| 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 energy | Fluorescence spectrometry/FLUOROMETRY / spectrofluorometry |
| Fluorescence spectrometry
PRINCIPLE | Certain 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 LIMITATIONS | STRENGTHS
• 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 / spectrofluorometry | An 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 spectrometry | 1) 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 molecule | RAMAN SPECTROSCOPY |
| RAMAN SPECTROSCOPY
APPLICATIONS | 1 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 suspension | TURBIDIMETRY 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 MEASURED | 1 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 ANTIBIOTIC | LESS |
