High-Performance Liquid Chromatography

(HPLC)
 Introduction
In 1941 Martin and Synge, described the discovery of liquid-
liquid partition chromatography and also laid the foundation of
Gas liquid chromatography and High performance liquid
chromatography.
The concept of the Height Equivalent to the Theoretical Plate ,
which has since been adopted as the measure of Chromatographic
efficiency.
In Classical Column Liquid Chromatography, the mobile liquid
phase flows slowly through the column by means of gravity. The
method is generally characterized by low column efficiencies and
long separation times.
Since about 1969, there has been a very marked revival of
interest in the technique of liquid column chromatography because
of the development of HPLC by Kirkland and Huber.
Column Chromatography
A mixture is applied to a solid support in a
chromatography column, and eluted by a solvent.
Elute with solvent

1 2 3 4

Absorbent
medium

tap
Cotton wool
plug

4
High-Performance Liquid Chromatography
They proposed high pressure systems capable of operating at
pressures up to 3000 psi. In HPLC, small diameter columns (1-3
mm) with support particle sizes in the region of 30 µm are used
and the eluent is pumped through the column at a high flow
rate.
It has been found that separation by HPLC may be effected
about 100 times faster than by the use of conventional liquid
chromatography.
Operating at high pressures these instruments for liquid
chromatography over come the effect of higher liquid viscosities
relative to gas viscosities and gave analysis times comparable
with GLC.
 Principle
When a mixture of components are introduced into a
HPLC column, they travel according to their relative
affinities towards the stationary phase. The component
which has more affinity towards the adsorbent, travels
slower. The component which has less affinity towards
the stationary phase travels faster.
HPLC Instrumentation

Liquid Mobile Phase

Pump

Injection Valve

Separation Column

Detector
HPLC
Instrumentation
1. Solvent delivery system
2. Pumps
3. Sample injection system
4. Column
5. Detectors
6. Recorders and Integrators
 1. Solvent delivery system and mobile phase

The solvents or mobile phases used must be passed

through the column at high pressure at about 1000 to 3000
psi. this is because as the particle size of stationary phase
is few µ (5-10 µm), the resistance to the flow of solvent is
high. Hence such high pressure is recommended.
Solvent delivery system
Mixing of solvents is done either with a static mixer or a
dynamic mixer.

Vacuum filtration, Helium purging, Ultrasonication systems are

needed in order to remove gas bubbles are formed during
pumping.

Normal phase chromatography: hexane, iso-octane, diethyl

ether are the mobile phases.

Reverse phase chromatography: water, methanol, acetonitrile

are the mobile phases.
Types of Solvent delivery system

Isocratic elution: A separation that employs a single solvent

or solvent mixture of constant composition.
Gradient elution: Here two or more solvent systems that
differ significantly in polarity are employed. After elution is
begun; the ratio of the solvents is varied in a programmed
way, sometimes continuously and sometimes in a series of
steps. Separation efficiency is greatly enhanced by gradient
elution.
Mobile Phase Composition Effect on Selectivity

30% MeCN 45% MeOH

70% Water 55% Water

Fast Slow and better separation

Some solvents used in HPLC and their polarity

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
I-propanol 3.9
2. Pumps
1. Reciprocating pump: Most widely used. Small internal volume
(35 ~ 400 μL), high-pressure (105 psi), gradient elution,
constant flow. Need pulse damper.
2. Syringe type displacement pump: Constant flow rate pump,
Non-pulsating flow , Isocratic flow only and Low flow rates (1 to
100 mL/min).
3. Constant pressure pump: This system provides pulse less and
continuous pumping. Inconvenient for gradient flow.
3. Sample Injection System
Several devices are available either for manual or auto injection
of the sample.

Different devices are:

1. Septum injectors

2. Rheodyne injectors (loop valve type)

Rheodyne injector is the most popular injector. This has a fixed

volume loop like 20 μl or 50 μl or more. Injector has 2 modes. Load
position and Inject mode.
Sample Injection System
Injector
 Sample Injection System

Sample size: 0.5 ~ 500 μL

Based on a sample loop: 1 ~ 100 μL
Reproducibility: 0.1%,
Pressure: < 7000 psi
Controlled temperature environment for derivatization
reaction.
 4. COLUMN
Stainless steel tubing for high pressure & Heavy-wall glass or
PEEK tubing for low P (< 600 psi)
Analytical column: straight, L (5~25 cm), dc (3~5 mm), dp (35 μm).
N (40 k~70 k plates/m)
Guard column: A guard column is introduced before the analytical
column to increase the life of the analytical column by removing not
only particulate matter and contaminants from the solvents but also
sample components that bind irreversibly to the stationary phase,
similar packing of analytical column.
Column
Column Packing Materials
Support Type
– Silica (or alumina) or polymer (cross-linked polystyrene)
Bonded groups
– C18, C8, C4, amino, cyano, phenyl
– Diethylaminoethyl (DEAE), sulfonate, quaternary ammonium
Particle size (dp): 3-, 5-, 7,- 10- or 20 μm
– Efficiency is inversely proportional to dp
– Column pressure is inversely proportional to (dp)2
Pore size (dpore): 60-300Ao
– Wide pore materials (300 Ao) are used for biomolecules or
polymers
Surface area: 90-400 m2/g
– High surface area maximizes solute interaction with bonded
groups
The Surface of Silica
For adsorption chromatography, the silica is used without
modification, but for most other types of HPLC, the silica is coated
with one of many types of stationary phases.
Precolumn (Guard Column)

 Remove impurities from

solvent

 Saturates mobile phase with

liquid of stationary phase
before the analytical column
5. DETECTORS
Detectors used depends upon the property of the
compounds to be separated. Different detectors
available are:
i. Refractive index detectors
ii. U.V detectors
iii. Fluorescence detectors
iv. Electro chemical detectors
v. IR detectors
vi. Mass Spectrometer
I. Refractive index detector
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
Nearly universal but poor detection limit
 II. U.V detector
Based on electronic transitions within
molecules.
Fixed wavelength, Hg lamp 254 nm (π => π*)
Tunable wavelength, selectable for specific
wavelengths, monochromators or filters. Still
limited to single wavelengths.
1 pg LOD
Solvent limitations with UV-vis abs.
Detectors Z-shape, flow-through cell (V,: 1~ 10
μL and, b: 2 ~ 10 mm)
Spectrophotometer: more versatile
III. Fluorescence detector
Based on emission of excited state molecules.
Detector.
LOD 10 fg
Hg or Xe lamp
Fluorometer and spectrofluorometer
Fluorescing species or fluorescent derivatives
IV. Electro chemical detector
Based on amperometric response of
analyte to electrode usually held at
constant potential.
If the analyte is electro active, can be
highly sensitive since response is based
on a surface phenomenon rather than
a solution bulk property (e.g. UV-Vis
absorbance)
Simplicity, convenience and wide-
spreading application
Thin-layer flow cell of Teflon : 50 μm
thick, 1 ~ 5 μL volume
Multi-electrode: Simultaneous
detection or sample purity indication.
 V. IR detectors
Filter instrument or FTIR
Similar cell (V: 1.5~10 μL and b: 0.2 1.0 mm)
FT-IR allows for spectrum records of flowing systems
analogous to the diode array system.
LOD 100 ng
Water/alcohols can be major interferences to solute
detection
6. Recorders and Integrators
Recorders: Are used to record the responses obtained from
detectors after amplification. They record the base line and all the
peaks obtained, with respect to time. Retention time for all the
peaks can be found out from such recordings, but the area of
individual peaks cannot be known.
Integrators: are improved version of recorders with some data
processing capabilities. They can record the individual peaks with
retention time, height and width of peaks, peak area, percentage
of area, etc. Integrators provide more information on peaks than
recorders. Now a days computers and printers are used for
recording and processing the obtained data and for controlling
several operations.
ADVANTAGES OF HPLC
1. Separations fast and efficient (high resolution power)
2. It can be applied to the separation and analysis of very complex
mixtures
3. Accurate quantitative measurements
4. HPLC is more versatile than GLC in some respects, because it has
the advantage of not being restricted to volatile and thermally
stable solute and the choice of mobile and stationary phases is
much wider in HPLC
5. A variety of solvents and column packing are available, providing
a high degree of selectivity for specific analyses.
6. It provides a means for determination of multiple components in
a single analysis.
Types of HPLC
1. Reverse-phase: polar mobile phase/non-polar
stationary phase/somewhat polar analytes

2. Normal Phase: non-polar mobile phase/polar

stationary phase/non-polar analytes

3. Adsorption: non-polar mobile phase/polar stationary

phase/non-polar analytes; isomer separation

4. Ion-Exchange: salts/ionic stationary phase

5. Size-exclusion: aqueous/gel for large MW solutes, >104

Normal-phase or Liquid-solid Chromatography

Used polar stationary phase pickings and low polarity

solvent

Non-polar compounds elute out first followed by

increasingly polar compounds

Elutents used – low to moderate polarity e.g. hexane,

CH2Cl2, CHCl3, etc.

Hexane ; dichloromethane; isopropanol; methanol

Increasing strength
 Normal Phase HPLC Columns
 Cyano: Rugged, moderate polarity,
general use
 -OH (Diol): More polar and retentive
 Amino: Highly polar, less stable
 Silica: Very rugged, low cost, adsorbent
(Unbonded)
Reversed-phase Chromatography (RPC)

Opposite of normal phase – stationary phase is non-

polar and mobile phase is polar

C18 columns (e.g. octadecyl, -C18H37)

Eluents used – aqueous solution of organic solvents

e.g. MeOH, ACN, THF, etc.

Gradient Elution
water ; methanol; acetonitrile; tetrahydrofuran (THF)
Increasing strength
 Reversed Phase HPLC Columns
• C-18, C-8: Rugged, general purpose, highly retentive

• C-3, C-4: Less retentive, used mostly for peptides

& proteins

• Phenyl: Greater selectivity than alkyl-bonded

• Amino: Weak retention, good for carbohydrates

Reversed phase stationary phase
• Most common; n-octyldecyl, C18

Si-O-Si-(CH2)17-CH3
CH3

CH3
CH3
CH3
Si-O-Si-(CH2)17-CH3 Si-O-Si-(CH2)17-CH3
CH3
CH3
Si-O-Si-(CH2)17-CH3
CH3

CH3
The stationary phase is less polar than the mobile phase
where a C3, C8, or a C18 chain length stationary phase is
used while the mobile phase is a polar solvent like
methanol, acetonitrile, etc or mixtures with water. In this
type of chromatography, the more polar compound is
eluted first while the less polar compound will be retained
more.
 3- 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 St. Ph
1- Anion exchange
resin
2- Cation exchange
resin
Matrix: is polymer of styrene
with divinyl benzene
Column Types in Ion Exchange LC
Stationary phase contains charged groups

 SAX (Strong Anion Exchange): NH3+

 WAX (Weak Anion Exchange): NR2H+ (DEAE)

 SCX (Strong Cation Exchange): SO3-

 WCX (Weak Cation Exchange): Carboxymethyl (CM)

 More highly charged analytes have stronger

retention
Ion Chromatography Applications

i. Inorganic Anions – tap water

 Fl-, Cl-, NO3-, PO43-, SO42-
ii. Cations and Transition Metals – tap water
 Li+, Na+, K+, Mg2+, Cu2+, M-CN complexes
iii. Organic Acids – fruit juice
 Oxalic, Maleic, Malic, Succinic, Citric
iv. Surfactants – soaps and detergents
 Sulfonates, long/short chain ammonium
2- Size exclusion chromatography.
The column is packed with material having controlled pore
sizes and the sample is screened or filtered according to its
molecular size, there is no interaction between solute and
stationary phase. The large molecules rapidly washed through
the column, the smaller molecules penetrate inside the pores
and elute later.

Large molecules

Small molecules
 Choice of separation technique
1- Sample molecular weight less than 2000
Reversed phase Chrom. (RPC)
Water soluble Ionic Ion exchnge Chrom.(IEC)

Non- ionic Reversed phase Chrom. (RPC)

Exclusion Chrm. (EC) if
soluble in tetrahydrofuran

Organic solvent soluble non polar solvent----Adsorption chromatography

Tetrahydrofuran------ Exclusion Chrm. (EC)

Polar solvent------Normal phase chromatography

------Reversed phase chromatography

2- Sample molecular weight greater than 2000

Reversed phase chromatography (RPC)
Water soluble
Ion exchnge Chrom. (IEC)
Exclusion Chrm. (EC)

Organic solvent soluble Exclusion Chrm. (EC)

Applications of HPLC in isolation and purification
of natural products

I. Purification
refers to the process of separation or extraction the
target compound from other compounds or
contaminants.
 1- Separation of quinine and quinidine
H H
4 4
7 7
H 5 3 5 3
HO S 6 H 8 6
9 2 9 2
8 N R N 1
H R 1 HO
5` 5` S H
MeO 4` R 4`

1
6` 6`

7`
3`
2` 2 7`
3`
2`
N N
8` 1` 8` 1`
1- Quinidine
2- Qinine
 2- Separation of vitamin B-1, 2, 6

• Column: Primesep
• 150x4.6 mm
• Flow rate: 1ml/min
• Detection: UV 280 nm
• Mobile phase: MeCN/H2O
(10/90)
• With H3BO4 buffer
• PH 3.0
 3- Separation of ascorbic acid and dehydro-
ascorbic acid

• Column: Primesep
• 50x4.6 mm
• Flow rate: 1ml/min
• Detection: UV
• Mobile phase: MeCN/H2O
(10/90)
• With HCOOH buffer
• 0.1%.
4- Separation of chloramphenicol from mixture
of antibiotics
 5- Separation of mixture of alkaloids

• 1- Codeine
• 2- Strychnine
• 3- Papaverine
• 4- Quinine
• 5- Quinidine
6. Food and Beverage
Mono-, Di- and Oligosaccharide Standards

3 5

7 1. Glycerol
4 6 2. Arabinose
3. Fructose
4. Glucose
9 11 5. Sucrose
1 6. Maltose
8 10 7. Maltotriose
8. Maltotetraose
9. Maltopentaose
2 10. Maltohexaose
11. Maltoheptaose

Column: Asahipak NH2-P50, 5µm, 250x4.6mm

Mobile Phase: A:Acetonitrile B: 0.0004N NH4OH
Gradient: (Time, %B)(0,15)(60,65)
Flowrate: 1.0mL/min
Col. Temp: 30ºC
Detector: Shimadzu ELSD-LT (Gain 5; T 40ºC; P 250kPa)
APPLICATIONS
HPLC is one of the most widely applied analytical separation techniques.

1. Pharmaceutical:
Tablet dissolution of pharmaceutical dosages.
Shelf life determinations of pharmaceutical products.
Identification of counterfeit drug products.
Pharmaceutical quality control.

2. Environmental:
Phenols in Drinking Water.
Identification of diphenhydramine in sediment samples.
Biomonitoring of PAH pollution in high-altitude mountain lakes through the
analysis of fish bile.
Estrogens in coastal waters - The sewage source.
Toxicity of tetracyclines and tetracycline degradation products to
environmentally relevant bacteria.
Assessment of TNT toxicity in sediment.
3. Forensics:
A mobile HPLC apparatus at dance parties - on-site identification and
quantification of the drug Ecstasy.
Identification of anabolic steroids in serum, urine, sweat and hair.
Forensic analysis of textile dyes.
Determination of cocaine and metabolites in meconium.
Simultaneous quantification of psychotherapeutic drugs in human plasma.

4. Clinical:
Quantification of DEET in Human Urine.
Analysis of antibiotics.
Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis.
Detection of endogenous neuropeptides in brain extracellular fluids.

5. Food and Flavor:

Ensuring soft drink consistency and quality.
Analysis of vicinal diketones in beer.
Sugar analysis in fruit juices.
Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits.
Trace analysis of military high explosives in agricultural crops.
Stability of aspartame in the presence of glucose and vanillin.