This document presents information on HPLC method development and validation. It begins with an introduction to analytical chemistry and chromatography. It then discusses the principles, types, and modes of HPLC, as well as factors to consider in method development such as column selection and mobile phase selection. The document concludes with a discussion of method validation parameters such as system suitability, specificity, linearity, precision, accuracy, limit of detection, limit of quantification, and robustness. References on the topic are also provided.

1. PRESENTED BY
MR.VILAS H.KAMBLE
GIUDED BY
DR.MRS SONALI MAHAPARALE
DR.D.Y.PATIL COLLEGE OF PHARMACY AKURDI,PUNE.
HPLC METHOD DEVELOPMENT AND
VALIDATION
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2. CONTENT
 Introduction
 Principle of HPLC
 Classification
 HPLC
 Types of HPLC
 Modes of HPLC
 Method development in HPLC
 Validation of developed method
 References
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3. INTRODUCTION
Analytical chemistry-
It plays a important role in
agriculture,chemicals,clinicals,environoment and are
critical component in QA/QC
 Qualitative Analysis
 Quantitative Analysis
Selection of Analytical method
 As simple as possible
 Most specific
 Most productive, economical and convenient
 As accurate and precise
 Fully optimized
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4. INTRODUCTION
Classification of analytical method
Analytic
al
Method
Classical
Volumetric
Gravimetric
Instrumenta
l
Spectroscopic
Electrochemical
Chromatographic
Hyphenated
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5. CHROMATOGRAPHY
 Chromatography is essentially a group of technique
for separation, identification and determination
compounds.
 This technique is based on difference in migration
rates of components trough stationary phase by
gaseous or liquid mobile phase.
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6. CHROMATOGRAPHY
Principle-
 When a mixture of components are introduced into a
HPLC column, they travel according to their relative
affinities towards the stationary phase.
 The component which has more affinity towards
stationary phase travel slower.
 The component which has less affinity towards
stationary phase travel faster.
 Since no two component have the same affinity
towards the stationary phase, the component are
separated.
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7. CHROMATOGRAPHY
FIG.SEPARATION OF COMPONENT BY HPLC
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8. CHROMATOGRAPHY
Classification
 By state of phases and mechanism
 By the polarity of phases
Mobile phase Stationary phase Mechanism
Gas
Solid Adsorption
Liquid Partition
Liquid
Solid Adsorption
Liquid Partition
Polarity type Stationary phase Mobile phase
Normal phase Polar Non polar
Reversed phase Non polar Polar
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9. CHROMATOGRAPHY
Term used in chromatography
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10. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 HPLC is a versatile analytical technology used for
analysis of pharmaceutical, biomolecules, polymers
and organic and ionic compound.
Advantages of HPLC
 Separation of mixed component
 Qualitative and quantitative analysis
 Automated operation
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11. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 Types
Adsorption
Ion exchange
Size exclusive
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12. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
 Modes of HPLC
LC Mode Packing
material
Mobile phase Interaction
Normal phase Silica gel N-hexane Adsorption
Reversed
phase
Silica-C18 Methanol/wate
r
Hydrophobic
Size exclusive Porous
polymer
THF Gel
permeation
Ion exchange Ion exchange
gel
Buffer solution Ion exchange
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13. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Stationary Phase
 The stationary phase can be a solid or liquid which
adsorbed on solid.
 It consist of particle and the separation based on
the surface interaction and depends on the types of
the adsorption site.
 Important feature of adsorbent parameter are
-particle size :3-10 micro meter
-pore size :70-300A
-surface area :50-250 m2/gm
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14. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Mobile phase
 It is the part of the system which carries solute
through the stationary phase.
 In HPLC the mobile phase present in liquid.
e.g. Acetonitrile,water,methanol,isopropyle
alchol,THF, buffer.
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15. METHOD DEVLOPMENT IN
HPLC
 Best column, best mobile phase, best detection
wavelength, efforts in their selection can make a
word difference while developing HPLC method for
routine analysis.
 Chromatography method selected according to the
sample properties
 The HPLC method is optimized with regard to
analysis time, resolution, selectivity and sensitivity.
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16. METHOD DEVLOPMENT IN HPLC
 HPLC analyst should have following knowledge
during method development
Sample
Origin
History
Component
Amount
Molecula
r weightFormula
pK
value
Solubilit
y
UV
spectrum
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17. STATERGY FOR METHOD DEVELOPMENT
Selection of
chromatographic
method
Reversed phase
Normal phase
Ion suppression by pH
control
Ion pair
Ion exchange
Go for other method
Not successful
Not successful
Ion forming organic
compound
Not successful
Not successful
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18. SELECTION OF COLUMN
Sample with
low
molecular
weight
MW≤1000
Soluble in
organic solvent Non-polar
Polar
Soluble in water
Non-ionic
Ion forming
Strong acid or
bases in
organic ions
Normal phase
Si,CN,Diol,NH2
Reversed Phase C8
C18
Non-ion
forming
Ion
formin
g
RP C8 C18
RP with pH
control C8 C18
RP ion pair
C8 C18
RP C18 Water
as eluent ,NH2
Organic eluent
With buffer as eluent
RP ion pair C8 C18
Ion exchange4/17/2017 18

19. SELECTION OF MOBILE PHASE
POLARITY SOLVENT MISCIBILITY WITH
WATER
Non polar Hexane No
Cyclohexane No
Carbon tetra chloride No
Chloroform No
THF Yes
Diethyl ether No
Acetone Yes
Acetonitrile Yes
IPA Yes
Methanol Yes
Water Yes
Acetic acid YesPolar 4/17/2017 19

20. VALIDATION OF DEVELOPED
METHOD
 System suitability
It testing originally to decide whether a
chromatographic system being utilised day to day
routine or not.
The parameter used in system suitability
test(SST) report are as follows.
No of theoretical
plates or
efficiency (N)
Capacity factor
(K)
Separation or
relative
retention (α)
Resolution (Rs) Tailing factor (T)
Relative
standard
deviation(RSD)
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21. VALIDATION OF DEVELOPED
METHOD
No of theoretical plates/Efficiency(N)
 It is defined as the measurement of degree of peak dispersion and it should
have the column characteristics.
 N=16(Ve/Wb)2 by sigma /tangential method
Where ,
N=No of theorotical plates
Ve=Retention time
Wb=Width of peak at the base line
Height equivalent to theoretical plate (HETP)
HETP=L/N
Where N=Plate No.
L=Length of column
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22. VALIDATION OF DEVELOPED
METHOD
Capacity factor/retention factor (k)
 It is the measure of extent of retention relating to an
analyte relative to an unretained peak
 Value of k is >2
 K=0 means that no compound is left in column
k=TR -T0/T0
Where TR=retention time of sample peak
T0=retention time of unretained peak.
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23. VALIDATION OF DEVELOPED
METHOD
 Resolution
Resolution is the capability of the column to
separate 2 drugs in 2 individual peaks or
chromatographic zones.
tR1 and tR2 are the retention times for the two peaks of
components. tw1 and tw2 = At the baseline lies
between tangents drawn to the sides of the peaks4/17/2017 23

24. VALIDATION OF DEVELOPED
METHOD
 Tailing Factor Or Asymmetry Factor
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25. VALIDATION OF DEVELOPED
METHOD
 Specificity
It refer to the ability of analytical method to
differentiate and quantify analyte in complex
mixture.
 Linearity
How well a calibration plot of response vs.
concentration approximate straight line.
 Precision
It represents the nearness of agreement
between a series of measurement got from multiple
sampling of the same homogeneous sample under
a similar analytical condition
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26. VALIDATION OF DEVELOPED
METHOD
 Accuracy
The closeness of measured value to the true
value.
 Limit of detection
It determine by the analysis of sample with
known concentration of analyte and by establishing
that minimum level at which the analyte can
reliably detected.
LOD = 3.3 δ/S
δ = standard deviation of intercepts of calibration
curves. S = the slope of linearity plot.
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27. VALIDATION OF DEVELOPED
METHOD
 Limit of quantification
It is the least concentration of drug in a sample which
is estimated with appropriate precision and accuracy
under the stated condition.
LOQ = 10 δ/S
δ = standard deviation of response. S = Mean of
slopes of the calibration curves
 Robustness
It is the capacity of analytical method to stay
unchanged by small deliberate change.
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28. REFERENCES
 Panchumurthy Ravishankar,Naga Navya et.al,a
Review On Step By Step Analytical Method
Validation, IOSR Journal Of Pharmacy ISSN-2250-
3013,07-19
 Bharadwaj Santosh kumar, Dwivedi K,et al A
Review On HPLC Method Development And
Validation, International Journal Of Analytical And
Bioanalytical Chemistry ISSN-2231-5012
 Gupta Vibha,Jajn Ajaydeep kumar et al
,Development And Validation Of HPLC Method A
Review, International Research Journal Of
Pharmaceutical And Applied Science ISSN 2277-
4149,17-25
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