PHYTOCHEMICAL SCREENING

OF NATURAL PRODUCTS
Dr. Md. Rageeb Md. Usman
(M. Pharm., Ph.D., FAPP, FICPHS, FSRHCP, FRSH, FSPER)
Associate Professor
Department of Pharmacognosy

Smt. Sharadchandrika Suresh Patil

College of Pharmacy,
Chopda, Maharashtra, India
• Extraction is defined as the process of isolation
of soluble material from an insoluble residue,
which may be liquid or solid, by treatment with a
solvent.
• On the basis of crude drug to be extracted the
extraction process may be liquid-liquid or solid-
liquid extraction.
• The process of extraction is controlled by mass
transfer. The driving force for mass transfer is a
difference in concentration; the random motion
of molecules causes a net transfer of mass from
an area of high concentration to an area of low
concentration until it reaches to an equilibrium.
 Extraction Methods
• Maceration : Involves immersion of
the crude drugs in a bulk of the
solvent or menstrum and allowed to
stand for at least 3-7 days in a warm
place with frequent shaking.

• It can be modified to a multistage

extraction unit.

• Disadvantage: Generally slow, time

consuming and inefficient extraction.
 Percolation
• Involves continuous flow of the
solvent through the bed of the crude
drug material to get the extract.
• 1st the drug is allowed to t/t with suff.
menstrum to make it uniformly wet.
• 2nd it is allowed to stand for 15 min.
• 3rd suff. Menstrum is added to
saturate the drug. Macerate for 24
hrs.
• 4th percolation continued.
• Advantage: It yields the product of
greater conc.
• It is modified to include evaporation.
 Continuous extraction
• Soxhlet Extraction:A Soxhlet extractor is a type
of laboratory glassware invented in 1879 by
Franz Von Soxhlet.
• It was originally designed for the extraction of
lipid from a solid test material, but can be used
whenever it is difficult to extract any compound
from a solid.
• The key advantage of this type of extraction;
only clean warm solvent is used to extract the
solid in the thimble. This increases the efficiency
of the extraction when compared with simply
heating up the solid in a flask with the solvent.
• At the end of an
extraction, the excess
solvent may be removed
using a rotary
evaporator, leaving Vapor
tube
behind only the
extracted constituent.
• Advantages: Extraction
can be continued until
complete exhaustion of
the drug.
• Less solvent is needed
• More conc. Products
obtained.
Large Scale Extraction
 Supercritical Extraction
• A supercritical fluid is any substance at a
temperature and pressure above its
thermodynamic critical point.
• It has the unique ability to diffuse through
solids like a gas, and dissolve materials like a
liquid. Additionally, it can readily change in
density upon minor changes in temperature or
pressure.
• These properties make it suitable as a
substitute for organic solvents in a process
called Supercritical Fluid Extraction.
• Carbon dioxide and water are the most
commonly used supercritical fluids.
 Supercritical extraction unit
CO2
H2O

d
b

a
c e

CO2
Product
f

a- Extractor, b- CO2 + Product, c- Washing tower, d- Heat exchanger, e-

Distillation chamber, f- Degassing chamber
 Uses
• Supercritical CO2 is forced through the green
coffee beans and then sprayed with water at
high pressure to remove the caffeine.
(decaffeination of coffee).
• Supercritical carbon dioxide is also becoming a
more common process for extracting volatile oils
and fragrance compounds from various raw
materials that are used in perfumery.
• The relatively low supercritical temperature and
reactivity of CO2 allows the fragrance
compounds to be extracted without extensive
damage or denaturing, which will alter their odor.
 Other uses
• Extraction of Acorone from Acorus
calamus.

• Matrisin and Bisabolol from Chamomile

flowers.

• Heat labile sesquiterpene hydrocarbons of

Valerian and Nardostachys.
 Types of Extracts
1. Aqueous extracts
i) Decoction : Crude drugs boiled in water for
15 min. filtered volume makeup
ii) Infusion: Periodic maceration of the drug with
either with cold or boiling water
iii) Digestion: Maceration with moderate heating
2. Tinctures: Alcoholic or hydro-alcoholic
3. Liquid Extracts
4. Soft Extracts
5. Dry Extracts
 Isolation & Purification
• It depends on the PHYSICAL & CHEMICAL
characteristics of the compound to be separated.
• Physical : Chromatographic techniques,
fractional crystallization, fractional distillation,
fractional liberation, and sublimation etc.
• Chemical: based on the chemical prop. Of
functional groups and moieties contained in the
comp. s/as aldehyde, phenols, -OH, acids,
alkaloids etc. the chemical derivatisation
techniques may be adopted to convert the
component in the form of derivative.
 Craig Counter current distribution
• Craig's first major application of this technique was in characterizing
penicillins and estimating the purity of benzylpenicillin, with an ether-
aqueous buffer solvent system.

• In 1950 he built a 200-tube CCD apparatus made from glass and

another in 1958 with 1000 tubes. He used his equipment to examine
substances such as gramicidin, bacitracin, insulin, bile acids,
tyrocidine, serum albumin, parathyroid hormone, ribonucleic acids,
and ribonuclease.

• CCD has also been used by other scientists to isolate hormones

such as oxytocin, vasopressin, adrenocorticotrophic hormone
(ACTH), growth hormone, lactogenic hormone, melanophore-
stimulating hormone, and parathyroid hormone.

• It has also aided in studies on the structure of the tobacco mosaic

virus protein, human hemoglobin and chains, angiotensin, and
other biologically active peptides.

• Also, CCD was used by Robert Holley at Cornell University to

fractionate crude RNA from liver to give a pure tRNA, which was
then fully sequenced for the first time.
Craig Countercurrent
Distribution Tubes

D
D

C A C

A
B
Distribution of two solutes in the tubes after different number
of transfers
a) After 5 transfers b) after 10 transfers c) After 15 transfers d)
after 20 transfers
 PHYTOCHEMICAL
RESEARCH
 Selection of plant and plant part
 dry
 Powder
 Successive extraction
 Concentration of extract
 Preliminary qualitative analysis of constituents
 Assessment of biological activity for the extracts
 Choose extract with good biological activity
 Separation of compounds from that extract by column
chromatography
 Chemical characterization of isolated compounds by IR, NMR, Mass
etc.
(Cont.)
 Nature of the Sample
• What chemical characteristics of your
compounds(s)?
– Neutrals, acids, bases
– Solubility
– pKa
– Stability
• How similar or “dissimilar” are the compounds in
your mixture?
– Similar structures (structural isomers)
– Differences or similarities in hydrophobicities
 Nature of the Sample
• How complex are your mixtures?
– How many?
• How “dirty” is your sample matrix?
– Need for sample prep (filtration and SPE)
• Biological
• Food
• Process development stream
• Instrumentation limitations?
– Volatile mobile phases (LC/MS)
 Chromatography
 Chromatography is a word used to encompass a range of
techniques in which mixtures of pure substances are
separated into the individual substances by using a
mobile phase (usually a liquid or gas) to push the mixture
along a stationary phase (usually a solid or liquid coated
on a solid).

 Because the individual substances have different

molecular structures, they interact differently with both
the stationary and mobile phases, and consequently are
"pushed" at different rates by the mobile phase. A
number of chromatographic techniques are summarized
in Table 1.
Modes of Liquid chromatography
v Normal Phase chromatography
v Reverse Phase chromatography
v Reverse Phase – Ion Pair chromatography
v Ion exchange chromatography
v Chiral chromatography
v Affinity chromatography
v Size exclusion chromatography
Normal Phase chromatography

POLAR NON-POLAR

H2 H2

H
O
H Mobile Phases: C H3
C
C
C
C
C H3
H2 H2

Water - Acetonitrile - THF - Hexane

 Chromatography

Supercritical fluid Liquid Gas

Planar liquid Column liquid

Paper TLC Packed columns Open tubular (capillary)

(dc < 350mm)

Analytical columns Preparative columns

1mm>dc>8mm dc>8mm
 Thin Layer Chromatography
Introduction
Thin Layer Chromatography (TLC) is a solid-liquid technique in which
the two phases are a solid (stationary phase) and a liquid (moving
phase).
Solids most commonly used in chromatography are silica gel (SiO2 x
H2O) and alumina (Al2O3 x H2O). Both of these adsorbents are polar,
but alumina is more so. Silica is also acidic. Alumina is available in
neutral, basic, or acidic forms.

Thin Layer Chromatography (TLC) is a sensitive, fast, simple and

inexpensive analytical technique.

It is a micro technique; as little as 10-9g of material can be detected,

although the sample size is from 1 to 100x10-6 g. TLC involves spotting
the sample to be analyzed near one end of a sheet of glass or plastic that
is coated with a thin layer of an adsorbent.
 Structure of Silica Gel

Silicon
Oxygen

It consists of silicon atoms bridged three dimensionally by

oxygen atoms
 The solvent rises by capillary action up through the adsorbent
 Differential partitioning occurs between the components of the
mixture dissolved in the solvent and the stationary adsorbent
phase.
The more strongly a given component of a mixture is adsorbed
onto the stationary phase, the less time it will spend in the mobile
phase and the more slowly it will migrate up the plate.
 The Process of TLC.
Performing a TLC analysis consists of

preparing a spotting capillary;

 marking the TLC plate;

 spotting the TLC plate;

 developing the TLC plate;

 drying the plate;

 visualizing the substance spots, and

 measuring the Rf values.

Spotting the TLC plate
 Problems in TLC.
Over-large Spots. Sample spots made using TLC capillaries should be no larger
than 1-2 mm in diameter, because component spots in the developed plate will
be no smaller than, and will usually be larger than, the size of the initial spot.

Uneven Advance of Solvent Front. A common problem in TLC is uneven

advance of solvent along the plate. Instead of a straight line, the solvent front
may appear to bow either up or down in the center. Uneven advance of solvent
leads to uneven advance of substance spots, and inaccurate Rf values result.

Streaking. Sometimes a substance will move along a TLC plate as a long streak,
rather than as a single discrete spot. This is the result of spotting the plate with
too much substance, more than the moving solvent can handle.
 TLC
• Detection methods: Destructive and Nondestructive
• Advantages:
1. Cost effective
2. Requires less training & knowledge
3. Easy scale up from analytical and preparative mode.
4. Flexibility of choice of mobile and stationary phases.
5. Separation may be readily optimized to “Zero in” on one component
and methods may be quickly developed.
6. Practically any separation can be achieved with the correct mobile
and stationary phase.
7. A large samples may be analyzed or separated simultaneously.
• Disadvantages:
1. Loading and speed are poor compared to flash chromatography.
2. There is poor detection and control of elution compared with
HPLC.
COLUMN CHROMATOGRAPHY
FOR ISOLATION OF ACTIVE
CONSTITUENTS
• Column chromatography (CC) is an extremely
valuable technique for purification of synthetic or
natural products.

• Compounds are separated by CC through the same

mechanism as TLC; by distribution between two
phases, one of which is stationary, and one of which is
moving.
 Column chromatography

• It is used for separation and isolation

of different constituents of extracts.
• Column chromatographic grade
adsorbents of choice is used as an
adsorbent for packing the columns
and various organic solvents are
used as eluting solvents.
• In order to fractionate the
components of varying solubility
“gradient elution technique is
followed.
1906- Tswett separated extracts on column
Chroma-color Tography-to write
“Separation of components in a sample
by distribution between two phases”

Stationary phase is a solid &

Mobile phase is a liquid

 Basic Separation Principle

• Introduce two solutes (A & B) onto a packed column through which a

mobile phase (i.e. solvent) or eluent is continuously pumped
 Mobile phase
Sample
Slurry

Mobile phase and analyte out

Polar component (b) adsorb more strongly to
the polar silica gel and elute after the less polar
component (a), which move more quickly with
the non-polar (relative to silica gel) solvent.
 COLUMN
CHROMATOGRAPHY:
General Procedure
• Prepare column

• Load column

• Develop column

• Collect fractions

• Analyze fractions
 COLUMN
CHROMATOGRAPHY:
Preparing and loading the column

• Efficiency of separation depends on how the column is

prepared and the sample is applied.
• A glass column is uniformly packed with a slurry of
silica gel in the mobile phase solvent system; it must
contain no holes or bubbles and the surface must be
even.
• The sample (dissolved in the mobile phase) is gently
applied with a pipette as concentrated band to the top
of the column (after the mobile phase has been drained
until the top is free of liquid).
 COLUMN
CHROMATOGRAPHY:
Developing the column, collecting,
and analyzing fractions.
• The mobile phase is applied to the top of the column, again taking
care not to disturb the top.

• The column is developed by allowing the mobile phase to pass

through the column at a flow rate of ~ 1- 3 ml/min.

• As compounds of interest elute through the column they are

collected in test tubes for TLC analysis.

• Fractions containing pure compound are combined and

concentrated for further characterization by IR, NMR and Mass
etc..
 COLUMN CHROMATOGRAPHY:
Factors Affecting the Success of Column
Chromatography

• Choice of adsorbent (silica gel or alumina are most common)

• When water is added to an adsorbent, it is deactivated.
• When water is removed by heating, it is activated.
• It is often suggested to that the wt. of the adsorbent be 25 - 30 times
greater than the wt. of the sample mixture.

• Size of the column (height and diameter)

For good separation it is often suggested that the height to diameter
ratio of the column be 8-10 : 1.
Factors Affecting the Success of
Column Chromatography (Cont.)
• Polarity of the solvent
Usually one solvent alone will not efficiently elute every
component of a mixture off the column. Often, a non
polar solvent is used first, and then a polar solvent is
used to elute remaining compounds.
• Elution rate neither too fast (no separation) nor too
slow (diffusion)
• Packing of adsorbent The adsorbent must be “packed”
into the column so that there are no irregularities such
as bubbles or cracks. For good separation, the bands in
the column must be as horizontal as possible.
 Procedure

 Before use, distill all the solvents

 Add suitable solvent and little adsorbent to the extract
and dry on butter paper
 Load the extract on top of the column of adsorbent
 Go initially by isocratic elution procedure i.e. use same
solvent
 Followed by Gradient elution technique i.e use in the
order of increasing polarity
 Collect the fractions until disappearance of the existing
spot by TLC
 Mix the suitable fractions and characterize by different
analytical techniques
 Applications
Adsorbent Chromatographic mechanism Applications
Silica gel adsorption steroids, amino acids,
alcohols, hydrocarbons,
lipids, oflotoxins, bile
acids, vitamins, alkaloids
Silica gel RP reverse phase Fatty acids, vitamins,
steroids, harmones,
carotenoids
Cellulose Kiesulguhr partition amino acids,
alcohols,carbohydrates,
sugar, carboxylic acids,
fatty acids
Alumina adsorption amines, alcohols,
steroids, lipids,
aflotoxins, bile acids,
vitamins and alkaloids
Magnesium silicate adsorption steroids, lipids,
alkaloids
 Solvents and Secondary metabolites
Petroleum ether
Steroids, fatty acids and hydrocarbons

Chloroform
Triterpenoids, alkaloids, aglycone and flavonols

Ethyl acetate
Flavonoids, Phenolics and coumarins

Methanol
Glycoside, Triterpene glycosides, saponins and alkaloids

Aqueous
Glycoside, saponins,alkaloids and flavonoids
 Solvent system for silica gel adsorbent
For essential oils -
For alkaloids- Toluene:
For anthracene derivatives-
For Coumarin aglycone-
For flavonoid- Ethyl acetate:
For cardiac glycosides-
For isothiocyanates-
Chloroform:Benzene (75:25)
Ethyl acetate: Diethylamine (70:20:10)
Ethyl acetate: Methanol: water
(100:17:13)
Toluene : ether (1:1 saturated with 10%
acetic acid)
Formic acid: Glacial acetic acid: Water
(100:11:11:27)
Ethyl acetate: Methanol: Water
(100:13.5:10)
n- butanol:n- propanol: glacial acetic
acid : water (30:10:10:10)
 Chromatographic and
spectroscopical methods
• HPLC
• HPTLC
• IR
• NMR
• MASS
• X-Ray crystallography
HPLC
 Basic Components of an HPLC System
1. Pump System. Mobil phase pressures up to 6000 psi are necessary to
achieve reasonable column elution times (~ minutes). Typical flow rates
are 0.1 to 10 mL/minute.
2. Injection System. Used to introduce small samples (0.1 to 500 µL) into
the carrier stream under high pressure.
3. Reservoirs (Solvents). Multiple solvents are necessary for performing
gradient elution's (i.e. changing the polarity of the mobil phase during a
run).
4. Chromatographic Column. Typically 10-30 cm in length containing a
packing of 5-10 µm diameter. Many types of columns are available,
depending on the type of liquid chromatography desired.
5. Detector. Many types are available including UV, IR, refractive index,
fluorescence, conductivity, mass spectrometry, and electrochemical.
Diode array detectors are used when wavelength scans are desired.
High Performance Liquid Chromatography
High Performance Liquid Chromatography
 Pump System
Desirable Features:
• Must generate pressures up
to 6,000 psi
– To allow for separation in
reasonable time frames
• Flow-rates range from 0.1 to
10 mL/minute
• Limited pulsing in the
system
– Many HPLC systems have a
dual pump system to minimize
pulsing
• Flow control and
reproducibility < 0.5%
• Corrosion resistance
 Sample Injection System
Used to introduce
small samples
(0.001 to 0.5 mL)
into the carrier
stream under high
pressure
 HPLC Detectors
• No universal or versatile detector
• Types
– General – respond to mobil phase bulk
properties which vary in the presence of solutes
(e.g. refractive index)
– Specific – respond to some propert of the solute
(not possessed by the mobil phase (e.g. UV
adsorption)
– “Hyphenated” detector – LC-MS
 Absorbance Detectors
• The UV/Vis source usually comes
from a monochromator so the
wavelength can be selected, or
scanned.
• Absorbance increases as eluate passes
through the cell.
• If wavelength scanning is desired, the
flow is stopped long enough for the
scan to take place.
• It’s possible to have the same setup
using IR light, although not as
common since many useful solvents
are not IR transparent.
 Diode
Array
Detector
HPLC Detectors
 HPLC Column
• Must operate in high pressure
– Usually constructed of metals
• Typical dimensions
– 10-30 cm long
– 1-3 cm ID
• Contains packing material
which holds the stationary
phase
– Many types exist
– Typical packing materials are 5-
10 µm in diameter
• Guard column used to extend
life of main column
HPTLC
 Spectoroscopical methods
• The chemical constituents present in the drug possess the
characteristic features because of which its
characterization becomes possible.
• Interpretation of the molecular spectra is generallly based
on empirical correlations of spectral data with reasonable
assurance to a particular group or arrangement of atoms in
the molecule.
• The instrumental methods which provides the spectral data
are complimentary to each other and there is no single
spectrometric method which can provide complete
information regarding the structural features of drug
molecules.
 Introduction to Spectroscopy “spectrum looking”

Instrumentally aided studies of the interactions between matter (sample being analyzed
and energy (any portion of the electromagnetic spectrum, EMS)

The four most common spectroscopic methods used in organic analysis are:
Method Abbrev. Energy used Units

Ultraviolet-Visible UV-Vis ultraviolet- nm

Spectroscopy visible
Infrared Spectroscopy IR infrared ? m or
cm-1
Nuclear Magnetic NMR radio Hz or ?
Resonance frequencies

Mass Spectroscopy Mass electron volts amu

Spec

Question: What actually happens to the sample during an analysis?

{How do the sample and energy “interact”?}
 Molecular Dynamics
Total energy of a molecule: Etotal =  Etranslation + Eelectronic + Evibration + Erotation
Etranslation = kinetic energy, 3-D movement
Eelectronic = electronic energy, excitation of electrons: ground state  excited state
Evibration = vibrational energy, bending and stretching of covalent bonds
Erotation = rotational energy

Chemists can use portions of the electromagnetic spectrum (EMS)

to selectively manipulate the energies contained within a molecule,
to uncover detailed evidence of its chemical structure and bonding.
 UV Visible Spectrum
The basic parts of a spectrophotometer are a light source (often an
incandescent bulb for the visible wavelengths, or a deuterium arc lamp in
the ultraviolet), a holder for the sample, a diffraction grating or
monochromator to separate the different wavelengths of light, and a
detector. The detector is typically a photodiode or a CCD. Photodiodes are
used with monochromators, which filter the light so that only light of a
single wavelength reaches the detector. Diffraction gratings are used with
CCDs, which collects light of different wavelengths on different pixels.

Diagram of a single-beam UV/vis spectrophotometer.

• The instrument used in ultraviolet-visible
spectroscopy is called a UV/vis
spectrophotometer. It measures the intensity of
light passing through a sample (I), and compares it
to the intensity of light before it passes through the
sample (Io). The ratio I / Io is called the
transmittance, and is usually expressed as a
percentage (%T). The absorbance, A, is based on
the transmittance:
• A = − log(%T)

The method is most often used in a quantitative way

to determine concentrations of an absorbing species
in solution, using the Beer-Lambert law:
 IR - Infrared Spectroscopy

• A general introduction to spectroscopic methods will be

followed by a more detailed presentation on the use of IR
Spectroscopy in structural analyses of organic compounds.
• Presence or absence of certain covalent bonds, especially
O-H and C=O, can be readily determined from IR spectra.
IR Spectroscopy  Functional Group Determination
• The atoms in a CH2 group, commonly
found in organic compounds can vibrate in
six different ways,
 Matter/Energy Interactions
• What happens when a sample absorbs UV/Vis energy?
p*
excitation of ground state electrons UV/Vis p  p*
(typically p and n electrons) sample transition
(200 nm)
Eelectronic increases momentarily
p

• What happens when a sample absorbs IR energy?

stretching and bending of bonds IR
(typically covalent bonds) -O-H -O —H
(3500 cm-1)
Evibration increases momentarily
 FTIR Instrumentation
Shimadzu 8300 FTIR Spectrometer

Infrared source

sample port IR
(closed)
Computer Workstation
running Hyper IR 1.57
 Perkin-Elmer
1600 FTIR

FTIR organic sample

Sample selectively absorbs
infrared radiation
Port
Infrared
IR radiation
to detector
salt plates positioned
on sample holder

calcium chloride
(CaCl2) desiccant
 IR Correlation Diagram
Region I Region II
3600-2700 cm-1 1800-1600 cm-1
100
O-H N-H C-H C=O
Transmittance (%)

80 bond stretching Fingerprint

alcohols
acid chlorides Region
60 anhydrides (below 1500 cm-1)
phenols
esters
carboxylic acids
ketones
40 amines aldehydes
amides carboxylic acids
amides
20 alkynes C-H
alkenes =C-H
alkanes -C-H
0
4000 3500 3000 2500 2000 1500 1000
2.5 3.0 4.0 5.0 6.0 10.0
Frequency (cm-1) / Wavelength (microns, mm)
NMR
• Nuclear Magnetic Resonance
Spectroscopy most commonly
known as NMR Spectroscopy is
the name given to the technique
which exploits the magnetic
properties of nuclei.

• Two very important techniques are

proton NMR and carbon-13 NMR,
although some other nuclei can be The NMR sample is prepared in a thin
measured as well. walled glass tube.
In its simplest form NMR allows identification of individual atoms in a pure
molecule. Much like using infrared spectroscopy to identify functional groups,
analysis of a 1D NMR spectrum tells the scientist what atom environments (like
a methyl proton), and in some cases how many atoms of each type, exist within
the sample.

When placed in a magnetic field, NMR active nuclei (like 1H or 13C)

resonate at a specific frequency, dependent on strength of the magnetic field.
For example, in a 21 tesla magnetic field, protons resonate at 900 MHz. It is
common to refer to a 21 T magnet as a 900 MHz magnet but it is important to
note that different nuclei resonate at a different frequency at this field
strength.
However, depending on the local chemical environment, different
protons in a molecule each resonate at slightly different frequencies.
Since this frequency is dependent on the strength of the magnetic
field it is converted into a field-independent value known as the
chemical shift.

The chemical shift is reported as a relative measure from some

reference resonance frequency (For the nuclei 1H, 13C, and 29Si, TMS
(tetramethylsilane) is commonly used as a reference.) This difference
between the frequency of the signal and the frequency of the
reference is divided by frequency of the magnetic field to give the
chemical shift.

By understanding different chemical environments, the chemical shift

can be used to obtain some structural information about the molecule
in question to assign signals to an atom or a group of atoms.
For example, in a proton spectrum for ethanol (CH3CH2OH) one would expect
three specific signals at three specific chemical shifts. One for the CH3 group, one
for the CH2 group and one for the OH. A typical CH3 group has a shift around 1
ppm, the CH2 attached to a OH has a shift of around 4 ppm and the OH has a shift
around 2–3 ppm depending on the solvent used.

Because the molecular motion at room temperature makes each of the three
methyl protons average out during the course of the NMR experiment (which
typically takes a few ms), the protons become degenerate and form a peak at the
same chemical shift.

Interestingly the shape and size of peaks are indicators of chemical structure too.
In the example above—the proton spectrum of ethanol—the CH3 peak would be
three times as large as the OH. Similarly the CH2 peak would be twice the size of
the OH peak but only 2/3 the size of the CH3 peak.

Modern analysis software allows analysis of the size of peaks to understand how
many protons give rise to the peak. This is known as integration—a mathematical
process which calculates the area under a graph (essentially what a spectrum is).
• The most reliable information for structure determination
in a one dimensional NMR spectrum is the spin-spin
coupling (also called J-coupling or scalar coupling)
between NMR active nuclei. This coupling arises from the
interaction of different spin states through the chemical
bonds of a molecule, which results in splitting of signals
into recognizeable patterns based on the pairing of spin
states. As a result, this coupling can tell us information
about the connectivity of atoms in a molecule.
 Proton NMR

• Typical chemical shifts of protons in 1H-NMR

•Proton NMR is the application of nuclear magnetic resonance in NMR
spectroscopy with respect to hydrogen.

•Simple NMR spectra are recorded in solution and solvent protons should
not interfere.

•Therefore a large range of deuterated solvents exist especially for NMR

such as deuterated chloroform CDCl3 and deuterated dimethyl sulfoxide
(CD3)2SO.

•These solvents contain small quantities of undeuterated solvent which may

give rise to a signal; CHCl3 is seen as a single peak at 7.27 ppm. Water
may be present as a contaminant, this gives a broad peak whose chemical
shift varies greatly with solvent; it occurs around 1.6 ppm in CDCl3. Spectra
are usually recorded against tetramethyl silane as the internal standard, set
as zero.

•Proton NMR spectra are characterized by chemical shifts in the range +12
to -4 ppm and by spin-spin coupling between protons. The integration curve
for each proton reflects the abundance of the individual protons.
MASS Spectrometry
Introduction to Mass Spectrometry
• General Characteristics and Features
– Used quantitatively and qualitatively (identification)
– Useful for both organic and inorganic compounds
– Can measure ~ 75 elements
– Rapidly evolving technology
– Expensive and complex

• Modification has couplings like:

– Inductively Coupled Plasma Mass Spectrometer (ICP-
MS)
– A gas chromatograph interfaced to a mass
spectrometer (GC-MS)
 Where are they used?
• Biotechnology:
analysis of proteins, peptides, oligonucleotides
• Pharmaceutical:
drugs discovery, combinatorial chemistry, pharmokinetics,
drug metabolism
• Clinical:
neonatal screening, haemoglobin analysis, drug testing
• Environmental:
water, food, air quality (PCBs etc)
• Geological:
oil composition
 Qualitative Identification – Basic
Principles
• Samples are ionized
– Some fragmentation usually occurs
• Sample components (and fragments) are
separated based on mass-to-charge ratio
• Output is a mass spectrum
Applications of Mass Spectrometry
1. Qualitative Identification
Identify organic compounds
2. Quantitative Analysis
Elemental analysis
Molecular compounds (GC-MS)
3. Isotope Ratio
– 206Pb/208Pb

4. Detector in “hyphenated” systems

GC-MS
LC-MS
 Qualitative Identification (cont.)
Mass Spectrum of CO2 (g) • Two main peaks are
100 observed
16-O – Masses 12 and 16
• The mass 16 peak is
Relative Abundance (%)

twice the size of the

mass 12 peak
12-C
– There are two oxygen
50
atoms for every carbon
atom in CO2
• Several minor peaks are
17-O 18-O observed at masses of
13-C
13, 17, and 18
0 – Minor isotopes of C and O
12 14 16 18
Mass-to-charge ratio (m/e-)
Qualitative Identification (cont.)
• The largest peak is
called the base
peak
• The base peak is
used to set 100%
relative abundance
• Ions created out of
the unfragmented
compound are
called the parent
ion
n-heptane
Qualitative
Identification
(cont.)
1-pentanol

n-heptanal
 Basic Components of a MS
Vacuum System

Volatilize Sample
Sample sample Ion Mass (m/e-)i
ions + Detector
Introduction Source fragments Separator
System
current
(g, l, s)

Signal
Processor

DC
Voltage

Computer
Interface
(Readout)
Mass Spectrum
 Purpose of Basic Components
1. Sample Introduction System
– Volatilizes the sample and introduces it to the
ionization chamber under high vacuum
2. Ion Source
– Ionizes the sample (fragmentation may occur) and
accelerates the particles into the mass analyzer
3. Mass Analyzer (or Mass Separator)
– Separates ionized particles based on their mass-to-
charge ratio (m/e-)
 Purpose of Basic Components
4. Detector - Ion Collector
– Monitors the number of ions reaching detector per
unit time as a current flow
5. Signal Processor
– Amplifies the current signal and converts it to a DC
Voltage
6. Vacuum Pump System
– A very high vacuum (10-4 to 10-7 torr) is required so
that the generated ions are not deflected by
collisions with internal gases
 A Basic Mass Spectrometer
Single Sector Magnetic Focusing Mass Spectrometer
X-Ray diffraction
• X-ray crystallography is a technique in crystallography in which the
pattern produced by the diffraction of X-rays through the closely
spaced lattice of atoms in a crystal is recorded and then analyzed to
reveal the nature of that lattice. This generally leads to an
understanding of the material and molecular structure of a
substance. The spacing in the crystal lattice can be determined
using Bragg's law.

• The electrons that surround the atoms, rather than the atomic nuclei
themselves, are the entities which physically interact with the
incoming X-ray photons.

• This technique is widely used in chemistry and biochemistry to

determine the structures of an immense variety of molecules,
including inorganic compounds, DNA and proteins.

• X-ray diffraction is commonly carried out using single crystals of a

material, but if these are not available, microcrystalline powdered
samples may also be used, although this requires different
equipment, gives less information, and is much less straightforward.
 Crystallisation

• In order to solve a crystal structure, you must first

crystallize the compound of interest. This is because a
single molecule in solution has insufficient scattering
power alone. A crystal can be considered to be an
(effectively) infinite repeating array of our molecule of
interest.

• Crystallization of small molecules has traditionally

followed three methods
• Diffusion gradient- solubility or temperature
• Concentration through evaporation
• Sublimation- not recommended due to low quality
crystals.
An X-ray diffraction image for the protein myoglobin.