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What is HPLC and how do you use it?
HPLC stands for high performance liquid chromatography. It is a liquid chromatography which involves the separation of the compounds on the basis of their polarity. It is used to analyze, identify, purify & quantify the compounds.
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In the laboratory, acetonitrile is used as a medium-polarity solvent that is miscible with water and has a convenient liquid range. With a dipole moment of 3.84 D, acetonitril…e dissolves a wide range of ionic and nonpolar compounds and is useful as a mobile phase in HPLC and LCMS.
it is used to check the efficiency of the chromatography column used
The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram. It is positioned immedi…ately posterior to the stationary phase in order to detect the compounds as they elute from the column. The bandwidth and height of the peaks may usually be adjusted using the coarse and fine tuning controls, and the detection and sensitivity parameters may also be controlled (in most cases). There are many types of detectors that can be used with HPLC. Some of the more common detectors include: Refractive Index (RI), Ultra-Violet (UV), Fluorescent, Radiochemical, Electrochemical, Near-Infra Red (Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR), and Light Scattering (LS). Refractive Index (RI) detectors measure the ability of sample molecules to bend or refract light. This property for each molecule or compound is called its refractive index. For most RI detectors, light proceeds through a bi-modular flow-cell to a photodetector. One channel of the flow-cell directs the mobile phase passing through the column while the other directs only the mobile phase. Detection occurs when the light is bent due to samples eluting from the column, and this is read as a disparity between the two channels. Ultra-Violet (UV) detectors measure the ability of a sample to absorb light. This can be accomplished at one or several wavelengths: 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) Diode Array measures a spectrum of wavelengths simulateneously UV detectors have a sensitivity to approximately 10-8 or 10 -9 gm/ml. Fluorescent detectors measure the ability of a compound to absorb then re-emit light at given wavelengths. Each compound has a characteristic fluorescence. The excitation source passes through the flow-cell to a photodetector while a monochromator measures the emission wavelengths. Has sensitivity limit of 10-9 to 10-11 gm/ml. Radiochemical detection involves the use of radiolabeled material, usually tritium (3H) or carbon-14 (14C). It operates by detection of fluorescence associated with beta-particle ionization, and it is most popular in metabolite research. Two detector types: A) Homogeneous- Where addition of scintillation fluid to column effluent causes fluorescence. B) Heterogeneous- Where lithium silicate and fluorescence caused by beta-particle emission interact with the detector cell. Has sensitivity limit up to 10-9 to 10-10 gm/ml. Electrochemical detectors measure compounds that undergo oxidation or reduction reactions. Usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential. Has sensitivity of 10-12 to 10-13 gm/ml Mass Spectroscopy (MS) Detectors- The sample compound or molecule is ionized, it is passed through a mass analyzer, and the ion current is detected. There are various methods for ionization: A) Electron Impact (EI)- An electron current or beam created under high electric potential is used to ionize the sample migrating off the column. B) Chemical Ionization- A less aggresive method which utilizes ionized gas to remove electrons from the compounds eluting from the column. C) Fast Atom Bombarbment (FAB)- Xenon atoms are propelled at high speed in order to ionize the eluents from the column. Has detection limit of 10-8 to 10-10 gm/ml. Nuclear Magnetic Resonance (NMR) Detectors- Certain nuclei with odd- numbered masses, including H and 13C, spin about an axis in a random fashion. However, when placed between poles of a strong magnet, the spins are aligned either parallel or anti-parallel to the magnetic field, with the parallel orientation favored since it is slightly lower in energy. The nuclei are then irradiated with electromagnetic radiation which is absorbed and places the parallel nuclei into a higher energy state; consequently, they are now in "resonance" with the radiation. Each H or C will produce different spectra depending on their location and adjacent molecules, or elements in the compound, because all nuclei in molecules are surrounded by electron clouds which change the encompassing magnetic field and thereby alter the absorption frequency. Light-Scattering (LS) Detectors- When a source emits a parallel beam of light which strikes particles in solution, some light is reflected, absorbed, transmitted, or scattered. Two forms of LS detection may be used to measure the two latter occurrences: A) Nephelometry- This is defined as the measurement of light scattered by a particulate solution. This method enables the detection of the portion of light scattered at a multitude of angles. The sensitivity depends on the absence of background light or scatter since the detection occurs at a black or null background. B) Turbidimetry- This is defined as the measure of the reduction of light transmitted due to particles in solution. It measures the light scatter as a decrease in the light that is transmitted through the particulate solution. Therefore, it quantifies the residual light transmitted. Sensitivity of this method depends on the sensitivity of the machine employed, which can range from a simple spectrophotometer to a sophisticated discrete analyzer. Thus, the measurement of a decrease in transmitted light from a large signal of transmitted light is limited to the photometric accuracy and limitations of the instrument employed. Near-Infrared Detectors- Operates by scanning compounds in a spectrum from 700 to 1100 nm. Stretching and bending vibrations of particular chemical bonds in each molecule are detected at certain wavelengths. This is a method which offers several advantages: speed (sometimes less than 1 second), simplicity of preparation of sample, multiple analyses from single spectrum, and nonconsumption of the sample.
caffein is a highly stable, this is show dual lamda max like 205 and 275 nm, it is not a degradable. this is cheep chemical.
Caffeineis dule absorbance,it gives multiwavelength responds like 205,245,273.
Caffeine having different wavelents it having 2 maximas and 1 minima. 1st maxima is 205nm 2nd maxima is 273nm minima is 245nm and it is primary reference standa…rd and also suggested in pharmacopiea. -Rajesh,Orchid
Caffeine responds in 3 wavelength regions i.e 196 - 208nm in which caffeine maxima at 203±2nm 240 - 248nm in which caffeine minima at 244±2nm 270 - 278nm in which caffeine m…axima at 273±2nm So it covers the operation range of HPLC UV detector to perform wavelength accuracy. Apart from this Caffeine elutes in approx 2 min when using any C18 column which helps in reducing run time. That s the caffeine is preferred than any other standards for HPLC calibration K S Badrinath
This is a difficult question to answer because there are so many uses for the HPLC. The HPLC is widely used to separate a complex mixture into individual compounds. Sc…ientists use standards in order quantify the amount of a particular compound they have by generating calibration curves. How all this is done... Choose an eluent that will separate your compounds - test out different eluents on a TLC plate to make sure that your compounds are separating. Set the temperature on the HPLC so that it is high enough to evaporate the eluent. Insert your sample into the injection port (typically around 20 uL). Wait for your peaks to all come out. There are different ways to miminimize the amount it takes to run samples, but this is unique to your experiment, and so you have to just try out different things.
Internal standard can be used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the analyte concentration of the sta…ndards. This is done to correct for the loss of analyte during sample preparation or sample inlet.
it used a mobile phase to carry a sample through a tube containing a stationary phase which is a liquid coated on solid particles.
higly unretainable and has high absorption at 260 nm
it is retained to a lesser extent....has dual wavelength detection (245nm, 350nm) and gives a sharp and consistent peak.
high performance liquid chromatography. Generally the stationary phase in HPLC is known as reversed phase as the stationary phase is non-polar while the mobile phase is polar.…
large columns, specifically for packing. * analytical columns, for quantitative analysis, usually accompanied by a UV-vis detector. * Narrow bore columns, for more sensiti…ve analysis * capillary columns, very this silica columns used almost exclusively with GC mass spectroscopy. * packed bed columns. with silica beads. and may have groups attached, e.g. C18.
It is very stable(3 days at room temp.) ,it is having multi wavelength like 2 maxi ma (205 ,273 nm and minima(245nm) and cheap cost.
propyl paraben being a stable compound with reproducible chromatographic parameters used as a internal standard for the calibration of the HPLC systems
Erbium has a strong absorption in uv and visible range, It is used in HPlc calibration for the wavelength accuracy verification of the PDA detector.