High Performance Liquid Chromatography- Dr. A. Amsavel Principle, Theory, Instrumentation, Application of HPLC Chromatographic Separation Definitions HPLC Techniques Instrumentation & Types of detectors Qualification & Calibration Method Development – Basics Column Chemistry Applications, Specifically Pharmaceutical QC

1. High Performance
Liquid Chromatography
Dr. A. Amsavel, M.Sc., B.Ed., Ph.D.
Principle, Theory, Instrumentation
and Application

2. An Overview
• Introduction
• Chromatographic Separation
• Definitions
• HPLC Techniques
• Instrumentation & Types of detectors
• Qualification & Calibration
• Method Development – Basics
• Column Chemistry
• Applications, Specifically Pharmaceutical QC

3. What is Chromatography?
• Chromatography is a separation technique used for Qualitative and
Quantitative Analysis.
• Essential for testing of chemicals in an Industries & academics
• Technique developed in 1941 by Martin & Synge and were awarded
Nobel Prize in 1952 (partition chromatography)
• In 1951 first GC experiment was performed by Martin & James
• Horvath & Lipsky built the first high-pressure liquid chromatograph.
End of the 70’s improvised – High Performance Liquid Chromatograph
instruments become familiar.
• Pharmaceutical industry made HPLC the workhorse beginning in the
1970s and become boom in 1980s
• Since 2006 advance models like UPLC, RRLC,UFLC, RSLC.. are available

4. Chromatographic Separation
Chromatography is based on a Physical
Equilibrium that results when a solute is
transferred between the mobile and a stationary
phase.
K = Distribution coefficient or Partition ratio
K = CS/CM
• CS -Molar conc. of the solute in the stationary phase
• CM -Molar conc. of the solute in the mobile phase.

5. Separation in Column
• In a mixture, each component has a Different distribution
coefficient in liquid Stationary phase and Mobile phase.
• The sample then has the opportunity to interact with the
stationary phase as it moves past it.
• Samples/ molecules that interact greatly, which move
slowly and weekly interact are move quickly.
Because of this difference in rates, the sample is Separated into
their components.
“Like Attracts Like – Opposites are Not Attracted”

6. Definitions
Gradient elution:
Continuously changing the solvent composition during the
chromatographic run is called gradient elution or solvent programming.
Retention factor (k):1 Also known as the “capacity factor (k)”
k = time spent by substance in Stationary phase
time spent by substance in mobile phase
k = (tR - tM) /tM
Hold-up time (tM): The time required for elution of an unretained component
Retention time (tR) ,

7. Definitions
 Number of theoretical plates (N): A measure of column efficiency.
For Gaussian peaks, it is calculated by: N = 16(tR/W) 2
where tR is the retention time of the substance, &W is the peak width at base
 Resolution (RS): The resolution is the separation of two components in
a mixture, calculated between peaks (1&2); tR - Retention time & W – peak
width
RS = 2 × (tR2 - tR1)/(W1 + W2)
 Symmetry factor (AS): Also known as the “tailing factor”, of a peak is
calculated by: AS = W0.05/2f (Fig-1)
Where W-0.05 is the width of the peak at 5% height from base and f is the
distance from the peak maximum to the leading edge of the peak.
Fig-1
Signal-to-noise (S/N) ratio:
is useful system suitability
parameter. (Sensitivity)
The S/N ratio is S/N ratio = 2H/h )
Signal
Noise

8. HPLC Techniques
Polarity
1. Normal Phase
2. Reversed Phase
Charge
3. Anion Exchange (SAX, WAX)
4. Cation Exchange (SCX, WCX)
Size
5. Size Exclusion Chromatography (SEC)
6. Gel Permeation Chromatography (GPC)

9. Normal Phase HPLC
• Polar stationary phase and non-polar mobile phase.
• Stationary phase is usually Silica, Cyanopropyl-
bonded, Amino-bonded etc.. Chiral columns
• Mobile phases are Hexane, Heptane, Methylene
chloride, chloroform, diethyl ether , ethyl acetate,
THF or mixture
• Polar samples are retained on the polar column
longer than less polar & non-polar samples.
• Used for: Water-sensitive compounds / geometric
isomers / cis-trans isomers/chiral compounds.

10. Reverse Phase HPLC
• Reverse Phase is opposite to normal phase: Stationary phase is
nonpolar ( C-18 Hydrophobic) and Mobile phase is a polar liquid;
eg. mixtures of water and methanol or acetonitrile.
• Lowering the amount of organic solvent in the mobile phase increases
the retention time.
• Retention is based on Van dar waal’s Interaction between the non-
polar stationary phase and the molecule (Fig)
• Nonpolar samples are retained longer than
polar molecules.
• Over 90% of chromatographers use RPC
• The technique is used for non-polar,
polar, ionizable and ionic molecules …
Neproxen

11. Separation and Polarity
• Chromatographic Retention Behavior
“Like Attracts Like – Opposites are Not Attracted”
• Polars attracted to other Polars
• Non-polars attracted to other non-polars
Polarity Compound Mobile Phase-
Solvent
Coulmn
Polar
Non-
Polar
Salts
Acids
Alcohols
Ketones
Ethers
Halogenated
compounds
Aliphatic
hydrocabons
Water
Alcohols
Acetonitrile
Ethylacetate
THF
Dichloromethane
Toluene
Hexane
Diol
Amine
Silica
Cyano
Phenyl
C8 (Octyl)
C18 (Octadecyl)

12. Ion-pair chromatography
• Ion-pair chromatography is a subset of reversed-phase
chromatography. Used for separation of complex mixtures of very
polar and ionic molecules.
• Organic salt containing a large organic counter-ion, such as a
quaternary ammonium ion or alkyl sulfonate is added to the mobile
phase as an ion-pairing reagent. Eg. Heptane or Octyl sulfonic acid (for
base) Tetrabutyl ammonium Hydroxide, certrimide (for acid)
• Ion-pairing reagents having a charge opposite to the analyte of interest
as well as a substantial hydrophobic region that allows interaction with
the stationary phase, plus associated counter-ions.
• This counter ion forms an uncharged ion pair with a solute ion of
opposite charge in the mobile phase. This ion pair then partitions into
the nonpolar stationary phase giving differential retention of solutes
based on the affinity of the ion pair for the two phases.

13. Ion-pair chromatography
Advantage of Ion-pair chromatography : Reduced separation times;
Highly reproducible results; Sharper peak shapes; Simultaneous
separation of ionized and non-ionized analytes; and Wide choice of
additives to improve separation
Example for Ion-pairing reagent (IPR) and interaction with molecule
Anionic ion-pairing agent: Adrenaline
with an ion-pairing agent in ODS column.
0.1 M sodium phosphate buffer/methanol
9:1 containing 0.02 % sodium
octanesulphonic acid (IPR)
Cationic ion-pairing agent : Ascorbic acid
with anion-pairing agent in ODS column.
0.1 M sodium acetate buffer pH 4.2,
Acetonitrile 95:5 containing 0.03 M
cetrimide (IPR)

14. Ion-Exchange HPLC
• The stationary phase is Ionically charged functional
groups on polymer
• The mobile phase is an aqueous buffer, where both pH
and ionic strength are used to control elution time.
• The mobile phase is an aqueous buffer (e.g. phosphate,
formate, etc.).
• Opposite charge of the sample ions are attracted and
elutes later.
• This technique is used almost exclusively with ionic or
ionizable samples.

15. Ion-Exchange Mechanism
• In ion exchange, the column packing contains ionic groups
(Eg. Sulfonic acid , tetraalkyl ammonium )
• Useful for separation of inorganic and organic anions and
cations in aqueous solution.
• Used to test Ionic dyes, amino acids, and proteins etc
Type of Exchanger
Functional
Exchanger Group
Cation
Strong acid
Weak acid
Sulfonic acid- SO3-,
Carboxylic acid
-COOH,
Anion
Strong base
Weak base
Quaternary ammonium
ion -NR3+
Amine group -NH2

16. Size Exclusion HPLC
• No interaction between the sample compounds and the
column.
• The column is filled with material having precisely
controlled pore sizes.
• Molecule /particles are separated according to its their
molecular size.
• Molecules larger than the pore opening do not diffuse
into the particles, while molecules smaller than the pore
opening enter the particle and are separated.
• Large molecules elute first. Smaller molecules elute later.
• Mainly for polymer characterization and for proteins.

17. SEC Retention Mechanism
Molecular Size in solution
• Analytes are dissolved in solution, injected into mobile phase
• Analytes are separated by their size once they are in solution
There are two modes:
• Non-aqueous SEC [Gel Permeation Chromatography (GPC)]
– Used in polymer separations
• Aqueous SEC [Gel Filtration Chromatography (GFC)].
– Used in biomolecule separations.

18. HPLC Instrumentation
HPLC consists:
1. Reservoir containing the mobile phase,
2. Pump to force the mobile phase through the
system at high pressure,
3. Injector to introduce the sample into the mobile
phase
4. Chromatographic column,
5. Detector
6. Data collection device.

19. HPLC–Typical Depiction

20. HPLC : Pump
• The role of the pump is to force the mobile phase at a specific flow
rate, (mL/min).
• Normal flow rates in HPLC are in the 1- to 2-mL/min range.
• Typical pumps can reach pressures in the range of 400 to 600 bar.
• During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing mobile
phase composition (gradient).
• Quaternary pump (low pressure) and binary pump (high pressure) are
available.
• A flow rate range of 1–2 mL/min on a 4.6 mm dia. column
will translate to a flow rate of ∼ 0.48 - 0.96 μL/ min for a 0.1 mm dia.

21. HPLC Pump Requirement
• The low flow rates obey Poiseuille’s law.
• A flow rate range of 1–2 mL/min on a 4.6 mm dia.
• Columns packed with 3- and 5-μm silica-based particles
required < 400 bar (5800 psi) pressure in HPLC.
• Pressure will increase to maintain same flow rate at lower
particle size. Pressure needed is inverse to the square of the
particle diameter. Eg Pressure required at a given flow rate is 1100%
higher when the column is packed with 1.5 μm particles than with 5 μm
particles.
• UHPLC operates with microbore column and pump pressure
designed to1300 bar (19,000 psi) and UPLC built for > 600bar
• Flow rate of ∼ 0.48 - 0.96 μL/ min for a 0.1 mm dia.

22. Mobile Phase Elution
Isocratic (ISO means Same)
• Solvent Composition remains Same during
the Entire Run.Eg. 60:40 Alcohol: Water
Gradient
• Solvent Composition Changes throughout
the HPLC Run time.
• Gradually Changed or Step Changes
o Analysis time is reduced by proper composition
& time
o Achieve good Peak Separation/ Resolution &
Peak shapes and Faster analysis of complex
molecules.

23. Mobile phase
• Mobile phase should be thoroughly degassed to remove
all dissolved gasses.
• Dissolved gas can be removed from solution by:
• Bubbling with helium
• Sonication / Vacuum filtration
If the mobile phase is not degassed, air bubbles can form
in the high-pressure system resulting in problems with
system instability, spurious baseline peaks etc.
• Do not use Online degasser while THF as mobile phase
due to degradation of Teflon in vacuum chamber.

24. HPLC: Injector
• The injector function is to introduce the sample into Column along
with mobile phase.
• Multiport value
• The injector must also be able to withstand the high pressures of the
liquid system.
• Mostly sample volumes used is 5 to 100 µL.
• Autosampler is available for automatic injection to analyze many
samples continuously by setting the program in the system
• Fill the vials with sample and keep in the order of injection in auto
sampler tray (100 samples) to inject automatically
– measures the appropriate sample volume,
– injects the sample automatically,
– then flushes the injector to be ready for the next sample and continue all
sample vials until all are processed …

25. HPLC Columns
• Considered the “heart of the chromatograph”
• High-purity spherical silica particles with low in trace metal content, of 2-10
μm diameter particles are coated with chemical stationary phase.
• Pore sizes of the particles are 60–150, 200–300, and 1,000–4,000°A,
used for separation of small molecules, polypeptides/proteins, and
very high molecular weight proteins respectively to allow the analyte to
penetrate the pores.
• Nonporous packings- of very small silica particle <1.5 μm
• Most columns for normal & reversed phase
are with length of 5 to 25 cm and ID of 3 to 5
mm and particle size (PS) 3-5µm.
• Particle size and pore size are different for
Ion-exchange & Gel-permeation
chromatographic columns.

26. HPLC Columns
• Materials of construction for the Column tubings;
– Stainless steel (gives high pressure capabilities and commonly used)
– Glass (mostly for biomolecules)
– PEEK polymer (biocompatible and chemically inert to most solvents)
• Analytical column : Length of 150 – 250 mm & ID 1.0 - 4.6-mm, PS.3 to 5µm
(40,000 to 70,000 plates/m)
• Preparative column: Lengths 50 – 250 mm & ID > 4.6 mm
• The below types of columns are advantage of speed and minimal
solvent consumption and high plates > 100,000 plates/m
– Microcolumn: Length 100 – 150 mm & ID 1.0 - 4.6-mm, PS.3 to 5µm
– Capillary: various lengths, ID 0.1 - 1.0 mm;
– Nano (i.d. < 0.1 mm, or sometimes stated as < 100 µm)

27. Guard Column & Ghost-Buster Column
• Guard column, is a short column packed with a similar
stationary phase as the analytical column.
• The purpose of the guard column is to prevent impurities, such
as highly retained compounds and particulate matter, system
debris from reaching and contaminating the analytical column.
• In gradient elution unexpected peaks ie Ghost Peaks may
appear.
• Ghost-Buster Column absorb the week polar and non-polar
impurities in the mobile phase and eliminate ghost. Also
baseline drift /fluctuation caused in the gradient is eliminated
and gives stable baseline. It is connected between mixture and
sample injection.

28. Column Oven
Temperature Control in HPLC: Uniform temperature throughout the run
give reproducible result. Column oven available with 5°C to 85°C.
Reproducibility:
• Retention of molecule is also temperature dependent
• If temperature varies, the peak areas/heights may vary for the specific
compounds. Peaks elutes faster if temperature is high (Ref Fig).
Solubility
• If compounds are low solubility in the mobile phase, in the flow stream they
may precipitate or forms salt in the column, by Increase the temperature or
maintain the column temperature high to overcome
• Stability:
Some of biological compounds such as enzymes
or proteins, may not be stable at room
temperature or higher. The temperature needs
to be much lower down to 4°C
25°C
30°C
35°C

29. Detectors
• Detectors sense the separated components and provide a
signal. Selection of detectors based on the compounds
• All the detectors are either concentration-dependent or mass
dependant. Can connect multiple detectors for good response
Commonly used detectors are;
 UV-spectroscopy: Dual wavelength & PDA
 Refractive Index
 Fluorescence,
 Mass Spectrometric
 Electrochemical detectors
 ELSD and etc

30. UV Detector
 A modified UV spectrophotometer equipped with a flow cell.
 UV light at selected wave length(s) is passes through a flow cell
 If a compound elutes from the column that absorbs this light energy,
 Absorbed energy is detected by the sensor, which converts as
electrical signal, which is amplified and directed to data system.
 Chromatogram is a plot of
absorbance as a function of
elution time (RT)
 UV absoption is based on the
chromaphores in the molecule
 Energy absorbed is proportional
to the amount of component
 The flow cell has a volume of 1–10
µL and a path length of 0.2–1 cm
Variable Wavelength Detector

31. Diode Array Detector
 This is working in the same principle of UV detector,
 In UV only single or dual wave length is absorbed, but in a diode
array spectrophotometer entire range of spectrum are recorded
 Shows three-dimensional chromatogram
 A plot of absorbance against elution time
 The flow cell has a volume of 1–10 µL and a path length of 0.2–1 cm.
Diode Array Detector PDA chromatogram

32. Refractive Index (RI) Detection
• Snell’s law of refraction – Sin i/Sin r
• It is a bulk property detector; Any change in its composition is reflected in the
RI.
• The ability of a compound or solvent to deflect light provides a way to detect.
• The RI is a measure of molecule’s ability to deflect light in a flowing mobile
phase in a flow cell relative to a static mobile phase contained in a reference
flow cell.
• The amount of deflection is proportional to concentration.
• The RI detector is considered to be a universal detector but it is not very
sensitive.
Limitations:
• Commonly used water methanol solvent
system
• RI changes considerably with temperature
• RI detection is generally incompatible with
gradient elution.
• Not suitable for trace analysis

33. Fluorescence Detection
• The fluorescence detector is most sensitive detector than
UV-Vis detectors
• It sense only detect those materials that will fluoresce or,
by appropriate derivatization can be made to fluoresce
• Used in quantify and identify compounds and impurities
in complex matrices at very low concentration levels
• Suitable for trace analysis/ genotoxic impurities
• Output :A plot of fluorescence
intensity as a function of time.
• Less popular than the UV
detectors since molecule
should be fluoresce

34. Mass Spectroscopy (MS)
• An MS detector senses a compound eluting from the HPLC
column first by ionizing it then by measuring it’s mass
and/or fragmenting the molecule into smaller pieces that
are unique to the compound.
• Mass spectrum is like a fingerprint and is quite unique to
that compound.
• The MS detector used to identify the compound with
library in application and also determine the quantity of
molecule.
• MS detector is sensitive and used for trace analysis

35. Data Processer
• Data system/ processing : The computer controls all the modules of the
HPLC instrument and converts the signal from the detector.
• Algorithms establish the time of elution (retention time) of the sample
components (qualitative analysis) and the peak area ie amount of
sample (quantitative analysis).
• Signals from the detector are processed based on the set of integration
events and displays the chromatograms for easy to read and interpret.
• Availability of Function for process chromatographic data based on the
application
• Computer with application available with many options like method
development, determine system suitability, calibration, auto calculation
etc. Also used to store, archive & back up of the data

36. Performance Qualification of HPLC
Component Parameter Acceptance Criteria
Pump Pump Flow Accuracy 0.5ml (0.475 to 0.525) 5.0ml (4.75 to 5.25)
Pump flow precision RT % RSD:NMT:0.50
Gradient composition in % 20, 40, 60, 80 + 2.0
Column oven Column Temperature Accuracy Column Oven: < 2.0
Column Temperature Stability Column Oven: < 1.0
Sample oven Sample temperature Accuracy Set temperature 4°C: > -2.00 and < 5.00
UV Detector Wavelength Accuracy (201nm to 209nm) 205 nm < 2
(241nm to 249nm) 245 nm < 2
(269nm to 277nm) 273 nm < 2
Noise and Drift Noise: < 0.040 mAU Drift: < 0.500 mAU/Hr
Signal to Noise > 3000
Response Linearity (Resp.Factors) Correlation coefficient: > 0.99900
Lamp Intensity 1000
Sampler /
Injectors
Injector Precision
Volume Delivery - Linearity
% RSD for Area: < 1.0 % RSD for Height: <
2.00
Correlation coefficient: NLT:0.99
Injection Carryover Carryover for Area: < 0.20
Carryover for Area: < 0.40
Ensure the below parameters covered in the Performance Qualification as
minimum, but not limited to;

37. Calibration of HPLC (UV)
Component Calibration test Acceptance Criteria
Pump Leak Test No leak
Flow rate Accuracy 0.5ml (0.49 to 0.51)
1.0ml (0.98 to 1.02)
2.0ml (1.96 to 2.04)
Flow stability RT % RSD:NMT:1.0
Gradient Delivery Accuracy in % 20, 40, 60, 80 ±2.0%
Column oven
&Sampler
Temperature Accuracy by Calibration of
Thermocouple and Air temperature
Column Oven:
25°C/40°C/60°C + 2.0
UV Detector Wavelength Accuracy(266nm to 276nm) 271 to 273nm
Dynamic Short-term Noise (Single to
Noise Ratio)
Noise:0.04 mAU or less
Drift:5.0mAU/hr or less
Response Linearity Correlation coefficient: NLT:0.99
Lamp energy Low intensity (> 200)
Average intensity (> 5000)
Highest intensity (> 10000)
Sampler /
Injectors
Volume Precision % RSD :NMT 1.0
Volume Delivery – Linearity Correlation coefficient:NLT:0.99
Injector Carryover NMT 0.1

38. Technique & Method Selection
• Methods can be
chosen based on
solubility and
molecular mass.
• Reversed-phase is
appropriate for
small molecules.
• Ion exchange is suitable
for strong anion & cation
• Size Exclusion is
appropriate for
high molecular mass
(>M 2000).

39. Method Development RP- Basics
Chemical nature and functional groups:

40. Method Development RP- Basics

41. Column Chemistry
Reverse phases:
Phenyl : R = -C6H5, moderate
C8 (octyl silane): R = -(CH2)7CH3, less hydrophobic
C18/ODS: R = -(CH2)17CH3, hydrophobic
Normal phases :
Cyanopropyl [R = (CH2)3CN], less polar
Diol: [R = -(CH2)2OCH2CH(OH)CH2OH],
Amino : [R = -(CH2)3NH2],
Dimethylamino, [R = -(CH2)3N(CH3)2] more polar
Silica based columns have limited lifetime at pH levels below 2 or above 8.

42. Column Chemistry
• Silanol in the silica, is bonded with Octyl- or
Octyldecyl group.
• The long-chain hydrocarbon groups are aligned
parallel to one another and perpendicular to the
surface of the particle,
• Present brush like, nonpolar hydrocarbon surface.
• Silica has limitation of stability
over strong basic or alkaline pH,
• Silanol (Si-OH) encaped to
increase the stability.
• Where required wider pH, Hybrid
columns are used

43. Column and pH
• Organic–inorganic hybrid silica particle with ethylene bridge
improves the stability to use wider pH range. Eg Propylene
bridged bidentate C18 silane (Fig).
• Chromatographic columns with Graphitic carbon, alumina,
titania, and zirconia are also available for wider pH
tolerance.
• pH Cross-linked polymeric particles. for example,
poly(methacrylate)s, and especially cross-linked
poly(styrene), can withstand the full range of pH.

44. Chiral Columns
• Silicamatrix bonded to β- and γ - cyclodextrins via a small hydrocarbon
chain /ether linkage.
• These cylindrically shaped ligands, which are oligosaccharides made of
five to seven molecules of glucose, possess a hydrophobic internal
cavity while the external part is hydrophilic.
• Central cavity is somewhat hydrophobic and the outer surface is
hydrophilic.
• This gives them a selective permeability and it forms reversible
diastereoisomer complexes at the surface and separates.
– Eg.Cyclofructans (CFs) based stationary phases are commonly used in the normal
separation mode of HPLC - to separate chiral primary amine enantiomers.

45. Selection of HPLC Columns
Bonded
Group
Polarity Retention
mechanisms
Comments
C18, C8,
C4
Nonpolar van der Waals does C8 does not retain hydrophobic
compounds as strongly as C18
Phenyl Nonpolar Hydrophobic and
pi-pi
Cyano Intermediate Hydrophobic,
dipole-dipole, and
pi-pi
Resolves polar organic compounds
by reversed-phase or normal-
phase chromatography
Amino Polar (NH2)
Ionic ( NH3
Dipole-dipole and
H-bonding
Normal-phase or ion-exchange
separations; separates ionic
carbohydrates, polar organic
compounds, and inorganic
ions; reacts with aldehydes and
ketones
Bare silica Very polar H-bonding Normal-phase separations

46. Factors Affects the Performance
• pH of the mobile phase shuold be close to the pKa value of
compound for better separation.
• Resolution is dependant on three variables, the column efficiency
(N), capacity factor (k’) & selectivity (α).
• Selectivity is a measure of the relative retention of two adjacent
peaks in a chromatogram
• Capacity factor is affected by changes in mobile phase, operating
temperature, analyte retention characteristics and changes to the
surface chemistry of the column.
• Increasing N increases resolution because peak width decreases.
• Decreasing k’ sharpens the peaks but decreases resolution.
Increasing α increases resolution.
• capacity factor decreases with an increase in temperature according
to the van’t Hoff equation

47. Adjustment Allowed for HPLC Condition
Property USP General Chapter 621 Ph.Eur. Gen. Chapter
2.2.46
Column length ±70% ±70%
Particle size Reduction by 50% Reduction by 50%
No increase No increase
Internal diameter Can be adapted as long as the
linear flow velocity remains the
same
±25%
Flow rate ±50% or more, provided the linear
flow velocity remains the same
±50%
Column temp. ±10 °C ±10 °C, maximum 60
°C
pH -mobile phase ±0.2 units ±0.2 units (±1% for
neutral subs)

48. Adjustment Allowed for HPLC Condition
Property USP General Chapter 621 Ph.Eur. Gen. Chapter
2.2.46
Injection
volume
Reduction allowed as far as
precision and detection limit
acceptable. No increase.
Reduction allowed as far as
precision and detection
limit acceptable. No
increase.
Salt conc. of the
buffer
±10%, as far as the allowed
change in pH value
±10%
Composition of
mobile phase
Minor components ±30%, if
not more than ±10% absolute
Minor components ±30%,if
not more than ±2%
absolute (greater value
accepted) *
Wavelength Not permitted , can be Max
±3nm based on the validation
*For gradient separation, a change of the mobile phase is not
recommended

49. Analytical Method Validation
• Ensure that Analytical method used is validated to the above
characteristics and suitable for its intended purpose (ICH Q2).
• Method should demonstrate Specificity, Precision (Repeatability
Intermediate Precision, Reproducibility ), Linearity, Range,
Accuracy, Robustness, Limit of Detection & Limit of
Quantification as appropriate.
• If test method is as per monograph, ensure that Analytical
method is verified for its suitability Eg USP General Chapter
<1226>
• Any change in the test method /condition shall be within the
allowable limit of Pharmacopeia methods (next page)

50. HPLC Application
• Qualitative Analysis – by comparing the retention time or
volume of the sample to the standard against the sample.
• Retention time or relative retention time can be used for
identification of eluted compound. Retention times are
characteristic of the compounds they represent (but are not
unique). Mass Detector gives the mass value to identify
precisely.
• Quantitative Analysis- Pear area / Peak height of elution peak
is proportional to the quantity / concentration. Peak response
is based of the detector used.
• Method used for Quantitative Analysis shall be validated
• Type of estimation- Area normalization, Internal standard,
Calibration and standard, Standard addition and etc

51. HPLC Application
Pharmaceutical Applications
• Testing of Quality of raw material, in-process , intermediates, APIs and drug
products
• Pharmaceutical quality control: Assay, related substances, content and,
trace analysis like genotoxic, nitrosamine impurities at ppm level.
• To perform drug stability.
• Testing the content of pharmaceutical dosages form, content uniformity
• Phytochemical & nutraceuticals- plant extracts, Ginseng, herbal medicines
Applications in Clinical Tests
• Bio-availability and bio-equivalency
• Toxicology, pharmacology, phatmacokinetics
• Urine analysis, analysis of blood, Bile acids, drug metabolites, urine extracts,
estrogens etc
• Detection of endogenous Neuropeptides in extracellular fluid of brain etc.

52. HPLC Application
Environmental Applications
• Detection of chemical compounds / contaminant in water and air quality
• Chemical Exposure in the workplace / environment
• Pesticides, herbicides, phenols, polychlorinated biphenyls (PCBs
Applications in Forensics
• Identification & determination of abuse drugs in blood, urine etc. Eg.
Cocaine, steroid, ketamine, amphetamine etc
• Quantification of Drugs, poisons, blood alcohol, narcotics.
• Forensic analysis like textile dyes, chemicals, etc
Industrial Application:
• Identification & determination of cosmetics- Active ingredient content,
purity, impurities and stability study.
• Analysis of Preservative, surfactants, propellants, dyes etc
• Organic chemicals like polymers (e.g. polystyrene, polyethylene)
• Artifcial sweeteners, antioxidants, aflatoxins, additives
• Thermally unstable compounds such as trinitrotoluene (TNT), enzymes

53. Tips for Good HPLC Practice
 Always use only Ultra pure / Milli-Q water for HPLC analysis
 Ultra pure HPLC water of 18MΩ resistivity
 Do not use RO water/de-ionised water for HPLC analysis. It will
have organic and in-organics impurities
 If water contains impurities, it will have higher absorption and
lead to poor baseline , drift , ghost peak and less accurate
• All reagents and solvents should be highest quality.
• HPLC grade reagents & solvents are high purity will have low
UV absorbance
• Low grade solvent contain impurities to produce spurious
peaks, poor peak , high baseline etc

54. Tips for Good HPLC Practice
 Do not store HPLC columns in buffers. A buffer may precipitate inside the
column, it will clog and affects the packing material.
 Mobile phases with 100% or close to 100% buffer may lead to bacterial
growth, which can block the column & frit and packing material.
 Bacteria may also affect the analyte, and organic products from the dead
bacteria may cause "ghost peaks" in chromatograms.
 Do not store HPLC columns in solvents that degrade easily tetrahydrofuran
(THF), triethylamine (TEA), trifluoroacetic acid (TFA).
 Unstabilized THF can form peroxides which may degrade the column
 All buffers should be washed out of the column before flushing with
Acetonitrile.
 Gradually start the column washing : Eg Starts the flow with 0.2 ml/min and
increases gradually to 2.0 ml/min and continues for 20min to 30 min or as per
procedure
 Have Dedicated Columns for each Method / each product

55. Integration of Peaks
• Do not integrate any peak by manually.
• Integrate all the peaks or else as per procedure
• Always use same processing method for processing of blank,
standard & sample chromatograms in case of Assay & related
substances, etc.
• Verify the processing parameters like
– Threshold,
– Width,
– System suitability,
– Peak names etc.
• Save the processing method
• Re-integration:
– Do not re-integrate the chromatograms without documenting.
– Document the reason for reintegration.

56. Common Problems in HPLC Analysis
• System failures may occur during analysis due to
– System over pressure
– Leakage
– Poor mobile phase
• dissolved gas, pH, solvent purity or composition, filtration,
– Communication error
– Failure of system suitability
– Peak splitting/ negative peak
– Spurious peak
– Bracketing standard failure

57. Handling of Deviation/Failure
 SOP shall be available and shall address the handling of Lab deviation/
incidents.
 SOP shall define clearly the deviation/ incident , reporting
investigation, CAPA and documentation
 Record all the deviation/ incident happened in chromatographic
analysis
 Process all the injections including the invalid injection and report and
store the data along with Raw data.
 Do not omit any injection
 Investigate the deviation/ incident and find the root cause for the
failure.
 Rectify the problem, take appropriate CAPA and document
 Repeat complete sample set of injections in case of sample injection
failure

58. References
1. Chemical Analysis: Modern Instrumentation Methods and
Techniques- Francis and Annick Rouessac and Steve Brooks - John
Wiley & Sons Ltd,.
2. Pharmaceutical Analysis David G. Watson
3. Analytical Chemistry for Technicians -John Kenkel
4. Analytical Chemistry - Gary D. Christian , Purnendu K. (Sandy)
Dasgupta & Kevin A. Schug
5. Quantitative Chemical Analysis- Daniel C. Harris
6. Vogel’s – Quantitative Chemical Analysis- 6th edition
7. Principles of Instrumental Analysis- 6th Edition- D.A. Skoog et al
8. United States Pharmacopeia
9. European Pharmacopeia

59. 59
About Author:
Dr. A. Amsavel, born at Begarahalli, Dharmapuri-Dist, Tamil Nadu, India.
Completed his M.Sc. in Dept of Analytical Chemistry, University of Madras.
B.Ed. in Annamalai University and Ph.D in Anna University, Chennai.
Worked as Lecturer and also worked in various Chemical & Pharmaceutical
Industries for the past 34 years. Presently working as Assistant Vice
President- Quality at Malladi Drugs & Pharmaceuticals Ltd.