ANNOUCEMENT

QUIZ 2 - ONLINE
• Week 14
• Date: 18/10/18 – 21/10/18
• Time: 2.00 PM – 2.00 AM
• Chapter: Chap 4 – 5

TEST 2 - WRITTEN
• WEEK 16
• Date: 2/11/18
• Time: 10.45 AM – 11.45 AM
• Chapter: Chap 4 - 6
 U N I V E R S I T I K U A L A L U M P U R

CHAPTER 6.1

CHROMATOGRAPHY
 SUBTOPIC

6. 1 High Performance Liquid Chromatography

6.1.1 High Performance Liquid Chromatography Introduction

6.1.2 Separation modes in HPLC
6.1.3 Developing a separation
6.1.4 Sample preparation and data evaluation
 HISTORY OF HPLC
• Early LC carried out in glass columns
- diameters: 1-5 cm
- Lengths: 50-500 cm Flow rates still low,
separation times long
• Size of solid stationary phase
- Diameters: 150-200 µm

Improved Technology
• Decrease particle size of packing causes increase in column
efficiency
-Diameters 3-10 µm
• This technology required sophisticated instruments.
-new method called HPLC
 ADVANTAGES OF HPLC

• Speed - 30 min or less

• Improved resolution - can adjust a variety of
parameters, including types of stationary phase
• Greater sensitivity - various detectors
• Reusable column - many analyses
• Ideal for ionic species and large molecules
(substances with low volatility
• Easy Sample recovery - fraction collectors
 HPLC Introduction
• High performance liquid chromatography can be applied to the
analysis of any compound with solubility in a liquid that can be
used as the mobile phase.

• Separate any compound with solubility in a liquid/ solvent that

can be used as a mobile phase.

• Analytical or preparative (highly purified compound in small or

large quantities).

• Applications include analysis of sugars, pesticide residues,

organic acids, lipids, amino acids, toxins, and vitamins.
 Basic Flow diagram of HPLC

Eluent

Column Detection cell F-

Cl-

Sample injection valve NO2-

NO3- SO42-
Br-
HPO42-
Pump

Detector

Pumping Injection Separation Detection Recording

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 HPLC Instrumentation Overview
Principle Pattern An Example

Solvent Reservoirs
Controller

Solvent Cabinet Vacuum Degasser

Binary Pump

Autosampler

Thermostatted
Column Compartment

Detector

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Composition of HPLC System
• Solvent
• Solvent Delivery System (Pump)
• Injector
• Sample
• Column
• Detectors
• Waste Collector
• Recorder/integrator/Data System
 Solvent Filters

Guard
column
Injector

Precolumn Analytical
Filter Column
Solvent Inlet Filter

Solvent Inlet Filer Precolumn Filter

• Stainless Steel or • Used between the injector and
glass with 10 micron guard column.
porosity. • 2 to 0.5 micron
• Removes particulates • Removes particulates from sample
from solvent. and autosampler wear debris.
• Must be well designed to prevent
10 dispersion.
 HPLC Pump
• function is to deliver mobile phase through the system » 1 ml/min - needs to be
controlled, accurate and precise
• 2 main type of pump: Constant volume and Constant pressure
• System and connecting lines are made from stainless steel (can handle
pressure and is resistant to corrosion by most mobile phases except mineral
acids and halide ions
• Pumps are sensitive to particles in the mobile phase and air - filter and degas
mobile phase
 Commercial HPLC pumping system and connecting lines are made of grade
AN51316 stainless steel.
 withstand the pressure generated and is resistant to corrosion by oxidizing
agents, acids, bases and organic solvents.
 Mobile phase filter using 0.45 or 0.22 µm porosity porous.
 Degassing HPLC eluent using vacuum/ultrasonication/sparging with helium.
• to prevent the problems that can be caused by air bubbles in a pump or
detector.
Functions of the Solvent Delivery System
The solvent delivery system has three basic functions:

1. Provide accurate and constant flow.

2. Provide accurate mobile phase compositions.

3. Provide the force necessary to push the mobile phase

through the tightly packed column.
 Injector
• To place the sample into the flowing mobile
phase.
• Valve injector
• trouble free, good precision and can change
loop for different volumes
• Place in load position and load sample into
an external fixed volume loop at
atmospheric pressure
• Rotate to inject position - loop becomes
part of the eluent flow stream and the
sample is carried to the column
• Auto samplers
• fully automatic injection systems enabling
greater productivity and the highest level of
precision.
• Small vials with a septum placed in a tray
• Needle penetrates septum and withdraws
the sample
Auto Samplers
 Sample Injectors

Requirements:
Reproducible introduction of the sample volume into
the mobile phase flow.

Two major designs:

•Automatic Injectors or
•Manual Injectors

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 Manual Injectors
Sample Loop

Load - Inject

Front View Rear View

Inject

15
16
 Manual Injectors
Sample Load
From Pump
Solvent in
Solvent out Sample in
To column

From Pump
Solvent in
Solvent out Sample in
To column

Sample Inject 17
Automatic Injectors

Step 1 Step 2

Step 3

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Column
 Columns
• Actual tool to separate molecules
• External hardware - stainless steel tube
with terminators for connection
• Precolumns - auxillary columns that
precede the analytical columns
• Presaturates -contains large particles
of bare silica and presaturate the
mobile phase with silica (slow down
dissolution of silica based packing
materials during the use of an
aqueous mobile phase)
• Guard columns - protect analytical
column (same diameter, small total
volume, and minimal dead space

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 ANALYTICAL COLUMNS CHARACTERISTICS:

– Made of Stainless steel tubing for high pressure

– Heavy-wall glass or PEEK tubing for low P (< 600 psi)

– Analytical column: straight, Length (5 ~ 25 cm), diameter column (3 ~ 5 mm),

diameter particle (35 μm). N (40 k ~ 70 k plates/m)

-Micro column: L (3 ~ 7.5 cm), d (1 ~ 5 mm), dp: 3 ~ 5 μm, N: ~100k plates/m,

high speed and minimum solvent consumption

– Guard column: remove particulate matter and contamination protect analytical

column, similar packing

– T control: < 150 °C, 0.1 °C

-Column packing: silica, alumina, a polystyrene-di vinyl benzene

synthetic or an ion-exchange resin
– Pellicular particle: original, Spherical, nonporous beads, proteins and large
biomolecules separation (dp: 5 μm)
– Porous particle: common used, dp: 3 ~ 10 μm. Narrow size distribution,
porous micro particle coated with thin organic films
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22
 Column and Solvent Filters

Guard
column
Injector

Precolumn Analytical
Filter Column
Solvent Inlet Filter

Solvent Inlet Filer Precolumn Filter

• Stainless Steel or • Used between the injector and
glass with 10 micron guard column.
porosity. • 2 to 0.5 micron
• Removes particulates • Removes particulates from sample
from solvent. and autosampler wear debris.
• Must be well designed to prevent
dispersion.
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 Packing material
• Silica based & polymeric
• General requirements
• Available in well defined particle sizes with narrow
distribution - small particles decrease the solute
distance between stationary and mobile phases -
facilitates equilibration and increases efficiency, but
more flow resistance and higher pressure
• Can withstand pressure
• Chemical stability

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 Silica based column packing

• Porous silica - half of volume consists of pores

• Bonded phases - silicon dioxide tetrahedron with
silanol groups on surface - (disadvantage is that silica
skelton slowly dissolves in aqueous solution
especially at pH >8 – remember use of presaturates
column)
• Pellicular packing - thin coating (polyamine is
physically absorbed to surfaces and then cross-linked
into a permanent polymer layer) on a
microparticulate core (core can be porous or
nonporous).
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 Polymeric column packing

• synthetic organic resins - have better chemical

stability and the potential to change properties by
direct chemical modification
• Microporous resins
• Macroporous resins

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 Types of Packing Materials Utilized in HPLC

A) Bonded-phase silica B) Pellicular packing

C) Microporous polymeric resin; C) macroporous polymeric resin

Detectors
• UV/VIS absorption detectors
• Refractive index detector
• Fluorescence detectors
• Electrochemical detectors
• Evaporative light scattering detectors
• Viscosity detectors
• Radioactive detectors
• Transport detectors
• Chemiluminescent nitrogen detectors

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UV cell

29
UV-Detectors
• Photodiode • Diode array

30
HPLC Detectors
Characteristics of Absorbance
Detectors:
• Eluent Measurement Cell
• Minimum Volume
• Reduces Extra Column
Broadening
• Volume : 1 to 10 mL
• Path Length (b): 2 to 10 mm
• Pressure Limited
• Maximum Typically 600 psi
• Usually Requires Pressure
Reducer

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32
33
Light path in a micro flow cell of a spectrophotometric detector.
Cells that have a 0.5-cm pathlength and contain only 8μL of liquid
are common.
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 Refractive Index
Refractive index detectors:

• Nearly universal but poor detection limit

• Passes visible light through 2 compartments,
sample & reference.
• When the solvent composition are the same
the light passed through the compartments the
light beam that passes through is recorded as
zero.
• When a solute is in the sample compartment,
refractive index changes will shift the light
beam from the detector.
• Limit of detection (LOD) 10 ng of solute
•The Refractive Index Detection is strongly
influenced by:
Pressure changes
Temperature changes
Flow pulse
Gradient elution is not possible!
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SEPARATION MODES
Separation Modes
• Based on physiochemical principles - adsorption,
partition, ion exchange, size exclusion, and affinity
• Two types of separation modes popular in HPLC
application:
• Normal phase
• stationary phase in polar,
• mobile phase in non polar
• Reverse phase
• stationary phase non polar and
• mobile phase is polar (more than 70% of all HPLC)

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Types of Separation Modes
 Normal phase
• Stationary phase
• Chemically attached polar nonionic functional group
(alcoholic hydroxy, nitro, nitrile, or amino)
• The terminal polar group is linked to the silica through a
short hydrocarbon spacer
• Mobile phase
• Nonpolar solvent (like hexane) with a more polar
modifier (like methylene chloride)
• Solvent strength and selectivity is controlled which
influences solute retention (weak solvents increase
retention and strong solvents decrease retention)

39
 Normal phase Application
• Best for compounds high soluble in organic solvent
(fat soluble vitamins) or that have low solubility in
an aqueous mobile phase
• Also can be used for compound classes, isomers,
and highly hydrophilic species like carbohydrates

40
 Reverse phase
• Stationary phase
• Silica
• Chemically bound to silica via surface silanol
• R3 group is usually an octadecyl (C18)
• Bonded phase (non polar ocatdecylsilyl) can be monomeric or
polymeric depending on interaction with silica surface
• Polymeric
• Highly cross-linked polystyrene-divinylbenzene
• Mobile phase
• Usually water mixed with methanol, acetonitrile, or a hydrofuran
• Solute retained due to hydrophobic interactions with the non polar
stationary phase

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 Reverse phase
• Solute eluted in order of increasing hydrophobicity (least
hydrophobic first, most hydrophobic last)
• Increasing polarity of the mobile phase increases solute
retention
• Increasing organic solvent content decreases solute retention
• Additives: an amine to deactivate silanols, aqueous buffers to
suppress ionization of sample components, ion-pair reagents
to neutralize charged solutes and make them more lipophilic

42
 Reverse phase Application
• Proteins - including cereal proteins
• Water soluble and fat soluble vitamins
• Carbohydrates using ion-pairing reagents
• Antioxidants, dyes, pigments, and phenolic flavor
compounds

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DEVELOPING
A SEPARATION
 Evaluating the sample and Define Goal of the
separation

• How many components need to be resolved?

• What degree of resolution is needed?
• Is qualitative and quantitative information needed?
• Molecular weight
• Polarity
• Sample Ionic character
 Choose a separation mode of sample

• Must select an appropriate column

• Elution conditions
• Detection method
• Trial experimental conditions may be based on
- literature search
-analyst’s previous experience
-recommendations from expert
 Resolution Optimization

• Manipulation of mobiles phase variables (nature and percentage

of organic components)
• pH
• Ionic strength
• Nature and concentration of additive
• temperature
SAMPLE PREPARATION
&
DATA EVALUATION
 Sample Preparation and Data
Evaluation
• General purpose is to remove interfering material prior to
chromatography
• Considerations - extraction efficiency, analyte stability, and
consistency of chemical and enzymatic pretreatment
• Peak identification - equivalent retention time does not
prove identity
• Identification techniques - spike, diode array detector,
rationing procedure, collect fraction and further analyze
• Quantitative validity - analyte recovery (addition of known
quantity before extraction), check sample (has known
quantitiy)

49
 Limit of Detection (LOD)
The limit of detection for a detector can be
characterized by its signal to noise ratio (S/N)
for an analyte under a given set of conditions.

Peak

Noise

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 Limit of Detection - Limit of Quantitation
Response

Linear range

Slope = sensitivity

MQL e.g.,RSD<10%, S/N > 20

MDL e.g., S/N > 3

Intercept

Amount
• Limit of detection (LOD) is a result of the whole chromatography system,
not only the detector performance
• Limit of quantification (LOQ) is a defined limit for a method used for a
specific purpose.
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 UV-Vis Detectors
Principles: The fraction of light transmitted through the detector
cell is related to the solute concentration according to Beer’s Law.

Detector Flow Cell

I0 c I

Log I0 = A = abc
I

Characteristics: Specific, Concentration Sensitive, good stability,

gradient capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).

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CHROMATOGRAPHIC TERMS
 CONCLUSION
HPLC is widely used for the analysis of

• small molecules and ions (eg: sugars, vitamins, and

amino acids)
• separation and purification of macromolecules, such
as proteins, nucleic acids, and polysaccharides.