LC-MS LC-FTIR

Principle, Instrumentation, Working, Applications of LC-MS and LC-FTIR Mpharm Pharmaceutical Chemistry 1st year SEM 2nd Advanced spectral analysis

1. 1 Department of Pharmaceutical Sciences Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440-033 Presented by: Dhanashree N. Sarwan M. Pharm. First Year (Pharmaceutical chemistry) LC-MS AND LC-FTIR

2. 2 Hyphenated technique Introduction to LC-MS Principle Instrumentation and Working Applications Introduction to LC-FTIR Principle Instrumentation and Working Applications References CONTENTS

3. 3 HYPHENATED TECHNIQUE The term "hyphenation" was first adapted by Hirschfeld in 1980. The technique developed from coupling of separation technique and spectroscopic detection technology. LIST OF HYPHENATED TECHNIQUES: GC-MS LC-MS LC-FTIR GC-IR GC-NMR ADVANTAGES: Fast and accurate analysis. Higher sample throughput. Better reproducibility.

4. 4 INTRODUCTION TO LC-MS LC/MS is a powerful analytical technique that combines the resolving power of LC with detection specificity of MS. LC separates sample components and then introduces them to MS, creates and detects charged ions. The LC/MS data used to provide information about molecular weight, structure, identity and quantity of specific sample components. In LC-MS we are removing detector from column of LC and fitting column to interface of MS. An interface is used to transfer liquid eluents from LC to MS. In most of cases the interface used are ionization source.

5. 5 PRINCIPLE: High Performance Liquid Chromatography [HPLC] principle is based on adsorption as well as partition phenomenon. MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. The ions are detected, usually by a quantitative method. The ion signal is processed into mass spectra.

6. 6 INSTRUMENTATION AND WORKING

7. 7 Reservoir :- Glass or stainless-steel containers capable of holding up to 1 liter mobile phase. Pump and Gradient controller Injector :- Septum Injector Stop Flow Rheodyne Injector Pre-column Analytical Column:- (Heart of the LC). Actual separation is takes place, made up of Stainless-steel tube. Ionization and Interface source:- Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI) Atmospheric pressure photo ionization (APPI) Mass analyzer : - Quadrupole Time-of-flight Fourier transform-ion cyclotron resonance (FT-ICR or FT-MS) COMPONENTS OF LC-MS

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9. 9 Molecular Weight Determination Structural Elucidation Pharmaceutical Applications Food and Environmental Applications Characterization and Identification of Compounds Proteomics Glycopeptides Characterization Peptide Mapping Bioanalysis of various Biological Samples APPLICATIONS OF LC-MS Pharmacokinetics: Bio analysis Drug development Toxicology Impurity detection Determination of isotopes Analysis of Complex Lipid Mixtures Phytoconstituents

10. 10 INTRODUCTION TO LC-FTIR LC-FTIR (Liquid Chromatography-Fourier-Transform Infrared spectrometry) LC and FTIR can be combined together for the detection and identification of certain separated compounds. The application of FTIR spectroscopy in LC is limited because the solvents used commonly in LC are strong IR absorbers, limiting both sensitivity and spectral information that may be obtained.

11. 11 PRINCIPLE: The light passes through beamsplitter , which sends light in two directions. One beam goes to a stationary mirror then back to the beamsplitter . The other goes to a moving mirror. When two meet up again at beamsplitter , they recombine, but difference in path lengths creates constructive and destructive interference: an interferogram. The recombined beam passes through the sample , absorbs all different wavelengths characteristic of its spectrum, and this subtracts specific wavelengths from interferogram. The detector now reports variation in energy versus time. Energy versus time is an odd way to record a spectrum, until you recognize the relationship between  time  and  frequency : they are reciprocals!

12. 12 The Fourier transform:                         A(r) and X(k) are the frequency domain and time domain points, respectively, for a spectrum of N points.  

13. 13 An interferogram  A spectrum

14. 14 LC setup - Interface - FTIR interface :- Flow-cell interfaces:- The effluent of the LC is passed directly through a flow cell and IR spectra are acquired in real time. Cell-window materials:- Choice for a specific window material is mainly determined by the properties of the LC eluent and the spectral region that has to be monitored B. Solvent-elimination interfaces:- The eluent is removed prior to detection. Eluent is directed to a nebulizer often aided with nebulizer gas. Types of Solvent Elimination Interfaces- Early LC-DRIFT Buffer memory Spray type interfaces :- Thermo-spray interface Particle beam interface Electrospray interface Pneumonic nebulizer Ultrasonic nebulizer COMPONENTS OF LC-FTIR

15. 15 APPLICATIONS OF LC-FTIR In trace analysis. Analysis of environmental pollutants such as polycyclic aromatic hydrocarbons, pesticides and herbicides. Analysis of Pharmaceuticals such as steroids and analgesics, and their impurities, drug metabolites, polymer additives, dyes, non-ionic surfactants and fullerenes. Distinction of isomers Separation of secondary structure of proteins such as beta globulin and lysozyme .

16. 16 Parasuraman S, Anish R, Balamurugan S, Muralidharan S, Kumar KJ, Vijayan V. An overview of liquid chromatography-mass spectroscopy instrumentation. Pharmaceutical methods. 2014 Jul 1;5(2):47-55. Kumar PR, Dinesh SR, Rini R. LCMS—a review and a recent update. J Pharm Pharm Sci. 2016 Mar 1;5:377-91. Stachniuk A, Fornal E. Liquid chromatography-mass spectrometry in the analysis of pesticide residues in food. Food Analytical Methods. 2016 Jun;9:1654-65. Kok SJ. Coupling of liquid chromatography and fourier -transform infrared spectroscopy for the characterization of polymers. Amsterdam: Universiteit van Amsterdam; 2004 Jan 1. Griffiths PR, de Haseth JA. Fourier Transform Infrared Spectrometry John Wiley & Sons. New York. 1986;340. Instrumental Methods of Analysis - By Skoog. REFERENCES

17. 17 THANK YOU