Scene 1 (0s)
. . HPLC. (HIGH PRESSURE LIQUID CHROMATOGRAPHY). KEERTHI THAKUR.
Scene 2 (9s)
. . WHAT IS chromatography?. technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid..
Scene 3 (27s)
. the Pyrtltj(.jt) On thc tasi; of bed Two Threo shape Iquid.
Scene 4 (34s)
. INTRODUCTION is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution..
Scene 5 (45s)
. PRINCIPLE + The principle of separation in normal phase mode and reverse phase mode is adsorption. *They travel according to their relative affinities towards the stationary phase •e The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster..
Scene 6 (1m 0s)
. Normal vs. Reversed Phase Chromatography Stationary phase Mobile phase Sample movement Separation based on Normal Phase Polar (silica gel) Non-polar (organic solvents) Non-polar fastest Different polarities (functionality) Reversed Phase Non-polar (C18) Polar (aqueous/organic) Polar fastest Different hydrocarbon content.
Scene 7 (1m 11s)
. TLC vs. TLC HPLC Type of Analysis Stationary Phase Instrumentation Sample Application Mobile Phase Movement Visualization of Results Form of Results qualitative only 2-dimensional thin layer plate minimal! spotting (capillary) capillary action (during development) UV lightbox spots, Rf's (retention factors) HPLC qualitative & quantitative 3-dimensional column much! with many adjustable parameters injection (Rheodyne injector) high pressure (solvent delivery) "on-line" detection (variable UV/Vis) peaks, RI's (retention times).
Scene 8 (1m 24s)
. *How can We Analyze the Sample? Carbohydrates 1. 2. 3. 4. 5. 6. fructose Glucose Saccharose Palatinose Trehalulose isomaltose mAU Glucose CH20H OH OH time.
Scene 9 (1m 33s)
. Rjrnps Separations Separation in based upon differential migration between the and Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column. Ill 1 Waste High Performance Liquid Chromatograph 7.
Scene 10 (1m 47s)
. ötup qdefimetuouqo P!nbn aoueuuopd qfi!H sdurnd nvtu SUO!JDb1Ddas.
Scene 11 (1m 54s)
. mAU Ptgnps time.
Scene 12 (2m 0s)
. Separations mAU Ptgnps time 10.
Scene 13 (2m 5s)
. Separations mAU mrnps 11 time.
Scene 14 (2m 11s)
. Separations mAU Pumps time.
Scene 15 (2m 17s)
. Separations mAU Pumps time.
Scene 16 (2m 23s)
. acup sdwnd nvtu.
Scene 17 (2m 28s)
. Pumps Separations mAU 11 time.
Scene 18 (2m 34s)
. Separations mAU Ptgnps time 16.
Scene 19 (2m 40s)
. Separations mAU Pumps time 17.
Scene 20 (2m 46s)
. Separations mAU Pumps time 18.
Scene 21 (2m 52s)
. atup 61 sdwnd nvtu su0!JDIDdas.
Scene 22 (2m 57s)
. . mAU Pumps 20 time.
Scene 23 (3m 3s)
. Separations mAU Pumps time.
Scene 24 (3m 9s)
. Separations mAU 01 Pumps time.
Scene 25 (3m 15s)
. The Chromatogram to - elution time of unretained peak tR- retention time - determines sample identity t t t Injection 24 Area or height is proportional to the quantity of analyte. time.
Scene 26 (3m 25s)
. ADVANTAGES 1. Separations fast and efficient (high resolution power) 2. Continuous monitoring of the column effluent 3. It can be applied to the separation and analysis of very complex mixtures 4. Accurate quantitative measurements. 5. Repetitive and reproducible analysis using the same column. 6. Adsorption, partition, ion exchange and exclusion column separations are excellently made..
Scene 27 (3m 43s)
. Adobe Stock | #305865526.