the slides in the ppt gives a brief review on product development and its validation in HPLC method. Contents are with advantages, disadvantages, application , classification and methods for development.

1. A brief review on development and validation of
HPLC method
By missBy. Adhira Sushil Jainsushil
jain

2. Content
 Introduction
 Principle of HPLC method
 Advantages , Application& Limitations of HPLC method
 Classification of HPLC method
 Instrumentation of HPLC method
 Method Development of HPLC
 Method Validation with it’s components of HPLC
 Conclusion
 References

3. Introduction
 CHROMATOGRAPHY: A technique for the separation of a mixture by passing it in solution or
suspension through a medium in which the components move at different rates.
 Chromatography is based on the principle where molecules in mixture applied onto the surface or
into the solid, and fluid stationary phase (stable phase) is separating from each other while moving
with the aid of a mobile phase.
 High Performance Liquid Chromatography (HPLC) is a form of column chromatography that
pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through
a column with chromatographic packing material (stationary phase).
 HPLC: It is an analytical technique used for the separation of components of an organic mixture of
compounds when such compounds are non-volatile, thermally unstable, and have relatively high
molecular weights.
 The High Performance Liquid Chromatography is more versatile than gas chromatography since (a) it
is not limited to volatile and thermally stable samples, and (b) the choice of mobile and stationary
phases is wider.
 High performance liquid chromatography (HPLC) is the most accurate analytical methods widely used
for the quantitative as well as qualitative analysis of drug product.

4. Principle of HPLC method
 The principle is that a solution of the sample is injected into a column of a porous material (stationary phase) and a
liquid (mobile phase) is pumped at high pressure through the column. The purification takes place in a separation
column between a stationary and a mobile phase.
 The stationary phase is a granular material with very small porous particles in a separation column.
 The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high pressure through the
separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless steel, the sample is injected
into the mobile phase flow from the pump to the separation column using a syringe.
 Subsequently, the individual components of the sample migrate through the column at different rates because they are
retained to a varying degree by interactions with the stationary phase
 After leaving the column, the individual substances are detected by a suitable detector and passed on as a signal to the
HPLC software on the computer. At the end of this operation/run, a chromatogram in the HPLC software on the
computer is obtained.
 The chromatogram allows the identification and quantification of the different substances. The chromatogram allows the
identification and quantification of the different substances.

5. Advantages Application Limitations
1. Speed. 1. Analysis of drugs 1. Cost: Despite its advantages,
HPLC can be costly, requiring large
quantities of expensive organics.
2. Efficiency. 2. Analysis of synthetic polymers. 2. Complexity
3.High Resolution. 3.Analysis of pollutants in
environmental analytics.
3. HPLC does have low sensitivity for
certain compounds, and some
cannot be detected as they are
irreversibly adsorbed.
4.High Sensitivity. 4. Determination of drugs in
biological matrices.
4. Volatile substances are better
separated by gas chromatography.
5.Good Repeatability. 5. Isolation of valuable products.
6. Small sample size. 6. Product purity and quality control
of industrial products and fine
chemicals.
7. Moderate analysis condition &
Easy to fractionate the sample and
purify.
7. Separation and purification of
biopolymers such as enzymes or
nucleic acids.
Advantages , Application & Limitations of HPLC method

6. Classification of HPLC Method
 Classification of HPLC method is based on various modes and factors.
1. Based on scale of operation: Preparative HPLC & Analytical HPLC.
2. Based on principle of separation: Affinity chromatography, Adsorption
chromatography, Size exclusion chromatography, Ion exchange
chromatography & Chiral phase chromatography.
3. Based on elution technique: Gradient separation and Isocratic separation.
4. Based on modes of operation: Normal phase chromatography and Reverse
phase chromatography.

7. DEFINITIONS OF VAROIUS CLASSIFIED TYPES OF HPLC METHODS
1.Preparative chromatography: Preparative chromatography is a type of chromatography used to isolate a
particular substance in a sample in large scale. Therefore, the main purpose of preparative chromatography is to
purify a particular substance.
2.Analytical chromatography: Analytical chromatography is the typical chromatography method used to identify
the components of a mixture and their proportions. The main purpose of analytical chromatography is the qualitative
and quantitative analysis of the components of a mixture.
3. Affinity chromatography: Affinity chromatography is a separation method based on a specific binding interaction
between an immobilized ligand and its binding partner. Examples include antibody/antigen, enzyme/substrate, and
enzyme/inhibitor interactions.
4.Adsorption chromatography: Adsorption chromatography can be defined as a technique for the separation of
the component in a mixture by adsorption from a mobile phase into the stationary solid surface. ... The selection of
the adsorbent. The choice of the solvent for the sample mixture. The rate of flow of the solvent.
5.Size Exclusion chromatography: Size-exclusion chromatography, also known as molecular sieve
chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in
some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as
proteins and industrial polymers.

8. DEFINITIONS OF VAROIUS CLASSIFIED TYPES OF HPLC METHODS
6.Ion Exchange chromatography : Ion chromatography separates ions and polar molecules based on their affinity to the ion
exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids.
7.Chiral phase chromatography : Chiral column chromatography is a variant of column chromatography that is employed for the
separation of optical isomers. The stationary phase contains a single enantiomer of a chiral compound.
8. Gradient separation chromatography : A separation method where the components are distributed between two phases, one
of which is stationary, while the other moves in a definite direction (the 'mobile' phase).
9. Isocratic separation chromatography : Isocratic elution is a term used in chromatography when the mobile phase has a
constant concentration. Here, the concentration of the mobile phase is constant throughout the chromatographic process. ...
Moreover, in isocratic elution, the selectivity does not change according to the column dimensions.
10. Normal phase chromatography : A separation method where the components are distributed between two phases, one of
which is stationary and polar, while the other is non-polar and moves in a definite direction.
11. Reverse phase chromatography : Reversed-phase chromatography is a technique using alkyl chains covalently bonded to the
stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less
polar compounds. The use of a hydrophobic stationary phase is essentially the reverse of normal phase chromatography, since the
polarity of the mobile and stationary phases have been inverted – hence the term reversed-phase chromatography.

9. Instrumentation of HPLC method
The Instrumentation of HPLC method has following components:
• PUMP
• DEGASSER
• INJECTOR
• COLUMN
• COLUMN HEATER
• DETECTOR
• RECORDER

10. Development method of HPLC
 A step involved in method development of HPLC is as follows:
1.Understanding the physiochemical properties of drug molecule
 Physical properties like solubility, polarity, Pka & pH.
 Helps to decide analyst the solvent and composition of mobile phase.
 Solubility of molecules can be explained with help of polarity of molecules.
 . pH and pKa plays an important role in HPLC method development.

11. 2. Selection of chromatographic conditions
 Selection of column( stationary phase)
 Selection of Buffer(governed by pH that is desired)
 Isocratic & gradient separation , Internal diameter
 Dimension of packaging material
 Selection of mobile phase
 Gradient HPLC , Column dimensions
 Selection of detectors ( UV wavelength , Fluorescence wavelength)
3. Developing approach for analysis
 Chromatographic parameters [ mobile phase, selection of column, selection of flow rate of mobile
phase, selection of pH of mobile phase]
 Selected on the basis of trials & considered the system suitability parameters { e.g.; retention time
should not be more than 5 minutes , theoretical plates should be more than 2000, resolution between
two peaks should be more than 5 ..like wise }
 Study to know the range of concentration upto which the drug follows the linear pattern.

12. 4. Sample preparation
 Essential part which provide homogenous and reproducible solution that is suitable for injection
onto the column.
 Aim is to avoid interferences which will not damage the column and compatible with intended
HPLC method.
 It begins with sample collection and extends upto the sample injection onto the HPLC column.
5. Method optimization
 Aim is to achieve adequate selectivity (peak spacing).
 To minimize the number of trial chromatograms involved, only the parameters that are likely to
have a significant effect on selectivity in the optimization must be examined.
 The optimization of mobile phase parameters is always considered first as this is much easier and
convenient than stationary phase optimization.
 This is used to find the desired balance between resolution and analysis time after satisfactory
selectivity has been achieved.
 The parameters involved include column dimensions, column-packing particle size and flow rate.
These parameters may be changed without affecting capacity factors or selectivity.

13. Validation method with its components of HPLC
METHOD VALIDATION :
 Validation is the confirmation by examination and the provision of objective
evidence that the particular requirements for a specific intended use are
fulfilled.
 A process of evaluating method performance and demonstrating that it meets a
particular requirement.
 Proper validation of analytical methods is important for pharmaceutical analysis
when ensurance of the continuing efficacy and safety of each batch
manufactured relies solely on the determination of quality.
 The ability to control this quality is dependent upon the ability of the analytical
methods, as applied under well-defined conditions and at an established level
of sensitivity, to give a reliable demonstration of all deviation from target criteria.

14. COMPONENTS OF VALIDATION METHOD
The following are typical analytical performance characteristics which may be tested
during method validation :
1. ACCURACY
2. PRECISION
3. LINEARITY
4. DETECTION TEST
5. QUANTITATION LIMIT
6. SPECIFICITY
7. RANGE
8. ROBUSTNESS
9. FORCE DEGRADATION STUDIES
10. SYSTEM SUITABILITY

15. DEFINITION OF COMPONENTS OF METHOD VALIDATION
1.ACCURACY:Accuracy is defined as the nearness of a measured value to the true or accepted value. Practically accuracy
indicates the deviation between the mean value found and the true value.
2.PRECISION:It expresses closeness of agreement (degree of scatter) between a series of measurements obtained from multiple
sampling of the same homogeneous sample under the prescribed conditions. Precision is a measure of the reproducibility of the
whole analytical method. It consists of two components: repeatability and intermediate precision.
3.LINEARITY:Linearity is the ability of analytical procedure to obtain a response that is directly proportional to the concentration
(amount) of analyte in the sample. Linearity is usually expressed as the confidence limit around the slope of the regression line.
4.DETECTION LIMIT:The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be
detected, not quantified. LOD is expressed as a concentration at a specified signal: noise ratio, usually 3:1.
5.QUANTITATION LIMIT:The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can
be determined with acceptable precision and accuracy under the stated operational conditions of the method.

16. DEFINITION OF COMPONENTS OF METHOD VALIDATION
6.SPECIFICITY:Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to
be present. Typically, these might include impurities, degradants, matrix, etc. Lack of specificity of an individual analytical procedure
may be compensated by other supporting analytical procedure(s). This definition has the following implications: Identification: to
ensure the identity of an analyte. Purity Tests: to ensure that all the analytical procedures performed allow an accurate statement of
the content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents content, etc. Assay (content or
potency): to provide an exact result which allows an accurate statement on the content or potency of the analyte in a sample.
7.RANGE:The range of the method is the interval between the upper and lower levels of an analyte that have been determined with
acceptable precision, accuracy and linearity. It is determined on either a linear or nonlinear response curve and is normally expressed
in the same units as the test results.
8.ROBUSTNESS:The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate
variations in method parameters and provides an indication of its reliability during normal usage. Robustness measures the capacity
of an analytical method to remain unaffected by small but deliberate variations in method parameters. It also provides some indication
of the reliability of an analytical method during normal usage.
9.FORCE DEGRADATION STUDIES:Forced degradation studies are undertaken to degrade the active drug deliberately. These
studies are used to evaluate an analytical method’s ability to measure an active ingredient and its degradation products without
interference. Samples or drug product (spiked placebo) and drug substance are exposed to acid, base, oxidizing agent, reducing
agent, and water. The degraded samples were then analysed using the method to determine if there are interferences with the active.
Thus, stability-indicating property was evaluated.
10. SYSTEM SUITABILITY: The purpose of the system suitability test is to ensure that the complete testing system, including
instruments, reagents, columns, analysts etc., is adequate for the intended analysis. The following parameters are usually
determined: theoretical plate count, tailing factors, resolution, and reproducibility

17. CONCLUSION
The method development and validation are continuous and interrelated processes that
measure a parameter as intended and establish the performance limits of the measurement.
The selection of Column, buffer, detector and wavelength and another conditions composition
(organic and pH) plays a dramatic role on the separation selectivity The advantages of HPLC
technique were high selectivity, sensitivity, economic, less time consuming and low limit of
detection. Final optimization can be performed by changing the gradient slope, temperature
and flow rate as well as the type and concentration of mobile-phase modifiers. Optimized
method is validated with various parameters (e.g. specificity, precision, accuracy, detection
limit, linearity, etc.) as per ICH guidelines

18. REFERENCES
1. Sethi PD. Introduction – High Performance Liquid Chromatography, 1st edn, CBS Publishers,
New Delhi. 2001; 1-28.
2. .A Guide to Validation in HPLC. http://www.standardbase.com
3. ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology. International
Conference on Harmonization, IFPMA, Geneva; 2005
4. Shah RS, Pawar RB, Gayakar PP. An analytical method development of HPLC. International
Journal of Institutional Pharmacy and Life Sciences. 2015; 5(5): 506-513.
5. Yadav Vidushi, Bharkatiya Meenakshi. A Review on HPLC Method Development and
Validation. RJLBPCS.2017;2(6):166-178
6. FDA Guidance for Industry (2000)-Analytical Procedures and Method Validation, Chemistry,
Manufacturing, and Controls Documentation, Center for Drug Evaluation and Research
(CDER) and Center for Biologics Evaluation and Research (CBER).
7. Kumar GS. et.al. Development and validation of RP-HPLC method for simultaneous
estimation of Atenolol and Chlorthalidone in Bulk and dosage form. Int Res J Pharma 2013;
3(10): 215-19.

19. THANK YOU