DETECTORS IN HPLC
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The document details a seminar on chromatography detectors presented by K. Tarun from the Department of Industrial Pharmacy, focusing on various types of detectors used in High-Performance Liquid Chromatography (HPLC). It describes the principles of HPLC, the characteristics and classifications of detectors, including UV-Visible, refractive index, fluorescence, electrochemical, and mass spectrometry detectors. The seminar emphasizes the selection of detectors based on the properties of compounds for accurate analysis in pharmaceutical education and research.
DETECTORS IN HPLC
- 1. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1 DETECTORS IN HPLC A seminar as a part of curricular requirement for I year M. Pharm I semester Presented by K. Tarun (20L81S0805) Dept. of Industrial Pharmacy Under the guidance/Mentorship of Dr. Hindustan Abdul Ahad M. Pharm, Ph.D, FAGE Life member of IPA & APTI Professor & Head, Dept. of Industrial Pharmacy
- 2. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2 Introduction Principle Types of detectors List of Detectors Conclusion Reference Contents:
- 3. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3 • A chromatography detector is a device to detect components of the mixture being eluted off the chromatography column. Properties: 1. Low drift and noise level. 2. High sensitivity. 3. Fast response. 4. Wide linear dynamic range. 5. Low dead volume (minimal peak broadening). 6. It should be non-destructive. Introduction:
- 4. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4 • HPLC is based on adsorption as well as partition chromatography. • It is depends on the nature of stationary phase. • Determination of volatile and non-volatile compounds. Principle: Stationary phase Solid Adsorption Stationary phase Liquid Partition
- 5. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5 Types of detectors Bulk Property Detectors: Refractive Index Detector Evaporative Light Scattering Detector Solute Property Detectors Electrochemi cal Detector UV- visible Detector Mass Detector (LC-MS) Fluoresc ence Detector
- 6. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6 1. UV-visible Detector a. Fixed / Single Wavelength Detector b. Variable Wavelength Detector c. Photodiode Array Detector / DAD 2. Refractive Index Detector 3. Fluorescence Detector 4. Electrochemical Detector 5. Conductivity Detector / Potentiometry 6. Evaporative Light Scattering Detector (ELSD) 7. Mass Detector (LC-MS) 8. IR Detector 9. Optical Rotation Detector (ORD) / Chiral Detectors 10.Corona Discharge Detectors List of detectors:
- 7. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7 1. UV-Visible Detector: • They give specific response to the class of compounds or particular compounds depending upon the functional group of eluting molecules. • When the light of a certain wavelength is directed at a flow cell, the substance inside the flow cell absorbs light. • These detectors are different types:
- 8. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8 Such type of detectors does not allow change in wavelength of the radiation. Absorbance of only one given wavelength is monitored by the system at all times (usually 254nm). Limited in flexibility. Simplest & Cheapest of the UV/Visible. a. Fixed Wavelength Detector:
- 9. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9 • These can be adjusted to work on any wavelength over full UV-Visible region. • The wavelength can be selected at 3nm or less. • More expensive. • More versatile. b. Variable Wavelength Detector:
- 10. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10 Light from the broad emission source such as deuterium lamp. It is collimated by an achromatic lens system. So that the total light passes through the detector cell onto a holographic grating. The dispersed light from the grating is allowed to fall on to a diode array. c. Photodiode Array Detector:
- 11. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11 • Refractive index detector measures the molecule’s ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase contained in a reference cell. • The amount of detection is proportional to the concentration of the solute in the mobile phase. 2. Refractive Index Detector:
- 12. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12 o The are different types are: Deflection refractometer Fresnel refractometer Interferometer detectors o Mostly used for detection of non-ionic compounds that neither fluorescence nor absorb in the UV region. o Draw backs: Less sensitive Temperature control Less suitability to gradient elution.
- 13. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13 Fluorescence is 10-1000 times sensitive than UV detector for strong UV absorbing compounds. Even a single analyte in the cell can be detected by the fluorescence detector. The system is easy to operate and relatively stable. Sensitivity depends on the fluorescence properties of the components in the elute. 3. Fluorescence Detector:
- 14. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14 Fluorescence Detector
- 15. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15 • There are 3 types of fluorescence detectors: Fluorescence Detector Single Wavelength Excitation Detector Multi Wavelength Fluorescence Detector Laser Induced Fluorescence Detector (LIFD)
- 16. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16 • Usually measure the current associated with the oxidation or reduction of solutes. • They are sensitive to changes in the flow rate or composition of the eluent. • The level of current is directly proportional to the analyte concentration. • Three electrodes are employed which are: Working electrode Auxiliary electrode Reference electrode 4. Electrochemical Detector:
- 17. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17 It is used in analytical applications for the detection of ionic compounds. Measures the ability of the mobile phase to conduct a current when placed in a flow-cell between 2 electrodes. Measured Value is directly proportional to the concentration of ions present in the solution. 5. Conductivity Detector:
- 18. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18 It is useful for large molecular weight molecules like surfactants, lipids and sugar. Detection is based on the scattering of a beam of light by particles of compound remaining after evaporation of the mobile phase. It is a universal detector Does not required a compound to have a chromophore for detection. There are 3 steps involved in detection: o Nebulization o Mobile phase evaporation o Detection 6. Evaporative Light Scattering Detector (ELSD):
- 19. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19 The mobile phase which must be volatile. The flow from the column is nebulized with a stream of inert gas. The particles are passed through a laser beam and they scatter the laser light. The scattered light is measured at right angles to the laser beam by a photodiode detector.
- 20. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20 The detection by mass spectrometers is based on molecular fragmentation by electric fields. Separation is based on the mass to charge ration of the fragmented molecule. It is a method that combines separation power of HPLC with detection power of mass spectrometry. They show high selectivity & sensitivity. In LC-MS technique advantage of resolution and sensitivity. The analyte for mass spectrometer must be in ionic form. 7. Mass Spectrometer:
- 21. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21 Mass Spectrometer
- 22. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22 Infrared detectors are chosen on the basis of property of compound to absorb infrared light. It is a sophisticated instrument and requires Cells or windows made of NaCl or CaF2. The wavelength scanning is provided by semicircular filter wedges, the wavelength range from 4000-690 cm-1. The detector is suitable for polymer analysis. Not very sensitive Mobile phase solvents absorb strongly in the IR region. 8. IR Detector:
- 23. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23 o Used for detection of optically active compounds such as amino acids, sugars & terpenes containing an asymmetric carbon. o There are 2 chiral detection techniques: Polarimetry / optical rotary dispersion (ORD) Circular dichroism (CD) o ORD detectors are based on differences in refractive index. o CD differentiate enantiomers by measuring differences between the absorption of light & left handed circularly polarized light due to existence of a chiral chromophore. 9. Optical Rotation Detector (ORD) / Chiral Detectors:
- 24. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24 Optical Rotation Detector
- 25. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25 Other name for this is Charged Aerosol Detection (CAD). It is a newer and unique technique. The HPLC column eluent is first nebulized with a nitrogen (or air) carrier gas to form droplets which are then dried to remove mobile phase, producing analyte particles. The analytes then are further transferred to a collector where it is measured by a highly sensitive electrometer. 10. Corona Discharge Detector:
- 26. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 26 Charged Aerosol Detection
- 27. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 27 The work of the detector is to detect and give the evidence to the recorder which shows it in a form of a chromatogram. It senses at a more rapidly. Each compound has its properties that are not the same as one another, thus arises a need to have different detectors for different compounds. Conclusion:
- 28. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 28 1. Swartz M. HPLC detectors: a brief review. Journal of Liquid Chromatography & Related Technologies. 2010 Jul 13;33(9-12):1130-50. 2. Sunil A, Anju G, Rajat V. HPLC Detectors, Their Types and Use: A Review. Organic & Medicinal Chemistry International Journal. 2018;6(5):143-6. 3. Sims JL, Carreira JA, Bragg RA. Optical rotation detection for reversed phase HPLC: Investigation of solvent effects. Chirality. 1997;9(2):122-5. 4. Sagliano Jr N, Hartwick RA. Micro-HPLC detectors: a review. Journal of chromatographic science. 1986 Nov 1;24(11):506-12. References:
- 29. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 29