Introduction to HPLC
High
Performance/Pressure
Liquid
Chromatography
Prepared By: Dr. Radwan Almofti
What is HPLC?

A separation technique based on the
differential affinity of sample components
between the solid stationary phase and the
liquid mobile phase

For the purposes of identification, and/or
quantification of these components
DEFINITIONS

Chromatography: a technique used to separate the components of mixtures of
chemical compounds based on different rates of travel through a stationary phase
resulting from the differential affinities of these components of mixtures for the
stationary and for the mobile phases. The aim is to identify and/or quantify these
components.

Differential Affinities: The difference between the attractions of the components of
a mixture to the mobile phase and to the stationary phase and the rate of the
migration of these components between the two phases.

Attraction examples are:
1- Polarity
2- Solubility
3- Ion Exchange
4- Size exclusion

Chromatograph: An equipment that uses chromatography

Chromatogram: The visual pattern of separated components of a mixture (sample)
obtained by chromatography
History of HPLC

Liquid Chromatography (LC) was firstly
defined in 1906 by the work of M.S. Tswett on
the separation of leaf pigments.

The revolutionary High Pressure LC (HPLC)
was firstly described by Prof. C. Horvath in
1970.

In 2004, Ultra High Performance LC (UPLC)
was introduced.
Advantages of HPLC
HPLC is the primary technique used in
Pharmaceutical Quality Control lab because
it is:
 Sensitive
 Selective
 Low sample consumption
 Moderate analytical conditions
 Superior capability and reproducibility
 Automation
Components of HPLC
Detector
Column
Pump
Mobile
phase
Injector
Drain
Data processor
Degasser
MOBILE PHASE (ELUENT)

Could be polar (water, buffer, methanol, acetonitrile),
non-polar (hexane and benzene) or mixture of both

Purity (HPLC grade)

Detector Compatibility (undetectable)

Solubility of Sample (to carry it throughout the system)

Low Viscosity (to avoid building pressure)

Chemical Inertness (Analytes stability)

Reasonable price (HPLC uses large volume)
DEGASSER

To remove dissolved air in the mobile phase

Dissolved air causes:
 Unstable delivery by pump
 More baseline noise and drift
There are two main types:
 Offline degasser:

Using ultrasonic along with decompression by aspirator
before connecting it to HPLC system
 Online degasser:
Using helium purge or gas-liquid separation membrane
while mobile phase is connected to HPLC system
PUMP
The role of the pump is to force the mobile
phase through the column. Two types:
1) Constant pressure (fluctuation flow, less use)
2) Constant flow (most common) because of its
ability to deliver:
- Constant mobile phase “ISOCRATIC”
- Varying mobile phase “GRADIENT”
INJECTOR
Sample is introduced to the HPLC system
through the injector.
There are two types of injectors:


Manual injectors
Autosamplers
Manual Injector
From pump
Loop
Loop
From pump
To column
LOAD
To column
INJECT
Autosampler
From
pump
To column
To column
From
pump
Needle
Sample vial
Measuring
pump
LOAD
INJECT
COLUMN

Type 316 Stainless Steel

Inner diameter range 2.6 – 5 mm

Length 10 – 30 cm

Packed with the stationary phase (usually silica gel
or its derivatives) of particle size range of 3 to 10 µm
and pore size range of 60 to 100 A°
FRITS
Frits are filters and have two main types:
1.Column frits: one at the beginning (inlet frit) and one at the end (outlet frit) of the column.
Made of Stainless Steel 316 grade to be inert, resistant to corrosion and withstand high pressure.
pore size range from 0.5 to 2 um (depending on the stationary phase particle size)
Primary functions are:
Filter particulate contaminants (microbial and inorganic) to protects the column
Retain packing (stationary phase) in column
Complete distribution of sample uniformly into column
Provide uniform flow of sample into column
2) Frit for purge valve:
Made of Polytetrafluoroethylene (PTFE) polymer.
Filter particulate contaminants
See this youtube: https://www.youtube.com/watch?v=mt3wcpEv1Wc
Types of Stationary Phases
1)
Normal Phase: Silica gel - Polar
2)
Reversed Phase: Silica derivatized - Non-polar
3)
Ion Exchange: Silica or resin derivatized to
have anionic or cationic active sites
4)
Size Exclusion: To separate polymers per their
MWs
NORMAL PHASE

Polar Stationary Phase
Silica gel: -Si-OH or its derivatives such as:


Non-Polar Mobile Phase
•
•

Amino type: -Si-CH2CH2CH2NH2
Basic solvent (hexane, benzene)
Additive solvent to adjust polarity to improve separation
(ethanol, methanol).
Used for Non or Low Polar Analytes

The highest polar analytes have the highest affinity to polar
stationary phase therefore they exit the column the last
REVERSED PHASE

Non or Low Polar Stationary Phase
•
•
Most common is silica gel bond to aliphatic chain
Most common is (ODS) silica bond to OctaDecyl (-C18H37)
Si
-O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3
C18 (ODS)

Polar Mobile Phase
•
•
•
Water has the highest polarity
Less polar solvents commonly used: methanol, acetonitrile,
tetrahydrofuran
Buffer is added if pH is important for separation
REVERSED PHASE (Continue)

Used for Polar Analytes



Most drugs are polar salts soluble in water.
Therefore, reserves phase HPLC is the most common in
pharma QC.
The highest polar analytes have the lowest affinity to the nonpolar stationary phase thus exit the column the first and vice
versa.
C18 (ODS)
OH
Weak
Strong
CH3
NORMAL PHASE VS REVERSED PHASE
Phase Type
Stationary
Mobile
Phase (SP) Phase (MP)
Separation - Affinity
Reversed
Phase
Non-polar
Polar
•Polar analytes have weak affinity to SP & strong
affinity to MP thus exit early
•Non-polar analytes have strong affinity to SP &
weak affinity to MP thus exit late
Normal
Phase
Polar
Non-polar
•Polar analytes have strong affinity to SP & weak
affinity to MP thus exit late
•Non-polar analytes have weak affinity to SP &
strong affinity to MP thus exit early
DETECTOR
1) Ultraviolet-Visible (UV-VIS):
Based on the principle that the emission absorbance is
proportional to the concentration of the absorbing analytes
Detection cell
C: Concentration
Ein
A
Eout
l
A = e·C·l = –log (Eout / Ein)
(A: absorbance, E: absorption coefficient)
C
DETECTOR (Continue)
Ultraviolet-Visible (UV-VIS) has three types:
A- Fixed one wavelength
B- Variable wavelength
Ability to select the wavelength at which the analyte
has highest absorbance
C- Photo Diode Array (PDA)
Ability to detect the absorbance across the entire UV
spectrum
Variable wavelength and PDA detectors are the most
common used ones in pharma QC.
DETECTOR (Continue)
2) Florescence:
For analytes that once excited emit
florescence proportionally to their concentration
3) Conductivity:
For ion-exchange chromatography
4) Electrochemical:
Detect the current generated from the
oxidation/reduction reaction of the analytes.
Data Collection and Processing

Using Software (Empower for Waters HPLCs)

Collect the output from the detector (HPLC: 1 to 5 data
points per second)

Plot the detector response (UV absorbance) over time
(Chromatogram)

Process data collected and present the required
results (identity, concentration)

Data are stored in local computers, servers or over
network (Lab Information Management System
“LIMS”)
Intensity of detector signal (mAU)
CHROMATOGRAM
tR
Peak
t0
t0: Non-retention time
h
A
Time
Injection
tR: Retention time
A: Peak area
h: Peak height
CAPACITY (K’)

The ratio of the elution time for the analyte (tR) to that
for the non-retained one (t0).

Its value depends on the affinity of the analyte to the
stationary phase compared to that to the mobile phase

Calculated as follows:
Detector Response
K’ = (tR – t0)) / t0
tR
t0
Time
tR: Retention time
t0: Non-retention time
SELECTIVITY (α)
 The ability of HPLC stationary phase to separate two
components in a sample
 Calculated as the ratio of the capacity factors k’
α = k2’/k1’ = (tR2 - to) / (tR1 - to)
EFFICIENCY (N)
 The width (sharpness) of the peak that represents the
suitability of HPLC stationary phase to perform separation
 Defined in terms of Number of Theoretical Plates (N) and
calculated as:
N = 16 X (tR / Wb)2
(tR is retention time and Wb is peak width at baseline)
RESOLUTION (R)
 The most important HPLC performance measurement
 Defined as the extent two compounds can be separated
 Calculated as:
R = 2 X (tR1 – tR2) / (Wb1 + Wb1)
 Required to be ≥ 1.5
tR1
tR2
Wb1
Wb2
PEAK TAILING
 A measure of peak symmetry
 Calculated as:
T = W0.05 / (2 X f )
(W0.05 is peak tail width and f is peak front width both at 0.05 height)
 T value for ideal symmetrical peak is 1
 Acceptable range 0.9 – 1.2
SYSTEM SUITABILITY
 To verify that HPLC system is adequate for the analysis
 Two parts:
1) Performance Measurements:
To ensure that capacity, resolution and tailing
values as determined by test method are adequate to
ensure successful analysis
2) System Reproducibility:
• The ability of HPLC system to reproduce the desired
injection volumes
• Relative standard deviation (RSD) ≤ 2% for 5 replicate
injections of primary standard (USP 621)
• For multiple samples, a check standard after 6 sample
injections with a recovery of 100.0 ± 3.0% (USP 621)
Chromatographic Troubleshooting
 Baseline fluctuation/noise
 Baseline drifting
 Ghost peaks
 Peak doublets
 Peak broadening
 Peak tailing
 Peak fronting
 Negative peaks
Baseline Fluctuation/Noise
Causes for baseline noise are:
 Detector problem (dirty flow cell, air bubbles, lamp
failing)
 Method (mobile phase change, insufficient degassing,
system not washed properly between runs, dirty solvent)
 Pump (mixing issue, pulse)
Baseline Drifting
 Could be upward or downward drifting
 Causes for baseline drifting are:
 Gradient elution issue
 Unstable temperature
 Mobile phase contamination
 Mobile phase not in equilibrium with column
 System bleed
Upward drifting
Ghost Peaks
 Peaks appears even no sample injection is made.
 Causes for ghost peaks are:
 Dirty mobile phase
 Dirty column
 System is not washed properly between runs
Peak Doublet
 All peaks doublets, possible causes are:
 Void volume in column
 Partially blocked frit
 One peak doublet, possible cause is:
 Co-eluting components
Peak Broadening
 All peaks broadening, possible causes are:
 Loss column efficiency
 Void volume in column
 Large injection volume
 High mobile phase viscosity
 Some peaks broadening, possible cause is:
 Late elution from previous sample
 High MW (Polymer)
Peak Tailing
 Small peak eluting on tail of larger peak
 Residual silanol interaction
 Column contamination
 Bad column
Peak Fronting
Usually due to sample overload
Negative Peak
Usually due to the absorbance of the sample being less
than the mobile phase
Pharmaceutical Quality Control
Guidelines
 Canada: Food and Drug Regulations
Part C, Division 2 (GMP guidelines)
Sections: C-02-009/010/016-019
 USA: Code of Federal Regulations, Title 21
Food and Drugs, part 211 (cGMP guidelines)
Subpart I (Laboratory Control) & Subpart J (211-194
Laboratory Records)
 The United State Pharmacopeia (USP), European
Pharmacopeia (EP) and Japanese Pharmacopeia (JP)
are approved guidelines by the regulators
Pharmaceutical Quality Control
Guidelines (continue)
Canadian GMP and US cGMP have similar main
guidelines for quality control
1) The timely and full permanent documentations of all
materials, equipment and procedures used, test
results, name of analyst and date of each entry
2) The timely and full documented qualification and
calibration of all equipment used (as applicable)
THANK YOU…