advanced molecular spectroscopy Flashcards
What kind of interferometer is used in FTIR?
Michelson interferometer
What is the output called?
Interferogram
Give 3 advantages to FTIR over dispersive spectroscopy?
Provides precise measurement method which requires no external calibration, can increase speed and sensitivity
What 3 things contribute to the increased sensitivity?
One second scans can be co-added together to ratio out random noise, greater optical throughput, mechanically simple - 1 moving part
what limitation of dispersive techniques was FTIR created to overcome?
slow scanning
How long does it take for a interferometer signal to be read?
1 second
What does a beam splitter do
Takes oncoming IR beam and divides it into 2 optical paths
What do the 2 resulting beams reflect off of?
One beam reflects off a flat mirror which is fixed in place, the other off a mirror on a mechanism which allows mirror to move a short distance from beamsplitter
Where are the 2 beams recombined?
when they meet back at the beamsplitter
What mathematical technique is used to decode the individual frequencies produced?
Fourier transform
What is fellgetts multiplex advantage?
info on all frequencies collected similtaniously, saves time by averaging number of interferometer scans
What is Jacquinots throughput advantage?
better sensitivity due to no monochromator, and light throughput not restricted
What is the Connes laser calibration advantage?
internally calibrated, gives precise frequency calibration without secondary spectral standards
What are the 2 disadvantages of FTIR over dispersive instruments?
single beam with long optical path, whole spectrum must be obtained from each transform
What problems might the single beam device lead to?
Cancelling atmospheric H2O and CO2
The high resolution work encountered with gaseous mixtures give complex spectras due to what?
superposition of vibrational and rotational bands
name 2 applications of FTIR
Study of samples with weak absorbancies, IR emission studies
What is evidence of bond between coupling agent and glass fibre surface vital in the development of?
optical fibres
Why are normal lenses not suitable for FTIR microscopy?
they absorb IR radiation
What is used instead?
metal coated relfecting optics instead
What 3 modes can samples be measured in?
transmittion, reflectance, or ATR modes
what is the range of Near IR?
0.75-2.5um
describe the vibrations in polyatomic molecules?
anharmonic
what are overtones?
vibrations due to energy transfer from ground level to second or higher levels
why do they occur at smaller wavelengths
require more energy
What do combination bands arise as?
summation fundamental bands (2 or more fundamental vibrations occur similtaneously)
how much weaker are Near IR absorption bands than corresponding fundamental mid IR absorption bands?
10-100x
How can this be used as an advantage
Allows direct analysis of strongly absorbing, highly light-scattering matrices
Give 3 advantages of Near IR?
Rapid, accurate and non-destructive
is it qualitative or quantitative
both
What is the benefit of it having a large pathlength
can analyse more sample
Name 3 disadvantages to near IR?
not suitable for trace analysis, complex spectra for interpretation, physical conditions of sample and environment add complexity to spectra
What are 2 applications of NIR in pharmaceutical industry
identification, information on crystalline state, powder size etc
What does UV-VIS stand for?
Ultravoilet and visible spectroscopy
What regions of radiation are UV and VIS?
200-400nm UV, 400-800nm VIS
What does absorption of this energy cause an excitation of?
an electron from a bonding to an antibonding molecular orbital
what does fluorimetry rely on?
the emission of electromagnetic energy by molecules
Which regions does the sample absorb and emit light?
absorbs in UV region, emits in visible region
The light emitted by the sample is always of …… wavelength (……. energy) than the light absorbed by the molecule
longer, lower
What is this called?
Stokes shift
what vibrational level does absorption always start at?
lowest (ground state)
what is required to excite molecules from the ground state?
high temperature
what is the most versatile spectrofluorimeter?
dual monochromator spectrofluorometer
What kind of lamp is used? Why?
Xenon lamp, as fluorescence intensity is proportional to I0
due to fluoresence signals being generally weak, what is used as a detector?
PMT
Why are all sides of a cell in fluoresence polished? (unlike in absorption)
fluoresence is emitted in all directions
What makes fluorimetry more specific than ordinary UV spec?
2 monochromators + not all molecules with a chromophore fluoresence
How much lower is fluorimetrys detection limit than UV/VIS>
100 x
What kind of analysis is it ideal for?
very small amounts of potent drugs
comment on the sensitivity of the analysis
among the most sensitive
What 3 factors affect fluorescence intensity?
source intensity, fluoresence efficiency, concentration
Name 3 more important factors?
quenching, self absorption, matrix effects
define quenching
a reduction in intensity of light emitted during fluoresence
what are the 2 types?
self and chemical quenching
in what environment does self quenching occur?
at high conc
What causes this?
Due to reabsorption and scattering of emitted light by other molecules/ions of sample
how can self quenching be solved?
by further dilution of sample
the detection system collects fluorescence emitted from which part of the exciting beam?
central
in large concentrations, what is absorbed significantly before reaching the central part of the cuvette?
incident light
How does the emitted light concentrated at the face of the cell rather than through the body of the solution affect the calibration?
causes it to curve
Chemical quenching incolves the ……. of the energy from an …….. molecule by another molecule usually as the result of a ………?
removal, excited, collision
why is this important in an analysis?
since fluoresence of the analyte might be quenched by molecules of some compound present in sample
what is this effect known as?
matrix effects
what are the following quenching mechanisms known as? A+Q=>A+Q and A+Q=>AQ=>AQ
collision, and complex formation
In the equations, what is A and what is Q?
A = fluorescent analyte molecule Q= quenching species
Regarding electrons, what type of compound act as efficient quenching agents? give example
compounds with unpaired electrons, eg. O2
how can this be removed from sample if necessary?
bubbling oxygen free N2 through the sample
How might halide ions affect fluoresence of quinine? for example in hydrochloric acid vs sulphuric acid?
70% reduction in fluorescence - referred to as heavy atom effect
what is this due to?
increased probablility of intersystem crossing. this leads to non-radioactive deactivation of excited state = no fluorescence
give 2 examples of ionsof inorganic species which can naturally fluoresce in solution?
lanthanides and actinides
what are the general requirements for organic compounds to be able to fluoresce? 3
a large molecule containing a conjugated system which has a rigid structure due to ring formation
what is the simplest example of a fluorescent derivative?
the complex formed between a metal ion and a donor organic molecule
ideally, both of these components should be …………. and form a ……….. complex?
non-fluorescent, fluorescent
What is the name of the most effective type of complex which can be formed by the metal forming bonds in 2 positions?
chelate ring
name 2 applications of fluorescence based assays in forensic laboratories?
quantitation of DNA and RNA yields in solutions, and DNA typing.
what are novel fluorescent reagents designed to enhance?
sensitivity while using simple methods and conserving precious samples.
Name the 2 derivative approaches available?
precolumn and post-column derivitisation
in precolumn, what are 3 requirements for the derivitisaing agent to be added to the sample before it is injected onto column?
useful where the reaction requires several reagents, heat treatment or time
What might this technique reduce?
efficiency of separation
When is the derivitising agent injected into the column in post-column?
injected continiously into eluent from column
in what instances is this method used?
when precolumn seriously affects the separation
What might need to be inserted to ensure the reaction is complete before components reach the detector?
delay coils
without them, what may occur?
diffusion and loss of resolution
