HPLC method development and validation

Table of Contents

Method development and validation refer to considering a set of parameters for better chromatographic performance and maximum purity. It is an umbrella term for all the factors that you need to consider from sample preparation to product detection while performing high-performance liquid chromatography.

 Proper HPLC method development and validation ensure efficient component identification and quantification. Therefore, in this article, we will discuss the key principles of HPLC method development and validation.

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What is HPLC method development

HPLC method development is a process of proving that the chromatographic method selected is suitable for its intended use of analyzing a particular class of targeted analyte components.

Why is HPLC method development important

High-performance liquid chromatography (HPLC) is a liquid column chromatographic technique that is performed to separate and analyze a diverse range of chemical compounds from complex sample mixtures. HPLC can be performed using different types of columns. The stationary phase material packed in the column can vary. Chromatographic mode of interaction; normal-phase, reverse-phase, ion exchange or size exclusion, etc., may vary.  Similarly, different mobile phase compositions, pH., flow rate, elution mode, and sample injection volume can be used as per requirement.

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Depending upon the wide variations present in all these parameters, HPLC method development is extremely important.

Steps of HPLC method development

The following steps are important in HPLC method development:

Selection of chromatographic mode

  • For non-polar compounds, biomolecules, plant metabolites, and pharmaceutical drugs: Reverse-phase chromatography is used.
  • For extremely polar compounds, proteins, nucleic acids, glycopeptides, and antibiotics: Normal-phase chromatography and/or hydrophilic interaction chromatography (HILIC) is employed.
  • For proteins and amino acids with hydrophobic side chains: Hydrophobic interaction chromatography (HIC) is used.
  • For bulk separation and purification of protein samples: size-exclusion chromatography is suitable.
  • For inorganic ions:  ion-exchange chromatography is preferred.
  • For enzyme assays, solute-specific analysis, and purification: affinity chromatography is used.

Depending upon the chromatographic mode, the column is packed with a relevant stationary phase material.

Column selection

Factors important to consider while selecting the HPLC column are:

  • Column length
  • Internal diameter of the column
  • Stationary phase support: different options are available such as silica gel, alumina, zirconia, and organic polymer matrices
  • Shape and size of stationary phase particles. Smaller particles are used for a higher column efficiency which is required for a sensitive, multi-component analysis
  • Column temperature

Selection and optimization of mobile phase  

Important factors to consider when choosing the right mobile phase for an HPLC separation are:

  • Solubility of the targeted analyte
  • pH of the mobile phase
  • Mode of elution: isocratic or gradient
  • Mobile phase composition based on a binary or tertiary mixture for gradient elution
  • Ratio of organic versus aqueous or polar versus non-polar solvents can be varied according to chromatographic separation requirements
  • Mobile phase flow rate inside the column

The mobile phase flow rate is highly crucial. It strongly influences chromatographic peak separation. The flow rate can be controlled by controlling the pressure inside the column. There are two main types of pumps used for HPLC: the constant flow pump and the constant pressure pump. The constant flow pump is a more viable choice. It maintains the mobile phase flow rate inside the column constant while varying the pressure. The mobile flow rate must be maintained below 2 mL/min to reduce any chance of back pressure inside the column.

Sample preparation and injection

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Sample preparation is another important step while considering HPLC method development.

  • Understanding the physicochemical properties of all components in the sample mixture is important including their chemical reactivity, thermal stability,  and solubility in aqueous and organic solvents.
  • The sample should be free of any impurities and/or interferences that can degrade the stationary phase packed inside the column by binding irreversibly to it.
  • A specific sample volume must be injected every time the HPLC is performed.
  • Three different modes of injection can be used: stop-flow injection, loop injection, and micro-volume injection.

Selection of detector

A plethora of detector choices are available for chromatographic identification and quantification. The detector is chosen keeping in mind the analyte of interest, for instance :

  • An ultraviolet detector is chosen if the analyte absorbs electromagnetic radiations in the ultraviolet (190-370 nm) range. Organic compounds with chromophores absorb in the ultraviolet region therefore the UV detector is most suitable for unsaturated and/or aromatic organic/drug molecules.
  • In the absence of a UV light absorbing functional group, the refractive index detector or an evaporative light scattering detector can be used.
  • The electrochemical detector can only be used for analytes that can undergo an oxidation or reduction reaction at the electrode.
  • Chiral detector can be used for optically active, enantiomeric analytes.
  • Analytes that emit native fluorescence can be detected via a fluorescence detector.

Before proceeding with a chromatographic analysis for a specific target compound, a proper literature survey based on all the factors given above is essential. Once all the experimental conditions are set, the method can then be validated by applying a test sample.

What is HPLC method validation

Validation is an exercise that is performed to gain confidence that the specific method you applied for a targeted chromatographic separation gives the best and the most consistent results under identical experimental conditions. HPLC method validation is an important quality control parameter, especially in the pharmaceutical sector to get rid of any possible errors.

Steps for HPLC method validation

The most important characteristics for validating an HPLC method are:

  • Limit of detection (LOD)

LOD is the minimum concentration of an analyte required for detection. A good HPLC method with a sensitive detector should have a LOD value of less than 0.2. The choice of detector controls LOD values for an HPLC method.

  • Limit of Quantification (LOQ)

LOQ is defined as the minimum concentration of analyte required for quantification i.e., for determining the amount of targeted analyte present in a sample. The lower the LOQ value, more sensitive the HPLC method developed for that analyte.

  • Precision

Precision refers to the consistency present in repeated readings. There should not be more than ± 2 variations in between readings. For a high-performance liquid chromatography, four types of precision are required:

  1. System precision: Repeated measurements of various dilutions of the standard solution should give consistent results. System precision proves that all the instruments used in HPLC are working in perfect alignment.
  2. Method precision: Repeated measurements of various dilutions of the sample solution should give consistent results. Method precision establishes that the HPLC method developed is suitable for the analyte of interest.
  3. Intra-day precision: If the same HPLC experiment keeping all the factors constant is performed in the same laboratory at three different time intervals and it gives consistent readings then it is called intra-day precision. Intra-day precision is also called repeatability.
  4. Inter-day precision: If the same HPLC experiment keeping all the factors constant is performed in different laboratories on different days then it is known as inter-day precision.

An HPLC method that fulfills the criteria of both an intra-day and an inter-day precision, such a method is considered reproducible. It can be reliably used for a targeted analyte separation with an adequate separation efficiency.

  • Accuracy

The average value of two sample results can be compared to the true value expected for marking the accuracy of the HPLC protocol. If this average value is close to the true value, then the HPLC method developed is accurate.

  • Recovery

A known concentration of analyte added to the sample must generate a linear detector response. This is called spiking and it is used to ensure that no unwanted excipient is interfering with the instrument response.

  • Linearity

The detector response should linearly increase with the increase in analyte concentration.

  • Relative standard deviation (RSD)                                                                         

RSD is a mathematical entity. In HPLC method validation, RSD denotes the deviation present in the results obtained in a test run, from the mean value. An  RSD value less than 2.5% guarantees the reliability of the developed HPLC method.

  • Coefficient of variation (CV)

The coefficient of variation is the ratio of standard deviation to mean. A lower CV value means a lesser spread of the data, away from the mean thus a more precise HPLC method.

  • System suitability

The system’s suitability for a particular analysis can be marked by calculating column efficiency, relative retention, and chromatographic peak resolution. Column efficiency can be calculated in number of theoretical plates per unit length of the column.

N= 5.54 (Ve/w1/2)2

Ve= retention time of analyte and w1/2 = Gaussian function of the peak width at half-height (1/2 h).

Relative retention (α)= t2-ta / t1– ta

t2= retention time calculated from the point of injection, ta= unretained peak time (retention time of an inert component not retained in the column), and t1=retention time from point of injection of reference peak.

The resolution of two adjacent chromatographic peaks can then be calculated as:

RS= (tR1– tR2)/0.5 (tW1+ tW2)

tR1 and tR2 are retention times for the two peaks of components tw1 and tw2.

The curves are reproduced  from chem. libretext.org

N > 2000 , α >1 and Rs>1.5 is the acceptance criteria for a good HPLC method.

The peak asymmetry/tailing factor however should be less than 2 to establish system suitability.

The curve is reproduced  from asdlib.org
  • Range

 An upper and a lower limit is set for the method validation parameters discussed above. This is called a range. If the results obtained from the HPLC protocol fall within this range, then they are accepted otherwise rejected. In this way, the chromatographer can decide for which kind of analyte mixtures is the specific method most suitable.

  • Robustness

An overall test performed keeping in mind all the factors discussed and the results obtained marks the robustness of your HPLC protocol.

  • Optimization

The developed method is optimized to keep a desirable balance between chromatographic resolution, analysis time, and cost of the method.

All the parameters discussed for HPLC method development and validation can be applied to other versatile column chromatographic techniques such as ultra-high performance liquid chromatography (UHPLC) and gas chromatography (GC).


Prior to all high-performance liquid chromatographic analyses, the HPLC method must be developed according to analyte requirements. HPLC method development includes all the chromatographic conditions, stationary and mobile phase selection, and/or type of detectors required. Once developed, the method must be validated against a series of parameters to test its reproducibility and robustness, for achieving optimum resolution in minimum time.

For more insightful information on the topic, you may like: Top Three HPLC method development tips.


1.Lakka, N. S. and C. Kuppan (2019). Principles of Chromatography Method Development.

2. Snyder, L. R., J. J. Kirkland and J. L. Glajch (2016). Practical HPLC Method Development.

3. Swartz, M. E. and I. S. Krull (2017). Analytical Method Development and Validation.

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