Hplc cont

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
September 29, 2014 1

CONTENTS
Introduction.
Modes of separation.
Instrumentation.
HPLC method development,optimization and
validation.
References.

INTRODUCTION
Most of the drugs in multicomponent dosage forms can be
analyzed by HPLC method because of the several
advantages like:
Improved resolution of the separated substances
Faster separation times and
The improved accuracy, precision, & sensitivity with
which the separated substances may be quantified.

MODES OF SEPARATION IN HPLC
There are different modes of separation in HPLC:
1.Normal phase mode.
2.Reversed phase mode.
3.Reversed phase ion pair chromatography.
4.Affinity chromatography.
5.Size exclusion chromatography

NORMAL PHASE MODE
Stationary phase-polar in nature.
Mobile phase-non polar in nature.
Generally used for separation of non polar compounds.

REvERSED PHASE MODE
Stationary phase-non polar .
Mobile phase-polar in nature.
Generally used for separation of polar compounds.

ION EXCHANGE CHROMATOGRAPHY :
The stationary phase contains ionic groups like NR⁺з, SO⁻з
which interact with the ionic groups of the sample
molecules.
This method is suitable for the separation of charged
molecules only.
ION PAIR CHROMATOGRAPHY :
This may be used for the separation of ionic compounds.
Strong acids & basic compounds may be separated by
reversed phase mode by forming ion pairs with suitable
counter ions.

AFFINITY CHROMATOGRAPHY:
• It uses highly specific biochemical interactions for
separations.
• The stationary phase contains specific groups of
molecules which can absorb the sample if certain steric &
charge related conditions are satisfied.
• This technique can be used to isolate proteins, enzymes,
as wel as antibodies from complex mixture.
SIZE EXCLUSION CHROMATOGRAPHY:
• Separates molecules according to their molecular mass.
• Largest molecules are eluted first and smaller molecules
last.

PRINCIPLE OF SEPARATION
The principle of separation is adsorption.
Separation of components takes place because of the
difference in affinity of compounds towards stationary
phase.

INSTRUMENTATION

vARIOUS COMPONENTS OF HPLC
1. A solvent delivery system including pump.
2. Sample injection system
3. A chromatographic column
4. Detectors
5. Recorders and integrators

A SOLvENT DELIvERY SYSTEM
A mobile phase is pumped under pressure from one or
several reservoir and flows through the column at a
constant rate.
For normal phase separation eluting power increases
with increasing polarity of the solvent but for reversed
phase separation, eluting power decreases with
increasing polarity.
A degasser is needed to remove dissolved air and other
Septegmbaesr 2e9,s 2 0f1r4om the solvent. 12

vARIAN SOLvENT DELIvERY SYSTEMS

PUMPS
The pump is one of the most important component of
HPLC, since its performance directly affects retention
time, reproducibility and detector sensitivity.
Three main types of pumps are used in HPLC.
1.Displacement pump
2.Reciprocating pump
3.Pneumatic or constant pressure pump

DISPLACEMENT PUMP: It produce a flow that
tends to independent of viscosity and back pressure and
also output is pulse free but possesses limited capacity
(250ml).
RECIPROCATING PUMP: It has small internal
volume (35-400μl), their high output pressure(up to
10,000psi) and their constant flow rates. But it produces
a pulsed flow.
PNEUMATIC OR CONSTANT PRESSURE
PUMP: They are pulse free . Suffer from limited capacity as well
as a dependence of flow rate on solvent viscosity and column back
pressure. They are limited to pressure less than 2000psi.

SAMPLE INJECTION SYSTEM
There are three important ways of
introducing the sample in to the
injection port.
Loop injection : in which a fixed amount
of volume is introduced by making use
of fixed volume loop injector.
Valve injection: in which, a variable
volume is introduced by making use of
an injection valve.
On column injection: in which, a
variable volume is introduced by
means of a syringe through a septum.

Varian 9010 Solvent Delivery
System
Rheodyne
Injector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
A
C
B
Ternary Pump
from solvent
reservoir
Column
to
detector
to column
through
pulse
dampener
to injector
through pump
load
inject

CHROMATOGRAPHIC COLUMN
• The column is usually made up of heavy glass or stainless
steel tubule to withstand high pressure .
• The columns are usually 10-30cm long and 4-10mm inside
diameter containing stationary phase at particle diameter of
25μm or less.
• Column with internal diameter of 5mm give good results
because of compromise between efficiency, sample capacity,
and the amount of packaging and solvent required.

DETECTORS
The function of detector in HPLC is to monitor the mobile
phase as it merges from the column.
Detectors are usually of two types:
1. Bulk property detectors: It compares overall changes
in a physical property of the mobile phase with and
without an eluting solute e.g. refractive index ,dielectric
constant or density.
2.Solute property detectors: It responds to a physical
property of the solute which is not exbited by the pure
mobile phase.e.g.UV absorbance,fluoroscence or
diffusion current.

TYPES OF DETECTORS
There are mainly 4 types of detectors are used in HPLC:
1. Photometric detectors.
 Single wavelength detectors.
 Multiwavelength detectors.
 Variable wavelength detectors.
 Programmable detectors.
 Diode array detectors .
2. Fluorescence detectors.
3. Refractive index detectors.
4. Electrochemical detectors.

PHOTOMETRIC DETECTORS
• These normally operate in the ultra violet region of the
spectrum .
• Most extensively used in pharmaceutical analysis.

SINGLE WAVELENGTH DETECTORS
Equipped with a low pressure mercury discharge lamp.
The absorbance is measured at the wavelength of
mercury at 254nm.

MULTIWAVELENGTH DETECTORS
• Employ mercury and other discharge sources.
• When used in combination with interference filters
,allow a no of monochromatic wavelengths to be
selected e.g. 206, 226, 280 , 313, 340 or 365 nm.

Multi-wavelength uv-vis absorption Detector
Deuterium
Lamp
Photodiode
Array

variable wavelength Detectors
• Use a deuterium light source.
• A grating monochromator to allow selection of any
wavelength in deuterium continuum (190-360nm).

variable wavelength Detector

ABS AUFS l RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min
Ready

prograMMable Detectors
Allow the automatic change of wavelength between and
during the chromatographic analysis.

DioDe arraY Detectors
They are microprocessor – controlled photodiode array
spectrophotometers in which light from an UV source passes
through the flow cell into a polychromator which disperses
the beam so that the full spectrum falls on the array of
diodes.

FLUORESCENCE DETECTORS
• These are essentially filter fluorimeter or spectro
-fluorimeters equipped with grating monochromators,
and micro flow cell.
• Their sensitivity depends on the fluorescence
properties of the components in the eluate.

Fluorescence Detector

REFRACTIVE INDEX DETECTORS
• Which respond to the change in the bulk property of the
refractive index of the solution of the component in the
mobile solvent system.
• The sensitivity of the refractive index detector is much
less than that of specific solute property detectors, they
are useful for the detection of substances(e.g
,carbohydrates& alcohols) which do not exhibit other
properties that can be used as the basis for specific
detection.

refractive inDex Detector

electrocheMical Detectors
• These are based on standard electrochemical principles
involving amperometry,voltametryand polarography.
• These detectors are very sensitive for substances that
are electroactive ,i.e. those that undergo oxidation or
reduction .
• They have found particular application in the assay of
low levels of endogenous catecholamines in biological
tissues,pesticides,tryptophan derivatives and many
drugs.

electrocheMical Detector

introDuction:
ANALYTICAL METHOD DEVELOPMENT:
• Method development usually requires selecting the
method requirements and deciding on what type of
instrumentation to utilize and why.
• The wide variety of equipment, columns, eluent and
operational parameters involved makes HPLC method
development .

There are several reasons for developing new methods of
analysis:
1. A suitable method for particular analyte in the specific
matrix is not available.
2.Existing methods may be too error or they may be
unreliable (have poor accuracy or precision)
3.Existing methods may be too expensive, time
consuming.

HPLC method development generally follows the following
steps:
Step 1-selection of the HPLC method and initial system.
Step2-Selection of optimum conditions.
Step3-selectivity optimization.
Step4-system parameter optimization.
Step5-method validation.

Step 3a
Initial HPLC
condition
Step 2
Sample
Step 1 preparation
Method goals and
chemistry
Step 3b
Optimize HPLC
separation
Step 4
Standardization
Step 5
Method validation
Fig 2.2 Pie diagram showing the time that should be spent on different steps of
the method development to meet the commended timeline. The sequence of events
and percentage of time allocated is only suggestive.

step1-selection of hplc MethoD anD
initial conDitions
SELECTION OF HPLC METHOD:
• When selecting an HPLC system it must have a high propability
of actually being able to analyse the sample.
• For example if the sample includes polar analytes then RP-HPLC
would offer both adequate retention and ressolution.
consideration must be given to the following ;
a. sample preparation
b.Types of chromatography
c. Column selection
d.Detector selection
e. Selection of mobile phase composition September 29, 2014 43

saMple preparation
Sample preparation is an essential part of HPLC
analysis, to provide a reproducible and homogenous
solution i.e. suitable for injection on to the column.
The aim of sample preparation is a sample that :
1. It is relatively free of interferences
2.Will not damage the column
3.Should compatible with the intended HPLC method ;

TYPES OF CHROMATOGRAPHY
Reversed phase is the choice for the majority of the
samples.
But if acidic or basic analytes are present reversed
phase ion suppression (for weak acids and bases) or
reversed phase ion pairing (for strong acids and bases)
should be used.
For low or medium polarity analytes normal phase
HPLC is used, particularly if the separation of isomers
is required.
For inorganic anion or cation analysis ion exchange
chromatography is best.

Size exclusion chromatography would normally be
considered for analyzing high molecular weight
compounds.
Gradient HPLC only a requirement for complex samples
with a large number of components (20-30) .
Reversed phase HPLC is commonly used in peptide and
small protein analysis using an acetonitrile –water
mobile phase containing 1% trifluoroethanolic acid.

COLUMN SELECTION
A column is chosen based on the
Knowledge of sample
On the expectation of how its components will interact
with the packing material.
 the properties of column packing material.

COLUMN SELECTION
1.Knowledge of the Sample: which influences the
choice of Column Bonded Phase characteristics
Knowledge of the sample
• Structure of sample components?
• Number of compounds present?
• Sample matrix?
• pKa values of sample components?
• Concentration range?
• Molecular weight range?
• Solubility?
• Other pertinent data?
Column Chemistry
(bonded phase, bonding
type, endcapping,
carbon load)
}

Pac king material:-
Most HPLC separations are performed on bonded
phase HPLC columns
Octadecyl-derivatized silica gel columns are the most
widely used bonded phase columns in the reverse phase
mode.
Commonly used polar bonded phases certain diol,cyano
or amino functional groups.
Silica based packing materials are used in about 75% of
all HPLC separations performed today.

• due to
 the physical stability
 the availability of bonded phase
 and to the high efficiency of silica based HPLC columns
• Many new resin packings have been introduced in recent
years particularly for biochemical analysis.
• The most well known resin based packings are formed by
the co polymerization of polysterene and divinyl benzene.
• Other packing materials such as alumina, titania and
zirconia have also been employed for bio polymer analysis.

COLUMN DIMENSIONS
Effect on chromatography
Column Dimension
• Short (30-50mm) - short run times, low backpressure
• Long (250-300mm) - higher resolution, long run times
• Narrow (£ 2.1mm) - higher detector sensitivity
• Wide (10-22mm) - high sample loading

DETECTOR SELECTION
Consideration must given to the following:
Do the analytes have chromophores to enable UV detection ?
Is more selective or sensitive detection required?
What detection limits are necessary ?
Will the sample require chemical derivatization to enhance
detect ability and /or improve the chromatography.

DETECTOR SELECTION
A HPLC detector will have a number of performance
characteristics that need to be specified and known before a
particular detector can be chosen for a specific application
and are listed as follows.
1. Dynamic range
2. Response index or linearity
3. Linear dynamic range
4.Detector response
5. Detector noise level
6.Detector sensitivity, or minimum detectable concentration.
7.Total system dispersion
8.Pressure sensitivity
9.Flow arte sensitivity.
10.Operating temperature range. 53
September 29, 2014

THE UV DETECTOR
Limited to the detection of those substances that absorb
light in the UV wave length range.
UV detectors detects all sample components that contain
chromophores.
Specifications:
S.NO CHARACTERISTIC FIXED WAVELENGTH
UV DETECTOR
DIODE ARRAY
DETECTOR
1 Sensitivity
(solute benzene)
5×10ˉ⁸gml 1×10ˉ⁷gml
2 Linear dynamic
range
5×10ˉ⁸ to
5×10ˉ⁴gml
10ˉ⁸to
5×10ˉ⁴gml
3 Response index 0.98 - 1.02 0.97- 1.03

THE FLUORESCENCE DETECTOR
It can detect eluted solutes on the basis of fluorescence
,but it can also provide their fluorescence spectra.
Fluorescence and electro chemical detectors should be
used for trace analysis.
Specifications:
S.No CHARACTERISTIC FLUOROSCENCE
DETECTOR
1. Sensitivity
(solute anthracene)
1×10ˉ⁹gml
2 Linear dynamic range 1×10ˉ⁹ to 5×10ˉ⁶gml
3 Response index 0.96 – 1.04

ELECTRICAL CONDUCTIVITY DETECTOR
It is usually employed with an ion suppressor column to
allow salts and buffers to be used in the mobile phase
without affecting the detector output.
Specifications:
S.NO CHARACTERISTIC CONDUCTIVITY
DETECTOR
1. Sensitivity(sodium
chloride)
5×10ˉ⁹gml
2. Linear dynamic range 5×10ˉ⁹to 1×10ˉ6gml
3. Response index 0.97-1.03

REFRACTIVE INDEX DETECTOR
It is one of the least sensitive LC detectors and is used
circumstances where other detector s are inappropriate.
For preparative HPLC it is preferred because it can
handle concentration without overloading the detector
Specifications:
S.NO CHARACTERISTIC RI DETECTOR
1. Sensitivity(solute
benzene)
1×10ˉ⁶g/ml
2. Linear dynamic range 1×10ˉ⁶ -1×10ˉ⁴g/ml
3. Response index 0.97-1.03

MOBILE PHASE SELECTION
The organic phase concentration required for the mobile
phase can be estimated by gradient elution method.
Gradient can be started with 5-10 % of the organic phase in
the mobile phase and the organic phase concentration can
be increased up to 100% within 30-45%.
The elution strength of a mobile phase depends upon its
polarity, the stronger the polarity higher is the elution.

Ionic samples(acidic or basic) can be separated if they
are present in undissociated form. Dissociation of ionic
samples may be suppressed by the selection oh pH.
If the retention times are too long an increase of the
organic phase concentration is needed.
When tailing or fronting is observed, it means that the
mobile phase is not totally compatible with the solutes.

SELECTION OF INITIAL SYSTEM
It could based on :
 assessment of the nature of the sample and analytes
together with literature data.
Experience
Expert system software and
Empirical approaches

STEP 2 : SELECTION OF INITIAL CONDITIONS
• This step determines the optimum conditions to adequately
retain all analytes ; i.e.
Ensures no analyte has a capacity factor of less than
0.5(poor retention could result in peak overlapping).
No analyte has a capacity factor greater than 10-15
(excessive retention leads to long analysis time and broad
peaks with poor detectability).
Determination of initial conditions:
• The recommended method involves performing two gradient
runs differ in only in the run time.
• A binary system based on either aceto nitrile/ water or
methanol/water should be used.

STEP 3: SELECTIVITY OPTIMIZATION
The aim of this step is to achieve adequate selectivity.
The mobile phase and stationary phase compositions
need to be taken in to account.
To select these the nature of the analytes must be
considered.
Once the analyte types are identified the relevant
optimization parameters may be selected.

STEP 4: SYSTEM PARAMETER OPTIMIZATION
This is used to find the desired balance between
ressolution and analysis time after satisfactory selectivity
has been achieved.
The parameters involve include column dimensions,
column packing particle size and flow rate.
This parameters may be changed without affecting
capacity factors or selectivity.

TYPES OF OPTIMIZATION
Two types :
Manually and
By using soft wares.
By Manual : separation then can be optimized by change in the
initial mobile phase composition and the slope of the gradient
according to the chromatogram obtained from the preliminary
run.

BY USING SOFT wARES :
Chemo metric protocols available for the development and
optimization of HPLC methods:
Experimental Design (ED)
 Factorial design
 Plackett-Burman design
 D-optimal design
 Two-level full factorial design
 Central composite design
 Box-Behnken design
 Doehlert design
Multi-Criteria Decision Making (MCDM)
 Overlay plots
 Pareto optimality
 Utility function
 Derringer’s desirability function

STEP 5 METHOD VALIDATION

The objective of validation of an analytical procedure is
to demonstrate that it is suitable for its intended
purpose.

A brief description of types of tests considered in this
document is provided below:-
Identification tests are intended to ensure the identity
of analyte in a sample. This normally achieved by
comparing the properties of sample with that of the
reference standard.
Testing for impurities can be either a quantitative test
or a limit test for the impurity in a sample.
Assay procedures are intended to measure the analyte
present in a given sample.

• Typical validation characteristics which should be
considered are listed below:-
1.Accuracy
2. Precision
a. repeatability
b. intermediate precision
3. specificity
4. Detection limit
5. Quantitation limit
6. Linearity
7. range.

ACCURACY
The accuracy of an analytical procedure expresses the
closeness of agreement between the value which is
accepted either as a conventional true or an accepted
reference value and the value found.
 Accuracy should be assessed using a minimum of 9
determinations over a minimum of 3 concentration
levels covering the specified range

LINEARITY
 The linearity of an analytical procedure is its ability
to obtain test results which are directly proportional to
the concentration of analyte in the sample.
 Linearity should be evaluated by visual inspection of a
plot of signals as a function of analyte concentration or
content.
For the establishment of linearity, a minimum of 5
concentrations is recommended.

PRECISION
The precision of an analytical procedure expresses the
closeness of agreement between the measurements
obtained from multiple sampling of the same
homogenous sample under the prescribed conditions.
repeatability
Precision intermediate precision
reproducibility

Repeatability:- Expresses the precision under the
same operating conditions over a short interval of time .
Repeatability is also termed intra- assay precision.
• a) a minimum of 9 determinations covering the specified
range for the procedure (e.g., 3 concentrations/3
replicates each);
or
• b) a minimum of 6 determinations at 100% of the test
concentration.

Intermediate precision:- intermediate precision
expresses within – laboratories variations: different days,
different analysts, different equipment e.t.c.
Reproducibility:- reproducibility expresses the precision
between laboratories.
• The standard deviation, relative standard deviation
(coefficient of variation) and confidence interval should
be reported for each type of precision investigated.

DETECTION LIMIT
 The detection limit of individual analytical procedure is
the lowest amount of analyte in a sample which can be
detected but not necessarily quantitated as an exact
value.
Several approaches for determining the detection limit
are possible, depending on whether the procedure is a
non-instrumental or instrumental. Approaches other than
those listed below may be acceptable

Based on Visual Evaluation
Visual evaluation may be used for non-instrumental
methods but may also be used with instrumental methods.
The detection limit is determined by the analysis of
samples with known concentrations of analyte and by
establishing the minimum level at which the analyte can
be reliably detected.
Based on Signal-to-Noise
This approach can only be applied to analytical
procedures which exhibit baseline noise.
Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known
low concentrations of analyte.
A signal-to-noise ratio between 3 or 2:1 is generally
considered acceptable for estimating the detection limit.

Based on the Standard Deviation of the Response
and the Slope
The detection limit (DL) may be expressed as:
DL = 3.3 σ /s
Where, σ = the standard deviation of the response
S = the slope of the calibration curve

QUANTITATION LIMIT
• The quantitation limit of an individual analytical
procedure is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accuracy.
• The quantitation limit is a parameter of quantitative
assays for low levels of compounds in sample matrices
and is used particularly for the determination of
impurities and or degradation products.

Based on Visual Evaluation
Visual evaluation may be used for non-instrumental
methods but may also be used with instrumental methods.
The detection limit is determined by the analysis of
samples with known concentrations of analyte and by
establishing the minimum level at which the analyte can
be reliably detected.
Based on Signal-to-Noise
This approach can only be applied to analytical
procedures which exhibit baseline noise.
Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known
low concentrations of analyte.
A signal-to-noise ratio between 10:1 is generally
considered acceptable for estimating the detection limit.

Based on the Standard Deviation of the Response
and the Slope
The quantitation limit (QL ) may be expressed as:
QL = 10 σ /s
Where, σ = the standard deviation of the response
S = the slope of the calibration curve

RANGE
The range of an analytical procedure is the interval
between the upper and lower concentration of the analyte
in the sample for which it has been demon started that the
analytical procedure has a suitable level of precision,
accuracy and linearity.
The following minimum specified ranges should be
considered:

for the assay of a drug substance or a finished (drug)
product: normally from 80 to 120 percent of the test
concentration;
 for content uniformity, covering a minimum of 70 to 130
percent of the test concentration, unless a wider more
appropriate range, based on the nature of the dosage
form (e.g., metered dose inhalers), is justified;
for dissolution testing: +/-20 % over the specified range;

ROBUSTNESS
The robustness of an analytical procedure is a
measure of its capacity to remain unaffected by small,
but deliberated variations in method parameters and
provides an indication of its reliability during normal
usage.

REfERENCES
High performance liquid chromatography; principles and
methods in biotechnology by Elena.d katz .
Practical pharmaceutical chemistry part 2 fourth edition by
A.H.Beckett, J.B.stenlake; p.no 157.
Instrumental methods of chemical analysis by
Gurdeep.R.Chatwal, Sham.K Anand, p.no 2.624-2.639.

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