Reverse-Phase High performance chromatography consists of any method of chromatography that utilizes a non-polar phase. In the early part of 1970s, non-modofied alumina or silica which has a greater attraction for polar elements and hydrophilic surface chemistry was used for majority of the liquid chromatography. Because of this, this method was said to be “normal”. The elution order was however reversed with the introduction of a covalently bonded alkyl chain which reinforces the surface.
In this method, the first to be eluted are the polar compounds while there is retention of the non-polar compounds hence the procedure is termed ‘reversed phase” Method validation It’s important to validate all various HPLC methods. Evaluation of suitability may not be necessary when methods from pharmacopoeia’s are utilizes given that the analyses are carried out with respect to the technique’s intended use. However, in situation where drug composition is being modified, a reevaluation of suitability of the HPLC method to its new intended use is necessary (Nagels etal, 2002).
The variables tested in the validation process as stated by FDA, ICH and USP as well as other health bodies include: Accuracy, linearity, limit of quantitation, limit of detection specificity & relativity, reproducibility & precision which includes repeatability, intermediate precision as well as ruggedness/reproducibility (Amersham, 1999). Role of rp-HPLC method for the measuring hydrolysis of ester Reverse phase high pressure liquid chromatography can be used in separation & quantitation of free fatty acids as well as its methyl esters obtained from tissue phosphopids of mineral.
In this method, mixtures of water & acetonitrile are utilized for elution of esters. For free acids to separate, aqueous phosphoric acid substitute for water. Quantification & detection of unsaturated compounds are done by absorption at 192nm . After methyl esters have been prefractionated based on its unsaturation by argentation TLC, there is total & rapid separation by elution with CH3CN. Reverse phase HPLC can both be applied as preparative & analytic method as well as in biochemical separation and purification. Precision
In rp-HPLC, precision is assessed by injection of several standards followed by measurement of the variability of the results. Three subcategories are obtained from the measured standard deviation . To assess repeatability, the operator executes the analysis in a laboratory over a short period of time. Then, the relative standard deviation is calculated from 5 or 6 determinates of two dissimilar matrices at 1 or 2 different concentrations. The ICH describes intermediate precision as long term variability & this is estimated by comparison of several results of a single laboratory over several weeks.
Variations of results gotten by different instruments, different batches of column and from operators with materials from dissimilar supplies are reflected in intermediate precision (Amersham, 1999) It is important to validate intermediate precision in rp-HPLC so that verification of same results being produced by same method in same laboratory following the phase of development could be possible. Reproducibility otherwise called ruggedness is simply inter-laboratory precision . It’s aim is to ensure that different laboratories will have same results using the method.
Variables that affect the reproducibility of rp-HPLC include the varying operator experience, varying ages of equipment, differential supplies of columns, variation in humidity & room temperature, different characteristics of equipment such as the delay capacity of HPLC system (Lebendiker, 2006). Accuracy is the degree in which test outcomes are proximate to the true value. This is obtained from outcome of quantitative assessment of a known sample. Whatever is measured is then compared to the known amount. Selectivity & specificity These are usually interchanged .
Selectivity of analytical method like rp-HPLC refers to its capability despite the presence of interference, to still be able to measure accurately (Guzzetta, 2010). Such interference includes enantiomers, degradation products and excipients. When selectivity of a method is verified, it becomes “Stability Indicating Method”. Limit of detection This refers to the least amount or concentration of analyte in a chromatographic sample that may not be quantifiable but is detectable. In rp-HPLC, the limit of detection is that amount which after injected gives a maximum height of 2 to 3 times as raised as the reference noise level.
Limit of quantitation refers to the least amount injected that results in precise measurement. In rp-HPLC, maximum heights of about ten to twenty times greater than reference noise are usually required at precision of less than 10-15% RSD between intervals of results. Linearity With injection of three to five series of four or more standards with spanning concentration of 80-120% expected range, the linearity of a rp-HPLC analytical method can be determined (Amersham, 1999). Whether directly or by calculation, it is expected that the response should be in proportion to the analytes’ concentration.
The intercept of linear regression formula applied to these results should not substantially differ from zero. An intercept of significant non-zero should be followed by validation that there is no impact on method accuracy (Alveldano etal, 2010). Principle This operates of the separation of substances based on hydrophobic binding between the stationary/immobile hydrophobic ligand (called the stationary phase) and the solute in the mobile phase (Amersham, 1999). The binding interaction is presumed to be due to favorable entropy effect.
The original mobile phase conditions in rp-HPLC are aqueous implying a highly organized water structure around stationary ligand & solute molecule . The available area exposed to solvent becomes diminished with binding of solute to the stationary hydrophobic ligand. With this, there is decrease in organization of water structure & increase system entropy (Amersham, 1999). In summary, rp-HPLC is based on “adsorption of hydrophobic molecules onto a hydrophobic solid support in a polar mobile phase”. Reverse phase HPLC has also been utilized in most analytical methods.