High Performance Liquid Chromatography HPLC- UV-VIS Detector Animation

high-performance liquid chromatography is a technique and analytical chemistry used to separate identify and quantify each component in a mixture as well as other forms of chromatography in HPLC there is a mobile phase which forced by a pump to pass through the system and there is a stationary phase called a column which located in an oven where the temperature can be controlled the sample can be automatically injected into an HPLC system by the use of HPLC auto sampler or can be manually introduced into the injector using a syringe there is also a detector attached to the HPLC system which measures the analytes after its separation in the column the pump forces the mobile phase through the column and then the detector under high pressures in the HPLC system a vacuum pump and a dagger sir are connected to the pump and used to remove dissolved gases from the solvents the pump drives each solvent to the mixing chamber where mixing takes place under higher pressures HPLC analysis commonly uses isocratic or gradient elution an isocratic mode the mobile phase composition remains constant throughout the procedure whereas in gradient mode the mobile phase composition is changed during the separation process sample introduction can be accomplished in various ways the simplest method is to use an injection valve in the load position the high-pressure el Yuting solvent flows to the column directly the loop is loaded at the atmospheric pressure from a syringe via the needle port excess sample exits the loop via vent port after loading the sample the valve is switched to the inject position the flow delivered by the pump flows through the loop forcing the sample ahead of it flowing to the column then the valve returns to the load position and the mobile phase moves the sample through the column the separation is based on differential partitioning of the sample components between the mobile and stationary phases the component which has more affinity to the mobile phase consequently less affinity to the stationary phase travels faster and eluded out first and the component which has more affinity to the stationary phase consequently more interaction travels slower and diluted later this separation can be carried out according to which type of HPLC used there are two main types of HPLC reverse phase and normal phase reversed phase has a non-polar stationary phase and moderately polar mobile phase one common stationary phase is a silica which has been modified by attaching a straight chain alkyl group to its surface such as the ITAT as'll group c18 or the octal group c8 in this case the more hydrophobic the analytes are the more retained they will be on the stationary phase the more polar they are the more they will prefer the mobile phase then normal phase chromatography the stationary phase is polar and the mobile phase is non-polar this method separates analytes based on their affinity for a polar stationary surface such as silica in this case the more polar the analytes are the more retained they will be on the stationary phase the more hydrophobic they are the more they will prefer the mobile phase as compounds ellyiot from the column they interact with the detector different types of detectors can be used such as the UV this detector which showing an absorption spectrum in the ultraviolet or visible region for UV detection a deuterium discharge lamp as a light source is used and four components detection and visible region a tungsten lamp is used then UV this detector we can also find entrance slit lens prism or diffraction grating exit slit flow cell and detector for absorption measurements light from the lamp is shown onto the prism and dispersed according to wavelength when the measurement is performed with a specific wavelength the angle of the prism is adjusted so that the light of this wave can shine on the flow cell as the compound zealot from the column they enter the flow cell where the bonding and non-bonding electrons of these compounds can absorb energy in the form of ultraviolet or visible light the manner in which the final data is displayed is based on the computer in software the number of Peaks present can indicate how many components are in the mixture usually the x axis of the HPLC chromatogram shows the amount of time taken for the analytes to pass through the column and reach the detector typically the y-axis or the area of the peak is a reflection of the amount of a specific analyte that's present