproducible. Thus, it a very reliable technique for positive identification of virtually
any sample. The sensitivity benefits enable identification of even the smallest of contaminants. This
makes FT-IR an invaluable tool for quality control or quality assurance applications whether it be
batch-to-batch comparisons to quality standards or analysis of an unknown contaminant. In addition,
the sensitivity and accuracy of FT-IR detectors, along with a wide variety of software algorithms, have
dramatically increased the practical use of infrared for quantitative analysis. Quantitative methods
can be easily developed and calibrated and can be incorporated into simple procedures for routine analysis.
Thus, the Fourier Transform Infrared (FT-IR) technique has brought significant practical
advantages to infrared spectroscopy. It has made possible the development of many new sampling
techniques which were designed to tackle challenging problems which were impossible by older
technology. It has made the use of infrared analysis virtually limitless.
7
5225 Verona Road • Madison, WI 53711-4495 • U.S.A.
TEL: 800-201-8132, 608-276-6100 • FAX: 608-273-5046
E-MAIL: nicinfo@thermonicolet.com • www.thermonicolet.com
A Thermo Electron business
P/N 169-707500 2/01
Lab. IV
FTIR Analysis of Aspirin
lab. V
Shimadzu
UV-V
Shimatzu 1800
UV-vis Spectrophotometer
“Demo”
SPECTROPHOTOMETRIC ANALYSIS OF ASPIRIN
Introduction:
A colored complex is formed between aspirin and the iron (III) ion. The intensity
of the color is directly related to the concentration of aspirin present; therefore,
spectrophotometric analysis can be used. A series of solutions with different
aspirin concentrations will be prepared and complexed. The absorbance of each
solution will be measured and a calibration curve will be constructed. Using the
standard curve, the amount of aspirin in a commercial aspirin product can be
determined.
The complex is formed by reacting the aspirin with sodium hydroxide to form
the salicylate dianion.
O
O C CH 3
C
O-
O
(s) + 3OH- (aq)
C O-
OH
O
(aq) + CH3C O - (aq) + 2H2O(l)
O
The addition of acidified iron (III) ion produces the violet
tetraaquosalicylatroiron (III) complex.
+
O-
O
Fe(H2O)4
+3
- + [Fe(H2O)6 ]
C O
C
O
O
O
+
+ H2O + H3O
Purpose:
The purpose of this lab is to determine the amount of aspirin in a commercial
aspirin product. This lab may also be used to determine the purity of the aspirin
produced in the Microscale Synthesis of Acetylsalicylic Acid lab.
Equipment / Materials:
6 - 125 mL erlenmeyer flasks
commercial aspirin product or aspirin the student has
made
10 mL graduated cylinder
acetylsalicylic acid
250 mL volumetric flask
1 M NaOH
100 mL volumetric flask
0.02 M iron (III) buffer
5 mL pipet
spectrophotometer
2 cuvettes
DI water
analytical balance (opt.)
Safety:

Always wear goggles and an apron in the lab.

Be careful while boiling the sodium hydroxide solution. NaOH solutions are
dangerous, especially when hot.
Procedure
Part I: Making Standards.
1. Mass 400 mg of acetylsalicylic acid in a 125 mL Erlenmeyer flask. Add 10 mL
of a 1 M NaOH solution to the flask, and heat until the contents begin to boil.
2. Quantitatively transfer the solution to a 250 mL volumetric flask, and dilute
with distilled water to the mark.
3. Pipet a 2.5 mL sample of this aspirin standard solution to a 50 mL volumetric
flask. Dilute to the mark with a 0.02 M iron (III) solution. Label this solution
"A," and place it in a 125 mL Erlenmeyer flask.
4. Prepare similar solutions with 2.0, 1.5, 1.0, and 0.5 mL portions of the aspirin
standard. Label these "B, C, D, and E."
Part II: Making an unknown from a tablet.
1. Place one aspirin tablet in a 125 mL Erlenmeyer flask. Add 10 mL of a 1 M
NaOH solution to the flask, and heat until the contents begin to boil.
2. Quantitatively transfer the solution to a 250 mL volumetric flask, and dilute
with distilled water to the mark.
3. Pipet a 2.5 mL sample of this aspirin tablet solution to a 50 mL volumetric
flask. Dilute to the mark with a 0.02 M iron (III) solution. Label this solution
"unknown," and place it in a 125 mL Erlenmeyer flask.
Part III: Making an unknown from the product of the Microscale Synthesis of
Acetylsalicylic Acid lab.
1. Mass all of the acetylsalicylic acid product and record the mass in the data
section. Place it in a 125 mL Erlenmeyer flask. Add 10 mL of a 1 M NaOH
solution to the flask, and heat until the contents begin to boil.
2. Quantitatively transfer the solution to a 250 mL volumetric flask, and dilute
with distilled water to the mark.
3. Pipet 10 mL sample of this aspirin solution to a 50 mL volumetric flask. Dilute
to the mark with a 0.02 M iron (III) solution. (When adding 0.02 M iron (III)
solution, the color should be obviously purple. If it is not, stop adding the 0.02
M iron (III) solution, and alert your instructor.) Label this solution "unknown,"
and place it in a 125 mL Erlenmeyer flask.
Lab. VI
UV Analysis of Aspirin
Part IV: Testing the Solutions.
1.
Turn on the spectrophotometer by twisting the front left knob clockwise.
Allow the spec to warm up for 15 minutes.
2.
Adjust the wavelength to 530 nm using the large knob on top of the spec.
3.
With the sample compartment empty, set the instrument to 0%T using the
zero control knob (front left knob).
4.
Using a Kimwipe, wipe off the cuvet containing the blank (0ppm), and place
this cuvet in the sample compartment, being sure to properly align it. (The
line on the cuvet should match up with the notch on the instrument.) Close
the cover.
5.
Set the mode to absorbance. Using the trans/abs control knob (front right
knob) set the absorbance to 0.000.
6.
Record the absorbance of the 0ppm solution – this should be a cuvet of iron
buffer.
7.
Obtain absorbance readings for each of the other standard solutions. Record
the results on the data sheet.
8.
Measure and record the absorbance of the unknown. Record it on the data
sheet.
Data:
Solution
A
B
C
D
E
unknown
Concentration (mg/L)
Absorbance
For commercial Product:
amount of aspirin in unknown
mg
accepted value
mg
percent error
%
For microscale lab product:
mass of impure product
mg
mass of aspirin in product
mg
% purity of product
%
Lab. VII
Chromatography measurements
HPLC
HPLC Basics
Fundamentals of Liquid
Chromatography (HPLC)
Courtesy of Agilent Technologies, Inc.
Detector
Compound B
Column and
column oven
Control and
data processing
Compound A
Compound C
0
Pump
Injector
2
4
6
8
10
12
14min
Chromatogram
HPLC Basics
Page 1
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
Page 2
Chromatographic Separation Techniques
Which separation technique for which compound?
HPLC Basics
Page 3
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.
• 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: The modern form of liquid chromatography is now referred to
as “flash chromatography”
Note: Look for the comparison with HPLC on page 7
HPLC Basics
Page 4
Principles of Liquid Chromatography
Load
sample
Add
solvent
Column
containing
stationary
phase
Collect
components
Time
HPLC Basics
Page 5
Then, What is HPLC?
• 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
HPLC in 1973
• Since beginning of the 80’s: “boom” in HPLC started
• Since 2006 new terms popped up like UPLC, RRLC,
UFLC, RSLC, …..
HPLC in 2009
HPLC Basics
Page 6
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.
• 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”.
 In principle, LC and HPLC work the same way except the speed,
efficiency, sensitivity and ease of operation of HPLC is vastly superior.
Note: Compare this description to that on page 4 about “Liquid Chromatography”
HPLC Basics
Page 7
What Does a Liquid Chromatogram Look Like?
Compound B
These are called chromatographic
peaks and each one represents a
separated compound
Compound C
Point of sample injection
into the column
0
2
Compound A
4
6
8
10
12
14
min
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.
HPLC Basics
Page 8
What does a high pressure LC look like?
(1) Describing the 5 major HPLC components and their functions …
Solvent reservoirs
and degassing
1
2
5
3
4
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.
HPLC Basics
Page 9
(2) Describing the 5 major HPLC components and their functions …
Solvent reservoirs
and degassing
1
2
5
3
4
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).
HPLC Basics
Page 10
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
 NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline,
etc.), gas chromatography is a better separation technique.
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
HPLC Basics
Page 11
What is HPLC used for?
Qualitative analysis
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.
Retention time
of compound B
Retention time
of compound A
Injection point
(time zero)
0
2.5
5
7.5
10
12.5
15
min
Time after injection
HPLC Basics
Page 12
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.
A
B
Peak area of Compound A
Peak height
of
Compound A
C
Peak area of Compound A
D
Peak area of Compound A
Peak area of Compound A
0
2
4
6
8
10
12
14 min
HPLC Basics
Page 13
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.
Start
Stop
Collect pure peak
from preparative
chromatography
column
Concentrate by
evaporation
Preparative LC Columns
Use for animal
testing
HPLC Basics
Page 14
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.
These are the main
substances in the
sample
These are the trace
substances in the sample
0
2
4
6
8
10
12
14 min
Time after injection
HPLC Basics
Page 15
Examples of Different Instruments and
Configurations
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)
HPLC Basics
Page 16
Introduction
HPLC Applications
High performance liquid chromatography (HPLC) is an important tool
for the analysis of pharmaceutical drugs, for drug monitoring and for
quality assurance. The method enables complex mixtures, for example,
herbal medicine plant extracts, to be separated into individual compounds, which can be identified and quantified by suitable detectors and
data handling systems. Separation and detection occurs at ambient temperature or slightly above. Therefore, the method is ideally suited for
compounds of limited thermal stability.
State-of-the-art HPLC equipment can automate HPLC separations, using
automatic samplers, injectors, microprocessor-controlled analytical conditions and ChemStations for data evaluation. Important requirements
for automation are:
•
•
•
•
•
excellent precision of the liquid chromatography system,
data evaluation with report printouts,
the possibility to store chromatograms and results,
the possibility to detect leaks and other errors for safety reasons, and
implemented OQ/PV tools in the HPLC system.
Automation not only increases the sample throughput in pharmaceutical
laboratories and companies, but also the precision of the results by
eliminating human errors.
An overview of the pharmaceutical drugs used in this guide is presented
on pages 5-6.
3
Agilent ChemStation Database
The client/server database for the Agilent ChemStation, for example,
the Agilent ChemStore C/S is a data organization system which
provides a solution to organize, manange and report chromatographic
results, as well as safeguard data. It supports important end-user tasks
such as reviewing and summarizing results for statistical evaluation,
archiving and restoring data and creating control charts and crosssample reports. These services also aid users in validating their
methods and doing on-going system suitability testing. Two examples
using the database for the Agilent ChemStation are described on pages
56-66.
Agilent 1100 Series
Combinatorial Chemistry
Analysis System
The new Agilent 1100 Series combinatorial chemistry analysis system is
a fully automated analysis solution designed to speed up the process of
drug discovery. The combination Agilent 1100 Series HPLC, Agilent
1100 Series LC/MSD detector and the Agilent 220 micro plate sampler
offers the full potential of well plate technology and is the ideal tool for
analyzing complex libraries. Details can be found on page 67.
4
Application Overview
Group
Examples
Analgesic drugs
Antipyrine, Hydroxyantipyrine, Acetaminophen
7
Androgen drugs
Testosterone Acetate, Testosterone
8
Antianginal drugs
Verapamil
9
Antiarrythmic drugs
Quinidine, Disopyramide, Procainamide, N-Acetylprocainamide 10
Antiasthmatic drugs
Antibacterial drugs
Penicillin-like
Tetracyclines
Miscellaneous
Caffeine, Theophylline, Enprofylline, Theobromine
11
Ampicillin, Amoxicillin, Penicillin G, Penicillin V
Minocycline, Tetracycline, Doxycycline
Hydroxybenzotriazole, Chloramphenicol, Trimethoprim
Sulfamethoxazole, Furazolidone, Nalidixic Acid
12
13
Anticoagulant drugs
Warfarin
17
Antidepressant drugs
Antiepileptic drugs
Antiestrogen drugs
Antihistaminic drugs
Bupropion, Trazodone, Maprotiline
18
Caffeine, Phenytoin, Methylphenylsuccinimide, Phenylethylmalonamide, Carbamazepinepoxide, Ethosuximide, Phenobarbital,
Carbamazepine, Primidone
20
Tamoxifen
26
Tetracaine, Promethazine, Chlorpheniramine, Tripelenamine
27
Antihypertensive drugs
Enalapril, Captopril
Antiinflammatory drugs
Naproxen
29
Antiprotozoal drugs
Metronidazole
30
Antitumor drugs
Paclitaxel (Taxol)
31
Antitussive drugs
Catecholamines
Dextromethorphan
33
Norepinephrine, Epinephrine, Dihydroxybenzylamine,
Dopamine
34
Beclomethasone Dipropionate, Prednisolone,
Prednisolone Acetate, Betamethasone, Betamethasone Valerate,
Hydrocortisone, Hydrocortisone Acetate
35
Glucocorticoid drugs
H2-Antagonists
Hypnotic drugs
Keratolytic drugs
Muscle-relaxing drugs
Sedative drugs
Sulfa drugs
Therapeutic peptides
Tricyclic antidepressant drugs
Vitamins
Fat soluble
Water soluble
Ranitidine, Cimetidine
Barbital, Allobarbital, Phenobarbital, Butabarbital, Butalbital,
Amobarbital, Mephobarbital, Flunitrazepam
Salicylic Acid, Phtalic Acid, Benzoic Acid
Papaverine
Diazepam, Oxazepam, Clonazepam, Flunitrazepam
Sulfanilamide, Sulfadiazine, Sulfathiazole, Sulfamerazine,
Sulfamethazine
Angiotensin II, Angiotensin I, Insulin, Oxytocin
Protriptyline, Nortriptyline, Doxepin, Imipramine,
Amitriptyline, Trimipramine
Vitamins A1, D3, E
Aminobenzoic Acid, Biotin, Folic Acid, Niacinamide,
Pantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine,
Riboflavine, Thiamine, Thiotic Acid
Page
14
28
37
38
41
42
43
44
45
46
47
48
5
Introduction
HPLC Applications
High performance liquid chromatography (HPLC) is an important tool
for the analysis of pharmaceutical drugs, for drug monitoring and for
quality assurance. The method enables complex mixtures, for example,
herbal medicine plant extracts, to be separated into individual compounds, which can be identified and quantified by suitable detectors and
data handling systems. Separation and detection occurs at ambient temperature or slightly above. Therefore, the method is ideally suited for
compounds of limited thermal stability.
State-of-the-art HPLC equipment can automate HPLC separations, using
automatic samplers, injectors, microprocessor-controlled analytical conditions and ChemStations for data evaluation. Important requirements
for automation are:
•
•
•
•
•
excellent precision of the liquid chromatography system,
data evaluation with report printouts,
the possibility to store chromatograms and results,
the possibility to detect leaks and other errors for safety reasons, and
implemented OQ/PV tools in the HPLC system.
Automation not only increases the sample throughput in pharmaceutical
laboratories and companies, but also the precision of the results by
eliminating human errors.
An overview of the pharmaceutical drugs used in this guide is presented
on pages 5-6.
3
Agilent ChemStation Database
The client/server database for the Agilent ChemStation, for example,
the Agilent ChemStore C/S is a data organization system which
provides a solution to organize, manange and report chromatographic
results, as well as safeguard data. It supports important end-user tasks
such as reviewing and summarizing results for statistical evaluation,
archiving and restoring data and creating control charts and crosssample reports. These services also aid users in validating their
methods and doing on-going system suitability testing. Two examples
using the database for the Agilent ChemStation are described on pages
56-66.
Agilent 1100 Series
Combinatorial Chemistry
Analysis System
The new Agilent 1100 Series combinatorial chemistry analysis system is
a fully automated analysis solution designed to speed up the process of
drug discovery. The combination Agilent 1100 Series HPLC, Agilent
1100 Series LC/MSD detector and the Agilent 220 micro plate sampler
offers the full potential of well plate technology and is the ideal tool for
analyzing complex libraries. Details can be found on page 67.
4
Application Overview
Group
Examples
Analgesic drugs
Antipyrine, Hydroxyantipyrine, Acetaminophen
7
Androgen drugs
Testosterone Acetate, Testosterone
8
Antianginal drugs
Verapamil
9
Antiarrythmic drugs
Quinidine, Disopyramide, Procainamide, N-Acetylprocainamide 10
Antiasthmatic drugs
Antibacterial drugs
Penicillin-like
Tetracyclines
Miscellaneous
Caffeine, Theophylline, Enprofylline, Theobromine
11
Ampicillin, Amoxicillin, Penicillin G, Penicillin V
Minocycline, Tetracycline, Doxycycline
Hydroxybenzotriazole, Chloramphenicol, Trimethoprim
Sulfamethoxazole, Furazolidone, Nalidixic Acid
12
13
Anticoagulant drugs
Warfarin
17
Antidepressant drugs
Antiepileptic drugs
Antiestrogen drugs
Antihistaminic drugs
Bupropion, Trazodone, Maprotiline
18
Caffeine, Phenytoin, Methylphenylsuccinimide, Phenylethylmalonamide, Carbamazepinepoxide, Ethosuximide, Phenobarbital,
Carbamazepine, Primidone
20
Tamoxifen
26
Tetracaine, Promethazine, Chlorpheniramine, Tripelenamine
27
Antihypertensive drugs
Enalapril, Captopril
Antiinflammatory drugs
Naproxen
29
Antiprotozoal drugs
Metronidazole
30
Antitumor drugs
Paclitaxel (Taxol)
31
Antitussive drugs
Catecholamines
Dextromethorphan
33
Norepinephrine, Epinephrine, Dihydroxybenzylamine,
Dopamine
34
Beclomethasone Dipropionate, Prednisolone,
Prednisolone Acetate, Betamethasone, Betamethasone Valerate,
Hydrocortisone, Hydrocortisone Acetate
35
Glucocorticoid drugs
H2-Antagonists
Hypnotic drugs
Keratolytic drugs
Muscle-relaxing drugs
Sedative drugs
Sulfa drugs
Therapeutic peptides
Tricyclic antidepressant drugs
Vitamins
Fat soluble
Water soluble
Ranitidine, Cimetidine
Barbital, Allobarbital, Phenobarbital, Butabarbital, Butalbital,
Amobarbital, Mephobarbital, Flunitrazepam
Salicylic Acid, Phtalic Acid, Benzoic Acid
Papaverine
Diazepam, Oxazepam, Clonazepam, Flunitrazepam
Sulfanilamide, Sulfadiazine, Sulfathiazole, Sulfamerazine,
Sulfamethazine
Angiotensin II, Angiotensin I, Insulin, Oxytocin
Protriptyline, Nortriptyline, Doxepin, Imipramine,
Amitriptyline, Trimipramine
Vitamins A1, D3, E
Aminobenzoic Acid, Biotin, Folic Acid, Niacinamide,
Pantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine,
Riboflavine, Thiamine, Thiotic Acid
Page
14
28
37
38
41
42
43
44
45
46
47
48
5
Fat-Soluble Vitamins
Retinol (A1)
Absorbance
[mAU]
1 Retinol (A)
2 Cholecalciferol (D3)
3 a-Tocopherol (E)
3
2
800
600
1
400
200
OH
0
HO
a- Tocopherol
0
2.5
5
7.5
12.5
10
15
17.5
20
Time [min]
Analysis of fat-soluble vitamins
O
Column
Mobile phase
Flow rate
Gradient
Column wash
UV detector
Column compartment temperature
Stop time
Post time
Injection volume
4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 µm
A = water, B = methanol
1.0 ml/min
at 0 min 90 % B
at 15 min 100 % B
at 20 min 100 % B
at 21 min 90 % B
variable wavelength detector
210 nm, standard cell
20 °C
21 min
5 min
5 µl
HPLC method performance
Limit of detection <4.0 mg/l (5-µl injection),
S/N=2
Precision of RT
10 runs, 1000 mg/l< 0.10 %
H
Precision of area
10 runs, 1000 mg/l< 0.2 %
HO
Instrumentation:
see configuration example 2 on page 77
Cholecalciferol (D3)
47