In most instances, calibration in fact requires a series of standards of. However, by using a mals, mw can be determined directly without the need of calibration curve. Difference between hplc and hptlc compare the difference. Take the print out of the chromatograms and attach to the calibration log. Figure 3 shows the hptlc densitogram for mixed standard. Make entry of the usage of the instrument and hplc column in the instrument usage log register and column usage log register respectively. The present study describes development and subsequent validation of stability indicating hplc and hptlc methods for simultaneous estimation of telmisartan tlm and atorvastatin atv in their combined formulation.
Stock solution1 weigh 500 mg of sodium salicylate in 250 ml volumetric flask dissolve and dilute with 96%vv alcohol. Band stability was assessed by inspecting the developed plates after 30 min and 1, 2, 4, and 12 h. Development and validation of hptlc method for determination of glycyrrhizin in herbal extract and in herbal gel varsha m. Development and validation of hptlc method for the. In addition to providing an introduction to hplc for pharmaceutical analysis it is intended that this book will be a useful resource. A number of enhancements can be made to the basic method of thinlayer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements. Calibration curve of apigenin was prepared by plotting peak area versus concentration of apigenin applied. Pdf development and validation of hptlc method for. Hptlc have similar approach and employ the same physical principles of tlc adsorption chromatography i. After development the chromatogram was evaluated for additional bands. The plate was developed and scanned as per the chromatographic conditions mentioned above. Department of pharmaceutical chemistry and analysis, indukaka ipcowala college of pharmacy, new vallabh vidyanagar388121, gujarat, india corresponding. The validation of developed hptlc method was carried out as per the international conference on harmonization ich guidelines q2 r1 for specificity, sensitivity, accuracy, precision and repeatability. Usually the linearized forms are used but the nonlinear regression analysis is therefore used in this paper.
For the sec analysis, mw of analyte is estimated from the calibration curve drown using a set of known standards. Hptlc method development and validation journal of. Quantitative analysis of oxyresveratrol in different plant. A calibration plot of peak area against concentration was prepared after chromatography of five different concentrations in the range 200 ng per band. Alumina glass plates sodium salicylate 96%vv alcohol preparation of stock solution. It is necessary that the stationary phase particles be as small as possible in order to provide a large surface area so that sorption and desorption of the solutes will occur frequently.
Validation and calibration of analytical instruments. Key difference hplc vs hptlc the key difference between hplc and hptlc is that hplc allows quantitative separation of components in a sample whereas hptlc does not allow quantitative separation of components in a sample chromatography is a physical separation method that is used to separate and identify the components in a mixture. Hptlc method for estimation of topiramate in solubility. The calibration curve was constructed by plotting the peak area versus the corresponding tpm concentration. Hplc and hptlc methods for simultaneous estimation of. Standard operating procedure to calibrate the high performance thin layer. The regression coefficient r2 for calibration curve of ramipril and telmisartan was 0. Development of tlc and hptlc method for determination. Development and validation of hptlc method for estimation of. Hplc detectors types comparison principles pdf ppt.
Calibration in tlchptlc using the michaelismenten function. The aims of this study were to compare hptlc and hplc, for qualitative and quantitative analysis of the major constituents of calendula officinalis. Validated hptlc method for quantification of luteolin and. Parameters validated by hptlc method accuracy% recovery fig.
A quantitative hptlc method was also developed using hexane. Hptlc journal of scientific and innovative research. The regression coefficient r2 for calibration curve of cefta and tazo was 0. Validation of hplc technique authorstream presentation.
The preferred method of choice for calibration in tlchptlc is external. Suited for object formats up to about 16 x 21 cm 20 x 10 cm and 10 x 10 cm tlc hptlc plates. Record the area and retention times of the of the benzene and toluene peaks in the calibration log. How to make a calibration standard for an hplc sciencing. Methods developed for the estimation of hca hptlc is a modern version of tlc with accurate spotting and quantification, which plays an important role in the quality control of food stuffs, usually with emphasis on the investigation of ingredients like lipids, carbohydrates, vitamins. Calibration is essential to improving a companys bottom line, by minimizing risk to product defects and recalls, and. Chromatography techniques such as hptlc and hplc are commonly used to produce a chemical fingerprint of a plant to allow identification and quantify the main constituents within the plant. Highperformance thinlayer chromatography wikipedia. Pdf quantitative analysis and validation of method using hptlc. A validated hptlc method for simultaneous estimation of. Hplc detectors types comparison principles power point pdf ppt multiangle light scattering detector. Each reading was the average of three determinations.
Simultaneous determination of metronidazole and miconazole nitrate in gel by hptlc 324 pak. Hplc standard operating procedure last revised march, 3, 2016 this document is intended to be a guide for operating the garner labs hplc systems and is only intended to provide instructions for users who are already generally familiar with hplc operation. The mixture of all plant extracts was applied on hptlc plate to check the presence of marker. Normal phase adsorption tlc on silica gel with a less polar mobile phase, such as chloroform methanol, has been used for more than 90% of reported analysis. Hptlc take place in high speed capillary flow rang of the mobile phase, there are three main step in hptlc 1 sample to analyzed to chromatogram layer volume precision and suitable position are achieved by use of suitable instrument. L syringe hamilton, bonaduz, switzerland which was programmed using. Typical data obtained in the calibration of a buret for the calibration below, assume that the temperature of the water is 23ec your water temperature may be different. A quality control for authentication of herbal phytochemicals article september 2011 with 20,048 reads how we measure reads. Srivastava editor the present edited book is the presentation of 18 indepth national and interna. Calibration basics and best practices individuals who view calibration as a necessary evil are not taking into account the bigger picture.
The mobile phase may be a pure liquid or a mixture of solutions e. The best fitting calibration model with singlelevel calibration or multilevel calibration via linear, polynomial or michaelismenten regression is selected. Hptlc densitometry method for simultaneous determination. Among the different mobile phase combinations tested, mobile phase consisting toluene. At this temperature,the conversion factor from mass of water to volume, corrected for buoyancy and glass expansion, is 1. Different r f values obtained in the hptlc method 0. In the example, i have chosen to calibrate the measurements using a doorway of three feet 3.
The standard solution of apigenin 50 to 250 ngband was applied in triplicate on hptlc plate. Sop for calibration of high performance thin layer. Calibration curve was constructed by plotting the peak area ratios of cmn and qcn versus the concentrations of the calibration standards. Kadam department of quality assurance, bharati vidyapeeths college of pharmacy, sector8, cbd belapur, navi mumbai400614, india. The calibration curve of the standard drug concentration xaxis over the average peak heightarea yaxis was prepared to get a regression equation by win cats software, which was used for the estimation of ascorbic acid in amla. Falgun a mehta, emanual michael patelia and pradip n bhoya. Proper calibration of an hplc instrument begins with the making of a suitable calibration standard. High performance thin layer chromatography hptlc and. Samples were applied using a linomat 5 camag, muttenz, switzerland semiautomated applicator with a 100. The linear regression analysis data for the calibration plots showed good. This hplc calibration standard operating procedure sop describes all the individual steps necessary for calibrating a hplc in accordance with cgmp current good manufacturing practices. Hptlc plates provide improved resolution, higher detection sensitivity, and improved in situ quantification and are used for industrial pharmaceutical densitometric quantitative analysis. When the developed method was applied to the sample, hptlc chromatograms were obtained for cmn and qcn, which were shown in fig. Development and validation of stability indicating hptlc.
Pharmaceutical formulation by hptlc desitometric method. Hptlc is the method of choice for many analytical tasks a broad range of application notes is available for download. As for any calibration in the pharmaceutical environment, the calibration of the hplc also needs to be described in a procedure and followed by the analyst in line with regulations. The michaelismenten function describes a non linear relationship and is a possible calibration curve in tlc. Ich the demonstration that a particular instrument or device produces results within specified limits by comparison with those produced by a reference or traceable standard over an appropriate range of measurements. The hptlc system camag, muttanz, switzerland with wincats software under window xp was used for the analysis of constituents. Simultaneous estimation of ambroxol hydrochloride and.
Simultaneous estimation of ambroxol hydrochloride and doxofylline in. The hplc and hptlc methods described here are simple, rapid, accurate and. Hptlc identification, quantification and validation of p. Hplc calibration standard operation procedure youtube. When working with high performance liquid chromatography hplc, good calibration is absolutely essential to ensure reliable, quality results. The plumbagin content in the roots were determined using both the methods. Calibration and validation of analytical instruments. Both hplc and hptlc methods exhibit baseline noise, so the detection limit is determined by the signaltonoise ratio. Development and validation of hplc and hptlc methods for. The proposed rphplc method utilizes a phenomenex luna c 18 column using acetonitrile.