CHROMATOGRAPHY

Brief History and Definition

Liquid chromatography was defined in the early 1900s by the
work of the Russian botanist, Mikhail S. Tswett. His pioneering
studies focused on separating compounds [leaf pigments], extracted
from plants using a solvent, in a column packed with particles.
Tswett coined the name chromatography [from the Greek words
chroma, meaning color, and graph, meaning writingliterally, color
writing] to describe his colorful experiment. [Curiously, the Russian
name Tswett means color.] Today, liquid chromatography, in its
various forms, has become one of the most powerful tools in
analytical chemistry.

Tswett filled an open glass column with particles.

Two specific materials that he found useful were
powdered chalk [calcium carbonate] and alumina. He
poured his sample [solvent extract of homogenized
plant leaves] into the column and allowed it to pass
into the particle bed. This was followed by pure
solvent. As the sample passed down through the
column by gravity, different colored bands could be
seen separating because some components were
moving faster than others. He related these separated,
different-colored bands to the different compounds
that were originally contained in the sample.

Column Chromatography

Separation Techniques
Separation processes are used to decrease the complexity of material
mixtures. Chromatography and electrophoresis are representative of this
field.

Figure: Separation of black ink on a thin layer chromatography plate.

Chromatography (Encyclopedia Britannica):

Technique for separating the components, or solutes, of a mixture on
the basis of the relative amounts of each solute distributed between a
moving fluid stream, called the mobile phase, and a contiguous
stationary phase. The mobile phase may be either a liquid or a gas,
while the stationary phase is either a solid or a liquid.

Chromatography
(IUPAC Compendium of Chemical Terminology):
A physical method of separation in which the components to be
separated are distributed between two phases, one of which is
stationary (stationary phase) while the other (the mobile phase)
moves in a definite direction.

CHROMATOGRAPHY
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient (equilibrium
distribution) of sample components between 2
different phases.
One of these phases is a mobile phase and
the other is a stationary phase.

Distribution Coefficient (Equilibrium Distribution )

Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of these 2 components to stationary
phase causes the separation.

Kinds of Chromatography

1. Liquid Column Chromatography

2. Gas Liquid Chromatography

3. Thin-Layer Chromatography

Schematic Presentation of a Chromatogram

Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

FOUR BASIC LIQUID CHROMATOGRAPHY

The 4 basic liquid chromatography modes are named according to the mechanism
involved:

1. Liquid/Solid Chromatography (adsorption chromatography)

A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)

Normal phase
In this column type, the retention is
governed by the interaction of the polar
parts of the stationary phase and solute. For
retention to occur in normal phase, the
packing must be more polar than the mobile
phase with respect to the sample

Structure of silica gel

Stationary Phase: Alumina

O

OH

OH

OH

OH

Al

Al

Al

Al

Al

Acidic: -Al-OH
Neutral: -Al-OH + -Al-OBasic: -Al-O-

LIQUID SOLID CHROMATOGRAPHY

Normal phase LS
Reverse phase LS

Si - O - H

Silica Gel

The separation mechanism in LSC is based on the

competition of the components of the mixture sample
for the active sites on an absorbent such as Silica Gel.

LIQUID SOLID CHROMATOGRAPHY

OH

HEXANE

Si - OH

CH3

OH CH
3
C-CH3
CH3

CH3- C
CH3

CH3

Reverse phase
In this column the packing material is relatively
nonpolar and the solvent is polar with respect to the
sample. Retention is the result of the interaction of
the nonpolar components of the solutes and the
nonpolar stationary phase. Typical stationary phases
are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the
solvents are polar aqueous-organic mixtures such as
methanol-water or acetonitrile-water.

LIQUID-LIQUID CHROMATOGRAPHY

ODPN(oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC

NCCH3CH2OCH2CH2CN(Normal)
CH3(CH2)16CH3 (Reverse)

The stationary solid surface is coated with a 2nd liquid (the

Stationary Phase) which is immiscible in the solvent (Mobile) phase.
Partitioning of the sample between 2 phases delays or retains some
components more than others to effect separation.

Reverse phase chromatography

Silica is alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.

Size exclusion
In size exclusion the HPLC column is
consisted of substances which have controlled
pore sizes and is able to be filtered in an
ordinarily phase according to its molecular
size. Small molecules penetrate into the pores
within the packing while larger molecules
only partially penetrate the pores. The large
molecules elute before the smaller molecules.

GEL-PERMEATION CHROMATOGRAPHY

Gel-Permeation Chromatography is a mechanical sorting of molecules

based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore,
retained longer than large molecules.

Ion exchange
In this column type the sample components
are separated based upon attractive ionic
forces between molecules carrying charged
groups of opposite charge to those charges
on the stationary phase. Separations are
made between a polar mobile liquid, usually
water containing salts or small amounts of
alcohols, and a stationary phase containing
either acidic or basic fixed sites.

MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS

pH2

SO3

Na

H3N

COOH

Ion-exchange Resin

SO3

H3N
Na

+
-

COO

pH4.5

Mechanism of
separation in
different
forms of
HPLC

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

(HPLC)

High

Performance
L iquid
C hromatography

High

Pressure
L iquid
C hromatography

High

Priced
L iquid
C hromatography

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High Performance Liquid Chromatography (HPLC) is one of the

most widely used techniques for identification, quantification and
purification of mixtures of organic compounds.

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.

There are a wide variety of stationary phases available for HPLC. In

all labs can be used a normal phase (Silica gel), although reverse
phase (silica gel in which a 18 carbon hydrocarbon is covalently
bound to the surface of the silica) columns are currently one of the
most commonly used HPLC stationary phases.

 HPLC is a form of liquid chromatography used to

separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column,
and a detector.
Compounds are separated by injecting a sample
mixture onto the column. The different component
in the mixture pass through the column at
differentiates due to differences in their partition
behavior between the mobile phase and the
stationary phase. The mobile phase must be
degassed to eliminate the formation of air bubbles.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html

General Schematic of LC

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

Chromatography: HPLC
Hewlett-Packard
Series 1100 HPLC
solvent

pump
injector

column
detector

Chromatography: HPLC
HPLC Column

Chromatography: HPLC
HPLC Pump

Chromatography: HPLC
HPLC Autosampler and Injector

Chromatography: HPLC
HPLC Detector
UV/Visible Spectrophotometer

Chromatography: HPLC
HPLC Waste Collection

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

TLC vs High Performance Liquid Chromatography (HPLC)

HPLC Optimization

http://www.labhut.com/education/flash/introduction07.php

Modes of HPLC

Bonding Phases onto the

Silica Support
Silica

OH

CH3
|
Silica -0-Si- CH3
|
CH3

CH3
|
Cl -Si- CH3
|
CH3

HCl
Could be many different
functional groups here

End Capping
Usually half or more of the silanol groups
remain unreacted after bonding with C18.
One method used to reduce the effects of
these residual silanol groups is a process
called endcapping.

End Capping
After bonding with C18 use small silane
molecules such as trimethylchlorosilane to
react some of the remaining OH groups

Steric Protection
CH3
|
CH2
|
Silica -0-Si- CH3
|
Hydrolysis may degrade
CH2
the column by breaking
|
off the bonded phase.
CH3

Comparison of Different C18 Columns

Column: Symmetry C18

Mobile Phase:
60% CH3CN
40% 50mM KH2PO4,
pH 3.2

Hypersil HyPURITY C18

1.
2.
3.
4.
5.
6.
7.

Sample:
Uracil
Pyridine
Phenol
Dimethyl phthalate
N,N-Dimethylaniline
4-Butylbenzoic acid
Toluene

Common RP Packings

Other Bonded Phases

Phenyl phases show weak dipole - induced
dipole interactions with polar analytes.
Usually this type of bonded phase is used
for separating closely related groups of
molecules.

Other Bonded Phases

The amino-phase is the most polar, and it can
also act as weak anion-exchanger, (protonated
at low pH). Amino columns are mainly used in
normal-phase mode, specially for selective
retention of aromatic compounds.

Other Bonded Phases

Diol columns are slightly polar and are used
for normal-phase separations.
Diols are
useful
for
samples
containing
many
compounds with different polarities , and
which usually have strong retention on
unmodified silica.

Non-Silica Supports
The most common polymer support material
for reversed-phase separation is made of
divinylbenzene cross-linked polystyrene
The main advantage of porous polymers is that
they can be used in the pH range from 1 to 13.
(silica supports tend to dissolve at pH greater
than 9).
Because of the surface acidity of silica
supports, polymer supports can be a better
choice for separating basic compounds.

All C18 Columns Are

Not Created Equal
Differences in:
Particle Size
Pore Size
Surface Area
Carbon Load
End-Capping
Silica Type
*Bonding Density

RP Column Properties

Hydrophobic Surface
Particle Size and Shape
Particle Size Distribution
Porosity, Pore Size and Surface Area

Particle Size
Columns have a distribution of particle
sizes
Reported particle diameter is an average
Broader distribution ---> broader peaks

RP Mechanism (Simple)

Reversed Phase Mechanisms

Classical measures of retention
retention factors
partition coefficients
Vant Hoff Plots

Give bulk properties only - do not give

molecular view of separation process

Proposed RP Mechanisms
Hydrophobic Theory
Partition Theory
Adsorption Theory
See Journal of Chromatography, volume 656.

Hydrophobic Theory
Chromatography of cavities in solvent
created by hydrophobic portion of analyte
molecule
Surface Tension
Interaction of polar functions with solvent
Stationary phase is passive

Partition Theory
Analyte distributes between aqueous
mobile phase and organic stationary phase
Correlation between log P and retention
organic phase is attached on one end
Does not explain shape selectivity effects

Adsorption Theory
Analytes land on surface - do not
penetrate
Non-polar interactions between analyte
hydrophobic portion and bonded phase
Weak interactions
dipole-dipole
dipole-induced dipole
induced dipole-induced dipole

None of these can completely explain all of

the
observed retention in reversed phase HPLC

Important Reversed Phase

Parameters

Solvent (mobile phase ) Strength

Choice of Solvent
Mobile Phase pH
Silanol Activity

HPLC Solvents
Mobile Phase

LC Mobile Phase Qualities

High purity
Reasonable cost (and disposal)
Boiling point 20-50 C above column temperature
Low viscosity
Low reactivity
Immiscibile with stationary phase
Compatible with detector
Safety limited flammability and toxicity

SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane

HPLC Solvents Properties

HPLC Solvents Groups

LC Mobile Phase Selection

k of 2-5 for two or three component mixture
k of 0.5-20 for multicomponent mixture
Match analyte polarity to stationary phase polarity
Mobile phase of different polarity
Normal Phase:
nonpolar solvent, polar stationary phase
least polar component elutes first
increasing mobile phase polarity decreases elution time

Reversed Phase:
polar solvent (water, MeOH, ACN), nonpolar stationary phase
most polar component elutes first
increasing mobile phase polarity increases elution time
most widely used

LC Pumping Systems
General Requirements:
Generate pressures up to 6000 psi
Pulse-free output
Flow rates from 0.1-10 mL/min
0.5% or better flow control reproducibility
Corrosion resistant

LC Pumping Systems
Reciprocating Pumps

Pulsed flow must be damped

Small internal volume
High output pressures
Adaptable for gradient elution
Constant flow rates independent of column back-pressure or solvent viscosity

Displacement Pumps

Flow independent of viscosity and back-pressure

Limited solvent capacity
Inconvenient to change solvents

Pneumatic Pumps

Inexpensive
Pulse free
Limited capacity and pressure
Dependent on solvent viscosity and backpressure
Not good for gradient elution

HPLC Pump Head

Piston HPLC Pump Head

Gradient
HPLC

Low Pressure

High Pressure

HPLC Chromatograph injectors

The function of the injector is to place the sample into the
high-pressure flow in as narrow volume as possible so that
the sample enters the column as a homogeneous, lowvolume plug. To minimize spreading of the injected volume
during transport to the column, the shortest possible length
of tubing should be used from the injector to the column.
When an injection is started, an air actuator rotates the valve:
solvent goes directly to the column; and the injector needle
is connected to the syringe. The air pressure lifts the needle
and the vial is moved into position beneath the needle. Then,
the needle is lowered to the vial.

HPLC columns
The column is one of the most
important components of the
HPLC chromatograph because
the separation of the sample
components is achieved when
those components pass through
the
column.
The
high
performance
liquid
chromatography apparatus is
made out of stainless steel
tubes with a diameter of 3 to 5
mm and a length ranging from
10 to 30 cm.

Normally, columns are filled with

silica gel because its particle
shape, surface properties, and pore
structure help to get a good
separation. Silica is wetted by
nearly every potential mobile
phase, is inert to most compounds
and has a high surface activity
which can be modified easily with
water and other agents. Silica can
be used to separate a wide variety
of chemical compounds, and its
chromatographic
behavior
is
generally
predictable
and
reproducible.

Reverse Phase HPLC

Skoog and Leary: Principals of Instrumental Analysis,

5th ed. Suanders, 1998

Alltech Chromatography Sourcebook, 2004-04 catalog

Alltech Chromatography Sourcebook, 2004-04 catalog

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

(TLC vs Normal Phase and Reverse Phase HPLC)
Normal Phase (SiO2) TLC

Normal Phase (SiO2)

a
a

c
Time

b
Reverse Phase (C18)
c
c

x
0

b
Time

RP-HPLC Stationary Phase

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html

Characteristics of Performance
The performance criteria affecting quality of the result include:

Accuracy
Precision (repeatability and reproducibility)
Sensitivity (LOD and LOQ)
Selectivity
Linearity
Dynamic range
Stability

The performance criteria for the economics include:

Cost of purchase, installation and maintenance

Analysis time
Safety aspects
Running costs supplies, gases, consumables
Training
Sample throughput

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 properties of the solute
(not possessed by the mobil phase (e.g. UV
adsorption)
Hyphenated detector LC-MS

Common LC Detectors
Bulk property detectors
Refractive index detector
Conductivity detector
Light scattering detector.
Analyte property detectors
UV detector
Fluorescence detector
Amperometric detector
Mass spectrometric detector
Combination detectors

Refractive Index Detector

1. The RI detector is one of the few universal detector available in LC
2. Principle:
The RI detectors measure a bulk property of the mobile phase leaving
the column: its ability to refract to bend light (i.e., its refractive index). This
property changes as the composition of the mobile phase changes, such as
when solutes from the column. By detecting this change, the presence of
solutes can be detected.
3. Detector
Design:
i. One of simplest of RI detectors is the deflection RI detector

ii. In this detector, light is created by a source and passed through flow-cells
containing mobile phase eluting from the column (sample stream) and a
reference stream (usually mobile phase with no solute in it). The light
passing through these flow/cells is passed through a second time using a
mirror and passed to a detector where its intensity is measured.
iii. When the refractive index of liquid in the sample and reference flow-cell
are the same, little or no bending of light occurs at the interface between the
low-cells. This allows the largest amount of light possible to reach the
detector.
iv. As solute elute from the column, the refractive index of the liquid in the
sample flow-cell will be different that that in the reference flow-cell and light
will be bent as it passes between them. This changes the amount of light
reaching the detector, producing a response.

4. Applications:
RI detector are universal applicable to the detection of any solute in LC.
This makes them useful in preliminary work in LC where the nature or
properties of a compound may not be known yet. They also the detector of
choice for work with carbonhydrates or in the separation of polymer by sizeexclusion chromatography.
Some disadvantages: (1) they do not have very good limits of detection,
(2) they can not used with gradient elution, where the composition of the mobile
phase is changing with time. (3) The temperature of the system must also be
controlled to avoid baseline fluctuations with these detectors.
5. Sensitivity
The response of a RI detector is approximately the same for all
compounds.
6. Limit of Detection: 10-5 to 10-6 M
7. Linearity/ Dynamic Range: The response of a RI is usually linear over a 104fold range in concentration.

HPLC Bulk Property Detectors

Refractive Index Detector
Plus:
1) Measures a bulk property
2) Nearly Universal (different RI than mobile phase)
3) Comparable response for different analytes
4) Detects species with no chromophores
Minus:
1) Temperature dependent
2) Poor sensitivity (LOD 100 ng)
3) No gradient elution

Conductivity Detector
1. A conductivity detector is an example of a universal detector for ionic
compound.
2. Principle:
i. This detector measures the ability of a solution to conduct a current when
placed in an electrical field. This ability depends on the number of ions or
ionic compounds present in the solution.
ii. The relationship between the current, electric field and conductivity of the
solution is shown as follows:

I=CE
I = Current
C = conductivity
E = electric field strength

3. Detector Design

4. Applications: for any compound that is ionic or weakly ionic. It is

widely used in ion chromatography.
5. Sensitivity:
The response of a conductivity detector depends on the charge and size
of the compound of interest. Small, highly charged compounds tend to
produce larger response that large, less charged compound.
6. Limit of Detection: 10-6 M
7. Linearity/Dynamic range: 104-fold

HPLC Bulk Property Detectors

Conductivity Detector
Plus:
1) Measures a bulk property
2) Nearly Universal (must be ionic)
3) Common for Ion Exchange LC
4) Detects species with no chromophores
5) Simple, robust
Minus:
1) Fair sensitivity (LOD 1 ng)
2) No salts or buffers in mobile phase
3) Gradients a challenge

Evaporative Light Scattering Detector

HPLC Bulk Property Detectors

Evaporative Light Scattering Detector
Plus:
1) Measures a bulk property
2) Nearly Universal (must be non-volatile)
3) Does not detect liquids (gradients are ok)
4) Detects species with no chromophores
Minus:
1) Signal not linear with concentration
2) Fair sensitivity (LOD 1 ng)
3) No salts or buffers in mobile phase

Absorbance Detector (UV/Vis)

1. The absorbance detector is the most common type of detector in LC.
2. Principle:
Absorbance detector measures the ability of solutes to absorb light
at a particular wavelength range. This absorbance is described by
the Beer-Lambert Law.

A = l c
where: A = Absorbance of light at a given wavelength
Molar absorption coefficient of the solute
l = path length of the flow-cell
c = concentration of solute

3. Detector design:
i. There are three types of UV-Vis absorbance detector: fixed wavelength
detectors, variable and diode array detector. They are generally based on the
following type of design:

ii. In a fixed wavelength detector, absorbance of only one given wavelength

is monitored by the system at all time. The wavelength is usually 254 nm.
A fixed wavelength detector is the simplest and cheapest of types of
detector, but is limited in terms of it flexibility and the types of compounds
it can used to monitor.

iii. In a variable wavelength detector, a single wavelength is monitored

at any given time, but any wavelength in a wide spectral range can be selected.
The wavelengths that can be monitored can vary from 190 nm to 900
nm. The ability to use one instrument for more than one wavelength is
achieved by adding in more advanced optics to the system.

Diode Array
Detector

HPLC UV/VIS Spectrophotometer

4. Applications:
Absorbance detector can be used to detect any compound absorbing at the
wavelength monitored. Absorbance detector can be sued with gradient elution.

5. Sensitivity:
The response of an absorbance detector depends on the molar absorption
coefficient. The larger this value is, the larger the response of the detector
6. Limit of detector: 10-8 M

7. Linearity/Dynamic range: 105-fold range

HPLC Specific Property Detectors

Multi-wavelength UV-Vis Absorption Detector
Plus:
1) Measures a specific property
2) Nearly Universal (must have chromophore)
3) Most common of all detectors (~75%)
4) Potential to provide qualitative info.
5) Simple, robust
Minus:
1) Fair sensitivity (LOD 1 ng)
2) Expensive with PDA, limited with Hg Lamp
3) Misses some important analytes

Fluorescence Detector
1.

A fluorescence detector is an example of a selective detector, with limits

of detection smaller than those by either RI or absorbance monitors.

2. Principle:
F = I (1-e- l c) = I l c (at low concentration)
F = Fluorescence intensity
I = intensity of the excitation light
= Fluorescence quantum yield
Molar absorption coefficient of the
solute
l = path length of the flow-cell
c = concentration of solute

3. Detector design

4. Applications:
It can be used to detect any compound absorbing and emitting light
at the given excitation and emission wavelength.

5. Sensitivity:

F = I (1- e- l c) = I l c (at low concentration)

6. Limit of detection: 10-10 M
7. Linearity/Dynamic Range: 103 to 104-fold

HPLC Specific Property Detectors

Fluorescence Detector
Plus:
1) Measures a specific property
2) Highly Selective (must fluoresce)
3) Second most common of all detectors
(~15%)
4) High sensitivity (LOD 0.01 ng)
5) Can interrogate very small volumes
Minus:
1) Not Universal
2) Limited Applications

Electrochemical detector
1. It can be used to detect an compound which can undergo an
electrochemical reaction
2. Principle:
i. This detector measure the ability of a solute to undergo either oxidation
(i.e., loss of electrons) or reduction (i.e. gain of electrons)
Oxidation: A A+ + eReduction: A + e- Aii. One way in which such a reaction can be monitored is by measuring the
change in current under a constant electric field. Another way is to
measure the change in the electric field produced when a constant current
is present.
3. Detector Design

4. Applications:
Electrochemical detectors can be used to detect any solute that can undergo
oxidation or reduction.
Detection by reduction: aldehydes, ketones, nitriles, conjugated acids
Detection by oxidation: phenols, peroxides, purines, diols
5. Sensitivity: It depends on the extent of oxidation or reduction that occurs at
given potential of the electrode.

6. Limit of detection: 10-11 M

7. Linearity/Dynamic Range: 106-fold
8. Disadvantages: destructive detector

HPLC Specific Property Detectors

Electrochemical Detector
Plus:
1) Measures a specific property
2) Highly Selective (depends on reduction potential)
3) High sensitivity (LOD 0.01 ng)
Minus:
1) Not Universal
2) Must have electrolyte in mobile phase
3) Mobile phase must be aqueous
4) Gradients not possible

Mass spectrometry
Mass spectrometry probably is the most versatile and comprehensive
analytical technique currently used by chemists and biochemists.

It measures the masses of individual molecules, fragments of molecules and

atoms.

It provides ultrahigh detection sensitivity requiring only a few picomoles of

a compound to obtain characteristic information regarding the structure
and the molecular weight of a compound.

In all cases, energy is transferred to the compound molecules to effect

ionization, causing the formation of the molecular ion of the compound.

The molecular ion fragments into a variety of fragment ions and the
resulting fragmentation pattern constitutes the mass spectrum.

The mass spectrum of each compound is unique and can be used as

General concepts of mass spectrometry

The first essential step in mass spectrometry analysis to convert the

analyte molecules by ionization into gas phase ionic species.

The excess energy transferred to the molecules leads fragmentation.

A mass analyzer separates the molecular ion and fragment ions according
to their mass/charge (m/z) ratio.

Data are recorded and then converted into a mass spectrum.

These steps are carried out under high vacuum [10

10-6 Pascal (Pa)].

(1 Pa = 0.0075 torr; 1 atm = 1.013 x 105 Pa)
-2

Basic components of a mass spectrometer

GC or LC

Ionization methods
1. Electron-impact ionization (GC/MS)
2. Chemical ionization (GC/MS)
3. Atmospheric pressure electrospray ionization (LC/MS)

Skoog and Leary: Principals of Instrumental Analysis, 4 th ed. Suanders, 1992

HPLC - Column Efficiency

van Deemter Equation

H = A + B/u +Cu

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

HPLC - Column Efficiency

H = A + B/u + Cu
A = 2dp
1.
2.
3.
4.

depends on particle size distribution, the

narrower the distribution the smaller the l
dp = particle size
Independent of mobile phase flow rate
Also known as eddy diffusion

Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

HPLC - Column Efficiency

particle size

Skoog and Leary: Principals of Instrumental Analysis, 5 th ed. Suanders, 1998

HPLC Column Efficiency

Longitudinal Diffusion (B)

H = A + B/u + Cu
B/u = 2DM/u

= constant depending
on quality of packing

2.

DM is the mobile phase

diffusion coefficient

3.

Inversely related to mobile

phase flow rate

HPLC Column Efficiency

Mass Transfer (Cs + Cm)

H = A + B/u + (Cs + Cm)u

CS = fS(k)df2 / DS
CM = fM(k)dp2 / DM

DM is the mobile phase diffusion

coefficient
DS is the stationary phase
diffusion coefficient
df is film thickness
dp is particle size
Directly related to mobile phase
flow rate
Skoog and Leary: Principals of Instrumental Analysis,
5th ed. Suanders, 1998

Flow Chart I: Small Molecules

1. m. wt.
2. solubility

Flow Chart II: Large Molecules

1. m. wt.
2. solubility

Uses of HPLC
This technique is used for chemistry and biochemistry research
analyzing complex mixtures, purifying chemical compounds,
developing processes for synthesizing chemical compounds,
isolating natural products, or predicting physical properties. It
is also used in quality control to ensure the purity of raw
materials, to control and improve process yields, to quantify
assays of final products, or to evaluate product stability and
monitor degradation.
In addition, it is used for analyzing air and water pollutants,
for monitoring materials that may jeopardize occupational
safety or health, and for monitoring pesticide levels in the
environment. Federal and state regulatory agencies use HPLC
to survey food and drug products, for identifying confiscated
narcotics or to check for adherence to label claims.

Area of application:
Separation and purification of substances and
Analysis
Chemistry
Biomedical and Clinical
Pharmaceutics
Agriculture and Food
Enviromental
Veterinary

RP-HPLC - Example

Alltech Chromatography Sourcebook, 2004-04 catalog

RP-HPLC - Example

Alltech Chromatography Sourcebook, 2004-04 catalog

RP-HPLC Gradient Elution

Alltech Chromatography Sourcebook, 2004-04 catalog

Chromatogram of Organic Compounds from Fermented Cabbage

Chromatogram of Orange Juice Compounds

Liquid chromatograph/mass spectrometer

Liquid
Liquid
Chromatograph
Chromatograph

Computer
Computer

Mass
Mass
Spectrometer
Spectrometer
UV
UV
Detector
Detector

Solvents
Solvents

Ion
Ion source
source

Column
Column

Pumps
Pumps

Interface
Interface

Sampler
Sampler
Injection
Injection port
port
Rough
Rough pump
pump