PHA6122 LEC| Molecular Emission Spectroscopy

FLUORESCENCE SPECTROPHOTOMETRY or FLUOROMETRY

Certain molecules, particularly those with a chromophore and a rigid structure (fluorophore), 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

chromophores

UV-Vis spectroscopy, we only need
______ for them to absorb UV-Vis
radiation

rigid structure

fluorescence, you need a _______ or fluorophore

excited state

initial absorption of a photon by a
molecule in the sample promotes an electron to an _______

emitting

excited electron returns to the ground
electronic state by ______ a photon

FLUORESCENCE

emission arising from an "allowed" transition that typically has a short lifetime between 1 ns and 10 ns

PHOSPHORESCENCE

emission arises from a "forbidden" transition that typically has a long lifetime between 1 ms and 1 s

FLUORESCENCE

does not change in electron spin

PHOSPHORESCENCE

there is a change in electron spin

polyatomic fluorophore

once electronically excited, experiences vibrational relaxation before emitting a photon, causing a red shift or Stokes shift of the fluorescence spectrum relative to the wavelength at which it was excited

Red Shift or Stokes Shift

change in energy or shift in longer wavelength

Xenon lamps

most common, high intensity and broad wavelength range (UV to NIR)

Lasers

are the highest-intensity source; used in applications where short collection times and small amounts of sample are required

Excitation Wavelength Selector

a filter or a monochromator with a known peak transmission wavelength and bandwidth

Excitation Wavelength Selector

enables fluorescence excitation spectra to be resolved

Sampling Device

includes all optics and other equipment needed to deliver the excitation beam to the sample, collect the emission from the sample, and hold the sample in place

Sampling Device

cuvettes, microwell plates, microarrays, microscope slides, and flow systems

Emission Wavelength Selector

to ensure that the emission wavelength region being detected does not overlap with the excitation wavelength profile

Emission Wavelength Selector

important for the rejection of stray light

- filters,
- monochromators, and
- grating polychromators

______ often are used for emission wavelength selection

Detector

a photomultiplier tube (PMT) or a charge-coupled device (CCD) array

right-angle or 0°/90° geometry

used to measure dilute solutions and other transparent samples

front-face geometry

used to measure optically dense samples

front-face geometry

fluorescence is collected at an angle ≤90°

front-face geometry

i.e., epifluorescence geometry - excitation beam and collected fluorescence are both on the same side of the sample, i.e., a 0°/0° geometry

0°/180° transmitting geometry

used in microscopy

- Wavelengths
- Bandwidths
- detector

These are Instrument-Based Factors

Instrument-Based Factors (wavelengths, bandwidths, and detector)

can introduce measurement uncertainty or bias that is particularly significant when measured values are compared between instruments

intensity of the excitation beam

can change significantly with excitation wavelength or with time

Instrument-Based Factors

linear intensity range of the detection system

Instrument-Based Factors

diffraction efficiency of gratings

optically dense samples (e.g., absorbance A >0.05 at a path length of 1 cm)

does not increase linearly with concentration because of significant absorption of the excitation beam and/or emission (reabsorption) by the sample

Sample-Based Factors

Depends on the nature and characteristics of the sample

optically dense samples

reduce the amount of fluorescence that reaches the detector

photobleaching and photodegradation

fluorescence intensity of a sample may decrease with time of exposure to light because of _____ and ______

temperature

fluorescence intensity of fluorophores is _____ dependent - increase with temperature can decrease in fluorescence intensity

intensity
peak position
spectral shape

fluorophore's fluorescence _____, _____, and sometimes ______, often depend on the environment, including changes caused by the solvent used, the solution's pH, or the species to which the fluorophore is bound

Raman signal

can introduce peaks into the fluorescence spectrum - sample's solvent or matrix

- Instrument-Based Factors
- Sample-Based Factors

What are the factors that affect Quantitation

low-dose

Application:
Determination of fluorescent drugs in _____ formulations in the presence of non- fluorescent excipients.

limit tests

Application:
In carrying out ______ where the impurity is fluorescent or can be simply rendered fluorescent.

binding

Application:
Useful for studying the ______ of drugs to components in complex formulations.

bioanalysis

Application:
Widely used in ______ for measuring small amounts of drug and for studying drug-protein binding

Strength of Fluorometry

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

complex molecules

Strengths of Fluorometry:
Can be used to monitor changes in _______ such as proteins, which are being used increasingly as drugs.

limited

Limitation of Fluorometry:
The technique only applies to a _____ number of molecules

- UV-absorbing species,
- heavy ions in solution, and
- affected by temperature

Limitation of Fluorometry:
Fluorescence is subject to interference by ______

Raman Spectroscopy

Change of wavelength occurs because of the molecules that deflect light and not because of relaxation

• It is not wavelength dependent
• Does not require the molecule to have a chromophore
• Energy shift in cm-1 (wavenumber) is measured instead of wavelength

The Raman effect is analogous to fluorescence except:

middle-IR

In Raman Spectroscopy: The shifts measured correspond to the wavenumbers of the bands present in the ______ spectrum of the molecule.

Lasers

are used to provide high-intensity radiation in the visible region, generally somewhere between 450 and 800 nm (NIR lasers)

Lasers

it does not excite fluorescence in molecules and has good penetration properties

weakly; strongly

bands that absorbed ____ in middle -IR region will absorb _____ in the Raman region and vice versa

FT-RAMAN AND FT-IR SPECTRA

they provide complementary information

Raman Spectroscopy

Application:
Has potential for identifying complex samples, e.g. drugs in formulations and in pack

Raman Spectroscopy

Application:
Samples such as peptide pharmaceuticals can be analysed for changes in their three-dimensional structure

Raman Spectroscopy

Application:
Provides additional fingerprint identity information complementary to middle-IR spectroscopy

Strengths of Raman Spectroscopy

Complementary to middle-IR spectroscopy but requires very little sample preparation since near-IR (NIR) radiation with its good penetration properties can be used for the analysis

Strengths of Raman Spectroscopy

Increasingly a readily available option on middle-IR FT-IR instruments.

Limitations of Raman Spectroscopy

Limitations:
- Not yet fully established as a quantitative technique

- The solvent may interfere if samples are run in solution