LIQUID

CHROMATOGRAPHY

HIGH PERFORMANCE
LIQUID
CHROMATOGRAHY (HPLC)
High-performance liquid chromatography (HPLC) is a technique in
analytical chemistry used to separate, identify, quantify or purify
the individual components from the mixture.
In HPLC, as in all chromatographic methods, components of a
mixture are partitioned between an adsorbent (the stationary
phase) and a solvent (the mobile phase).
The stationary phase is made up of very small particles contained
in a steel column. Due to the small particle size (3-5 um), pressure
is required to force the mobile phase through the stationary phase.
 HISTORY OF HPLC
Liquid chromatography was initially discovered as an analytical technique in the early
twentieth century and was first used as a method of separating colored compounds.
A Russian botanist named Mikhail S. Tswett used a rudimentary form of chromatographic
separation to purify mixtures of plant pigments into the pure constituents.
1940s Martin and Synge developed the theory of partition chromatography and used
mathematics to describe the separation process resulting from the use of a liquid-coated
solid phase and a moving liquid phase.
1969 .The first commercial HPLC was manufactured by Waters Corporation, and was known
as the ALC100 HPLC.
The chromatographic process has been significantly improved over the last hundred years,
yielding greater separation efficiency, versatility and speed.
Instrumentation

Mobile Phases
(Solvent reservoirs)

Flow Rate
Composition (pump)

Injection Volume (injection system)

Column
Oven Temperature

Wavelength
Time Constant (detector)
SCHEMATIC DIAGRAM OF HPLC
PRINCIPLE OF HPLC

Analytes are separated based on their differential affinity between

a solid stationary phase and a liquid mobile phase.
Kinetics of distribution of solutes between the stationary phase and
mobile phase is largely diffusion-controlled.
Diffusion coefficient of analytes in liquids is 1000-10000 x slower than in
gases.
PRINCIPLE

To minimize time required for interaction of analytes between mobile

phase and stationary phase:
a) The packing particles should be small and as uniformly and densely
packed as possible. (affect A – van Deemter)
b) The stationary phase should be effectively a thin uniform film with no
stagnant pools and provide a small C (van Deemter)
PRINCIPLE

General Rule of Thumb:

• Polarity of analytes ≈ polarity of stationary phase ≠ polarity of
mobile phase
• To achieve good separation, the analytes should interact with the
stationary phase,
but not too strongly or the retention time will become very long.
MODE OF HPLC OPERATION

NORMAL PHASE

RESEARVE PHASE
NORMAL PHASE

Characteristics
• Highly polar stationary phase
• Silica or alumina oxides
• Relatively non-polar solvent
• e.g. hexane or i-propylether
• Least polar solutes elute first
• Increasing mobile phase polarity decreases elution times (i.e. polar
compounds remain in the mobile phase longer)
NORMAL PHASE (PARTITION) CHROMATOGRAPHY

Partition chromatography in which the stationary phase has a

high polarity (hydrophilic) and the mobile phase has a low
polarity (hydrophobic)
Essentially based on the same separation mechanism as
adsorption chromatography in which the stationary phase has
a hydrophilic base, such as silica gel

11
STATIONARY PHASE AND MOBILE PHASE USED IN NORMAL
PHASE MODE
Stationary Phase
Silica gel: -Si-OH
Cyano type: -Si-CH2CH2CH2CN
Amino type: -Si-CH2CH2CH2NH2
Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
Mobile Phase
Basic solvents: Aliphatic hydrocarbons,
aromatic hydrocarbons, etc.
Additional solvents: Alcohols, ethers, etc.

12
 RELATIONSHIP BETWEEN HYDROGEN BONDING
AND RETENTION TIME IN NORMAL PHASE
MODE

SiOH HO
Strong
SiOH
Weak
Very weak
OH

Steric hindrance
13
RELATIONSHIP BETWEEN ELUENT POLARITY AND RETENTION
TIME IN NORMAL PHASE MODE

Eluent: Hexane/methanol

100/0

98/2

95/5

14
REVERVED PHASE

Characteristics

• Non-polar stationary phase

• e.g. a hydrocarbon

• Relatively polar mobile phase

• e.g. water, methanol or acetonitrile

• More polar solutes elute first

• Increasing the mobile phase polarity increases elution time

POLARITY OF SUBSTANCES
Miscibility of solvents

Polarity Solvents of similar polarities

can be easily dissolved
Property of a substance together.
whereby the positions of the
electrons give rise to Polar and nonpolar
positive and negative poles molecules have a similar
relationship to that of water
Water: Polar and oil.
Methane: Nonpolar H –
H O
O
C H C C
H H O
–
H H + H
H
Methane Water Acetic acid 16
NONPOLAR (HYDROPHOBIC) FUNCTIONAL GROUPS AND POLAR
(HYDROPHILIC) FUNCTIONAL GROUPS

Polar Functional Groups

Nonpolar Functional Groups
-COOH
-(CH2)n CH3
Carboxyl groups
Alkyl groups
-NH2
-C6H5
Amino groups
Phenyl groups
-OH
Hydroxyl groups

17
REVERSED PHASE CHROMATOGRAPHY

Stationary phase: Low polarity

Octadecyl group-bonded silical gel (ODS)
Mobile phase: High polarity
Water, methanol, acetonitrile
Salt is sometimes added.

18
SEPARATION COLUMN FOR REVERSED PHASE CHROMATOGRAPHY
C18 (ODS) type Phenyl type
C8 (octyl) type TMS type
C4 (butyl) type Cyano type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)

19
EFFECT OF CHAIN LENGTH OF STATIONARY PHASE

C8

C18 (ODS) Medium

Strong
C4

Weak

20
HYDROPHOBIC INTERACTION
H2O H2O H2O
H2O
H2O
H2O Nonpolar solute
H2O
H2O
H2O If a nonpolar H2O
H2O H2O substance is added... H2O H2O
Network of hydrogen bonds …the network is broken and...

H2O H2O
H2O
H2O H2O …the nonpolar substance
H2O H2O is pushed to a nonpolar
location.
Nonpolar solute

Nonpolar stationary phase 21

RELATIONSHIP BETWEEN RETENTION TIME AND POLARITY

C18 (ODS) OH

Weak
Strong
CH3

22
BASIC SETTINGS FOR ELUENT USED IN REVERSED PHASE MODE

Water (buffer solution) + water-soluble organic solvent

Water-soluble organic solvent: Methanol
Acetonitrile
Tetrahydrofuran etc.
The mixing ratio of the water (buffer solution) and organic solvent has the
greatest influence on separation.
If a buffer solution is used, its pH value is an important separation
parameter.

23
DIFFERENCE IN SOLUTE RETENTION STRENGTHS FOR WATER
AND WATER-SOLUBLE ORGANIC SOLVENTS

Tightly packed network Loose network

H2O H2O CH3OH CH3OH

H2O
H2O
H2O CH3OH
H2O H2O CH3OH CH3OH

CH3OH
Nonpolar solute CH3OH
Nonpolar solute

Nonpolar stationary phase

24
RELATIONSHIP BETWEEN POLARITY OF ELUENT
AND RETENTION TIME IN REVERSED PHASE MODE

Eluent: Methanol / Water

60/40

70/30

80/20

25
COMPARISON OF NORMAL PHASE AND REVERSED PHASE

Normal Phase Reversed Phase

Effective for separation Wide range of applications
of structural isomers
Effective for separation of
Offers separation
homologs
selectivity not available
with reversed phase Stationary phase has long
Stabilizes slowly and is service life
prone to fluctuations in
Stabilizes quickly
retention time
Eluents are expensive Eluents are inexpensive and
easy to use
26
NORMAL PHASE / REVERSED PHASE

Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (hydrophilic)

27
Normal phase VS reversed phase
 Basic Components of HPLC System

1. Pump System.
2. Injection System.
3. Reservoirs (Solvents).
4. Columns.
5. Detector.
6. Data acquisition.
 Pump System
Desirable Features:
• Must generate pressures up to 6,000 psi
Column

◦ To allow for separation in reasonable

time frames (~ min) L| Pulse |

I damper j

• Flow-rates range from 0.1 to 10 mL/minute v Ball

VW VSii' y-

\ check

Limited pulsing in the system ' valves

Reciprocating
◦ Many HPLC systems have a dual pump system to

minimize pulsing
Solvent
• Flow control and reproducibility < 0.5%
A reciprocating pump for HPLC
• Corrosion resistance
 Pump System
• The pump is the most critical piece of equipment for a successfully
operating HPLC.
• Performance parameters for HPLC pumps:

- Flow precision
- Flow range
- Delay volume
- Pressure pulse
- Composition precision
 Sample Injection System

Used to introduce small samples

(0.001 to 0.5 mL) into the carrier
stream under high pressure

Figure . A sampling loop for liquid chromatography.(Courtesy of Beckman Instru¬

With the valve handle as shown on the left, the loop is filled from
ments, Fullerton, CA.)
the syringe, and the mobile phase flows from pump to column. When the valve is
placed in the position on the right, the loop is inserted between the pump and the col
umn so that the mobile phase sweeps the sample onto the column.
 Sample Injection System

Requirements:

Reproducible introduction of the sample volume into the mobile

phase flow.

Two major designs:

Automatic Injectors or Manual Injectors
 Manual Injectors

Front View Rear View

Flow Connections

Inject
 Auto Injectors

• Continuous injections operator free

• Comparable precision and accuracy to manual

• Much more expensive initially

• Much more convenient

• Up 100 samples and standards with microprocessor control

 Reservoirs (Solvents)

Multiple solvents are necessary for performing gradient elution’s

(i.e. changing the polarity of the mobile phase during a run).

 Mobile Phase Reservoirs

• Inert container with inert lines leading to the pump are required.

• Reservoir filters (2-10 mm) at reservoir end of solvent delivery lines.

• Degassed solvent
- Vacuum filtration
- Sparge with inert gas (N2 or He)
• - Ultrasonic under vacuum
• Elevate above pumps
 Solvent Filters

JO —DU
Analytical column

Pre column Filter

Solvent Inlet Filer
• Used between the injector and guard column.
• Stainless Steel or glass • 2 to 0.5 micron
with 10 micron porosity.
• Removes particulates from sample and
• Removes particulates autosampler wear debris.
from solvent.
Must be well designed to prevent
dispersion.
 Mobile Phase Reservoirs
Isocratic elution:
• A separation that employs a single solvent or solvent mixture of
constant composition.

Gradient elution:
• Here two or more solvent systems that differ significantly in polarity
are employed.

• After elution is begun; the ratio of the solvents is varied in a

programmed way, sometimes continuously and sometimes in a series
of steps.
• Separation efficiency is greatly enhanced by gradient elution.
 Mobile Phases

• Many types are available

◦ In contrast to GC

• The mobile phase interacts with sample components

◦ In contrast to GC
• Polarity is important for solute, mobile phase and stationery
phase
 Liquid-Chromatographic Columns

• Liquid-chromatographic columns are ordinarily constructed from

smooth-bore stainless steel tubing, although heavy- walled glass
tubing is occasionally encountered.
• The latter is restricted to pressures that are lower than about 600 psi.
 Liquid-Chromatographic Columns

• Liquid-chromatographic columns range in length from 10 to 30cm.

• Normally, the columns are straight, with added length, where needed,
being gained by coupling two or more columns together.
• The inner diameter of liquid columns is often 4 to 10mm; the most
common particle size of packings is 5 or 10pm.

• The most common column currently in use is one that is 25 cm in

length,4.6mm inner diameter, and packed with 5pm particles.

• Columns of this type contain 40,000 to 60,000 plates/meter.
 Guard Columns
• Introduced before the analytical column to increase the life of the
analytical column by removing not only particulate matter and
contaminants from the solvents but also sample components that bind
irreversibly to the stationary phase.
• The guard column serves to saturate the mobile phase with the
stationary phase so that losses of this solvent from the analytical
column are minimized.
• The composition of the guard-column packing is similar to that of the
analytical column; the particle size is usually larger.
• When the guard column has become contaminated, it is repacked or
discarded and replaced with a new one.
 Column
• Must operate in high pressure
• ◦ Usually constructed of metals
Typical dimensions
◦ 10-30 cm long
• Contains packing material which holds the
stationary phase
◦ Many types exist
◦ Typical packing materials are 5-10 ^m in diameter
• Guard column used to extend life of main column

column end fitting

Guard Column
Analytical Column
 Column - Stationary Phase
H-Bonded Vicinal
Microporous Silica Particles Silanol Groups

Aggregate of Particles

Isolated

Silanol

Groups

■ Particles are permeable to solvent

■ 3.5 microns, ~100 m2/g of particles,
■ Only use for pH < 8.0
■ Use polystyrene particles for pH 8-12

Gem inal Silanol

Groups
 Column - Stationary Phase
• The particles are high-purity silica, low in trace metal content, and are
typically 5 to 10 mm in diameter, although 3-mm particles are finding
more use for high-speed chromatography.

• Pore sizes are in the 60 to 100 A range, although pore sizes of 300 A or
larger are used for larger biomolecules to allow them to penetrate the
pores.
 Column - Stationary Phase

• Most HPLC is performed in the liquid-liquid (partition

chromatography) mode, but adsorption chromatography is useful
for many applications.

• Liquid stationary phases are either coated on the particles or

are chemically bonded.
REPRESENTATIVE HPLC DETECTORS

UV-VIS absorbance detector

Photodiode array-type UV-VIS absorbance detector
Fluorescence detector
Refractive index detector
Evaporative light scattering detector
Electrical conductivity detector
Electrochemical detector
Mass spectrometer

48
DETECTION CONDITION REQUIREMENTS

Sensitivity
The detector must have the appropriate level of sensitivity.
Selectivity
The detector must be able to detect the target substance without, if
possible, detecting other substances.
Adaptability to separation conditions
Operability, etc.

49
 UV DETECTOR 

There are three types of UV detectors: fixed wavelength, variable

wavelength, and photodiode array [Link] detector is a very
commonly used detector for HPLC analysis.
During the analysis, sample goes through a clear color-less glass cell,
called flow cell. When UV light is irradiated on the flow cell, sample absorbs
a part of UV light. Thus, the intensity of UV light observed for the mobile
phase (without sample) and the eluent containing sample will differ. By
measuring this difference, the amount of sample can be determined. Since
the UV absorbance also differs depend on what wavelength is used, it is
important to choose an appropriate wavelength based on the type of
analyte. A standard UV detector allows user to choose wavelength between
195 to 370 nm. Most commonly used is 254 nm.
Compared to a UV detector, a VIS detector uses longer wavelength
(400 to 700 nm). There are detectors that provide wider
wavelength selection, covering both UV and VIS ranges (195 to
700 nm) called UV/VIS detector.
PDA detects an entire spectrum simultaneously. UV and VIS
detectors visualize the obtained result in two dimensions (light
intensity and time), but PDA adds the third dimension (wavelength).
This is convenient to determine the most suitable wavelength
without repeating analyses.
SCHEMATIC DIAGRAM
Refractive-Index
Detector
RI detector measures change in reflex
index. A glass cell is divided into two
chambers (cells). The effluent from LC
column flow through the "sample cell",
while other cell called "reference cell" is
filled with only mobile phase. When the
effluent going through the sample cell
does not contain any analyte, the solvent
inside both cells are the same (Figure 1A)
. When a beam is irradiate on the cells,
the observed beam will be straight in this
case. However, in a case the effluent
contains any components other than
mobile phase; bending of the incident
beam occurs due to the reflex index
difference between the two solvents
(Figure 1B). By measuring this change,
the presence of components can be
observed.
 Evaporative Light Scattering Detector

ELSD provides good sensitivity for non-volatile analytes at ng level.

The column effluent is nebulized and then evaporated to make it form
fine particles. The analyte is then radiated with a laser beam and the
scattered radiation is detected. The target sample includes lipids,
sugar, and high molecular weight analytes. It is used in the similar way
as a RI detector, but can provide more sensitive detection with stable
base line. Another advantage is that ELSD can be used for the gradient
method whereas RI cannot.
SCHEMATIC DIAGRAM
 Fluorescence Detector
The advantage of fluorescence method is its high sensitivity for selective
groups of compounds at ~fg level. By using a specific wavelength, analyte
atoms are excited and then emit light signal (fluorescence). The intensity of this
emitted light is monitored to quantify the analyte concentration. Most
pharmaceuticals, natural products, clinical samples, and petroleum products
have fluorescent absorbance. For some compounds which do not have
fluorescence absorbance or low absorbance, they can be treated with
fluorescence derivatives such as dansylchloride. The system is easy to operate
and relatively stable.
SCHEMATIC DIAGRAM
 Chemiluminescence Detector

Similar to FL, but instead of using a light source to excite

the analyte atoms, the excitation is initiated by chemical
reaction. Since it is not relied on the external excitation
source, the noise is small, results in high signal to noise
ratio, i.e. it provides even higher sensitivity than FL.
SCHEMATIC DIAGRAM
 Electro Chemical Detector

There are several different types of ECs. The detection is

based on amperometry, polarography, coulometry, and
conductrometry. They offer high sensitivity, simplicity,
convenience, and wide-spread applicability. It is especially
suitable for the use with semi-micro or capillary type system.
SCHEMATIC DIAGRAM
 Optical Rotation Detector

Specific for the optical isomer measurement. The column

can separate R- and L- type optical isomers, but the
general detectors (e.g., UV) cannot distinguish which is R
nor L. OR detector provides this information.
THALIDOMIDE BABIES
SCHEMATIC DIAGRAM
Mass Spectrometry
 What is Mass Spectrometry?

Mass Spectrometry is a collection of techniques

used to generate ions from either inorganic or
organic compounds by suitable method, to separate
these ions by theirmass-to-charge ratio (m/z) and
to detect them qualitatively and quantitatively by
their respectivem/z and abundance
 MASS SPECTROMETRY

Molecular weight can be obtained from a very small sample.

It does not involve the absorption or emission of light.
A beam of high-energy electrons breaks the molecule apart.
The masses of the fragments and their relative abundance reveal
information about the structure of the molecule.

Mass spectrometry concerned with the

separation of matter according to atomic and
molecular mass.
 History of Mass Spectrometry
1886:E. Goldstein discovers anode rays (positive gas ions) in gas discharge
1897:J.J. Thomson discovers the electron and determines its m/z ratio. Nobel
Prize in 1906.
1898:W. Wien analyzes the anode rays by magnetic deflection, and establishes
that they carry a positive charge. Nobel Prize in 1911.
1909:R.A. Millikan & H. Fletcher determine the elementary unit of charge.
1912: First Mass Spectrometer(J.J. Thomson).
1919: Electron ionization and magnetic sector MS(A.J. Dempster)
1942: First commercial instrument
1953: Quadrupole and the ion trap(W. Paul & H.S. Steinwwdel). Nobel Prize to
Paul 1989.
1956: First GC-MS
1968: First commercial quadrupole
1975: First commercial GC-MS
1990s: Explosive growth in biological MS, due to ESI & MALDI
2002: Nobel Prize toFenn & Tanaka for ESI & MALDI.
What does a mass spectrometer do ?
1. It measures mass better than any other technique.
2. It can give information about chemical structures.

What are mass measurements good for?

To identify, verify, and quantitate: metabolites, recombinant
proteins, proteins isolated from natural sources,
oligonucleotides, drug candidates, peptides, synthetic
organic chemicals, polymers
 Define mass in MS

□Atomic / molecular weight

• Atomic and molecular weight are generally expressed

in terms of atomic mass units(amu) or dalton.

• amu is based upon a relative scale in which the

reference is the carbon isotope C, which is

assigned

a mass of exactly 12 amu. That is 1amu = 1/12

of the

mass of one neutral C atom.

 Define mass in MS
□Exact mass and nominal mass
• In mass spectrometry, people are interested in the exact
mass of particular isotopes of an element or the exact
mass of compounds containing a particular set of
isotopes. Normally, exact masses are quoted to 3 to 4
figures to the right of the decimal point. e.g. 12C,
12.0000; 13C, 13.0033(High-resolution MS)
• The term nominal mass implies a whole number
precision in mass measurement. The nominal mass is
calculated by summing the integral atomic-mass values
of the lightest (most abundant) isotopes of all atoms
present in a given ion.
 Exact and nominal mass for methane CH 4

Mass in amu Abundance (%)

12Q 12.0000 98.90
13C 13.003355 1.10
1H 1.007825 99.98
2H 2.014102 0.015
The exact mass of CH is C*H= 16.031; CH=

17.038

The nominal mass of CH is 12 + 1 x 4 = 16

C1H32H= 17.038
12
 Mass-to-Charge Ratio (m/z )

• Mass-to-charge ratio is obtained by dividing the

atomic or molecular mass of an ion m by the number

of charges z that the ion bears.

• Ions are often singly charged. As a result, the term

mass-to-charge ratio is often shortened to the more

convenient term mass.

• But ions can be multiple charged, especially those

ions

generated by electrospray ionization.

 Mass spectrometry (MS)
Basic Principles of MS •

• The resulting species - a molecular ion - is positively charged as it has lost

an electron.
• The dot (•) represents the unpaired electron remaining from an electron
pair
when the other electron has been expelled.
• The molecular ion can fragment even further forming new ions, molecules
or radicals.
• Radicals are neutral species containing an unpaired electron.
 Forming a radicals
H H
e" + H:C:H —* 2e" + H = C|H
H H ^-unpaired
electron methane electron
M+, radical cation
Copyright © 2006 Pearson Prentice Hall, Inc.

Radicals (free radicals):

Atomic or molecular species with unpaired electrons on an otherwise
open shell configuration. These unpaired electrons are usually highly
reactive
The Physics Behind the Technique
Mass spectrometry is based upon the motion of a charged

particle, called an ion, in an electric or magnetic field.

“The mass to charge ratio(m/z) effects this motion”

 The Physics Behind the Technique
The physics behind mass spectrometry is that a charged
particle passing through a magnetic field is deflected along a
circular path of radius that is proportional to the mass to charge

ratio, m/z. electromagnet

mixed j j- _
ion stream C
ion stream

ion sfream B
ion stream A

All commonly used mass analyzers use electric and magnetic fields to apply
a force on charge particles (ions).
 How Does
Mass Spectrometry
Works?
 General Operation of Mass Spectroscopy
1. Create gas-phase ions.

2. Separate the ions in space or time based on their

mass-to-

charge ratio.

3. Measure the quantity of ions of eachmass-to-charge

ratio.

The separation power is described by the resolution (R),

M sometimes called Resolving power.

R=-
AM
M = is the ion mass (lighter)

AM = is the difference in mass between two resolvable peaks in mass

spectrum
 How does it works?
Mass Spectrometer Block Diagram
 Vacuum Requirement in MS

All mass spectrometers required vacuum system to maintain the low

pressure (high vacuum) needed for operation.......Why?
To: minimize ion-molecule reaction, scattering, and neutralization of
the ions.
An outline of what happens in a mass spectrometer
 Stage 1: Ionisation (the formation of ions)

The atom is ionized by knocking one or more electrons off to

give a positive ion (a radical cation) . This initial ion is called the
molecular ion (M+.), because it contains all the atoms present in
the starting molecule (same molecular weight).
 Ionisation

The need for a vacuum

It’s important that the ions produced in the ionization chamber
have a free run through the machine without hitting air molecules
 Stage 2: Fragmentation
Excess vibrational energy is imparted to the molecular ion by

collision with the electron beam

1. Some of the collisions are energetic enough to knock one or

more electrons out of the sample particles to make positive

ions

2. Most of the positive ions formed will carry a charge of+1

(because it is much more difficult to remove further electrons

from an already positive ion)

3. The positive ions are persuaded out into the rest of the

machine by ion repeller(another metal plate carrying a

slight

positive charge)
 Fragmentation
The fragmentation pattern is highly characteristic of the structure
of the molecule

F + neutral molecule
+

F| +¥2
+

F|* + F 2
+
What the mass spectrometer output looks like?
•A mass spectrometer produces a spectrum of masses based
on the structure of a molecule.
•A mass spectrum is a plot of the distribution of ion masses
corresponding to the formula weight of a molecule and/or
fragments derived from it
• The x-axis of a mass spectrum represents the masses of
ions produced
• The y-axis represents the relative abundance of each ion
produced
• The pattern of ions obtained, and their abundance is characteristic of the
structure of a particular molecule
fragment ions

m/e
 What the mass spectrometer output looks like?
relative
abundance
m olybdenum

M o (95.94 g/m ol)

rn/z

• The output is simplified into stick diagram

• It shows the relative current produced by ions of varyingm/

z ratio

• Vertical axes: labeled as either relative abundance or relative

intensity (the greater the current, the more abundant the ion)
 Mass Spectra

Blue = lightest

Red = middle

Gray = heaviest

% Red % Blue
 THE MASS SPECTRUM
Masses are graphed or tabulated according to their
relative abundance.
base peak (strongest)

100

80

60

40

20

0
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
m/z
Copyright © 2005 Pearson Prentice Hall, Inc.
IN A MASS SPECTRUM
COMPARISON OF DETECTORS

Possibility of
Selectivity Sensitivity
Gradient System
Light-absorbing
Absorbance ng Possible
substances

Fluorescence Fluorescent substances pg Possible

Differential
None µg Impossible
refractive index
Evaporative light
Nonvolatile substances µg Possible
scattering
Electrical
Ionic substances ng Partially possible
conductivity
Oxidizing / reducing
Electrochemical pg Partially possible
substances
Note: The above table indicates general characteristics. There are exceptions. 93
 HPLC Applications
Chemical Bioscience
polystyrenes
proteins
dyes
peptides
phthalates
nucleotides
tetracyclines
Pharmaceutical corticosteroids
antidepressants
barbiturates

Environmental
Consumer Products
polyaromatic hydrocarbons
lipids
Inorganic ions antioxidants
herbicides Clinical sugars
amino acids
vitamins
homocysteine