High Pressure Liquid
Chromatography
(HPLC)
1
High Pressure Liquid
Chromatography (HPLC)
What is HPLC?
Types of Separations
Columns and Stationary Phases
Mobile Phases and Their Role in Separations
Injection in HPLC
Detection in HPLC
2
Introduction
High pressure liquid chromatography (HPLC) is an
advanced form of liquid chromatography used to
separate the components of a mixture (Analytes).
HPLC is an automated and sophasticated type of
column chromatography.
In HPLC chromatography: the 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.
3
The separation occurs because each component in
the mixture
Interacts differently with the stationary phase.
Interacts differently with mobile 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.
The start to the detector
4
- Why high pressure?
Thus pressure from 1000 to 5000 psi, pound per square inch
(68 to 340 atm.) is applied to overcome the obstructive effect
of the fine particles, especially in analytical HPLC
To overcome back pressure
In HPLC the stationary phase has two characters :
- - small particles size (5-10 µm).
- - packed under high pressure.
Reduction of the particle size of the stationary phase leads
to:
- Leaving less space for the mobile phase to pass through.
- Decrease the flow rate of the liquid mobile phase.
5
High Pressure Liquid Chromatography
(HPLC)
Other names for HPLC
1. High Performance Liquid Chromatography (HPLC)
High performance is the result of many factors:
high number of theoretical plates
- Smaller particles of the stationary phase,
- uniform pore size,
- high pressure column slurry packing technique,
- accurate low volume of the sample injected,
- sensitive detector, and
- good pump system
2. High Speed Liquid Chromatography (HSLC)
- As the separation is completed within few minutes.
3. High Resolution Liquid Chromatography (HRLC ) 6
The advantages of HPLC
1- High speed
2- High resolution
3- High sensitivity
4- Re-usable column
5- No destruction of the components
6- Analysis of thermally labile analytes
7- The instrumentation are automatic,
computerized
8- Sample is recovered completely
9- Quantitative work is more easily and most
sensitive 7
HPLC Advantages vs GC
Not limited by sample volatility
or thermal stability
Two interacting phases
Both stationary and mobile
phases are important
On GC, only the stationary
phase is important
Room temperature analysis
Ease of sample recovery 8
Classification of LC
I- Types of LC according to the
mechanism
of separation
1- Adsorption chromatography
The stationary phase is
an adsorbent and
the separation is based on adsorption
strength.
Normal phase chromatography
The stationary phase is strongly
polar (e.g. silica gel) and the mobile
phase is non polar such as (hexane
or tetrahydrofuran).
Polar sample retained longer on the
column.
9
2. Partition chromatography
Liquid as a stationary phase
Reversed Phase chromatography
The stationary phase is strongly non-polar ( e.g.
C-18 silica , hydrpophobic)
while the mobile phase is polar (as a mixture of
water and methanol or acetonitrile).
10
3. 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,
The large molecules rapidly washed through the
column, the smaller molecules penetrate inside the
pores and elute later.
Large molecules
Small molecules 11
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 St. Ph
1- Anion exchange resin
2- Cation exchange resin
Matrix: is polymer of styrene with divinyl
benzene
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Columns and Stationary Phases
13
II- HPLC can be divided into two
main types according to the
uses:
1- Analytical type: which is used
a. In identification and assay of the
components in a mixture .
b.To know the number of components
in a mixture (screening).
2- Preparative or semipreparative type:
used in isolation and purification. 14
The difference between
analytical and preparative HPLC
a. Dimensions of the column.
Analytical, 1-6 mm i.d.
Preparative up to 3 cm i.d.
b. Flow rate of mobile phase (pump).
For analytical HPLC pumps should
has flow rates that range from 1
to 5 ml/min.
but for preparative HPLC, flow
rates in excess of 10 ml/min.
c. Injected volume of the sample
in analytical HPLC range from 20 uL to
1 mL,
but in preparative or semi preparative
from 1 ml to 5 ml or more. 15
d. Size of the loop of injection port.
Chromatographic process
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
16
on a chart or
Select the correct type of separation
Select an appropriate
column (stationary phase) and
mobile phase
appropriate detector based on whether
universal or compound-specific
Optimize the separation using standard
mixtures
17
Instrumentation of HPLC
HPLC instrument includes:
A- Reservoir for solvents (mobile phase)
B- High pressure pump
C- Sample inlet device
D- Column
E- Detector
F- Recorder
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(A)- Reservoir for solvents
(mobile
phase)
- Mobile phase is
placed in bottles of
glass
reservoir
- Mobile phase is
usually
organic or
aqueous or
mixture of both.
- Mobile phase should
19
Mobile phase
Characters of mobile phase:
1. Pure
2. Low viscosity
3. Chemically inert
4. Low price
5. Compatible with detector
Miscible with water, such as
6. Solubility of the sample -acetonitrile,
7. The solvents should be -methanol, or
miscible -isopropanol.
20
Elution Techniques (Programming)
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)
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Isocratic and gradient elution curves
22
Isocratic versus Gradient Elution
Isocratic elution has a constant mobile phase
composition
Can often use one pump!
Mix solvents together ahead of time!
Simpler, no mixing chamber required
Limited flexibility (no change in polarity
index), not used much in research
23
Isocratic versus Gradient Elution
Gradient elution has a varying mobile phase
composition
Uses multiple pumps whose output is mixed
together
often 2-4 pumps (binary to quaternary
systems)
Changing mobile phase components changes the
polarity index
Better separation
Column has to re-equilibrate to original
conditions after each run (takes additional time)
24
Advantages of gradient elution
technique
1- Shortening the time of
analysis.
2- Reduces tailing, gives sharp
peak.
3- Increases the sensitivity of
analysis.
4- Decreases the retention of the
later-eluting components so
that they elute faster. 25
The selectivity of HPLC is affected by :
1- Type of mobile phase, organic or aqueous.
2- The composition of the mobile phase,
whether one solvent or more.
3- The pH of the mobile phase.
PH of mobile phase
The pH of the solvent (water) may be
adjusted using
phosphate or
perchlorate or
trifloroacetate acid or
sulphate buffer. 26
Effect of buffer used in separation
of xanthene alkaloids
Primesep B column: it is a strong basic column, where it retains
acid residue in the stationary phase in equimolar amount.
TFA
Incomplete
separation
HClO4
No separation for 2
and 3
H2SO4 27
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 use
of acetonitrile and water give fast and bad separation 28
% of Mobile phase B (MeOH) and
separation selectivity
High % of B gives Low % of B
gives
fast and bad
separation slow and slightly
better separation
29
Some solvents used in HPLC and
their polarity
Polarity Solvents
10.2 Water
7.2 Dimethyl sulfoxide
6.9 Ethylene glycol
5.8 Acetonitrile
5.1 Methanol
5.1 Acetone
4.8 Dioxane
4.3 Ethanol
4.0 Tetrahydrofuran
I-propanol
3.9
N.B. Chlorinated solvents do not
used in HPLC to prevent rusting of
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stainless parts of the instruments
Treatment of mobile
phase
A- Filtration before entering the column.
B- Degassing using degasser
To remove gases dissolved in the mobile
phase by
1- Heating with stirring
2- Applying vacuum,
3- Passing nitrogen or helium
4- Ultrasound
C- Pre-saturation with the stationary phase in case of
liquid-liquid chromatography
To avoid stripping (washing off) 31
The Mobile Phase in HPLC...
Must be:
compatible with the stationary phase
readily available (often use liters/day)
of adequate purity
Analytical grade!
compatible with the instrument (pumps,
seals, fittings, detector, etc)
Not too compressible (causes pump/flow
problems)
Free of gases (which cause compressibility
problems and disturb repeated equilibration
process) 32
33
Optimization of Mobile Phase Polarity…
- Changing the mobile phase composition alters the
separation.
34
35
(B)- Pump
Function of the pump:
Pump is used for forcing the
mobile phase through the
column
To column
There are two types of pump:
1- Constant pressure pump
It is free from pulsation
resulting in smooth baseline
For trace sample analysis
2- Constant flow pump
It is able to give constant Mobile phase
flow rate of mobile phase
For normal HPLC run 36
The main criteria for the
pump
1-The pump should be capable of
delivering
accurate and
pulse free flow rate (e.g. 5 ml/min).
2-The pump should be capable of
delivering
high volume of solvent.
3-The pump should be capable of
37
delivering
(C)- Sample inlet device
(Injection port)
1- Manual 2-Automated
injection injection
38
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.
39
Manual Injection in HPLC
Usually 20 to 1000 L volumes, all directly onto
the column
not much worry about capacity since the columns
have a large volume (packed).
A source of poor precision in HPLC
errors of 2-3 %RSD are due just to injection
Two positions, load and inject in the typical
injector
Injection loop internal volume determines
injection volume.
40
Manual injection
Valve-type injectors
- Six port fixed volume (Rheodyne)
reproducible injection volumes
fixed loop size (variable injection is impossible
without loop change)
easy to use, reliable
- Six port variable volume (Waters)
variable injection volumes without loop change,
operator skill required
more expensive
41
LOAD (the sample loop) Inject (move the sample
loop into the mobile
phase flow)
42
43
Automated Injectors
Operator free injection
Comparable precision and
accuracy to manual
Much more expensive initially
Much more convenient Up 100
samples and standards with
microprocessor control
44
(D)- Column
Column in HPLC is either
1- Analytical column
1-6 mm i.d.
2- Preparative column
up to 3 cm i.d.
Made from: Stainless
Shape: Straight
Length: Variable Different shapes
for columns used
in HPLC 45
Other types of columns used in
HPLC
Guard column:
1- Also known as
Pre-column
2-Protect the
analytical
column
3- Organization of
separation in 46
HPLC
Columns and Stationary Phases
HPLC is largely the domain of packed columns
Molecules move too slowly to be able to reach
and therefore “spend time in” the stationary
phase
Stationary phases
Particle size which are usually about 5
to 10 m in average diameter (often
irregularly shaped for packed column)
Particle size affect the efficiency of the stationary
phase
47
Effect of particle size on separation
48
Stationary Phases
Polar (“Normal” Phase):
Silica, alumina
Cyano, amino or diol terminations on the
bonded phase
Non-Polar (“Reversed Phase”)
C18 to about C8 terminations on the
bonded phase
Phenyl terminations on the bonded phase
Mixtures of functional groups can be used!!
49
(E)- Detectors
It is referred as the Brain of HPLC
It is where the individual analyte detects
Characters of detectors
1- High sensitivity
2- Low noise (straight base line)
3-Wide range of response to different compounds
4- Unaffected by temperature or mobile phase
5- Non-destructive to the compounds
6- Provides qualitative and quantitative information
about the detected sample
50
Detection in HPLC
Numerous Types – UV/vis; RI, MD etc
Must be solvent -compatible, stable, etc.
Universal
respond to all analytes
Analyte Specific
respond to specific properties of analytes
Non-destructive
UV/Vis, RI
Destructive
MS and a few others.
51
HPLC Detectors
52
Types of Detectors
1-UV (absorbance) detector
- It is one of the most sensitive
detector,
- sensitive to pg (pico gram) of
compound.
- The most widely used,
- it measure the UV absorption of the
solute.
53
UV Detector
(a). Single wavelength (filter)
with only one fixed wavelength
(b). Standard absorbance detector
One device with variable wavelength
(monochromator), but it measures one at a
time
(c). Diode Array detector (DAD) or photodiode
array detector (PDA)
Multiple wavelengths
(a). Single wavelength (filter)
available with only one fixed wavelength
Old UV detector 54
(b). Standard Absorbance Detector
Usually utilize typical UV-VIS lamps and 254 nm default
wavelength
Can be set to other wavelengths (most)
Simple filter detectors no longer widely used
adjustable wavelength units are cost-effective
Can use any UV-VIS with a special flow cell
Extra connections lead to band-broadening if UV-VIS
is far from HPLC column exit.
Non-destructive, not-universal
not all compounds absorb light
can pass sample through several cells at several
different wavelengths
55
56
(c)- Photodiode array detector
(PDA)
Is also known as diode array detector
(DAD)
It is series of detectors each is
responsible for receiving a different
wavelength.
Scans multiple wavelength at a time
57
Photodiode Array Detector (PDA)
PDA scans a range of wavelengths every second
or few seconds.
At each point in the chromatogram one gets a
complete UV-VIS spectrum!
Huge volumes of data
Detailed spectra for each peak and each region of
each peak
Non-destructive, non-universal
58
2 - Refractive index detector
It measures the difference in RI between the
column eluate (mobile phase + solute) and
pure mobile phase.
Often used with isocratic elution
Not often used in case of gradient elution
Less sensitive
59
Refractive Index Detector
Responds to analytes changing the RI of the
mobile phase
requires a separate reference flow of mobile phase
One of a very few Universal HPLC detectors
Non-destructive
Extremely temperature sensitive, usually
heated
sensitive to temp changes of +/- 0.001 °C
No longer really widely used
Absorbance detectors are relatively cheap. 60
3- Fluorescence detector
Based on emission of compound, not absorption
More sensitive than UV detector (1000 fold as
UV)
It is used with compounds which are naturally
fluorescent. Or compound which can be
converted to fluorescent derivative.
61
4- Mass spectrometer
detector
It is used with capillary column in
analytical HPLC (LC-MS)
It gives information about nature of
the material by giving the mass
spectrum of the material.
62
5-Flame ionization detector
Not common
Used for detection of substances whose boiling
point is higher than that of the mobile phase.
It is more sensitive than refractive index detector
63
Optimization of Separations in HPLC
Correct choice of column so the equilibrium has
some meaningful (non-infinity, non-zero)
equilibrium constants (KD).
Correct choice of mobile phase
Decision on the type of mobile phase
composition
constant composition = isocratic
varying composition = gradient elution
Determination of appropriate flow rate
Decision on heating the column (column temp.)
heating HPLC columns can influence the
equilibrium constant 64
Applications of HPLC
1- Isolation and purification of
biologically active natural products
2- Control of synthetic reactions
Identification of intermediates and
target compound.
3- Biosynthesis study
Detection of biogenetic
intermediates and enzymes involved.
4-Control the microbiological process
Used for separation of antibiotic
from broth mixture 65
5- Pharmacokinetics study
Pharmacokinetic study comprises the
measurement of drug metabolites
concentration in body fluids, absorption,
bioavailability and elimination of drugs
HPLC determines the drug and its
metabolites in one step.
6- Stability test
Rapid method of analysis in stability test.
7- Quality control
HPLC is used to know the identity, purity
and content of the ingredients (drugs, raw
and pharmaceutical products,
8- Drugs metabolisms
66
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.
67
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
6` 4` R
6` 4`
3`
2` 2 3`
2`
1
7` 7`
N N
8` 1` 8` 1`
1- Quinidine
2- Qinine
68
2- Separation of Xanthines
alkaloids
O O O
H
N 6 5 N 5 N
6 5 N 7 6
HN
1 7 1 N
1 7
8 8 8
2 9 2 9 2 9
3 3 4 N 3 4
4 N O N O N
N
O N H
1- Theobromine 3- Theophylline
3,7 Dimethylxanthine 2- 1,7 Dimethylxanthine 69
1,3 Dimethylxanthine
3- 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
70
4- 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%.
71
5- Separation of
chloramphenicol from mixture
of antibiotics
72
6- Separation of mixture of alkaloids
1- Codeine
2- Strychnine
3- Papaverine
4- Quinine
5- Quinidine
73
II- Quantitative (assay) and
qualitative determination of
natural products
Quantification of compounds by HPLC
Is the process of determination of
the unknown concentration of a
compound in a known solution.
By external (internal) standard
methods
Identification of compound by HPLC
through
Comparison of retention time with
authentic
Comparison of UV spectrum of the 74
Quantification of oroidin in
Axinella damicornis sponge
(assay)
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.
it was quantified by external
calibration method.
75
Steps of assay
1- Quantification was done by injection of
different known conc. of oroidin
(authentic).
2- Determination of the peak area for each
concentration
3- Followed by drawing the standard curve
(area under the peak against conc.).
4- Injection of known weight of the sponge
extract and find the area under the peak.
5- From the standard curve find the
corresponding concentration of oroidin in
the injected weight.
6- Calculate the weight of oroidin in the 76
Standard curve of oroidin
160 mg was found to be the conc. of oroidin in one gram
of the sponge 77
HPLC
TROUBLESHOOTING
POSSIBLE CAUSES
SOLUTION
78
NO PEAKS OR VERY SMALL PEAKS
What you expect
What you got
79
NO PEAKS OR VERY SMALL PEAKS
Possible causes Solution
Detector off Check Detector
Broken connections to recorder Check connections
No sample/Wrong sample Check sample. Be sure it is not
deteriorated.
Check for bubbles in the vials
Wrong settings on recorder or Check attenuation
detector
No flow Check pump, line (connection lines)
80
Retention times are not constant
What you expect
What you got
81
Retention times are not constant
82
Peak tailing
What you expect
What you got
83
Peak tailing
84
Peak fronting
What you expect
What you got
85
Peak fronting
86
Base line drift
What you expect
What you got
87
Base line drift
88
Broad peaks
What you expect
What you got
89
Broad peaks
90
Negative peaks
What you expect
What you got
91
Negative peaks
Possible Causes Solution
Refractive index of mobile Change mobile phase
phase higher than that of
solute
Mobile phase more absorptive Use mobile phase that is transparent at the
than sample components wavelength used
Recorder connections Check polarity
Vacancy peaks. Originate from great difference in
composition between sample solvent and
mobile phase
- Dissolve sample in mobile phase
92
Ghost peaks
What you expect
What you got
93
Ghost peaks
94
Base line noise
What you expect
What you got
95
Base line noise
96
Peak splitting
What you expect
What you got
97
Peak splitting
98
