Introduction

Chromatography
High Performance Liquid
Chromatography (HPLC)
Assoc. Prof. Pham Van Hung
TLC CC HPLC GC

Content of this lecture Chromatography Theory

• Introduction
• Even though each method utilizes different
• Components of HPLC techniques to separate compounds, the
– Pump principles are the same.
– Injector
j
• Common to all:
– Column
– Detector
– Stationary phase
– Data system • a solid or a liquid supported on a solid
• Classification – Mobile phase
• A liquid or gas
• Qualitative and Quantitative analysis

1
 Chromatography Theory Introduction
¾ As the mobile phase passes through the
HPLC: improved form of column
stationary phase, it carries the components of
chromatography
the sample mixture with it.
¾ The components of the sample will be attracted ¾ Instead of the mobile phase moving through
to the stationary phase,
phase but there will also be a the column as a result of gravity, it is forced
competing attraction for the mobile phase. through the column under high pressure.
¾ Each component will have its own characteristic • Typical operating pressures: 500-6000psi
balance of attraction to the mobile/stationary
¾ A smaller sized packing material (<10µm) is
phase.
required to get improved separation.
¾ So the components will not move at the same
speed and are separated.

HPLC History Diagram of HPLC Apparatus

1966: Horváth • Mobile phase pumped
• Built the first HPLC instrument and gave it its name through column at high
pressure.
– HPLC = High Pressure Liquid Chromatography.
• Sample is injected into the
1970’s: HPLC became popular with an increase in system.
technology
gy
• S
Separation
ti occurs as the
th
• Improved columns and detectors mobile phase and sample
• Production of small silica packing material are pumped through the
– By 1972 particle sizes less than 10µm were introduced column. • The response of the
• This allowed for more precise and rapid separations. • Each component will elute detector to each component
from the column, one at a eluted will be displayed on a
• As new technology continued to develop, HPLC
time, and will be detected by chart or computer screen.
became more efficient.
one of several possible
– HPLC = High Performance Liquid Chromatography Known as a chromatogram.
detector types.

2
 Running HPLC Data analysis
¾ Each compound eluted will show up as a peak on the
A mixture is injected into a “steel-jacketed” chromatography chromatogram.
column and eluted with solvent at high pressure (4000psi or
approx 130 atm).
Inject sample A solid component can be dissolved in solvent but a solvent
peak will also be seen.

Elute with solvent UV detector

Retention Time- tR An HPLC system

Solvent reservoir

• The elapsed time between the time of analyte Solvent manager

injection and the time which the maximum peak Sample manager
height for that compound is detected. Column manager

• Different compounds will have different retention Detector

times.
times Read-out
Read out device

– Each compound will have its own characteristic

balance of attraction to the mobile/stationary phase.
» So they will not move at the same speed.
• Running conditions can also effect tR:
– Pressure used, nature of the stationary phase,
mobile phase composition, temperature of the
column

3
 Diagram of HPLC system Design & Operation of an HPLC Instrument

1) Mobile phase degassing:

• Dissolved gases in the mobile phase can come out of
solution and form bubbles as the pressure changes
from the column entrance to the exit.
– May block flow through the system
• Sparging is used to remove any dissolved gas from
the mobile phase.
– An inert and virtually insoluble gas, such as helium, is forced
into the mobile phase solution and drives out any dissolved
gas.
• Degassing may also be achieved by filtering the
mobile phase under a vacuum or using sonicator.

Design & Operation of an HPLC Design & Operation of an HPLC

Instrument Instrument
2) Solvent reservoirs: 3) Mobile phase mixing:
• Individual reservoirs store the mobile phase • Solvent proportioning valve can be programmed
components until to mix specific
they are mixed amounts of solvent
and used. 2.
from the various
• May also manually reservoirs to
prepare the mobile produce the
phase mixture and
desired mobile 3.
store in a single
reservoir. phase composition.

4
 Design & Operation of an HPLC Design & Operation of an HPLC
Instrument Instrument
3) Mobile phase mixing: 4) HPLC pump:
• Isocratic elution: • Fill stroke: mobile phase is pulled from the
– operate at a single, constant mobile phase composition solvent side
• Gradient elution: • Exhaust stroke:
– Vary the mobile phase composition with time the mobile phase 4.
– If there is a wide polarity range of components to be eluted. is pushed from the
– Allows for faster runs. injector to the
– Ex: mobile phase composition can be programmed to vary column head.
from 75% water: 25% acetonitrile at time zero to
25% water: 75% acetonitrile at the end of the run. – This is where the
• More polar components will tend to elute first.
high pressure is
• More non-polar components will elute later in the gradient. generated

Design & Operation of an HPLC Design & Operation of an HPLC

Instrument Instrument
5) Injector: 6) Column:
• Introduces the sample • Usually constructed of stainless steel
into the mobile phase – glass or Tygon may be
stream to be carried used for lower pressure
into the applications (<600 psi)
psi).
column. • Length: 5-100cm
• Syringe = impractical – 10 to 20cm common
for use in highly • Diameter:
pressurized systems.
– Typical: 2.1, 3.2, or
• Rotary injection 6.
5. 4.5mm
valve is used. – Up to 30mm for preparative applications

5
 Design & Operation of an HPLC Design & Operation of an HPLC
Instrument Instrument
• Column packing: 6) Column:
– Usually spherical silica particles of
uniform diameter (2-10µm) • Guard column: Protects the analytical column
• The smaller p
particles yyield higher
g – Particles
separation efficiencies. http://hplc.chem.shu.edu/NEW/HP

– The silica particles are very porous

LC_Book/Adsorbents/ads_part.html
– Interferences
• Allows for greater surface area for – Prolongs the life of the analytical column
interactions between the stationary
phase and the analytes. • Analytical column: Performs the separation
– Other packing materials may also be
used:
• Zirconia (ZrO2)
http://www.lcresources.com/resources/getstart/3a01.htm

Design & Operation of an HPLC Detection in HPLC

Instrument
*There are six major HPLC detectors:
7) Detector:
• Refractive Index (RI) Detector
• The component that
emits a response due • Evaporative Light Scattering Detector (ELSD)
to the eluting sample • UV/VIS Absorption Detectors
compound and • The Fluorescence Detector
subsequently signals
• Electrochemical Detectors (ECDs)
a peak on the
chromatogram. • Conductivity Detector
• A wide variety of 7. * The type of detector
detectors exist.
utilized depends on the
• Must have high sensitivity- characteristics of of
small sample sizes are used the analyte interest.
with most HPLC columns
http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4

6
 Advantages of HPLC Application in Food System
• Speed – many analyses can be Carbohydrates
accomplished in 30 min or less. Amino acids, proteins
• A wide variety of stationary phases Vitamins, A, D, E, K
Nucleosides (purines and pyrimidines)
• Improved resolution
Fatty acids, fats
• Greater sensitivity – various detectors Aflatoxins
can be employed. Antioxidants
• Easy sample recovery – less eluent Contaminants of packaging materials
volumn to remove. Carotenoids, chlorophylls
Saccharines

Classifications of HPLC
Normal phase HPLC
• There are numerous types of HPLC which • The stationary phase is a polar adsorbant.
vary in their separation chemistry. – Silica attached with polar nonionic functional
– All chromatographic modes are possible: groups: hydroxyl, nitro, cyano, or amino.
• Ion-exchange
Ion exchange
• Size exclusion • The mobile phase is a nonpolar solvent.
• Also can vary the stationary & mobile – Hexane added with a more polar modifier such
phases: as methylene chloride to control solvent
strength and selectivity.
– Normal phase HPLC
– Reverse phase HPLC

7
 Normal phase HPLC
• Analysis
– Fat-soluble vitamins
– Biologically active polyphenols from natural
plant sources such as grape and cocoa
cocoa.
– Polar vitamins: A, D, E and K.
– Natural carotenoid pigments
– Highly hydrophilic components such as
carbohydrates.
Reversed phase HPLC
• Analysis
– Plant proteins
– Cereal proteins
– Water- and fat-soluble vitamins
– Antioxidants: bytylated hydroxyanisole (BHA)
and butylated hydroxytoluene (BHT).
– Phenolic flavor compounds: vanillin
– Pigments: chlorophylls, carotenoids,
anthocyanins
Reversed phase HPLC
• The stationary phase is non-polar adsorbant
– Octadecylsilyl (ODS) bonded silica matrix: An
octadecyl (C18) chain; Octyl (C8) or Butyl (C4).
– Phenyl groups
• The mobile phase is polar solvent.
– Water mixed with methanol, acetonitrile or
tetrahydrofuran.
Ion exchange HPLC
• The stationary phase is functional organic
resins.
– Macroporous resins are most effective due to
their rigidity and permament pore structure.
– Pellicular packings also are used.
• The mobile phase is an aqueous buffer.
• Gradient elution is frequently employed.
8
 Ion exchange HPLC Size exclusion HPLC
• Analysis • Size-exclusion chromatograpy (SEC)
– Simple inorganic ions fractionates solutes solely on the basis of
– Carbohydrates size, with larger molecules eluting first.
– Amino acids • Prepacked columns of microparticulate
– Preparative purification of protein media are available in a wide range of
oligosaccharides pore sizes
• Aqueous buffers are used for biopolymers
such as proteins and polysaccharides

Size exclusion HPLC Sample analysis

• Determination of average molecular • In most cases,
sample peaks
weight and molecular weight range of on the
polysacchrides: amylose, amylopectin, chromatogram
other soluble gums and water-soluble can be used to
estimate the
cellulose
ll l d
derivatives.
i ti amount of a
compound
present.
• The more
concentrated,
the stronger
the signal, the
larger the
peak. http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4

9
 Selectivity Resolution Equation
™ Ratio of Net Retention Time of 2 components (Distribution
Coefficient). Resolution is the
™ Selectivity represents the separation power of particular V2 - V1
parameter R=
adsorbent to the mixture of this particular components. 1/2(W1 + W2)
describing the
R esp on se
separation power
X 2
of the complete
Response

α=
X2 - X0
chromatographic
system relative to V2

X1 - X0 X1
the particular
X0 components of the V1

mixture.
1 3 6 W W2
1
R e te n t io n T im e W1 W2

Column selectivity refers to the distance, or relative Volumes

separation, between two peaks

Resolution General Factors Increasing Resolution

• Increase column length
• Decrease column diameter
• Decrease flow-rate
• Pack column uniformly
• U uniform
Use if stationary
t ti phase
h (packing
( ki material)
t i l)
• Decrease sample size
• Select proper stationary phase
• Select proper mobile phase
• Use proper pressure
• Use gradient elution

10
 What is Ultra Performance Liquid
Chromatography?
• 2004: Further advances in column technology
and chromatography instrumentation
– Utilized even smaller packing particle sizes
(1 7µm)
(1.7µm)
– Higher pressures (15000psi)
Th end!
The d!
– Allowed for significant increases in LC speed,
reproducibility, and sensitivity.
• New research utilizing particle sizes as small
as 1µm and pressures up to 100,000psi!

WHO KNOWS WHAT THE FUTURE MAY BRING!

11