HPLC and its relation with mass spectroscopy

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1. MAULANA ABUL KALAM AZAD UNIVERSITY OF TECHNOLOGY CA-4 : POWERPOINT PRESENTATION TOPIC : HPLC AND ITS RELATION WITH MASS SPECTROSCOPY. GROUP 4: B.SC. BIOTECHNOLOGY(4 TH SEMESTER) DEBJANI SAHA DEBANSHU GHOSH ANKITA SHOME RAJDIP GHOSH SREEJIT DAS DOYEL PAL SALMAN HOQUE B.SC BIOINFORMATICS(4 TH SEMESTER) MEGHA GHOSH SAYARI PAL

2. INTRODUCTION High Performance Liquid Chromatography (HPLC) is a type of chromatography used to separate, identify, and quantify compounds in a mixture. It is widely used in the pharmaceutical, food, and environmental industries. HPLC utilizes a high pressure pump to push a mobile phase (a liquid or a gas) through a column containing a stationary phase (a liquid or solid). The compounds in the sample are separated based on their interactions with the stationary and mobile phases. The separated compounds are then detected and quantified using a variety of detectors. This technique is highly sensitive, reliable and reproducible, making it one of the most widely used chromatography techniques. The Liquid Chromatography-Mass Spectrometry (LC-MS) is hyphenated analytical technique which is combination of Liquid Chromatography (LC) and Mass Spectrometry (MS).

3. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC) Aim: HPLC separates a mixture components based on their polarity. Stationary phase: Dimethylpolysiloxane (Hydrophobic). Mobile phase: Polar solvent like borate buffer/acetic acid(Hydrophilic). Principle: 1)The sample is injected into the mobile phase flow from the pump to the separation column using a syringe. 2)Subsequently, the individual components of the sample migrate through the column at different rates because they are retained to a varying degree by interactions with the stationary phase. 3) After leaving the column, the individual substances are detected by a suitable detector and passed on as a signal to the HPLC software on the computer. 4) At the end of this operation/run, a chromatogram in the HPLC software on the computer is obtained. 5) The chromatogram allows the identification and quantification of the different substances.

4. There are three types of detector in HPLC:- (i)Refractive Index Detector (ii) UV-Vis spectrophotometer(Scanning and variable wavelength detector). (iii) Fluorescence Detector. More hydrophobicity, less polarity, retention time increases. Less hydrophobicity, more polar, retention time decreases.

5. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY (HPLC-MS) High performance liquid chromatography-mass spectrometry (HPLC-MS) is an effective analytical technique for determining the composition and purity of chemicals. The combination of high performance liquid chromatography (HPLC) and mass spectrometry (MS) offers great capabilities in physical separation and mass analysis, providing accurate data on sample composition.

6. OPERATING PRINCIPLE OF HPLC-MS In HPLC-MS, the two techniques (HPLC and MS) are connected by an interface that transfers the separated components from the liquid chromatograph column into the mass spectrometer ion source. An interface is needed since HPLC operates at high pressure and the MS system has a high vacuum. HPLC includes a mobile phase and a stationary phase, both of which can be modified to suit the sample matrix and the desired properties to be determined. Usually, the mobile phase is adjusted to suit the sample, and the stationary phase is adjusted to work well with the mobile phase. The degree of compound separation is based on the compound’s affinity for the mobile phase. HPLC methods can be divided into two main categories based on the properties of the stationary and mobile phases. A combination of polar stationary phase and non-polar mobile phase is called "normal-phase chromatography" and the opposite of that, the combination of non-polar stationery and polar mobile phase is called "reverse-phase chromatography".

7. COMBINING HPLC WITH MASS SPECTROSCOPY Once compounds have been separated using HPLC, they are identified and their contents are determined by mass spectrometry (MS), which creates a mass spectrum that is unique for every compound. In mass spectrometry, the compounds and their fragments are ionized using either electron or chemical ionization. The sample is then accelerated through a mass analyzer, which includes either a quadrupole or an ion trap, and the ions are identified based on their mass-to-charge (m/z) ratios. It is also possible to combine HPLC with detectors other than the mass spectrometer. In HPLC-DAD analysis, for example, a diode-array detector is used instead. The choice of the most appropriate detector depends on the analyte and the goal of the analysis. In general, HPLC-MS is more suitable for identifying unknown components, while the DAD-detector works well in quantifying known components.

8. APPLICATION OF HPLC-MS IN FOREINSIC SCIENCE: LC-MS is used for determination of toxicity, in drug analysis and also in trace analysis. IN DOPING TEST: The LC/ESI-MS with positive mode can be used for detection in urine of 4-Methyl-2-hexaneamine doping agent. IN PHARMACOKINETICS: LC-MS is used in the study of absorption, metabolism, and excretion of drugs. IN BIOAVAILABILITY AND BIOEQUIVALENCE STUDY: Comparative bioequivalence studies in which quantitative determination of drugs or metabolites is measured in biological matrix, pharmacodynamics, clinical trials and In-vitro dissolution tests.

9. IN DETERMINATION OF MOLECULAR WEIGHTS: LC-MS is used for determination of molecular weights of known and unknown compounds. IN DETERMINATION OF ASSAY OF DRUG AND INTERMEDIATES: LC-MS is used in pharmaceutical industry for determination of assay of drug substances, drug products, intermediates and their related compounds. IN AGROCHEMICAL AND PESTICIDES INDUSTRY: It is used in determination of different components present in the fertilizers and pesticides. ENVIRONMENTAL APPLICATIONS: LC-MS is used for detection of phenyl urea herbicides, detection of low level of carbaryl in food.

10. SOME RESEARCH WORK ON HPLC-MS The development and use of HPLC-MS for the study of metabonomics is reviewed. To date the technique has been applied to the analysis of urine samples obtained from studies in rodents in investigations of physiological variation (e.g., factors such as strain, gender, diurnal variation, etc.) and toxicity. Examples are provided in the form of graphs to the use of conventional HPLC, capillary methods and the recently introduced high-resolution systems based on a combination of high pressure and small particle size (“UPLC”). Comparison is also made of the use of 1HNMR spectroscopy and HPLC-MS for the analysis of biofluid samples and the advantages and limitations of the two approaches are assessed. Likely future developments are considered.

11. CONCLUSION High-performance liquid chromatography or commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture. The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy. The LC-MS is a hyphenated technique used in combination with separation power of HPLC with detection power of Mass spectrometry. It is widely used in pharmaceutical, chemical, food, agrochemical industries, environmental and forensic applications. LC-MS is used for qualitative and quantitative determination of drug substances and biological samples. Also it is commonly used in drug research and quality control .

12. ACKNOWLEDGEMENT We would like to thank Mr. Arnab Kumar Ghosh , our respected professor for introducing us to such a nice topic and for his valuable guidance in completing our presentation. I would like to take this opportunity to express my gratitude to all of my group members Debanshu Ghosh, Rajdip Ghosh, Ankita Shome, Sreejit Das, Doyel Pal, Salman Hoque, Megha Ghosh and Sayari Pal for their valuable comment and suggestions on this presentation. We all have equally contributed in this PowerPoint presentation. by Debjani Saha

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