Introduction Mass spectrometry (MS) is a method used to analyse a sample and measure the mass-to-charge ratio thereof. It can be used to determine the mass of samples as well as the composition of the sample. Liquid chromatography (LC) or more specifically known as high-performance liquid chromatography (HPLC) is a technique that makes use of chromatography to separate a mixture of complex compounds into its constituent molecules and can further be used to identify, quantify and purify these components. The combination of MS and LC is a highly specialised analytical technique with high sensitivity and combines the separation capability of HPLC with the mass analysis of MS (COVEY, et. al., 1986). Liquid chromatography-mass spectrometry (LC-MS also known as HPLC-MS) can be used for the specific detection of a certain chemical substance in the presence of numerous other chemicals closely related to the required substance. Thus the identification of a certain substance in a complex mixture can be done (ARDREY, 2003). Chromatography decreases analysis speed but offers two main benefits. Metabolites with identical masses, but different retention times, can be distinguished and the separation of metabolites from interfering substances allows for improved quantitative accuracy (LAST, et. al., 2007). 2. LC-MS 2.1 Principles of LC-MS and LC-MS/MS The combination of the separation technique of LC allows pure compounds to enter the identification by mass spectrometry and therefore increases the chance of successful identification of an unknown substance. This is based on the principle that many compounds with identical retention characteristics have different mass spectra and in this way can be differentiated. The... ... middle of paper ... ...of sulfonamides. RSC publishing. 30:471-478 GATES, P. 2005. High performance liquid chromatography-mass spectrometry (HPLC/MS). Organic Chemistry Unit-Bristol Research Centre LAST, R.L., JONES, A.D. & SHACHAR-HILL, Y. 2007. Towards the plant metabolome and beyond. Nature Reviews Molecular Cell Biology. 8:167-174 NANDAKUMAR, R., MADAYIPUTHIYA, N. & FOUAD, A.F. 2009. Proteomic analysis of endodontic infections by liquid chromatography-tandem mass spectrometry. Oral microbioligal immunology. 24:347-352 PRZYBOROWSKA, A. 2002. Introduction to Liquid Chromatography/ Mass Spectrometry and Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS). King's College London, United Kingdom ROSEN, J. & HELLENAS, K.E. 2008. Determination of the neurotoxin BMAA in cycad seed and cyanobacteria by LC-MS/MS. The Analyst. 133:1785-1789
Performing this experiment, we used the technique called Acid-Base extraction to isolate Eugenol, which is one of the main ingredients of clove oil. Acid-Base extraction is the most efficient method for isolating organic component; it is efficient because it purifies the acid and base mixture based on their chemical identities. We have seen throughout this experiment that acid and base play an important role, when it comes to solubility in water. Our basic knowledge of acid and base is acid is a proton donor and base is a proton acceptor. This ideology helps us to understand why organic compounds are not soluble in water. When compounds tend to be insoluble, we have to use acid and base reaction, to change its solubility. The changes that occurred
This experiment involves performing various techniques, including heating under reflux, separation, drying, distillation, gas chromatography (GC), infrared spectroscopy (IR spectroscopy), and nuclear magnetic resonance (1H NMR). Heating under reflux is important to overcome any activation barrier of energy that may be present in order to complete the reaction.
Determining the Identity of Unknown Substances by using the Rƒ Factor found with Chromatography Paper
Note: The revised version of the method (9.1.) differs in one major point from reference 9.4.: Preparation of samples and standards for analysis. It is now ...
The history and theory of the gas chromatography started over forty years ago with the invention of the capillary column. The gas chromatograph offers rapid and very high-resolution separations of a very wide range of compounds, with the only restriction that the analyzed substance needs to have sufficient volatility. The theory behind the mass spectrometer is to use the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate them from each other. Mass spectrometry is therefore useful for quantitation of atoms or molecules and also for determining chemical and structural information about molecules. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components.
Chromatography has been developed over the past century and has an important contribution in many areas of modern science. However the main original work of M.S.Tswett was published in a book Chromatographic Adsorption Analysis.
Paper chromatography is the ability to separate specific parts of a mixture, in order to identify its content. There are many forms of chromatography, but paper chromatography tends to work with substances such as dyes, inks, or any colored chemical. In the fields of biology, paper chromatography benefits police that need to test blood. It can also be used by chemists to test substances in their labs. Lastly, it can be used to identify compounds that may be in a plant substance.
UV-visible diode-array detectors and mass spectrometers play are employed during this phase of the method validation. In case of the assay, demonstrating specificity requires the procedure to prove that it is unaffected by the presence of impurities or excipients. In practice, this can be done by spiking the drug product with the appropriate levels of impurities or excipients, and check if the obtained peaks are representative of only one compound or not.
Due to the versatility and inexpensiveness of thin layer chromatography, it is one of the most widely used techniques in laboratories today. Thin layer chromatography has many uses in the realms of pharmaceuticals, forensics, industry and most especially organic chemistry.
HPLC (High Performance Liquid Chromatography) is an analytical technique which separates a complex mixture of components into its specific individual components. It is a powerful tool in analysis, as it combines high speed with extreme sensitivity compared to traditional methods of chromatography because of the use of a pump which creates a high pressure and forces the mobile phase to move with the analyte in high speed. It is been used as a principle technology in various automated analyzers used for diagnostic purpose.
The ways of analyzing drugs and identifying them are microcrystalline tests, gas chromatography, mass spectrometry, and spectrophotometry. Microcrystalline tests are more specific than the color tests and is uses the polarizing microscope. Gas chromatography is when the sample is separated into its different components based on size and chemical structure. Mass spectrometry fragments the molecules in the sample and that pattern of fragmentation helps with the identification of the substance when compared to a known standard. Spectrophotometry identifies substances by measuring how it absorbs the different wavelengths of light including; UV, visual, and infrared. There are all used to decipher the chemicals compositions of the sample to determine what kind of drug is contained in the substance, including how pure the substance is as well.
Office of Pharmaceutical Science (2005). Process Analytical Technology (PAT) Initiative. Retrieved on October 08, 2005 from http://www.fda.gov/cder/OPS/PAT.htm
Furthermore, an additional method to use other hydrochloric acids that have different concentration levels such as 1 M and 2.5 M ones, can improve the outcome of the results. This increases the variation of the independent variable, which accordingly increases the precision of results.
...e retention time under particular conditions is considered a reasonably unique identifying characteristic of a given compound and can be used to identify the compound through HPLC-MS or HPLC-DAD which are the most powerful techniques in this respect (Ahuja and Dong, 2005).
Kuhn and A. Winterstein) published a paper (Ettre & Sakodynskii, 1993) on purification of xanthophylls on CaCO3 adsorption column following the process described by Tswett. In the year 1941, partition chromatography was discovered by R. L. M. Synge and A. J. P. Martin at Cambridge University in the UK, (Martin & Synge, 1941) for that in 1952 they were awarded the Noble Prize. In 1952, Martin and Synge published a seminal paper (Martin, 1941) which, along with the paper of A.T. James and A. J. P. Martin (Martin & Synge, 1952), laid a foundation for the quick growth of chromatographic techniques that shortly followed. Prior to the 1970's, few good chromatographic methods were commercially obtainable to the laboratory scientist. During 1970's, most chemical separations were performed using different techniques including open-column chromatography, TLC (thin-layer chromatography) and paper chromatography. However, these chromatographic techniques were insufficient for resolution between similar compounds and quantification of compounds. During this time, to decrease flow-through time pressure liquid chromatography began to be used, thus reducing time taken for purification and separation of compounds being isolated by column chromatography. As flow rates were Inconsistent, the question if it was good to have a constant flow rate or constant pressure was debated (Chatrabhuji et al., 2015). In the mid-1970's, High pressure liquid chromatography (HPLC) was developed and improved rapidly with the development of different column packing materials and the additional suitable detectors. Some new methods including reverse phase liquid chromatography allowed for better separation between very similar molecules. By 1980's, for the separation of chemical molecules, HPLC was widely used. New techniques enhance purification, separation,