Pharmaceutical Guidelines

What is Delay Volume in HPLC? : Delay volume, dead volume, dwell volume and void volume are well know terms in HPLC analysis. These are useful for an accurate analysis of pharmaceutical products  by HPLC. The delay volume in HPLC is often a topic where most people not only industry specialists in analytical chemistry but also other unrelated spheres  seek clarification or seek to understand what it entails. This article, therefore, is aimed at enlightening anyone in need of having a solid understanding  of the concept be it in the pharmaceutical aspect or the general public intending to have a basic understanding of the idea for knowledge purposes. Giving a concise and clear explanation of the delay volume in HPLC, therefore, will leave everyone who reads the article with a satisfactory and  candid comprehension of the concept. HPLC is an abbreviation for high-performance liquid chromatography. It is a technique used in analytical chemistry to separate, quantify and ...

Theoretical Plates 'N' and their Determination in HPLC Analysis Learn about the theoretical plates N and their calculation in HPLC using the retention time and peak width. This is a helpful parameter to determine the system suitability. Theoretical plates are known as a measuring tool of HPLC column efficiency . Any chromatography column doesn't have any physical plate but it is a result of a mathematical calculation. The columns having a high number of theoretical plates are considered more efficient in HPLC separation than the columns having less number of theoretical plates. A more efficient HPLC column will have a narrower peak than a less efficient column with less theoretical plates at the same retention time. Related: Principle of HPLC High column efficiency is needed to resolve the narrow peaks in the drug analysis. Hence the resolution of peaks also depends upon the column efficiency i.e. theoretical plates. Theoretical plates are calculated per meter length of the...

System Suitability in HPLC Analysis System suitability is to prove that system is working perfectly before the analysis on HPLC, GC, TOC analyzer or any other system. It is required to done before every  sample analysis. HPLC, short for High-performance liquid chromatography is a technique used for separating the components in a mixture. In the HPLC technique, a  liquid sample is passed over an absorbent material to test its efficacy. HPLC chromatography technique is used in pharmaceutical industries for research and testing purposes. Most of the pharmaceutical companies  follow a three-step approach to check the functioning of their High performance liquid chromatography systems- Initial System Qualification, Method  Validation and Suitability Testing (SST). System Suitability Test (SST) Selecting a proper System Suitability Testing mixture is essential to check the specifications of a liquid chromatographic system. System Suitability  Testing limits are accep...

Steps for HPLC Method Validation It is required to validate the HPLC methods used for analysis of pharmaceutical products. It is a regulatory requirement to verify all analytical methods. In the pharmaceutical industry, validation is an essential part of quality control and quality assurance. Various regulatory authorities give particular emphasis on the validation of all the processes used in the industry. Validation is a formal and systematic way to demonstrate the suitability of the assay method to provide useful data to ensure that the process or the technique gives satisfactory and consistent results within the scope of the process. The analytical techniques refer to the way of performing the analysis. Validation is the procedure by which it is set up, by research facility considers, that the execution qualities of the strategy meet the prerequisites for the proposed application. All the investigative procedures that are planned for examining any specimen should be approved. The p...

Steps for HPLC Method Development Know about the different steps of the HPLC analytical method development in pharmaceutical analysis. Analytical method development is considered as a critical process in pharmaceuticals. Availability of the different types of columns, operating  parameters, mobile phase composition, diluent and pH values make it critical to develop an analytical method. A good analytical method should be  simple, used column, mobile phase and buffer should be common. It can be done easily step by step. Following are the common HPLC method development steps. 1. Selection of HPLC Analytical Method 2. Selection of Chromatographic Conditions 3. Parameter Optimization 1. Selection of HPLC Analytical Method: First of all consult the literature that is available on the product. It will help you to understand the nature of  the product that will help to select the different parameters. A. Sample Preparation: Select method to prepare the sample according to its ...

Solvents used in HPLC Mobile Phase Different types of solvents are used in HPLC mobile phase used for different types of chromatography. These solvents are chosen on the basis of the stationary phase  used for the analysis of the product. Perhaps we have already known HPLC, the high-performance liquid chromatography process that isolates, marks and quantifies the constituents of  a mixture. HPLC is extensively involved in the pharmaceutical and other chemical industries. A pressurized liquid mixture contained in a solvent is  passed under significant high pressure through a solid adsorbent column. Principally the process has two physical phases, solid and liquid. The solvent carrying the mixture is the mobile phase since it moves along the  stationary phase or the absorbent column. We will be discussing the solvents used in the HPLC mobile phase as their selection determine how the  analytes can be separated. The solvents used in the HPLC mobile phase a...

Resolution Factor, Tailing Factor, Theoretical Plates and Capacity Factor in HPLC Formula and calculation for resolution factor, tailing factor, theoretical plates and capacity factor in HPLC analysis of pharmaceutical products as per usp chromatography. Resolution factor: Resolution factor shows the accuracy of the quantitative analysis and resolution factor should be greater than 1.5 or specified in the individual  monograph. Resolution factor can be calculated by following formula: R =                   2(t2-t1)                               2.70 (W1h/2+W2h/2) W = Peak width h = Hight t1 = Retention time of first peak t2 = Retention time of second peak Related: Calculation of Relative Response Factor (RRF) Tailing factor: It should meet the requirements of the individual monograph and can be calculated by following formula: T = W ...

Relative Response Factor (RRF) and its Calculation in HPLC Analysis Know about the relative response factor and its calculation in system suitability parameters in HPLC analysis of pharmaceutical substances and pharmaceutical products. During the manufacturing process of active pharmaceutical ingredients, some unwanted substances are produced those are known as impurities.  Some impurities are raised due to the degradation of the actual drug. Impurities in pharmaceutical drug substances and drug products are required to be analyzed for the quantity of the impurities. To determine the  actual quantity of the impurity it is required to have impurity standard. In the absence of the impurity standard relative response factor (RRF) may be  used to calculate the actual quantity of the impurity. Q3B R2 of ICH Guidelines also says that if the response factor is determined correctly, it can be used to measure the actual amount of impurity.  Identification threshold, ...

Principle of HPLC (Liquid Chromatography) Learn how High Performance Liquid Chromatography works (HPLC principle) and how chromatographic column separates the compounds from a mixture. High-performance liquid chromatography is an effective type of column chromatography which is widely used in pharmaceuticals. It is very useful to  determine the assay and related substances in drug substances. In general, HPLC is used to separate the components of a mixed drug substance. In HPLC chromatography, column plays a significant role in the separation of different compounds because it contains stationary phase. The  stationary phase is a bad of polar or non-polar particles according to the type of column. Polar and non-polar columns are used according to the  nature of the sample to be analyzed. Related: HPLC Column Performance Evaluation and Column Care Mechanical pumps are used to pump the mobile phase into the system and injector introduces the sample into the mobile ...

Mechanism of Separation of Components by HPLC HPLC is used in various industries to analyze different products by their separation into its components. Separation technique is described in this article. HPLC stands for High Performance Liquid Chromatography system. This is an analytical chemistry technique that is used to separate, quantify and  identify every component that is in a mixture. The technique depends on pumps to push some constrain liquid solvent which contains a mixture of the  sample through a column containing a solid absorbent material. Each of the components in the sample will interact with the absorbent material in a slightly different way. This will cause different flow rates in the  various components hence causing the components to separate as they flow out of the column. High Performance Liquid Chromatography system is  used mainly for manufacturing, medicine and research purposes. In manufacturing, HPLC is mostly used in the process...

Insight on Data Integrity in Chromatography Chromatography like HPLC and GC are used to analyzed the finished pharmaceutical products. Assay and purity of the products have direct impact on the product quality therefore the analysis must be done accurately and carefully. The core objective of any analytical work is to produce reliable and scientifically accurate data. When the process is compromised the analyst is bound to obtain inaccurate results. This, in turn, affects the integrity of the data. Data integrity in the chromatography analysis is a key area of concern in the pharmaceutical industry. Technicians have linked it to the ease of data manipulation when conducting chromatographic analysis. Since chromatographic data is susceptible to manipulation, regulation must be put in place to control and ensure accurate data is obtained. This is crucial as every laboratory is expected to conform to the conditions and regulations of ISO 17025. This article seeks to outline and e...

HPLC System Software Validation Plan HPLC software are required to be validated to assure the performance of the system. The same should be validated before its use in analysis. Introduction Softwares are used to operate and interpret the analysis results in HPLC systems. There are many softwares those are used in different HPLC systems. All softwares are required to validate to ensure the accuracy of the produced data. Software Validation is a requirement of GxP system . This software validation is intended to provide details of activities carried out and results therein to support the qualification and operation of Computer System, which are                                                                         connected with the HPLC systems . Purpose This validation plan is to outline the a...

HPLC Column Void Volume Void volume in High Performance Liquid Chromatography system analysis plays an important role in accuracy of the analysis. void volume affects the resolution of the HPLC system. High-Performance Liquid Chromatography system is a test used mainly in analytical chemistry for identification, quantitative and control of related substances in chemical mixtures. The technique depends mainly on the uniqueness of the molecular weights and the polarities of elements. Compounded mixtures are passed through the HPLC column and depending on the unique features stated above; they are separated before they can leave the HPLC column. The HPLC set up is composed of two main parts, a mobile phase and a stationary phase. The stationary phase is a solid packed in a column, usually consisting of a silica gel while the mobile phase is a liquid solvent mixture. The mobile phase is pumped up the column where separation takes place followed by detection. There are several volum...

HPLC Calibration Procedure : Know the procedure to calibrate the High Performance Liquid Chromatography ( HPLC) including leakage test, flow rate, reproducibility and linearity, lamp energy and pump pressure drop in Pharmaceutical Quality Control. Check HPLC chromatography (Pump) for the following: Checkpoint: Leakage test (By Pressure Drop) . Flow rate calibration CHECKPOINT: LEAKAGE TEST (BY PRESSURE DROP): 1. Ensure that, the instrument is ready for calibration and Start-up procedure is followed. 2. Place inlet tubing of the Pump into the Water HPLC grade through a suction filter. 3. Allow the mobile phase to flow for about 5 min. 4. Block Pump outlet with the block screw. 5. The pressure rises and on crossing the 300 bar, “ERROR P-MAX” appears on the display window. Note the time. Press “CE” key and observe the pressure drop for 5 min. 6. After 5 min., record the pressure in the Calibration Log. 7. Make entry of the HPLC column usage in the Column Usage...