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Method Development and Validation Parameters of HPLC- A Mini Review

L.R.D.Bhavani1*, Durga aruna R2

1Dr.C.S.N Institute of Pharmacy, Bhimavaram, Andhra Pradesh, India

2Dept of Pharmaceutics, Vagdevi College of Pharmacy, Gurajala, Guntur District, Andhra Pradesh, India


*Corresponding Author:
Dr.C.S.N Institute of Pharmacy,
Bhimavaram, Andhra Pradesh, India.

Received: 04 May 2015 Accepted: 27 May 2015 Published: 02 June 2015

Visit for more related articles at Research & Reviews: Journal of Pharmaceutical Analysis


High-Performance Liquid Chromatography (HPLC) is an uncommon branch of section chromatography in which the portable stage is constrained through the segment at fast. Accordingly the examination time is lessened by 1-2 requests of extent with respect to established segment chromatography and the utilization of much littler particles of the adsorbent or backing gets to be conceivable expanding the section productivity generously.


Chromatography, Chromatogram, Method Development, Validation


Adsorption Chromatography or Normal Phase Chromatography

In ordinary stage chromatography, the stationary stage is a polar adsorbent and the portable stage is by and large a blend of non-fluid solvents.

The silica structure is immersed with silanol bunches toward the end. These Gracious gatherings are factually aggravated over the entire of the surface. The silanol gatherings speak to the dynamic destinations (extremely polar) in the stationary stage. This structures a powerless kind of bond with any particle in the region when any of the accompanying collaborations are available [1-5].

Dipole-induced dipole


Hydrogen bonding

-Complex bonding

These circumstances emerge when the particle has one or a few molecules with solitary pair electron or a twofold bond [5-10]. The retention qualities and thus k' values (elution arrangement) increment in the accompanying request Immersed hydrocarbons < olefins < aromatics < organic halogen mixes < sulfides < ethers< esters < aldehydes and ketones < amines < sulphones < amides < carboxylic acids. The quality of associations depends not just on the useful gatherings in the specimen particle additionally on steric variables [10-15]. On the off chance that an atom has a few useful gatherings, then the most polar one decides the response properties [15-20].

The amino propyl and cyanopropyl stages give chances to particular connections between the analyte and the stationary stages and accordingly offer extra choices for the enhancements of partitions. Different preferences of reinforced stages lie in their expanded homogeneity of the stage surface [21-27].

Reversed Phase Chromatography

Countless fortified stationary stages in view of silica are accessible monetarily. Table -2.1 rundowns a percentage of the useful gatherings fortified in artificially adjusted silica. Silica based stationary stages are still most prominent in turned around stage chromatography however different sponges in view of polymer (styrene-di-vinyl benzene copolymer) are gradually making strides.


Table 1: required characteristics

The maintenance diminishes in the accompanying request: aliphatic > incited dipoles (i.e. CCl4) > perpetual dipoles (e.g.CHCl3) > frail Lewis bases (ethers, aldehydes, ketones) > solid Lewis bases (amines) > powerless Lewis acids (alcohols, phenols) > solid Lewis acids (carboxylic acids). Additionally the maintenance increments as the quantity of carbon iotas increments. In turned around stage frameworks the solid alluring strengths between water atoms emerging from the 3-dimentional bury sub-atomic hydrogen fortified system, from a structure of water that must be mutilated or disturbed when a solute is broken up. Just higher polar or ionic solutes VAL associate with the water structure. Non- polar solutes are crushed out of the versatile stage and are moderately insoluble in it however with the hydrocarbon moieties of the stationary stage [28-31].

Performance calculations

Computing the accompanying qualities (which VAL be incorporated in a custom report) used to get to general framework execution.

1. Relative retention

2. Theoretical plates

3.Capacity factor

4. Resolution

5. Peak asymmetry

6. Plates per meter

Relative retention (Selectivity):
= (t2 - ta) / (t1 - ta)

Theoretical plates:
n = 16 (t / W)2

Capacity factor:
K' = (t2 / ta) - 1

R = 2 (t2 - t1) / (W2 + W1)

Peak asymmetry:
T = W0.05 / 2f

Plates per meter:
N = n / L


t2 = Retention time of the second peak measured from point of injection.

t1 = Retention time of the first peak measured from point of injection.

ta = Retention time of an inert peak not retained by the column, measured from point of injection.

n = Theoretical plates.

t = Retention time of the component.

W = Width of the base of the component peak using tangent method.

K' = Capacity factor.

R = Resolution between a peak of interest (peak 2) and the peak preceding it

W2 = Width of the base of component peak 2.

W1 = Width of the base of component peak 1.

T = Peak asymmetry, or tailing factor.

W0.05 = Distance from the leading edge to the tailing edge of the peak, measured at a point 5 % of the peak height from the baseline.

f = Distance from the peak maximum to the leading edge of the peak.

N = Plates per meter.

L = Column length, in meters.

HPLC Method Development

A decent system improvement technique ought to require just the same number of exploratory keeps running as important to accomplish the sought last result [32-37]. At long last technique improvement ought to be as basic as could be expected under the circumstances, and it ought to permit the utilization of refined devices, for example, PC demonstrating. The critical elements, which are to be considered to get dependable quantitative examination [38-43], are

Careful sampling and sample preparation

Choice of the Column

Choice of the operating conditions to obtain the adequate resolution of the mixture

One methodology is to utilize an isocratic versatile period of some normal natural dissolvable quality (50%). A superior option is to utilize an extremely solid versatile stage initial (80-100%) then lessen %B as vital. The starting division with 100% B brings about quick elution of the whole specimen; however few gatherings will isolate [44-49]. Diminishing the dissolvable quality demonstrates the fast partition of all parts with an any longer run time, with a widening of recent groups and diminished maintenance affectability. Objectives that are to be accomplished in system improvement are quickly outlined a roughly in order of decreasing importance but may vary with analysis requirements [50-53].

The time needed for a division (runtime = maintenance time for base band) ought to be as short as could be expected under the circumstances and the aggregate time spent on system advancement is sensible (runtimes 5 to 10 minutes are attractive) [54-56].

HPLC Method Development

Method Validation VAL be characterized as (ICHQ.2B) "Building up recorded proof, which gives a high level of certification that a particular movement will reliably create a fancied result or item meeting its foreordained details and quality qualities".

Strategy approval is a basic piece of the technique advancement; it is the procedure of exhibiting that expository techniques are suitable for their expected utilization and that they bolster the character, quality, immaculateness, and intensity of the medication substances and medication items. Basically, technique acceptance is the procedure of demonstrating that a systematic strategy is satisfactory for its proposed reason.

Strategy Approval, then again, is by and large a one-time procedure performed after the system has been produced to exhibit that the technique is logically solid and that it fills the planned logical need.
For chromatographic methods used in analytical applications there is more consistency in validation practice with key analytical parameters including

(a) Recovery (b) Response function (c) Sensitivity (d) Precision
(e) Accuracy (f) Limit of detection (g) Limit of quantitation
(h) Ruggedness (i) Robustness (j) stability (k) system suitability.


Without a doubt the recovery of scientific technique is measured as the reaction of a prepared spiked lattice standard communicated as a rate of the reaction of unadulterated standard, which has not been subjected to test pretreatment and demonstrates whether the strategy gives a reaction to the whole measure of analyte that is available in the specimen. It is best settled by contrasting the reactions of removed examples at low, medium and high fixations in repeats of no less than 6 with those non-separated guidelines, which speak to 100 % recovery.

Absolute recovery = response of an analyte spike into matrix (processed) X 100 response of analyte of pure standard (unprocessed)


The system is said to be delicate if little changes in fixation bring about expansive changes accordingly work. The affectability of a diagnostic strategy is dead set from the slant of the alignment line. The breaking points of measurement (LOQ) or working element scope of bio explanatory system are characterized as the most astounding and least fixations, which VAL decided with satisfactory exactness. It is recommended that, this be set at ± 15% for both the upper and lower point of confinement of quantitation separately. Any specimen fixation that falls outside the adjustment range VAL not be inserted from the alignment line and extrapolation of the alignment bend is debilitated. In the event that the fixation is over range, the example ought to be weakened in medication free network and re-tested.


The motivation behind doing a determination is to acquire a legitimate appraisal of a "genuine" worth. At the point when one considers the criteria as per which an explanatory system is chosen, precision and exactness are typically the first run through to ring a bell. Precision and exactness together focus the mistake of an individual determination. They are among the most critical criteria for judging diagnostic systems by their outcomes.

Precision alludes to the reproducibility of estimation inside of a set, that is, to the dissipate of scattering of a set about its focal worth. The expression "set" is characterized as alluding to a number (n) of autonomous imitate estimations of some property. A standout amongst the most well-known factual terms utilized is the standard deviation of a populace of perception. Standard deviation is the square foundation of the entirety of squares of deviations of individual results for the mean, partitioned by one not exactly the quantity of results in the set. The standard deviation S, is given by


Relative standard deviation is the standard deviation communicated as a small amount of the mean, i.e., S/x. It is a few times reproduced by 100 and communicated as a percent relative standard deviation. It turns into a more dependable articulation of accuracy. % Relative standard deviation = S x 100/ x


Calibration is the most critical stride in bioactive compound investigation. A decent accuracy and exactness VAL just be acquired when a decent adjustment technique is received [57-60]. In the spectrophotometric routines, the convergance of an example. VAL not be measured straightforwardly, however is resolved utilizing another physical measuring amount "y" (absorbance of an answer). An unambiguous exact or hypothetical relationship VAL be demonstrated between this amount and the centralization of an analyte. The adjustment between y = g (x) is specifically valuable and yields by reversal of the diagnostic estimation capacity.


Exactness ordinarily alludes to the distinction between the mean x****, of the arrangement of results and the genuine or right esteem for the amount measured [61-63]. As indicated by IUPAC exactness identifies with the distinction between results (or mean) and the genuine worth. For logical routines, there are two conceivable methods for deciding the precision, supreme technique and relative strategy.

Precision is best reported as rate predisposition, which is figured from the expression


Since for genuine specimens the genuine quality is not known, an estimate is acquired in light of spiking medication – free network to an ostensible fixation [64,65]. The precision of logical system is then decided at every focus by evaluating the assention between the deliberate and ostensible convergances of the analytes in the spiked medication – free framework sample.


To create reproducible and dependable results, the specimens, benchmarks and reagents utilized for the HPLC system must be stable for a sensible time (e.g. one day, one week, one month and so forth, contingent on need). For instance, the examination of even a solitary example may oblige ten or more chromatographic rushes to focus the framework suitability, including standard focuses to make a working logical bend and copy or triplicate infusions of the specimen to be tested [66-70].