HPLC

From Liquid Chromatography to High
Performance Liquid Chromatography
Higher degree of separation!
 Refinement of packing material (3 to 10 µm)
Reduction of analysis time!
 Delivery of eluent by pump
 Demand for special equipment that can withstand high pressures
The arrival of high performance liquid chromatography!

What is HPLC?
HPLC is really the automation of
traditional liquid chromatography
under conditions which provide for
enhanced separations during shorter
periods of time, utilizing very small
particles, small column diameters, and
very high fluid pressures.

HPLC principle
It is a technique by which a sample mixture
is separated into components for
identification, quantification and
purification of mixtures

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.

Partitioning
Separation is based on the analyte’s relative solubility between two liquid
phases
Stationary PhaseMobile Phase
Solvent Bonded Phase

HPLC instruments consist of a reservoir of mobile phase,
a pump, an injector, a separation column, and a
detector.
Compounds are separated by injecting a sample mixture
onto the column.
The different component in the mixture pass through
the column and differentiates due to differences in their
partition behavior between the mobile phase and the
stationary phase.
The mobile phase must be degassed to eliminate the
formation of air bubbles.

Column
The heart of a HPLC system is the column.
The column contains the particles that contains the stationary phase.
The mobile phase is pumped through the column by a pump

COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
Degasser
Solvent Reservoir
Solvent Delivery System (Pump)
Pre gaurd column
Sample Injector System
Column
Detectors (Diode Array)
Recorder (Data Collection)and Integrators
Waste Collector

Pump
The role of the pump is to force a liquid (mobile phase) through the liquid
chromatograph at a specific flow rate.
A pump can deliver a constant mobile phase composition (isocratic) which the
mobile phase composition remains unchanged during the analysis.
or (gradient) in which the mobile phase changed during the analysis..

Pump
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 (5-10µ), the resistance to the flow of solvent is high.
Hence such high pressure is recommended.
The choice of mobile phase is very important in HPLC
and the eluting power of the mobile phase is determined by
its overall polarity, the polarity of the stationary phase and
the nature of the sample components.
Mixing unit is used to mix the solvents in different
proportions and pass through the column.

Injector
•The injector serves to introduce the
liquid sample into the flow stream of
the mobile phase.
May be auto-sampler or manual

HPLC columns
The column is one of the most
important components of the HPLC
chromatograph.
Because the separation of the
sample components is achieved
when those components pass
through the column.

Column has a diameter of 3 to 5mm
Normally, columns are filled with silica gel because its
particle shape, surface properties, and pore structure
help to get a good separation.
Silica is wetted by nearly every potential mobile
phase, is inert to most compounds and has a high
surface activity which can be modified easily with
water and other agents.
Silica can be used to separate a wide variety of
chemical compounds, and its chromatographic
behavior is generally predictable and reproducible.

Bonded Phases
C-2 Ethyl Silyl -Si-CH2-CH3
• CN Cyanopropyl Silyl -Si-(CH2)3-CN
• C-18 Octadecyl Silyl -Si-(CH2)17-CH3
• C-8 Octyl Silyl -Si-(CH2)7-CH3

Solid Support - Backbone for bonded phases.
Usually 10µ, 5µ or 3µ silica or polymeric particles.
Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
Extremely stable
Reproducible
Guard - Protects the analytical column:
Particles
Interferences
Prolongs the life of the analytical column
Columns Characteristics

Selectivity Factor
K’ values tell us where bands elute relative to the void volume.
These values are unaffected by variables such as flow rate and column
dimensions. The value tell us where two peaks elute relative to each other.
This is referred to as the selectivity factor or separation factor.

Selectivity factor, α , which describes the
separation of two species (A and B) on the
column.
α = k 'B / k 'A
Where k’ is the retention factor

Sample Preparation
Dissolve the sample in the mobile phase or in a solvent
weaker than the mobile phase.
The sample volume should be kept as small as possible.
47
Sample in Mobile Phase Sample in Stronger Solvent

U.V detectors:
• Based on electronic transitions
within molecules.
• Most common type of detector
for LC
• Fixed wavelength, Hg lamp 254
nm (π => π*)
•Tunable wavelength, selectable
for specific wavelengths,
monochromators or filters. Still
limited to single wavelengths.
•Solvent limitations with UV-vis
abs. Detectors
•Z-shape, flow-through cell.

Solvent UV-Cutoff/Transparency
53
Solvent UV Cutoff (nm)
Acetonitrile 190
Water 190
Cyclohexane 195
Hexane 200
Methanol 210
Ethanol 210
Diethyl Ether 220
Dichloromethane 220
Chloroform 240
Carbon Tet 265
Tetrahydrofuran 280
Toluene 285
UV cutoff is the
wavelength at which
absorbance equals 1,
measured in a 1 cm cell
with air as a reference.

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

Fluorescence detectors:
• Review - based on emission of excited state molecules.
• Fluorescing species or fluorescent derivatives

Electro chemical detectors:
•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 :
50μm thick, 1 ~ 5 μL volume
• Indictor E: Pt, Au, C
• Multi-electrode: simultaneous
detection or sample purity
indication.

Evaporative light scattering
detectors:
• Responds to any analyte
that is significantly less
volatile than the mobile
phase.
• Eluate is mixed with N2(g)
and forms a fine mist.
• Solvent (m.p.) evaporates
leaving fine particles of
analyte. The particles
themselves are detected by
light scattering.
• Response is proportional to
analyte mass.

IR detectors:
• Filter instrument or FTIR
• Similar cell as that of UV-Vis
• Limit: no suitable solvent, special optics
• FT-IR allows for spectrum records of flowing systems analogous to the
diode array system.
• Water/alcohols can be major interferences to solute detection
• LOD 100 ng
Photo diode array detector:
This is a recent one which is similar to U.V detector which
operates from 190-600 nm. Allows for the recording of the entire
spectrum of each solute as it passed through the diode array detector.
The resulting spectra is a 3-D or three dimensional plot of Response Vs
Time Vs Wave length.

WHAT AFFECTS SYSTEM
Column Parameters
Column Material
Deactivation
Stationary Phase
Coating Material
Instrument Parameters
Temperature
Flow
Signal
Sample Sensitivity
Detector

• Broad peaks occur due to the more conc. of sample,
large injection volume, column deterioration.
• Ghost peaks occur due to the contamination of the
column, compound from earlier injections.
• Negative peaks occur if mobile phase absorbance is
larger than sample absorbance.
• Peak doubling occurs due to the co- elution of
interfering compound, column over load, channeling
in column.
• Base line spikes occur due to the air bubbles in the
mobile phase and/or detector, column deterioration.
Peaks:

TYPES OF HPLC TECHNIQUES:
A. Based on modes of chromatography
1. Normal phase mode
2.Reverse phase mode
B. Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Ion pair chromatography
4.Size exclusion(or)Gel permeation chromatography
5. Affinity chromatography
6. Chiral phase chromatography

C. Based on elution technique
1. Isocratic separation
2. Gradient separation
D. Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
E. Based on the type of analysis
1. Qualitative analysis
2. Quantitative analysis

Several types of column
Normal phase
Reverse phase
Size exclusion
Ion exchange

Normal phase
In this column type, the retention is governed by the interaction of the polar
parts of the stationary phase and solute.
For retention to occur in normal phase, the packing must be more polar than
the mobile phase with respect to the sample

Reverse phase
In this column the packing material is relatively nonpolar
and the solvent is polar with respect to the sample.
Retention is the result of the interaction of the nonpolar
components of the solutes and the nonpolar stationary
phase.
Typical stationary phases are nonpolar hydrocarbons,
waxy liquids, or bonded hydrocarbons (such as C18, C8,
etc.) and the solvents are polar aqueous-organic mixtures
such as methanol-water or acetonitrile-water.

Common Reverse Phase Solvents
Methanol CH3OH
• Acetonitrile CH3CN
• Tetrahydrofuran
• Water H2O

Size exclusion
In size exclusion the HPLC column is consisted of
substances which have controlled pore sizes and is able
to be filtered in an ordinarily phase according to its
molecular size.
Small molecules penetrate into the pores within the
packing while larger molecules only partially penetrate
the pores.
The large molecules elute before the smaller molecules.

Ion exchange
In this column type the sample components are
separated based upon attractive ionic forces
between molecules carrying charged groups of
opposite charge to those charges on the
stationary phase.
Separations are made between a polar mobile
liquid, usually water containing salts or small
amounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.

RECORDERS
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.
INTEGRATORS

PARAMETERS USED IN HPLC
1.Retention time
2.Retention volume
3.Seperation factor
4. Resolution
5. Height Equivalent to a Theoretical
Plate (HETP)
6. Asymmetry factor

1.Retention time:
Retention time is the difference in time between the point of
injection and appearance of peak maxima. It is also defined as time
required for 50% of a component to be eluted from a column. It is
measured in minutes and seconds.
2.Retention volume:
Retention volume is the volume of carrier gas required to
elute 50% of the component from the column. It is the product of
retention time and flow rate.
Retention volume = Retention time × flow rate

4. Resolution:
Resolution is the measure of extent of separation of 2
components and the base line separation achieved.
Rs = 2 (Rt1-Rt2) / w1+w2
5. Height Equivalent to a Theoretical Plate (HETP):
A theoretical plate is an imaginary or hypothetical unit of a
column where distribution of solute between stationary phase and
mobile phase has attained equilibrium. It can also be called as a
functional unit of the column.

6. Asymmetry factor:
A chromatographic peak should be symmetrical about its
centre and said to follow Gaussian distribution. But in
practice due to some factors, the peak is not symmetrical
and shows tailing or fronting.
Fronting is due to saturation of stationary phase and can
be avoided by using less quantity of sample.
Tailing is due to more active adsorption sites and can be
eliminated by support pretreatment.
Asymmetry factor (0.95 to 1.05) can be calculated
by AF = b/a (b, a calculated by 5% or 10% of the peak
height).

DERIVATISATION IN HPLC:
In order to increase the detectability of various
classes of compounds ( for which sensitive detectors are
not available ) derivatisation is carried out in HPLC. A good
amount of work has been performed on the labelling of
compounds with chromophores and flurophores for
detection using UV spectrometers and fluorimeters
respectively. There are 2 important types of derivatisation.
These are,
1. Pre column derivatisation
2. Post column derivatisation

PRE COLUMN DERIVATISATION:
•In pre column derivatisation there are no restrictions on
the solvents, reagents, or reaction rates chosen and excess
of reagents can be removed before the injection.
•However, artifact formation, if present, can be checked by
positive identification of the eluted peaks.
•For example, in the derivatisation of a triketone with more
than one functional group capable of being derivatised
there is a possibility of range of derivatives being formed
from one solute.

Examples of pre column derivatisation to form UV
chromophores include the treatment of ketosteroids
with 2,4, DNP
Benzoylation of hydroxy steroids
Esterification of fatty acids.
Similarly, fluorophores have been introduced into
amino acids, biogenic amines, and alkaloids by
treatment with dansyl chloride.

POST COLUMN DERIVATISATION
It is carried out on the separated solutes as they emerge from the
chromatographic column. In HPLC, this places serious restriction on the
derivatisation reactions, because dilution of the eluent peak must be
minimized. Consequently, very fast reactions must be used and the
reagents and mobile phase must be compatible.
Examples of post column derivatisation reactions for use with UV detectors
include:
A. Reaction of amino acids with ninhydrin and fluorescamine.
B. Reaction of fatty acid with ortho nitro phenol.
C. Reaction of ketones with 2, 4, DNP.
D. Thermal or acid treatment of carbohydrates.
An oxidation detector for the fluorimetric analysis of carbohydrates in
body fluids using Ce (III) flourescence has also been reported.

Parameters of HPLC
1- Qualitative analysis
the most common parameter for compound is retention time
(the time it takes for that specific compound to elute from the column
after injection)

Capacity Factor (k’):
Is a measure for the position of a sample peak in the chromatogram.
k’ = (tR1-to)/to

2- Quantitative Analysis
The measurement of the amount of compound in a sample (concentration)
1.determination of the peak height
2.determination of the peak area

Resolution (RS)
Resolution of a column provides a quantitative measure of its
ability to separate two analytes
Rs = 2(TR2- TR1 ) / W2+W1

The factors which influence the HPLC
performance
1. Internal diameter of column
- the smaller in diameter, the higher in sensitivity
2. Pump pressure
- the higher in pressure, the higher in separation
3. Sample size
4. The polarity of sample, solvent and column
5. Temperature
- the higher in temperature, the higher in separation

Chromatograms
A B
Conditions: A: 150mm x 4.6mm, 5µ.
Flow Rate: 1.5 mL/min
Conditions: B: 50mm x 4.6mm, 3µ.
Flow Rate: 3.0 mL/min

Uses of HPLC
This technique is used for chemistry and biochemistry research
analyzing complex mixtures, purifying chemical compounds,
developing processes for synthesizing chemical compounds,
isolating natural products, or predicting physical properties.
It is also used in quality control to ensure the purity of raw
materials, to control and improve process yields, to quantify assays
of final products, or to evaluate product stability and monitor
degradation.
In addition, it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational safety or
health, and for monitoring pesticide levels in the environment.
Federal and state regulatory agencies use HPLC to survey food and
drug products, for identifying confiscated narcotics or to check for
adherence to label claims.

Application of HPLC
1. Pharmaceuticals industry
To control the drug stability
Quantity of drug determination frompharmaceutical dosage forms, ex.
Paracetamol determination in panadol tablet
Quantity of drug determination from biological fluids, ex: blood glucose
level
2. Analysis of natural contamination
- Phenol & Mercury from sea water
.

3. Forensic test
Determination of steroid in blood, urine &
sweat.
Detection of psychotropic drug in plasma
Forensic analysis of textile dyes.
Determination of cocaine and metabolites.
4. Clinical test
Monitoring of hepatic cirrhosis patient through
aquaporin 2 in the urine.
Quantification of DEET in Human Urine.
Analysis of antibiotics.
Detection of endogenous neuropeptides in brain
extracellular fluids.

5. Food and essence manufacture
• Sweetener analysis in the fruit juice
•Preservative analysis in sausage.
•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 aspartamine in the presence of glucose and
vanillin.

6. 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.

ADVANTAGES OF HPLC
1. Separations fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent.
3. It can be applied to the separation and analysis of
very complex mixtures.
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same
column.
6. Adsorption, partition, ion exchange and exclusion
column separations are excellently made.

ADVANTAGES OF HPLC
7. High separation capacity, enabling the batch analysis
of multiple components.
8. Superior quantitative capability and reproducibility.
9. Moderate analytical conditions.
◦ Unlike GC, the sample does not need to be vaporized.
10. Generally high sensitivity.
11. Low sample consumption.
12. Easy preparative separation and purification of
samples.
105

13. HPLC is more versatile than GLC in some aspects,
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
14. Both aqueous and non aqueous samples can be
analyzed with little or no sample pre treatment
15. A variety of solvents and column packings are
available, providing a high degree of selectivity for specific
analyses.
16. It provide a means for determination of multiple
components in a single analysis.

Disadvantages
1. Need a skill to run the instrument.
2. Solvents consumption is more.

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