High Performance Liquid

Chromatography

Chem. 331
High
Performance
L iquid
Chromatograph
y
High
Pressure
L iquid
Chromatograph
y
High
Priced
L iquid
Chromatograph
y
 Introduction
 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.
HPLC system
 FOUR TYPES OF LIQUID
CHROMATOGRAPHY
 Partition chromatography

 Adsorption, or liquid-solid

 chromatography

 Ion exchange chromatography

 Size exclusion, or gel, chromatography

 Partitioning

 Separation is based on the analyte’s relative

solubility between two liquid phases

Mobile Stationary
Phase Phase

Solvent Bonded Phase

 COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
 Solvent
 Solvent Delivery System (Pump)
 Injector
 Sample
 Column
 Detectors (Diode Array)
 Waste Collector
 Recorder (Data Collection)
Picture of HPLC instrument
 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.
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, low-volume 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 5mm and a length
ranging from 10 to 30cm.
 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.
Picture of an HPLC column
 WHAT AFFECTS SYSTEM
Column Parameters Instrument Parameters

 Column Material  Temperature

 Deactivation  Flow
 Stationary Phase  Signal
 Coating Material  Sample Sensitivity
 Detector
 WHAT AFFECTS SYSTEM
Sample Parameters

 Concentration
 Matrix
 Solvent Effect
 Sample Effect
 Several column types
(can be classified as )

 Normal phase

 Reverse phase

 Size exclusion

 Ion exchange
 HPLC - Modes
 Normal Phase.
- Polar stationary phase and non-
polar solvent.

• Reverse Phase.
- Non-polar stationary phase and a polar solvent.
 Common Reverse Phase
Solvents
CH3OH
 Methanol
• Acetonitrile CH3CN

• Tetrahydrofuran

• Water H2O
 Columns
 Solid Support - Backbone for bonded phases.
 Usually 10µ, 5µ or 3µ silica or polymeric particles.
 Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
 Extremely stable
 Reproducible
 Guard - Protects the analytical column:
 Particles
 Interferences
 Prolongs the life of the analytical column

• Analytical - Performs the separation.

 Bonded Phases

 C-2 Ethyl Silyl -Si-CH2-CH3

•C-8 Octyl Silyl -Si-(CH2)7-CH3

• C-18 Octadecyl Silyl -Si-(CH2)17-

CH3
•CN Cyanopropyl Silyl -Si-(CH2)3-
CN
 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
 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.
 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.
 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.
 Selectivity Factor

 K’ values tell us where bands elute relative

to the void volume. These values are
unaffected by such variables as flow rate
and column dimensions. The value tell us
where two peaks elute relative to each
other. This is referred to as the selectivity
factor or separation factor (now and then
as the chemistry factor).
 Types of Liquid Column
Chromatography
(LCC)
 LLC (Liquid Liquid)

 LSC (Liquid Solid -

adsorption)

 SEC (Size Exclusion)

 Types of Detectors
 Absorbance (UV  Evaporative Light
with Filters, UV with Scattering Detector
Monochromators) (ELSD)

 IR Absorbance  Electrochemical

 Fluorescence  Mass-
Spectrometric
 Refractive-Index
 Photo-Diode Array
UV detector The light source is a D2 lamp. This detector is used mainly to
detect components having an absorption wavelength of 400 nm or
less in the ultraviolet region.

UV-VIS detector A D2 lamp and a W lamp are used as the light source. This
detector is effective in the detection of coloring components such
as dyes and stains because of coverage of the visible light region.

Diode array detector Data on the spectrum from the ultraviolet to visible light range is
(DAD) also collected.

Fluorescence (FL) Fluorescent substances can be detected specifically with high

detector sensitivity.

Differential Change in the refractive index is detected. Components absorbing

refractive index (RI) no ultraviolet light can also be detected despite low sensitivity.
detector
Conductivity detector Mainly inorganic ions are detected by monitoring the
conductivity.
EVALUATION PARAMETERS
 EFFICIENCY
 RESOLUTION
 INERTNESS
 RETENTION INDEX
 COLUMN BLEED
 CAPACITY FACTOR
 Chromatograms

A B
Supelcosil LC-PAH
Columns.
Conditions: A: 150mm x 4.6mm, 5µ. Conditions: B: 50mm x 4.6mm,
Flow Rate: 1.5 mL/min 3µ.
Flow Rate: 3.0 mL/min
Quantitative analysis  
  The height and area of a peak are  
proportional to the concentration of the
corresponding component. A calibration
curve is created using the standard sample.
The concentration of aspartame in the
beverage can be determined from the peak
area of the detected aspartame.
   
   
Separation unit
 
A column is selected to suit both the sample
and the purpose of separation. The column
oven is used to maintain a constant column
temperature. If the column temperature
were allowed to vary during qualitative or
quantitative analysis, the elution time of the
components would change, so that an
accurate analysis could not be performed.
An analysis temperature between 25 and
50℃ is often selected
 References
 http://192.215.107.101/ebn/942/tech/techfocus/1071main.html
 http://www.chem.usu.edu/~sbialk/Classes/565/opamps/
opamps.html
 Skoog, Holler, and Neiman. Principles of Instrumental Analysis.
5th ed. Orlando: Harcourt Brace & Co., 1998.
 http://weather.nmsu.edu
 http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
 http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html
 http://weather.nmsu.edu/Teaching_Material/SOIL698/
Student_Material/HPLCHP1090/HPLCINJ.HTM
 http://test-equipment.globalspec.com/LearnMore/
Labware_Scientific_Instruments/Analytical_Instruments/
Chromatographs/HPLC_Columns
 http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-
col.htm