In the name of God

High Performance Liquid

Chromatography
(HPLC)

Presented by:
M.T.Naseri
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Get Some Good Books for
Reference
 Introduction to Modern Liquid Chromatography, 3rd Edition,
Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan,
John Wiley and Son.
 Practical High-Performance Liquid Chromatography Fifth
Edition . Veronika R. Meyer 2010 John Wiley and Sons.
 HPLC FOR PHARMACEUTICAL SCIENTISTS , YURI
KAZAKEVICH WILEY-INTERSCIENCE 2007.
 Get a Good Online Training System
LCGC’s http://www.chromatographyonline.com/

2
 Classification based on physical states of phases
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 Liquid chromatographic techniques
4

 Paper chromatography (PC, Whatman no. 1 filter

paper)
 Thin-layer chromatography (TLC, Silica gel 60 )
 Column chromatography

Photographs taken by scanning

electron microscopy showing-
HPTLC LiChrospher® Si 60
 Thin-layer chromatography (TLC)
5

Retardation factor
 The principle of chromatographic separation
6

Adsorption Chromatography Technique

Russian, Mikhail Tswett
1901, botanist plant scientist . petrol ether/ethanol mixtures
Plant leaves in solvents such as solid calcium
carbonate
ether and ethanol

Chlorophyll and
carotenoid pigments
 What is HPLC used for?
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Applications: Large Molecule Separations (MW > 2,000)
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 Simple Enzymatic Digests (< 12 proteins)

 Nucleosil
 Complex Enzymatic Digests (≥ 12 proteins)
 Biomolecules (5‐10,000 MW)
 Biomolecules (>10,000 MW)
 Antibodies
 Oligonucleotides Nucleosil
 Oligosaccharides
 Hormones

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 Outline
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 Introduction
 Instrumentation
 Solvent Reservoirs
 Online degassing system
 Pump
 Injector
 Column
 Detectors
 Operating conditions
 Analysis methodology
 Quantization methods
 Application
Separation Mechanisms in
HPLC
 Chromatographic Modes:

 Normal-phase (NPC) or adsorption chromatography

NPC is used mainly for water-insoluble samples, preparative HPLC, and the
separation of isomers (achiral isomers).
 ‘‘Aqueous normal-phase chromatography.’’ Hydrophilic Interaction Liquid
Chromatography (HILIC): separate small polar compounds on polar stationary
phases

 Reversed-phase (RPC),
 Ion-exchange (IEC),
 Size-exclusion (SEC)

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Stationary Phase Separation Mechanism
Solid Adsorption
Liquid Layer Partition (hydrophobicity)
Ion exchange resin Ion exchange
Microporous beads Size Exclusion
Chemically modified resin Affinity

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Stationary Phase
Silica Type - Fully Hydroxylated and
Metal Free Silica Reduces Acidity
99.995% purity

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1) Surface Adsorption & Adsorption Chromatography

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 Normal phase chromatography
•
• Column packing is polar: silica (strongest)>amino>diol>cyano (weakest)
• Mobile phase is non-polar: hexane, iso-octane, methylene chloride, ethyl
acetate, etc.
• Polar compounds are more retained
• Retention decreases as polarity of mobile phase increases
Choose normal phase for:
• Resolution of strongly-retained hydrophobic samples
• Isomer separations
• Sample injection solvents that are non-polar and/or not water miscible.
• Recovery in non-polar solvents

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4.4 µm

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Partition Phase

Normal phase Column packing is polar:

silica (strongest)>amino>diol>cyano (weakest)

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 The Partition Coefficient (log P) and
Reversed-Phase Method Development Phase Selection

The resulting log P value indicates the

extent to which the measured compound
is hydrophobic or hydrophilic. A log P > 0
indicates a more hydrophobic analyte, while a
log P < 0 signifies a more hydrophilic analyte.

(THC is ∼ 10000000 times more non-polar than urea)

9-Tetrahydrocannabinol (THC)
9-Tetrahydrocannabinol (THC) is the principal
psychoactive constituent of the cannabis products marijuana
and hashish.
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 The Distribution Coefficient (log D)
and Acid Dissociation Constant (pKa)

Log D is always expressed as a function of pH

hydrophobic or hydrophilic?
Atenolol ( beta-blocker):
pKa of 9.63

> 99%

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Ion Exchange and Ion Exchange Chromatography

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 Size-Exclusion chromatography (SEC),
Gel filtration chromatography (GFC). Gel permeation chromatography (GPC),

cross-linked polystyrene stationary, cross-linked polydextran gels

four different
polystyrenes
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 Size Exclusion Chromatography (SEC)
Gel permeation chromatography (GPC) Gel-filtration chromatography (GFC):

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Applications of liquid
Chromatography Methods :
•Solubility
• Molecular mass

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 HPLC solvent delivery systems

 A modern HPLC solvent delivery system consists of:

1) Solvent reservoirs
2) Degassing system
3) One or more pumps

Polypropylene
polyethylene
fluorocarbon tubing
(e.g., Teflon®) :
transport of solvents
to the pump

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• Ideally, solvents used as HPLC mobile phases should have these
characteristics:

 High solubility for the sample components

 Noncorrosive to HPLC system components
 High purity
 Detector compatibility
 Low viscosity
 Solvent miscibility
 No particle
 No organic contaminate
 No ions
 Price

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Common solvent in RPLC

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 E0(Al2O3)=1.3 E0 (SiO2)]

Column backpressure is directly proportional to solvent

viscosity

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 Viscosity mix of organic solvent /water

Methanol
Acetonitrile

Low-viscosity mobile phases generally provide better efficiency than less-viscous

ones because diffusion coefficients are inversely related to solvent viscosity.
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 High purity

0.22 µm filtration

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 RESERVOIRS AND SOLVENT FILTRATION

Store mobile phases in borosilicate

glass reservoirs.
Borosilicate glass should be type 1,
class A or type 3.33.

Commercial HPLC-grade solvents are Frit porosity of ≥10 μm

filtered through submicron filters
(generally 0.22 μm) prior to packaging.

NOTE: If using bottled water, pay attention to the expiration date suggested by the
manufacturer, and discard the water after that date.
CAUTION: DO NOT USE PARAFILM® OR OTHER PLASTIC FILMS TO COVER
SOLVENT RESERVOIRS.
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Solvent Filters with vacuum-filter apparatus
 No ions

Siemens per centimeter (S/cm)

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 total failure of pump
Unstable flow
High system pressure
cell window dirty:
Higher noise levels

Phosphate buffer PH-4 to 7

>90-95% AQUEOUS MOBILE PHASE

How to prevent and reduce the algae problem

1-Always use freshly prepared solvent especially use demineralized water which was
filtered through about 0.2 µm filters
2-Never leave mobile phase in the instrument for several days without flow
3-Always discard ”old” mobile phase
4-Use amber solvent bottle for your aqueous mobile phase
5-Use aluminium foil to cover the reservoirs
6-If possible add a few mg/l sodium azide (0.0001-0.001 M) or a few percent organic
solvent (5-10%) to the aqueous mobile phase
7-CAUTION: DO NOT STORE THE SYSTEM IN WATER OR >90% AQUEOUS
MOBILE PHASE. MICROBIAL CONTAMINATION MAY RESULT.
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 Elutropic strength of the mobile phase

Normal Phase Reversed Phase

Normal phase with bare silica (adsorption)

Normal phase with bonded phase silica
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 For good resolution in separations, the three
terms can be optimized
Poor
Rs ~ 0.8
ACN-H2O
20 : 80

 1) Increasing k (retention factor) Increase k

– Change mobile phase composition ACN-H2O Rs > 1.5
(LC) 15 : 85

 2) Increasing  (selectivity factor)

Change 
– Changing mobile phase MeOH: H2O
Rs > 1.5
30 : 70
– Changing stationary phase
Increase N
 3) Increasing N (number of plates) Rs > 1.5
– Decrease particle size (LC)
L2= 3* L1
– Increase Column Length
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 Mobile Phase Factors for Improved Peak Shape

• Buffers (pH and ionic strength)

• Organic modifiers (Methanol, Acetonitrile, Tetrahydrofuran)
• Additional mobile phase modifiers (TEA: triethylamine, TFA:
triflouroacetic acid)

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pH and resolution

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Amphetamine

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Use Buffered Mobile Phases for Best Peak Shape and Retention

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Organic modifiers
Mobile Phase Organic Modifier
Acetonitrile vs. Methanol

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CAUTION: IF YOU ARE USING A
MASS SPECTROMETER, AVOID
USING NON-VOLATILE
ADDITIVES SUCH AS SODIUM (Na+),
POTASSIUM (K+), OR PHOSPHATE
(PO4-3).

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Solubility of the buffer in the organic cosolvent

• Avoid precipitation of the buffer salt

• Use very low concentration of buffer salt (10 mM) in the aqueous portion of the
eluent.
• low buffer capacity and ionic strength can lead to slow solvation equilibration,
irreproducible retention, and poor peak shapes.
• Ammonium salts are generally more soluble in organic/water mobile phases than
potassium salts, and potassium salts are more soluble than sodium salts
(NH4+ > K+ > Na+).
• Some salt buffers are hygroscopic. If an analyst makes a 20 mM buffer and the
original buffer salt contains 20 w/w% water, then the buffer concentration would be
16 mM.
• Therefore it is recommended to wash the HPLC system with acetonitrile/water
(20:80) for at least 20 column volume before the system is shut down to remove
any potential buffer residues.

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use a buffer concentration: 10-50mM 44
Mixing and degassing

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Different mobile phase preparation
O O O
O N O
N N N N
N O O N N O
O N
N N N
O N O N
O O O

HMX, octogen RDX, hexogen

O O
N O O O
O N N N
N O O O
N
O
N
O
O N
O O
tetryl

O TNT, trinitrotoluene
O N
O O
N
O O O O
N
O
O
N O
O
PETN, nitropenta

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 Solvent Degasser

 Heating the solvent

 Degassing by stirring or ultrasonication
 using vacuum (in a vacuum flask)
 helium sparging is inconvenient and expensive. Since helium has a very
low solubility in common solvents, after sparging the solvents are nearly
gas-free.

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 Vacuum and In-line membrane Degassing

In-line vacuum degassers: in-line

evacuation from a tube made of a gas-
permeable membrane such as
polytetrafluoroethylene (PTFE).

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 Pipe Material
• Low-Pressure Tubing, pressures less than ≈100 psi fluorocarbon tubing (e.g., Teflon®)
polymers (e.g., polypropylene and polyethylene)
• Teflon (reaction coil, drain tube)
• Strong chemical resistance
• Withstand up to only 5 kg/cm2 pressure

• “High-Pressure Tubing” 6000 psi (400 bar) between the pump and detector

Stainless Steel (whole connection can be applied)

• Withstand up to thousand kg/cm2 pressure
• Corrosion in the acidic condition ( particularly less than pH 2) or high concentration
of salt conditions

• PEEK (whole connection can be applied)

• Withstand up to 7000 psi pressure and whole pH range (pH 1-14)
• Week against high soluble organic solvent such as chloroform, THF

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 PUMPS constant volume pumps

• The pump – the heart of an LC system

 Typical requirements of an analytical HPLC pumps are:

 Provide precise and pulse-free delivery of solvents at typical flow rates range of
0.1–10 mL/min and pressure up to 6000 psi (400 bar, 42MPa )
 Compatible with common organic solvents, buffers, and salts
 Reliable operation with long pump seal life

1. Reciprocating-piston pump
2. Hydraulic-amplifier pumps
3. Syringe pumps
4. Piston-diaphragm pumps

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Dual piston reciprocating pump
two pistons are 180° out of phase

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Binary pumps mix solvents at the high-
pressure side of the pump and are usually
able to mix just two solvents

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53
 “System reference pressure" -
Estimating Pressure “Method reference pressure”

150 mm × 4.6
mm, 5-µm particle size (d)
C18 column,
Column
Mobile phase: 50:50 (v/v)
pressure
Water- Methanol
Flow: 2 mL/min
Temperature: 30 °C,

HPLC operated at pressures of 7–40 MPa (70–400 bar, 1000–6000

pounds/inch2) to attain flow rates of 0.5–5 mL/min.

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 Estimating Pressure

B: Methanol
A: Water
 UHPLC

Acetonitrile generates
approximately
60% of the pressure of
methanol, which
is one reason it is
favored for UHPLC
mobile phases.
Isocratic and Gradient Elution

Water 95%-5% in 10 min

Acetonitrile 5%-95% in 10 min

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Injectors And Autosampler

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Accuracy of large metal loops is ±5%, intermediate loops ±10%, small loops ±30%

Accuracy of large PEEK loops is ±14%, intermediate loops ±21%, small loops
±65%
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Split Peaks from Injection Solvent Effects

Tip: Injecting in a solvent stronger than the mobile phase can cause peak shape
problems such as peak splitting or broadening
Trick: Keep Organic Concentration in Sample Solvent < Mobile Phase

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Autosampler

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66
Typical column dimensions in HPLC

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Column re-equilibration

Pump pressure profile

Column re-equilibration

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Calculating column volume
 HPLC Column

Tubing
Male nut Ferrule

 1/4″ o.d.

 Most HPLC columns are made of 316 grade stainless steel, which is austenitic chromium–
nickel–molybdenum steel, Resistant to the usual HPLC pressure and also relatively inert to
chemical corrosion (chloride ions and lithium ions at low pH being important exceptions

 For bio-separation or ion-chromatography, the stainless steel column hardware can

be replaced by titanium or polyetheretherketone (PEEK) for more corrosion
resistance. 72
What Happens If the Connections Poorly Made ?

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Column Connectors Used in HPLC

0.228 cm

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Column Selection
 Column packing
• Rigid solids (silica matrix) or hard gels (polystyrene
crosslinked with divinylbenzene)
• Porous or pellicular (superficially porous particles)
• Spherical or irregular particles
• Particle size (dp)

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 fully porous

Superficially-Porous Particles

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78
 1. Column Length

 The length of a column determines the overall separation time and influences the resolution of
the peaks. For the same particle size:

 3 ‐ 7.5 cm length columns provide faster analysis run times

 10 – 25 cm length columns provide better resolution

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Figure 5: Effect of column
length.3

Column: Hypersil GOLD

Mobile phase: H2O/MeCN
(50:50) + 0.1% formic acid
Temperature: 30 °C
Flow rate: 0.2 mL/min
Detection: UV 254 nm
Injection volume: 1 µL
Methyl paraben
Ethyl paraben
Propyl paraben
Butyl paraben

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2. Internal Diameter of column

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Narrow bore columns compared with 4.6-mm columns with
the same stationary phase material will:

• Operate at higher backpressures (for the same column length)

• Operate at reduced flow rates: Reduce solvent consumption
• Require a smaller injection volume
• Exhibit increased ‘in-peak’ analyte concentration

This optimum value for u is

independent of column dimensions.

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 Effect of particle size on H
Small Particles Give High Efficiency but Require High Pressure

27 times higher pressure 83

 Bonding Chemistries

• These bonded phases suffer from several known

disadvantages:

1) At pH < 2, the Si-O bonds are subjected to acidic hydrolytic

cleavage, causing the loss of the bonded phase.

2) At pH > 8, the silica structure is prone to dissolution.

3) Any unbonded acidic silanols might lead to peak tailing of basic

analytes.

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 End-capping

only ∼50%
∼ bonding efficiency, the remaining residual silanols are further reacted or
“end-capped” with a smaller silane such as trimethylchlorosilane

One limitation of silica-based bonded phases is the operating pH range of 2–8, which
has been extended by recent innovations in bonding and support chemistries.

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 Support Type: Polymer

 Polymer support materials such as cross-linked polystyrene-

divinylbenzene, polyethers, and polymethacrylates have been used
successfully for many years, mostly for ion-exchange chromatography.

 Major advantages are wider pH range (1–14) and an absence of active

silanols groups.

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 Column Oven

 A column oven is required for most automated assays to improve retention time precision.
Column temperatures of 30–50°C are typical.
 Temperatures higher than 60°C are often used to increase flow and column efficiency (High
temperature liquid chromatography (HTLC) (T>60 C)).
 Subambient operation is used in chiral separations to enhance selectivity.

 Solvent preheating is achieved by passing the mobile phase through a long coiled tube
embedded onto the heating element before the column.

 New trends are toward wider temperature ranges (e.g., 4–100°C) using Peltier devices.

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Higher Temperature:

1) Provides more rapid mass transfer:

• analysis time – faster separations with no loss in resolution
• Improves Efficiency – enhances resolution

2) Decreases Mobile Phase Viscosity

• Lowers backpressure – allows for higher flow rates, faster separations,
greater efficiency and use of sub 2-micron columns
Drawbacks
1) First, the limited availability of stable high temperature-resistant packing
materials is a problem.
2) Second, a potential degradation of unstable compounds could occur.

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 Tips on Column Use
• Keep the pH of bonded-phase silica column between 2.0 and
8.0 (better is pH 2.5–7.5).

• Do not switch from organic solvents to buffer solution or vice

versa.
• Do not shock the column bed by rapid pressure changes.
• Pressure increases are caused by compound accumulation, by
column plugging with insoluble materials, or by solvent
viscosity changes.
 Tips on Column Use
• It is poor practice to run silica-based columns above 4,000psi.
• Solvents mixtures such as water/methanol, water/isopropanol, and
DMSO/water undergo large viscosity changes during gradient runs and
washouts.
• Adjust your flow rates and overpressure setting to accommodate these
increases so the systems does not shut down or overpressure columns.
• Use deoxygenated solvents for running or storing amine or weak anion
exchange columns.
• Wash out buffer, ion pairing reagents, and any mixture that forms solids on
evaporation before shutting down or storing columns.
• Store capped columns in at least 60% organic solvent (preferably 100%
MeOH or acetonitrile) to prevent bacterial growth.
Column Storage Recommendations
1. Long-term storage of silica-based, bonded-phase columns
should be in a pure organic solvent, preferably an aprotic
liquid such as 100% Acetonitrile.
2. If the column was previously used with a buffered mobile
phase, the buffer should first be removed by purging the
column with 20–30 column volumes of a 50/50 mixture of
methanol or acetonitrile and water, followed by 20–30
column volumes of the pure solvent.
3. Before storing the column, the end-fittings should be tightly
capped with end-plugs to prevent the packing from drying
out.

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Troubleshooting example: broadening or splitting caused by high pH

Basically, pH 11 dissolved the silica and formed a void,

thereby causing catastrophic band broadening.
A new column was the only option.
 Pressure Issues

• Check pressure with/without column - many pressure problems are due to blockages in the
system or guard column.
•Remove Column - Pressure Still High?
•Remove Guard – Pressure Still High?
•If Column pressure is high:
• Back flush column – Clear “dirty” frit surface
• Wash column – Eliminate column contamination and plugged packing – high
molecular weight/adsorbed compounds – precipitate from sample or buffer
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 The Cleaning and Regeneration of
Reversed-Phase HPLC Columns

• A recommended column washing system for a typical bonded-silica

column and a mobile phase without buffer salts is to use
• • 100% methanol,
• • 100% acetonitrile,
• • 75% acetonitrile–25%isopropanol,
• • 100% isopropanol,
• • 100% methylene chloride and
• • 100% hexane.
• When using methylene chloride or hexane, the column must be flushed with
isopropanol before returning to an aqueous mobile phase because of solvent
immiscibility
• A minimum of 20 column volumes of each wash solvent should be passed
through a column.
 Prevention Techniques - A Better
Choice! •
• Use column protection
• In-line filters
• Guard columns
• Filter samples
• Filter buffered mobile phases
• Sample clean-up (i.e. SPE)
• Appropriate column flushing

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 Guard columns

Guard columns usually are only about 1–3 cm in

length, they can be inverted and backwashed
without causing them to void.
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Cartridge and Holder for guard column

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 In-Line Filter

 In-line filters are designed to capture particulates that may come off the pump or be introduced by
samples.

 In-line filters generally contain Frits Available in 0.2, 0.5 and 2.0µm Porosity
to catch fine particulates that would otherwise collect in the column over time causing blockages.

 In-line filters should be replaced when they contributes excessive back pressure to the HPLC
system.

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 Sample filtration
• Injection of samples containing even small amounts of
particulate will clog the column inlet, cause
• high column backpressure
• retention time shift
• loss of resolution
• subsequently shorten the normal column lifetime.

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101
 Filtration impact on sub-2 micron column

Sample filtration prior to their introduction into an HPLC system was

demonstrated to make significant improvement on the column usage life time.

102
Detectors
The chromatographic detector is a transducer that converts a
physical or chemical property of an eluted analyte into an
electrical signal that can be related to analyte concentration.
 Overview of LC Detectors

 Common HPLC detectors

– UV/Vis (190-950 nm)
 Fixed Wavelength
 Variable Wavelength
 Diode Array
– Fluorescence Detector
– Refractive Index
– Evaporative light-scattering detection (ELSD)
– Conductivity (Electrochemical)
– Mass-spectrometry (MS)
– Nuclear magnetic resonance spectroscopy (NMR)
– inductively-coupled plasma (ICP)

104
If I and I0 are equal, then it follows that the intensity of the emergent
beam is equal to the incident beam and that I0/I equals 1. The log10 of 1 is
0, which corresponds to an absorbance of zero.
Now, an absorbance of 1 means that (I0/I) equals 10 (i.e. 10/1) and that 90% of the
light is being absorbed.
– UV/Vis (190-950 nm)

Beer–Lambert law

105
106
 Cut off point for HPLC grade solvent
or transparent mobile phase

Wavelength 190 195 200 205 210 215 220 230 235 240 245 250 254
Acetonitrile 1.000 0.150 0.070 0.040 0.020 0.010
1-Butanol 1.000 0.500 0.200 0.025
Chloroform 1.000 0.320 0.150
Cyclohexane 1.000 0.880 0.670 0.014
Ethanol 1.000 0.650 0.350 0.040
Ethyl Acetate 1.000
Ethyl Eter 1.000 0.070
Heptane 0.750 0.200 0.014
Hexane 1.000 0.250 0.080 0.014
Methanol 1.000 0.300 0.150 0.025
Methylene Chloride 1.000 0.700 0.200 0.100
Pentane 1.000 0.300 0.100 0.014
2-Propanol 1.000 0.250 0.130 0.025
THF 1.000 0.600 0.300 0.100

107
 Fixed Wavelength UV-Vis Detectors

Monochromatic detection
Measurement at one wavelength,
usually 254
The fixed nm
types use a lamp which emits at a certain wavelength.
 Mercury vapor lamps are probably the most common and emit intense light at 253.7 nm.
 Typical atomic vapor sources include mercury with emission lines at 254, 313 and 365 nm (among others),
cadmium at 229 and 326 nm and zinc at 308 nm .
 Compounds containing carbonyl groups, multiple double bonds, or aromatic rings can be detected at
this wavelength.

108
 UV/Vis absorbance detectors

 They are the most common detectors since most analytes of interest have UV absorbance. A
UV/Vis detector consists of a deuterium lamp, a monochromator, and a small flow cell
 A flow cell has typical volume of 2–10 μL and path lengths of 2–10 mm, with quartz lenses
serving as cell windows.
 UV detector is a concentration-sensitive device.

Polychromatic light

109
Selective or General Detectors?

Figure 4.8 Wavelength selectivity for UV

detection. (a) Absorbance spectra for two
hypothetical compounds X and Y. Chromatograms
at (b) 280 nm, (c) 260 nm, and (d) 210 nm.

110
180-210 nm

111
112
 Aflatoxin B1 is the most
common and toxic compound
of this class.

The oral LD50 value of aflatoxin B1 in humans is

estimated to be 4 mg/kg.
carcinogen

113
Optical arrangement of variable-wavelength (left) and diode-array (right) detectors

Polychromatic light from a

deuterium (UV) or tungsten
(visible) lamp

– UV/Vis (190-950 nm)

114
 Multiple Wavelength UV-Vis Detectors

Multiple Wavelength Detector (MWD) allows you to collect UV-Visible data in different
wavelengths (eight different wavelengths) simultaneously.
wavelength range from 190 to 950 nm

Simultaneous Data Acquisition

115
 Diode Array Wavelength UV-Vis Detectors

Diode array detector (DAD) also called photodiode-array, PDA

Detects full spectra and 8 signals with up to 80-Hz sampling rate for ultimate confidence
in ultra-fast LC.

Chromatogram but also provides spectral information

116
 Peak RT
uracil 1.4
phenol 1.6
4-chloronitrobenzene 2.2
Toluene 2.8

117
λmax 273 nm
 Absorbance and fluorescence spectra
Mirror image of the absorbance
spectrum

Fluorescence spectra
for 1 ppm anthracene in
alcohol:
(a) excitation spectrum;
(b) emission spectrum.

Photoluminescence“: fluorescence and phosphorescence

 Fluorescence Detector (FLD)
The fluorescence detector is a specific and
concentration-sensitive detector.

continuous spectrum
of light from 200 nm
to 900 nm

120
 Separation of Aflatoxins by HPLC

These naturally occurring toxins are found in several

types of food crops such as peanuts,
walnuts, almonds, pistachios, pecans, cottonseed, corn
and millet.

121
 Refractive Index Detector (RID)
• A refractive index detector measures the refractive index change between the
sample cell containing the eluting analyte and the reference cell purged with pure
eluent.
It offers universal detection and is used commonly for analytes of low chromophoric
activities such as
• alcohols, sugars,
• fatty acids: triglycerides,
• pharmaceutical excipients,
• polymers.
• It is the standard detector in GPC.
• • Low sensitivity (2–3 orders of magnitude lower than UV) least sensitive LC
detectors
• Very sensitive to pressure, flow, and temperature fluctuations
incompatibility with gradient elution are still the biggest disadvantages.

122
123
 HYPHENATED AND SPECIALIZED
SYSTEMS
LC/MS, LC/MS/MS

• Two common LC/MS interfaces are the electrospray ionization (ESI) and atmospheric
pressure chemical ionization (APCI).

• LC/MS/MS using a triple quadrupole analyzer can further increase sensitivity and specificity
for quantitation of trace analytes in complex matrices.

124
Components of a mass spectrometer

125
Electrospray ionization (ESI)

126
127
 Mass Analyzers
mass-to-charge ratio of ions

• Scanning (scan) mode

• Selected ion monitoring
(SIM) mode

128
LC/MS/MS

129
LC-DAD-MS
DAD

Mass spectrometry

130
chiral

WADA prohibited : Specified Stimulants

Ephedrine&Pseudoephedrine is prohibited
when its concentration in urine is greater
than 150 micrograms per milliliter.

[MH-H2O]+

[M+H]+

132
 QUALITATIVE ANALYSIS

• Retention Time ±0.02–0.05 min

• compare the retention time tR of the analyte
to that of a reference standard
• retention time is characteristic, but not
unique; more than one compound can have
the same retention time.
• Co-injection of a reference standard
• On-line Qualitative Analysis e.g. LC-MS
• off-line analysis; preparative HPLC

133
 Sensitivity and Detection Limit

Detection limit (DL) or limit of detection (LOD) : peak-to peak signal-to-noise ratio
(p-p S/N) S/N= 2 (or 3)
Limit of Quantitation (LOQ) : S/N=10
S/N ≥ 50 may be chosen for high-precision methods

where k is called the confidence

factor and m is the calibration
sensitivity.
The factor k is usually chosen to
be 2 or 3.

Figure 8-14 Calibration curve of

response R versus concentration c.
The slope of the calibration curve is
called the calibration sensitivity m. 134
 50 ppm : S/N=200mm/1mm=200
? ppm : S/N=2

Concentration=50 ppm

(S = 200mm)

(N = 1mm)
? =0.5ppm

135
 Limit of detection (LOD) for melamine at the
concentration of 0.05 mg/L

The calibration points include 0.5, 1.0, 5.0, 10, 50, and 100 µg/mL.

Sensitivity is the
slope of a
calibration plot

136
 Linearity

y = ax+b
 Saturation of the detector
y = 0.999x + 0.1312 y = 0.923x + 12.069

Although UV detector manufacturers often advertise that their detectors are linear to >2
AU (>2000 mAU), I’m a bit skeptical and don’t recommend operating a UV detectors
for quantitative analysis with peaks larger than 1 AU.
138
 Quantitative analysis

• Area normalization (Area Percent Method)

• External standard calibration
• Internal standard calibration
• Standard addition calibration” blank sample is not available e.g.
urine & plasma

139
Area normalization (Area Percent Method)

• Purity analyses, standards for calibration do

not exist.
• All analytes must be eluted.
• All analytes must be detected.
• All analytes must have the same sensitivity
(response/mass).

140
 Calibration-curve samples
•Linear dilution or
•Exponential (sometimes incorrectly
called ‘‘logarithmic’’) dilution scheme.

For example, with a standard curve

covering a method range of 1 to 100
ng/mL, standards might be prepared at 0,
1, 20, 40, 60, 80, and 100 ng/mL for a
linear dilution,
Or 0, 1, 2, 5, 10, 20, 50, and 100 ng/mL
for an exponential dilution.

141
 External standard

y = ax+b

3.50E+04

3.00E+04

2.50E+04

2.00E+04
y = 10000x
R² = 1 x= y/1000
Area

1.50E+04

1.00E+04

5.00E+03

0.00E+00
0.5 1 1.5 2 2.5 3 3.5
Concentration 142
 Single Point External Standard

• A second technique for determining the concentration of unknown samples

uses response factors.
• A response factor, RF (sometimes called a sensitivity factor), can be
determined for each standard as follows:

y = ax+b x= (y-b)/a
If the intercept does not differ significantly from zero, you can also use a straight line through
zero.
143
Example: Measurement of
DMSO in waste water by Concentration ppm Peak Area

LC-MS 3.16
7.9
7553
16489
15.82 26367
39.55 51004
79.11 86632

Sample Peak Area Concentration

P 80031 70.8949mg/l
PDP 31341 22.9339mg/l
PD 36039 27.56156mg/l

100000
90000 y = 1015.2x + 8058.5
80000 R² = 0.9926
70000
Peak Area

60000
50000
40000
30000
20000
10000
0
0 10 20 30 40 50 60 70 80 90
Concentration ppm
144
 Example 1
• A calibration solution with a concentration of 12 mg L-1 yields
a peak area of 6000 area units. The peak area of a sample
solution containing the same analyte is 8000 area units.
• What is the concentration of the analyte in the sample?

145
 3.5
3 y = 0.3331x + 0.0005
2.5 R² = 1

Signal ratio
2
1.5
1
0.5
0
0 2 4 6 8 10 12
{A] mg/ ml
 I.S. substances must satisfy the following conditions and
consequently selection can sometimes be difficult.

1) Its peak must be completely separated from the peaks for other constituents contained in the
sample.
2) It must not already be contained in the sample.
3) It must be eluted close to the target constituent.
4) Its chemical structure must be similar to that of the target constituent.
5) It must be chemically stable and easy to obtain in pure form.
(If the purpose is only to compensate for inconsistencies in injection O
volume, condition (4) is not required.) N N
OH

compatible detector response N N

O

147
Example 2 (internal standard method)
In a calibration, injecting an analyte solution at a concentration of 8 mg L-1
yields a peak area of 8000 area units. The peak area of the internal standard
is 4000 area units. Injecting the sample with the same internal standard
concentration results in an internal standard area of 4200 area units and an
area of the unknown analyte concentration of 5200 area units.

• What is the concentration of the analyte in the sample?

148
Method of standard additions

149
 Method of standard additions
We use the method of standard additions when it is difficult or impossible to duplicate
the sample matrix.

150
 Highly polar and Ionic species
1. Derivatization
2. ION-PAIR CHROMATOGRAPHY (IPC)
Ion-pairing reagents such as alkylsulfonates R–SO3 for amins & tetraalkylammonium
salts R4N+ (R+) for organic acid and organic phosphate (typically 30–100mM added to the
mobile phase) as well as strong (normally ionized)
carboxylic acids (trifluoroacetic acid, TFA; heptafluorobutyric acid, HFBA [R ]),
and so-called chaotropes (BF4 , ClO4 , PF6 ).

3. Hydrophilic Interaction Liquid Chromatography (HILIC) – sometimes called ‘aqueous

normal phase’ (ANP)
4. Ion Exchange Chromatographic Method

ION-PAIR
CHROMATOG
RAPHY (IPC)
 Derivatization for detection

• Postcolumn Derivatization – good detection

• Precolumn Derivatization - good separation

Sample itself Reagent

Column Detector

Derivatized Sample

Column Detector

152
Example: Amino Acid Analysis

NH2 UV-210nm

R C COOH

153
 Postcolumn Derivatization

Amino Acid itself Reagent

Column Detector

Column : Cation Exchange Column

Reagent : Ninhydrin method --- UV/VIS detector

OPA method --- Fluorescence detector

154
 Ninhydrin Method

Primary and Secondary amino (imino acid) acid

Detection : 570nm
Reaction Temperature : 130 C
Reaction speed : Fast

155
 Column Oven
Reaction
Amino Acid itself 130 C Heater

UV/VIS
Cation Exchange Column detector

Ninhydrin Reagent

156
 Precolumn Derivatization

Derivatized Amino Acid

Column Detector

Column : Revered Phase Column (ODS)

Reagent : OPA (Fluorescence)
FMOC (Fluorescence)
Fluorescamine (Fluorescence)
Dabcyl (Fluorescence)
PITC (UV)

157
 OPA Method
(o-phthaldialdehyde)

Primary amino acid

Detection : Fluorescence (Ex=380nm, Em=420nm)
UV at 338 nm
Reaction Temperature : Ambient
Reaction speed : Fast 158
159
160
 HPLC Applications
• Drug and compound discovery
• Proteomics and other biologicals
• Metabolites, Impurities, degradations
• Toxicology: Drugs of Abuse
• Clinical: Therapeutic Drug monitoring
• Water and pesticides analysis

161
ion-pairing reagent with reversed-phase chromatography

162
 Ion Exchange Chromatographic Method

Separation of melamine in milk powder sample

SCX is used for cation exchange high-performance liquid chromatography (HPLC)

163
 Analysis of Diet Cola Additives
O

N N
O
O N N
HO O
NH
O NH 2 O

 Conditions:
– Cartridge column 200 x 2.1mm C18
– 0.2 mL/min
– 15% acetonitrile/ 10mM phosphate buffer
(pH 2.2)
– Detector: 210nm

164
Carbohydrates

165
Antidepressants

Column: C18 , 4.6 x 150mm, 5μm

166
 Peptides/Proteins

Column: ZORBAX 300SB-C8 , 4.6 x 150mm, 5μm

167
B Vitamins

168
Herbicides

169
170