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level: Level 1

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level questions: Level 1

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a type of ELECTROMAGNETIC SPECTROSCOPY which ANALYZES FLUORESCENCE from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energyFluorescence spectrometry/FLUOROMETRY / spectrofluorometry
Fluorescence spectrometry PRINCIPLECertain molecules, particularly those with a chromophore and a rigid structure, can be excited by UV/visible radiation, and will then emit the radiation absorbed at a longer wavelength. The radiation emitted can then be measured.
Fluorescence spectrometry APPLICATIONS•Determination of FLUORESCENT DRUGS in low-dose formulations in the presence of nonfluorescent excipients. • In carrying out LIMIT TESTS where the IMPURITY IS FLUORESCENT or can be simply rendered fluorescent. • Useful for STUDYING THE BINDING OF DRUGS to components in complex formulations • Widely used in BIOANALYSIS for measuring small amounts of drug and for studying drug-protein binding
Fluorescence spectrometry STRENGTHS AND LIMITATIONSSTRENGTHS • A SELECTIVE DETECTION method and can be used to QUANTIFY A STRONGLY FLUORESCENT compound in the presence of a larger amount of non-fluorescent material. • Can be used to MONITOR CHANGES IN COMPLEX MOLECULES such as proteins, which are being used increasingly as drugs. LIMITATIONS • The technique only applies to a LIMITED NUMBER of molecules. • Fluorescence is SUBJECT TO INTERFERENCE by UV-absorbing species, heavy ions in solution, and is affected by temperature.
THEORY Fluorescence spectrometry/FLUOROMETRY / spectrofluorometryAn emission phenomenon e- of a molecule prefer to reside at the lowest vibrational level of the ground electronic state e- promoted to excited electronic energy state reside in one of these vibrational energy levels Time of residence is short (10-8 to 10-4 s) Reverted back to ground state: collision deactivation and radiative process (fluorescence and phosphorescence)
Reverted back to ground state:collision deactivation and radiative process (fluorescence and phosphorescence)
General Procedure FLUORESCENCE spectrometry1) Illuminate sample w/ monochromatic UV radiant energy 2) Measure photometrically the fluorescent light produced in the sample (treated by oxidizing agent)
ANALOGOUS to fluorescence except that it is NOT WAVELENGTH DEPENDENT and does NOT require the molecule to have a CHROMOPHORE The shifts measured correspond to the wavenumbers of the bands present in the MIDDLE-IR SPECTRA of the moleculeRAMAN SPECTROSCOPY
RAMAN SPECTROSCOPY APPLICATIONS1 Has potential for IDENTIFYING COMPLEX SAMPLES (drugs in formulation and in pack) 2 Samples such as PEPTIDE PHARMACEUTICALS can be ANALYSED for changes in their three-dimensional structure 3 PROVIDES ADDITIONAL FINGERPRINT IDENTITY information complementary to middle-IR spectroscopy.
RAMAN SPECTROSCOPY Strength: Limitations:STRENGTH: 1 Requires VERY LITTLE SAMPLE PREPARATION 2 Increasingly a readily available option on middle-IR FT–IR instrument Limitations: 1 NOT YET fully established as A QUANTITATIVE TECHNIQUE 2 The SOLVENT MAY INTERFERE if samples are run in solution
IN RAMAN SPECTROSCOPY, What is the instrumentation?LASERS are used to provide high-intensity radiation in the visible region, generally somewhere between 450 AND 800 NM.
Branches of spectrometry in which TRANSMITTED OR REFLECTED LIGHT, respectively, is MEASURED after radiant energy passes through a TURBID solution or suspensionTURBIDIMETRY AND NEPHELOMETRY
1 LIGHT TRANSMITTANCE as a measure of turbidity. THE INTENSITY OF LIGHT TRANSMITTED THROUGH THE SAMPLE IS MEASURED. 2 Measurement of the BRIGHTNESS of light REFLECTED/ SCATTERED by a cloud of finely divided particles suspended in a liquid. THE INTENSITY OF THE SCATTERED LIGHT IS MEASURED1 Turbidimetry 2 Nephelometry
If a beam of light is passed through a turbid sample, its intensity is reduced by scattering, and the QUANTITY OF LIGHT scattered is __ upon the CONCENTRATION and size distribution of the particles.DEPENDENT
Turbidimetric methods are used in official assays of:1. Antibiotics 2. Calcium pantothenate 3. Vitamin B12 4. Assay for chloride and sulfate VACA
Antibiotic dilution containing microbiological culture: The MORE TURBID, THE __ ACTIVITY OF THE ANTIBIOTICLESS