CHM 260

Faculty of Applied Sciences,

UiTM (Perlis)
 High performance liquid chromatography (HPLC) is an
advanced form of liquid chromatography used to
separate the components of a mixture (analytes).

Why it is called ‘high performance’?

The high performance is the result of many factors:

➢ Smaller particles of the stationary phase

➢ Uniform pore size, high pressure column slurry packing
technique
➢ Accurate low volume of the sample injected
➢ Sensitive detector
➢ Good pump system
 The sample mixture is dissolved in a solvent (mobile
phase) and then forced to flow through a
chromatographic column under a high pressure. In the
column, the mixture is resolved into its components.
• The separation occurs because each component in the
mixture interacts differently with the stationary phase.
• Molecules that interact strongly with the stationary
phase (yellow component) will move slowly through the
column, while the molecules that interact less strongly
(blue component) will move rapidly through the
column.
HPLC instrument includes:
 Reservoir for solvents (mobile phase)
 High pressure pump
 Sample inlet device
 Column
 Detector
 Recorder
 Mobile phase
• Mobile phase is usually organic or
aqueous or a mixture of both.
• Mobile phase is placed in glass
bottles . Solvent A Solvent B
• Characteristics of mobile phase: Water Organic solvent
Miscible with water,
1- Pure such as acetonitrile,
methanol or
2- Low viscosity isopropanol.

3- Chemically inert
4- Low price
5- Compatible with detector
6- Solubility of the sample
 Solvents Polarity
Water 10.2
Dimethyl sulfoxide 7.2
Ethylene glycol 6.9
Acetonitrile 5.8
Methanol 5.1
Acetone 5.1
Dioxane 4.8
Ethanol 4.3
Tetrahydrofuran 4.0
1-propanol 3.9

Chlorinated solvents do not used in HPLC to prevent

rusting of stainless parts of the instruments
1. MP is filtered through a membrane/milipore filter
(0.45µm) to remove any particulate matter.
2. Removing dissolved air bubbles in MP by:
a) Heating with stirring ➔ passing nitrogen or helium
gas (Sparging)
Sparging: A process in which dissolved gasses are
swept out of a solvent by bubbles of an inert,
insoluble gas OR
b) Ultrasonic
3. Pre-saturation with the SP in case of liquid - liquid
chromatography.
 What are the function of the treatment of the
MP/sparging?

➔ To prevent the particulate matter/formation of air

bubbles which interfere with the pump function
(decrease pump efficiency)
➔ To remove particulate matter/air bubbles which
generate peaks when they pass through the detector
1- Isocratic elution:
The mobile phase composition remains constant
throughout the separation procedure.

2- Gradient elution:
The mobile phase composition is changed during the
separation process.

*Gradient elution is divided into two types:

A- Continuous (linear)
B- Discontinuous (stepwise)
% of B
Linear (continuous)
70%

60%
Gradient elution
50%
Stepwise (discontinuous)
40%

30%

20%

10% Isocratic elution

0 5` 10` 15` 20` 25` 30` Time

1- Shortening the time of analysis.
2- Reduces tailing and gives a sharp peak.
3- Increases the sensitivity of analysis*
4- Decreases the retention of the later eluting components
so that they elute faster.
 *Mobile Phase Composition Effect on
Selectivity
30% MeCN 45% MeOH
70% Water 55% Water

Fast Slow and better separation

**Methanol and water give slow and better separation

while acetonitrile and water give fast and bad separation
*Mobile Phase at Different Composition
 Function of the pump: Pump is used for forcing the
mobile phase through the column
 Main criteria of pump:

o The pump should be capable of delivering accurate and

pulse free flow rate (E.g: 5 ml/min).
o The pump should be capable of delivering high volume
of solvent.
o The pump should be capable of delivering high
pressure up to 5000 psi.
o Constant pressure pump ➔ free from pulsation ➔
resulting a smooth baseline
o Constant flow pump ➔ provide a constant flow rate of
mobile phase
There are three types of pump:
1) Screw driven syringe type (pulse-free delivery but lack of
capacity)
2) Reciprocating pump
• Used in 90% of HPLC's
• Advantages: Small internal
volumes (35 to 400 mL), high
output pressures (10,000 psi),
useful for gradient elution,
flow rate independence from
back pressure (constant flow
rate), solvent viscosity
• Disadvantages: Pulsed
output, produces baseline
noise
3) Pneumatic/constant pressure pump
• Simplest design
• Advantages: Inexpensive, pulse free
• Disadvantages: Limited solvent capacity, pressure
output limitations, flow rate dependence on solvent
viscosity, back pressure, not useful for gradient
elution
• Maximum Pressures ~2000 psi
1- Manual injection 2- Automated injection
The injection port consists of:
A: The injection valve
B: The sample loop

Manual injection
1- The sample is typically dissolved in the mobile phase.
2- It is drawn into a syringe and injected into the loop via
injection valve.
• Packed with various particles.
• Are often unidirectional.
• Are often preceded by a preparative or “guard” column.
• Usually have 3-5 mm diameter but of micro-columns or
capillary columns ranges from 3 µm to 200 µm.
Different shapes for columns
1- Analytical: 1-6 mm i.d. used in HPLC
2- Preparative up to 3 cm i.d.
 Made from: Stainless steel
 Shape: Straight
 Length: Variable
1) Guard column:
 Function: Increase the life of the
analytical column by removing
particulate matter and
contaminants from the solvent.
 Serves to saturate the mobile
phase with SP so that losses of
the SP from analytical column are
minimized

2) Column thermostat
- most modern commercial column
that can control temperature
 Guard Columns Column Thermostats

 Extend analytical column life  Temperature Control

 Removes: Particulate matter,  Unimportant for most
applications
solvent contaminents, sample
 Constant temperature
components which bind
improves chromatograms
irreversibly to SP
 Liquid-Liquid Chromatography  Modern HPLC's have column
 Saturates MP with SP
heaters
 Range: Ambient to 100° to
 Minimizes analytical SP loss
150° C
 Control: a Few 0.1° C
 Composition Matched to
Analytical  Column water jackets
 Pressure drop minimized with  Provides Heating or Cooling
larger particle size  Uses Standard Constant
 Repacked or discarded when Temperature Baths
contaminated
Characteristics of IDEAL detector
1. High sensitivity, good Stability and reproducibility
2. Short response time
3. Low noise (straight base line)
4. Unaffected by temperature or mobile phase
5. Non destructive
6. Provides qualitative and quantitative information about the
detected sample
7. Smaller operational temperature range
8. Minimum possible internal volume for LC
9. Either:
▪ Similar response to all analytes
▪ Selective response to some analytes
1. UV absorption detector (100 pg)
2. Refractive index detector (100 ng)
3. Mass spectrometer detector
4. Fluorescence detector (1 pg)
5. Flame ionization detector
1 - UV absorption detector
 It is the most sensitive
 The most widely used, it measure the UV absorption of
the solute.
 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
2 - Refractive index detector
- Not used in case of gradient elution

3 - Mass spectrometer detector

- Used with capillary column in analytical HPLC to give
information about nature of the material by giving the
mass spectrum of the material.

4 - Fluorescence detector
- More sensitive than UV detector (1000 fold as UV)

5 - Flame ionization detector

- Used for substances whose boiling point is higher
than that of the mobile phase
 HPLC

Based on mechanism
or types of stationary Uses (Function)
phase

2. 3. Size 4. Ion Analytical

1. Partition Preparative
Adsorption exclusion exchange

Normal Reverse
phase phase
1 - Partition Chromatography
The most common type of chromatography whereby the
stationary phase is thin film of liquid bonded to solid
(immobilized liquid)

➔ be further classified as “normal-phase” HPLC or

“reverse-phase” HPLC
 Normal-Phase HPLC utilizes a non-polar mobile
phase with polar stationary phase

 Reverse-Phase HPLC utilize a polar mobile phase in

combination with a non-polar stationary phase
 Normal Phase Reversed Phase

1. The SP make use of a 1. The SP is strongly non

polar liquid phase polar (e.g. C-18 silica
chemically bonded to (C18H37) hydrophobic)
strongly polar particles
(e.g. silica gel)

2. The MP is polar solvent

2. The MP is non polar to (e.g a mixture of water
low polarity such as and methanol or
(hexane or THF or CH2Cl2, acetonitrile).
benzene)
 Normal Phase Reversed Phase

3. Polar component of the

sample retained longer
on the column. Non-
polar compounds elute
out first followed by
increasingly polar
compounds

4. Application: 4. Application:
Ideal for samples with Amino acids, homologs,
low/moderate polarity, herbicides, etc.
water insoluble and
non-ionic
• successful partition chromatography requires a proper
balance of intermolecular forces among the three
participants in the separation process (analyte, SP and
MP).
• these intermolecular forces are described in terms of
the relative polarity possessed by each of the three
components.
• Polarities of common organic functional groups:
Aliphatic hydrocarbon < olefins < aromatic hydrocarbon
< halides < sulfides < ether < ester, aldehyde, ketone <
alcohol, amine < carboxylic acid < water
• As a RULE: Matching the polarity of the analyte to that
of the stationary phase
Applications of Partition Chromatography
• Typical applications of bonded-phase partition
chromatography for separating soft-drinks additives and
organophosphates insecticides.
2- Adsorption chromatography
• Stationary phase (SP) is the surface of a finely divided
polar solid. Mobile phase (MP) is usually organic solvent.
• Separation is due to a series of adsorption-desorption
steps.
• Applications:
1) Separation of relatively nonpolar, water-insoluble
organic compounds with molecular mass that
are less than 5000.
2) Speciality: Ability to
resolve isometric mixtures
such as meta and para
substituted benzene
derivatives.
3- Size Exclusion Chromatography (Gel Permeation)
• Used for large molecular weight compound such as
protein and polymers
• The column is packed stationary phase made of porous
silica or polymers particles.
• The sample is screened or filtered according to its
molecular size
• There is no interaction between solute and stationary
phase.
• The large molecules are washed through the column
(elute first), the smaller molecules penetrate
/permeate inside the pores and elute later.
large molecules

small molecules
• Gel Permeation:
• Gel Filtration:

(refer GC Notes)
Gel Permeation Gel Filtration

Mobile phase Non aqueous Aqueous

Packing Hydrophobic Hydrophilic

Applications Separation for high Separation for high

molecular weight, molecular weight,
non polar Polar compound
4- Ion Exchange Chromatography
• The stationary phase has an ionically charged surface
of opposite charge to the sample ions
• This technique is used only for ionic or ionisable
samples.
Types of stationary phase.
1- Anion exchange resin
2- Cation exchange resin
• Cation-exchanger resin - has a replaceable H+ ion that can
be exchanged with metal from the mobile phase.
[cation- resin]SO3-H+ + M+ [cation-resin]SO3M + H+

H+ ,
Cu2+ , Fe2+ Cu2+
 Cation exchange resin Anion exchange resin

Strong- sulfonic acid strong anion - quaternary

ammonium form Matrix-
Matrix-(SO3)– H+
(NR3)+ -Cl-
Weak- Matrix-COO- H+
weak anion as Matrix-
NH2(CH3)-Cl-

The stronger the charge on the sample, the stronger it will

be attached to the ionic surface and thus the longer it will
take to elute.
 Analytical
 Used to identify and to
know the number of
components in a mixture
(screening).
 Dimensions of the column:
1-6 mm i.d.
 Flow rate of mobile phase
(pump): range from 1 to 10
ml/min
 Injected vol of the sample:
range from 20 uL to 1 mL
Preparative
 Used in isolation and
purification.
 Dimensions of the
column up to 3 cm i.d.
 Flow rate of mobile
phase (pump): in excess
of 100 ml/min.
 Injected vol of the
sample: from 1 ml to 5
ml or more.
The process begins by:
• Injecting the solute onto the column (zero time).
• The separation occurs as the analyte and mobile
phase are pumped through the column.
• Detection of components by detector is
displayed on a chart or computer screen
(chromatogram).
1- High speed
2- High resolution
3- High sensitivity
4- Re-usable column
5- No destruction of the components
6- The instrumentation are automatic, computerized
7- Sample is recovered completely
8- Quantitative work is more easily and most sensitive
1. Isolation and purification of biologically active natural
products
2. Biosynthesis study: Detection of biogenetic
intermediates and enzymes involved
3. Control the microbiological process - Used for
separation of antibiotic from broth mixture
4. Quality control
HPLC is used to know the identity, purity and content
of the ingredients (drugs, raw and pharmaceutical
products)
 Isolation and Purification
- Refers to the process of separation or extraction the
target compound from other compounds or contaminants.
example : Separation of mixture of alkaloids from natural
product

1- Codeine
2- Strychnine
3- Papaverine
4- Quinine
5- Quinidine
 Column: Primesep
50x4.6 mm
 Flow rate: 1mL/min
 Detection: UV 280 nm
 Mobile phase: MeCN/H2O
(10/90)
 With H3BO4 buffer
 pH 3.0
Quantification of compounds by HPLC
Is the process of determination of the unknown
concentration of a compound in a known solution.
 Identification of compound by HPLC through :
- Comparison of retention time with authentic
- Comparison of UV spectrum of the compound with
that of the authentic.
- Comparison of the Mass spectrum with that of the
authentic.
 Oroidin is known alkaloid isolated from sponge Axinella
damicornis and it was identified by HPLC through the
comparison of retention time and UV absorption with
data base on HPLC.

Br
3
4
H
12 N
7
13 NH2
11
2 5 H 9 +
Br 6 N
N1 10
NH
14
H 8 15
O
Heptane and toluene were separated with
retention times of 15.4 min and 16.5 min,
respectively on a 1.0 meter packed column. An
unretained species passed through the column in
1.8 min. The peak widths measured at the base
were 1.15 min for heptane and 1.20 min for
toluene.
a) Calculate the resolution between the peaks
(Ans: 0.94)
b) Calculate the average number of plates for the
column (Ans: 2947.12 or 4081.76)
c) Calculate the average plate height. (Ans: 3.39 x
10-4 m or 2.45x10-4 m)