High-performance liquid chromatography originally referred to as high-pressure liquid chromatography, is an extremely important technique in the world of analytical chemistry. It is normally utilized for the separation, identification as well as quantification of every component present in the mixture. The method depends on pumps to pass a pressurized liquid solvent that contains the sample mixture via a column full of a solid adsorbent material.
Normally, each element present in the sample mixture interacts a bit differently with the adsorbent material. This, in turn, causes different rates of flow for the varying components and thus resulting in the separation of elements as they leave the column. This incredibly important technique has been utilized for manufacturing – in the production of pharmaceutical as well as biological products. It has also been used in other vital areas such as legal processes (in the determination of performance improvement drugs in urine), in research and even in the medical field such as during the determination of levels of Vitamin D in blood serum tests.
On the other hand, the response factor is defined as the ratio between the concentration of a chemical under analysis and the response of the detector to the concentration of the analyte. The concentration is divided by the peak area to obtain the response factor. For example, suppose you are handling a solution with three substances. The simplest way of separating these elements is to immerse it in an adsorbent material and then inject it into the HPLC. The chromatogram being used delivers a response from the detector as a peak of the varying components. In Gas Chromatography, as well as analysis methodology, these variations are to blame for the different variations witnessed. Upon finding a response-factor of a given chemical, the concentration of the chemical in the future samples can easily be computed from the detector response of the said sample.
Undoubtedly, response factors are extremely critical in the application of Gas Chromatography as far as quantitative analysis is concerned. However, there is also an urgent need to pursue a technique that can help get rid of any potential variations in response factor. A simple, yet practical means of achieving this is through the use of relative response factors (RRF) and internal standards in the calibration of Gas chromatography. The RF is obtained through calibration of chromatography using known standard solutions.
Response factor in HPLC analysis is largely used in the production of pharmaceuticals. For the impurity standard to be obtained, it is important that impurities in the ingredients of pharmaceuticals are generated and their analysis conducted. Apparently, this can only happen if RF is obtained and then RRF is employed. The latter comes in an alternative approach to determining the amount of impurities present in pharmaceutical products.
The process entails monitoring and evaluation of the peak area of every component. To obtain the RRF, one is required to divide the response factor of the impurity by that of the active pharmaceutical ingredient (RFimpurity/RFAPI). This means that the accurate response factor can be gotten through a quantitative analysis of the API and impurity standard. To obtain accurate RRF, the slope of the area concentration must be calculated as well as the impurity standard.