JSPM’SCharak College Of Pharmacy & ResearchGat no.720/1&2, Wagholi, Pune-Nagar Road,wagholi, pune -412207 Detection of HPLC Presented By, Guided By,Miss. Shradha Jedge Dr. Rajesh J Oswal Prof.Sandip KshirsagarDepartment Of Pharmaceutical Chemistry
Detection of HPLC
INDEX INTRODUCTION TYPES WORKING OF HPLC
WORKING OF HPLC
WHAT IS DETECTORAn instrument use to detect or observe. selection of detector is on the basis of analyze or sample under detection. visualize components of the mixture being eluted off the chromatography column.
Properties of detector Low drift and noise level (particularly crucial in traceanalysis). High sensitivity, Fast response, Operational simplicity and reliability, non-destructive, inert (non-reactive) Produce uniform, reliable and reproducible detection and analytic data. Compatible for all types of compounds under testing. Wide linear dynamic range (simplifies quantization). Low dead volume (minimal peak broadening). Cell design which eliminates remixing of the separated bands. Insensitivity to changes in type of solvent, flow rate. It should be tunable so that detection can be optimized for different compounds.
1) UV DETECTORUltra-Violet (UV) detectors measure the ability of a sample toabsorb light. This can be accomplished at one or several wavelengths. sensitivity to approximately 10-8 or 10 -9 gm/ml. TYPES OF UV- DETECTOR A) Fixed Wavelength measures at one wavelength, usually 254 nm B) Variable Wavelength measures at one wavelength at a time, but can detect over a wide range of wavelenths. C) simulateneously wavelengths Diode Array
UV detectors Depends on absorption of UV ray energy by the sample. When the UV rays emitted by lamp pass through gratings, rays split into different wavelengths. One specific wavelength rays are passed through sample. Some amount of light is absorbed by sample and the unabsorbed rays which fall on photo cell. These rays on collision on photo cell produces electrons whose current is recorded. This is indicative of nature and quantity of sample. This UV wavelength range of absorption is specific for sample . They are capable to detect very wide range of compounds. The sensitivity ranges till microgram quantity of estimation.
2) FLUORESCENCE DETECTORfluorescence rays emitted by sample after absorbing incident light is measured as afunction of quality and quantity of the sample Xenon arc lamp is used to produce light for excitation of sample molecules. These light rays excite the sample molecules. These excited molecules emit florescence, which pass through gratings. These gratings pass the florescence at specific wavelength to photo cell which is recorded. The detector is suitable for compounds which can produce florescence. they have high precision and sensitivity (with less noise in data).
3) ELECTROCHEMICAL DETECTORThis detector is specially suitable to estimate oxidisable & reduciblecompounds. principle :when compound is either oxidized or reduced, the chemical reactionproduces electron flow. This flow is measured as current which is thefunction of type and quantity of compound.This electrode is suitable for compounds which cant be assayed by UVdetector especially due to their similarities in light absorptionproperties ex: monoamines.This detector has super sensitivity which ranges till picogramsmeasurement.
4) THERMAL CONDUCTIVITY DETECTOR(TCD) chemical specific detector commonly used gas-liquid chromatography.This detector senses changes in the thermal conductivity of the column effluent and compares it to a reference flow of carrier gas.Since most compounds have a thermal conductivity much less than that of the common carrier gases of helium or hydrogen, when an analyte elutes from the column the effluent thermal conductivity is reduced, and a detectable signal is produced. Operation The TCD consists of an electrically heated filament in a temperature- controlled cell. Under normal conditions there is a stable heat flow from the filament to the detector body. When an analyte elutes and the thermal conductivity of the column effluent is reduced, the filament heats up and changes resistance. This resistance change is often sensed by aWheatstone bridge circuit which produces a measurable voltage change.The column effluent flows over one of the resistors while the reference flowis over a second resistor in the four-resistor circuit.
Since all compounds, organic and inorganic, have a thermalconductivity different from helium, all compounds can be detectedby this detector. The TCD is often called a universal detectorbecause it responds to all compounds. Also, since the thermalconductivity of organic compounds are similar and very differentfrom helium, a TCD will respond similarly to similarconcentrations of analyte. Therefore the TCD can be used withoutcalibration and the concentration of a sample component can beestimated by the ratio of the analyte peak area to all components(peaks) in the sample.
5) flame ionization detector (FID)As the name suggests, analysis involves the detection of ions. The source of theseions is a small hydrogen-air flame. Sometimes hydrogen-oxygen flames are used due to an ability to increasedetection sensitivity, however for most analysis, the use of compressedbreathable air is sufficient. The resulting flame burns at such a temperature as to pyrolyze most organiccompounds, producing positively charged ions and electrons.In order to detectthese ions, two electrodes are used to provide a potential difference.The positive electrode doubles as the nozzle head where the flame is produced.The other, negative electrode is positioned above the flame. When firstdesigned, the negative electrode was either tear-drop shaped or angular piece ofplatinum.Today, the design has been modified into a tubular electrode, commonly referredto as a collector plate. The ions thus are attracted to the collector plate and uponhitting the plate, induce a current. This current is measured with a high-impedance picoammeter and fed into an integrator How the final data is displayed is based on the computer and software. Ingeneral, a graph is displayed that has time on the x-axis and total ion on the y-axis.The current measured corresponds roughly to the proportion of reducedcarbon atoms in the flame.Specifically how the ions are produced is not necessarily understood, but theresponse of the detector is determined by the number of carbon atoms (ions)