High Performance Liquid
Chromatography
Introduction
• HPLC is a form of liquid chromatography used to separate compounds that are
dissolved in solution.
• 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.
HPLC system
Picture of HPLC instrument
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.
HPLC Chromatograph injectors
• The function of the injector is to place the sample into the high-pressure flow
in as narrow volume as possible so that the sample enters the column as a
homogeneous, low-volume plug.
• To minimize spreading of the injected volume during transport to the
column, the shortest possible length of tubing should be used from the
injector to the column.
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.
• The High performance liquid chromatography collumns are made out of stainless steel
tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm.
• 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.
Picture of an HPLC column
Packing materials in HPLC Column
• HPLC columns are usually packed with pellicular, or porous particles.
• Pellicular particles are made from polymer, or glass beads.
• Pellicular particles are surrounded by a thin uniform layer of silica,
polystyrene-divinyl-benzene synthetic resin, alumina, or other type of ion-
exchange resin.
WHAT AFFECTS SYSTEM
Column Parameters Instrument Parameters
• Column Material • Temperature
• Deactivation • Flow
• Stationary Phase • Signal
• Coating Material • Sample Sensitivity
• Detector
Sample Parameters
• Concentration
• Matrix
• Solvent Effect
• Sample Effect
Types of HPLC
• 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 (stationary phase) 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.
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.
Types of Detectors
• Absorbance (UV with Filters, • Evaporative Light Scattering
UV with Monochromators) Detector (ELSD)
• IR Absorbance • Electrochemical
• Fluorescence • Mass-Spectrometric
• Refractive-Index • Photo-Diode Array
EVALUATION PARAMETERS
• Retention Time
• Retention Volume
• Separation Factor
• Resolution
• Height Equivalent To A Theoretical Plate (HETP)
• Efficiency
• Retention Index
• Column Bleed
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/solvent 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
3. Resolution:
Resolution is the ability of the column to separate peaks on the chromatograph.
It is the measure of extent of separation of 2 components and expressed as the
ratio of the distance between two peak maxima to the mean value of the peak
width at the base line
R
Where, tR1 and tR2 are retention time of component 1 and 2. tw1 and tw2 are the
component peak widths. If Rs is equal to or more than 1, then components are
completely separated but if Rs is less than 1, then components overlap.
4. Height Equivalent to a Theoretical Plate (HETP):
•Plate theory concept was introduced to explain efficiency of columns.
•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.
•Any analyte spends a finite time in each plate and this is the equilibrium time.
•A theoretical plate can be of any height, which describes the efficiency of separation.
•If HETP is less, the column is more efficient.
•If HETP is more, the column is less efficient.
HETP = length of the column ÷ no. of theoretical plates (H = L/N)
5. Efficiency:
Efficiency of a column is expressed by the theoretical plates.
n = 16 RT2/ w2
Where, n = no of theoretical plates
RT= retention time
w = peak width at base
If the no. of theoretical plates is high, the column is said to be highly efficient.
For GLC, a value of 600/metre is sufficient. But in HPLC, high values like
40,000 to 70,000/ meter are recommended.
Column efficiency is also affected by column capacity ratio, K.
K = (TA-T0) / T0
TA= retention time of concerned analyte
T = Retention time of unretained peak or void volume peak
Selectivity Factor
• The selectivity (or separation) factor (α) is the ability of the chromatographic
system to 'chemically' distinguish between sample components.
• It is usually measured as a ratio of the retention (capacity) factors (k) of the
two peaks in question and can be visualized as the distance between the
apices of the two peaks.
• K’ values tell us where bands elute relative to the void volume. These values
are unaffected by 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.
• Q1. What are the main differences between High Performance Liquid Chromatography and Gas
Chromatography?
• A:
• In HPLC the mobile phase is a liquid whereas in GC the mobile phase or carrier is a gas.
• HPLC is useful for analysis of samples which are liable to decompose at higher temperatures. GC involves high
temperatures so compounds are stable at such temperatures.
• GC is applied for analysis of volatile compounds whereas non volatile compounds can be easily analyzed on HPLC
• GC cannot be used for analysis of high molecular weight molecules whereas HPLC has applications for separation and
identification of very high molecular weight compounds
• HPLC requires higher operating pressures than GC because liquids require higher pressures than gases for transport
through the system
• HPLC columns are short and wide in comparison to GC columns
• Q 2. Which type of High Performance Liquid Chromatography technique is most widely used?
• A. Reverse phase Chromatography has the widest range of applications. The stationary phase comprises non
polar organic chains bound to inert silica surface and mobile phase comprises of aqueous or aqueous-
organic mixtures comprising of polar solvents of varying degrees of polarity. The elution sequence is polar
followed by less polar and least polar or non polar compounds eluting last through the column.
• Q3. What is the separation principle in Size Exclusion Chromatography?
• A. In size exclusion chromatography the separation does not involve chemical interactions between eluting
molecules and stationary phase. The separation takes place on the basis of molecular size with larger
molecules eluting first and small molecules in the end. Small molecules are retained longer in the pores of
the stationary phase therefore they get eluted last.
• Q4. Why is it necessary to degas the mobile phase?
• A. Mobile phases entrap air from the atmosphere and this trapped air gets released as small bubbles
under high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in detector
response or hinder flow of mobile phase through columns. In order to overcome such problems
degassing of mobile phase becomes essential.
• Q5. Which is the most commonly used detector in High Performance Liquid
Chromatography and why?
• A. The most commonly used detector in HPLC is the UV-VIS detector. The reason for its predominant
use is that it gives specific response to a particular compound or class of compounds. Most of the
organic compounds absorb at specific wavelengths covered in the available wavelength range of the
detector.
• Q6. What do you understand by a bulk property detector and a specific property detector?
• A. A bulk property detector responds to some property of mobile phase and sample combination passing
through it at any point of time such a Refractive index or Electrochemical detector whereas a specific
property detector is responsive only to the characteristic property of the eluting molecule and is
independent of changes in mobile phase composition such as UV-Vis and Fluorescence detectors.
• Q7. What do you understand by Isocratic and Gradient elution?
• A. When the composition of the mobile phase is not changed through the chromatographic run the
operation is termed as isocratic. It can involve a single solvent or a mixture of two or more solvents mixed
in a fixed proportion. In gradient operation the composition at start of run is programmed to change at a
predetermined rate and the composition at the end of run is different from the composition at the start.
• Q8. What are the desirable features of a High Performance Liquid
Chromatography detector?
• A. The desirable features of a detector are:
• Sensitivity towards solute over mobile phase.
• Low dead volume to eliminate memory effects
• Low noise
• Low detection limits
• Large dynamic linear range
• Q9. What are the benefits of Fast LC or UHPLC?
• A. Fast or UHPLC technique makes use of small particles below 2 μ size
Use of such particle sizes result in high resolution and as small columns
can be used it results in completion of analysis in much less time thereby
reducing consumption of expensive solvents.
Method and Validation basics
Content of HPLC test procedure
Any analytical procedure submitted should be described in sufficient detail, includes:
• Preparation of mobile phase
• Chromatographic condition:
Column: type (e.g., C18 or C8), dimension (length, inner diameter), particle
size (10μm, 5 μm)
Elution procedure: isocratic or gradient
Detector: wavelength
elution
Injection volume Preparation of standards and samples
column T Operation procedure: sequence of injections
flow rate System suitability testing (SST) and criteria
Calculations
Compendial methods
When claim a compendial method, there should be no change in:
• The type of column i.e the stationary phases
• Detector wavelength
• Components in Mobile phase
• System suitability testing and criteria
Adjustments to ratio of components in mobile phase, flow rate, column temp,
dimension of column, particle size, may be necessary to achieve the system
suitability criteria.
System suitability testing (SST)
• Precision:
– Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5
– Impurities: in general, RSD ≤ 5% at the limit level, up to 10% or higher at
LOQ, n ≥ 6
• Resolution (R): >2
• Tailing factor/peak asymmetry: (≤ 2)
• Number of theoretical plates (N): column efficiency ≥ 2000
The Theoretical Plate
Theoretical plate is a term coined by Martin & Synge.
•It is based on a study in which they imagined that chromatographic columns were
analogous to distillation columns and made up of numerous discrete but connected
narrow layers or plates.
•Movement of the solute down the column then could be treated as a stepwise
transfer.
Theoretical plates (N) measure how efficiently a column can separate a
mixture into its components. This efficiency is based on the retention time of the
components and the width of the peaks.
= Number of theoretical plates (a measure of e
tR 2
N 16( )
wb
tR
wb
tR is the retention time; it is measured from the injection peak (or zero) to the
intersection of the tangents.
wb is the width of the base of the triangle; it is measured at the intersection of the
tangents with the baseline.
tR
tR
wb wb
Larger N Smaller N
tR 2
N 16( )
wb
When the retention time, tR, is held constant, the column that produces peaks with
narrower bases, wb, will be more efficient – have a greater N value.
Likewise a column that produces wider peaks will be less efficient – have a smaller N
value.
System suitability testing (SST)
• System suitability is a set of pre-defined criteria that evaluate the performance of the
HPLC system, encompassing factors such as resolution, peak symmetry/ tailing factor,
retention time, and peak area reproducibility.
• Adhering to these criteria is essential for consistent and precise analyses, as they confirm
that the instrument is operating optimally and capable of generating dependable data.
• A SST should contain:
• For Assay:
precision + one or more other parameter
• For impurity test:
resolution + precision + one or more other parameter
Validation – compendial methods
Assay – API
No validation generally required. Exception: specificity for major impurities not in
the monograph.
Assay – FPP
Specificity, accuracy and precision (repeatability).
Purity – API and FPP
Full validation for specified impurities that are not included in the monograph
(specificity, linearity, accuracy, repeatability, intermediate precision, LOD/LOQ).
Non-compendial methods
Full validation is required for purity, assay and dissolution methods
(HPLC, UV) :
• Specificity
• Linearity
• Accuracy
• Repeatability
• Intermediate precision
• LOD/LOQ (not required for assay, dissolution)
• Robustness (recommended)
Specificity
• Blank solution to show no interference
• Placebo to demonstrate the lack of interference from excipients
• Spiked samples to show that all known related substances are resolved from
each other
• Stressed sample of about 10 to 20% degradation is used to demonstrate the
resolution among degradation products
– Check peak purity of drug substance by photodiode array detector (PDA)
– Representative chromatograms should be provided with time scale and
attenuation indicated
Linearity / Range
• The working sample concentration and samples tested for accuracy should be in the linear
range (concentrations Vs. Peak areas)
• Minimum 5 concentrations
• Dilute of stock solution or separate weighings
• Assay : 80-120% of the theoretical content of active
• Content Uniformity: 70-130%
• Dissolution: ±20% of limits; eg if limits cover from 20% to 90% l.c. (controlled release),
linearity should cover 0-110% of l.c.
• Impurities: reporting level to 120% of shelf life limit
• Assay/Purity by a single method: reporting level of the impurities to 120% of assay limit
Correlation coefficient (r): API: ≥ 0.998, Impurities: ≥ 0.99
Accuracy
Assay
API: against an RS of known purity, or via an alternate method of known accuracy; analysis in triplicate.
FPP: samples/placeboes spiked with API, across the range of 80-120% of the target concentration, 3
concentrations, in triplicate each.
Report per cent recovery (mean result and RSD): 100±2%
Impurities: API/FPP spiked with known impurities
Experienced in PQ:
Across the range of LOQ-150% of the target concentration (shelf life limit), 3-5 concentrations, in
triplicate each. (LOQ, 50%, 100%, 150%)
Per cent recovery: in general, within 80-120%, depends on the level of limit
Precision
• System precision:
– by multiple injections (n ≥5) of a homogeneous sample (standard solution).
– RSD ≤ 1% is recommended for assay;
– RSD ≤ 5% is recommended for related substances (reference standards at
the limit)
– Indicates the performance of the HPLC system
– As a system suitability test
Precision
• Repeatability (method precision)
– Multiple measurements of a sample by the same analyst
– A minimum of 6 determinations at the test concentration (6 times of a single
batch), or
– 3 levels (80%, 100%, 120%) , 3 repetitions each (combined with accuracy)
– For Assay: RSD ≤ 2.0%
– For individual impurity above 0.05%, in general, RSD ≤ 10%
Precision
• Intermediate precision (part of ruggedness)
– Test a sample on multiple days, analysts, equipments
– Repeat the method precision by different analyst in different equipment
using different lot of column on different days
– RSD should be the same requirement as method precision
• Reproducibility (inter-laboratory trial)
– Not requested in the submission
LOD/LOQ
• signal to noise ratio: LOD 3:1 , LOQ 10:1
– May vary with lamp aging, model/manufacturer of detector, column
• standard deviation of the response and the slope of the calibration curve at levels
approximating the LOD /LOQ
σ = the standard deviation of the response, based on
– the standard deviation of the blank
– The calibration curve
should be validated by analysis of samples at the limits.
LOD/LOQ
• LOD: below the reporting threshold
• LOQ: at or below the specified limit
Not required for assay/dissolution methods.
• Applicant should provide
– the method of determination
– the limits,
– chromotograms
Robustness
• The method's capability to remain unaffected by small but deliberate
variations in method parameters
– Influence of variations of pH in a mobile phase
– Influence of variations in mobile phase composition
– Different columns (different lots and/or suppliers)
– Temperature
– Flow rate
Robustness
Conclusion
• HPLC methods play a critical role in analysis of pharmaceutical product
• Validation of HPLC should demonstrate that the method is suitable for its
intended use
• Data for acceptance, release, stability will only be trustworthy if the
methods used are reliable