Instrumental Analysis
Sheet#7
Dr.Emad Hamdan Done by:Noor Aswad
Last lecture we’ve talked about difference spectroscopy. We took the example of different pHs,
but difference spectroscopy is also applicable in other cases like in different oxidation states of
analyte.
Relevant definitions:
1- Chromophore: chemical entity (group) which has conjugation (high electron density)
which induces the ability of the molecule to absorb light. Typical example: benzene ring.
2- Auxochrome: chemical group that does not have strong absorption on its own but can
enhance the absorption of adjacent chromophore. (e.g. NO2, OH). (OH on benzene ring
will increase the absorption).
3- Bathochromic shift (Red shift): shift of the lambda max to a longer wavelength. Can be
caused by changes in pH, solvent chemical reaction.
4- Hypsochromic shift (blue shift): shift of the lambda max to shorter wavelength. Can be
caused as above.
Visible spectroscopy
All what we’ve said about UV (applications: qualitative and quantitative) is also applicable on
visible spectroscopy EXCEPT the range of wavelength that we are dealing with.
In UV, we’ve said that the cell must be made from quartz (to avoid absorbing UV light and
interference), but in visible we may use plastic or glass cells (they do not interfere with the
visible range).
Colorimetry: color measurement.
This analysis method measures within the visible range. The substances that are analyzed here
are colored substances. It is not necessary to use colored substances, but this technique may
induce color in colorless substances (i.e. by reacting the colorless substance with colored
derivative to induce color in this substance). The aim of this is to increase selectivity. This is
illustrated in the measurement of blood substituents by UV (remember when we said that
measurement of blood substituents need high selectivity because there are so much substances
in the blood). Cholesterol, glucose, lipids, steroids, and amino acids are colorless, only
hemoglobin is the colored one. Remember in biochemistry lab, we converted the glucose to a
colored substance which is measured within the visible range (wavelength around 520nm) in
order to measure the glucose alone without the interference of other substances.
But, why glucose made such colored compound? Why not other substances did?
Because of the selectivity of the reaction between glucose and the colored derivative. This
selectivity of such reaction can be achieved by specific enzymes (e.g. here glucose oxidase
which is specific for glucose).
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Instrumental Analysis
Sheet#7
Dr.Emad Hamdan Done by:Noor Aswad
Note: 99.9% of pharmaceutical substances have NO COLOR.
Optimum conditions for UV spectroscopy
1- Wavelength
Usually we choose lambda max unless we have exceptional cases
2- Slit width
remember when we talked about spectrophotometer
light source → prism system → monochromatic light (I°) → sample
- Not all of the light that is going out of the light source will go through the sample
but we make a slit where the light can pass. The width of this slit can be adjusted.
- What is the effect of slit width on the UV spectroscopy?
When we use larger slit width then we allow larger amount of light to pass through
the sample, and the ultimate result to this is the INCREASED SENSITIVITY (more
molecules will be excited).
-
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When we increase the split width we face many problems:
As we increase the slit width, then we have a higher risk of not having a
monochromic light. (I.e. the principle of the prism system is to give a light of certain
wavelength at the angle of the split, but when we increase the split width too much
then we have higher space and angles for other wavelengths to pass through the
sample). This will reduce the resolution of the spectrum, because we have now
fewer points on the spectrum (fewer points because when we made the slit larger→
allow more wavelengths to enter but we treat them as one wavelength → fewer
wavelengths on the spectrum).
Remember: Resolution is the ability to give the exact specifications of the spectrum
(as in the resolution of the camera, higher resolution → higher number of points in
the same area).
So, as we increase the slit width we increase the sensitivity and decrease the
selectivity.
When we want to measure a very slight concentration of a substance then the
selectivity is not so important to us (to have an exact spectrum) but we want to
increase the sensitivity, so here we’ll increase the split width.
Instrumental Analysis
Sheet#7
Dr.Emad Hamdan Done by:Noor Aswad
The same thing applies when we want a very specific spectrum (exact), then we
need high selectivity rather than high sensitivity, so here we reduce the split width.
Resolution and scan speed
-
We talked about this when have compared between single and double beam
spectrophotometers, and we said that the higher the speed the lower the resolution
(because of the reduced number of points on the spectrum).
Inverse proportion between resolution and scan speed.
Stray light effect
SL is a light with any other lambda that of interest, which reaches the detector.
SL is a common source of error that comes for example from uncovered spectrophotometer or
poorly fitted sample, etc.
Effect of the sample on the absorbance
1- If the sample itself naturally is a fluorescent (leave this factor for next lectures).
2- Turbid sample: this means that there are many insoluble (undissolved) particles in the
sample, and this will increase the absorbance.
I°
Absorbance= log It . I° Will not be affected by the turbidity (because it is measured from
the light source from the beginning before getting through the sample) BUT It will be
affected, high proportion of the It will not pass through the sample because of the
collision with the turbid particles (much of the transmitted light is deflected away from
the detector) → ↓It → ↑ absorbance.
Deviations from Beer’s Lambart’s law
Beer’s Lambart’s law must be validated (LINEAR) experimentally because deviations may occur
due to the following reasons (unlinear from the beginning of the curve):
1- Instrumental
Here it is not our fault, but it is the manufacturer’s
fault, even though we must investigate this.
How can we investigate this?
Take one of the concentrations you’ve measured and
measure it again on the same spectrophotometer BUT
at different bath lengths. Then plot against b. If the
plot was linear, then the problem is not an
instrumental problem and it is a chemical problem.
If curvature still presents then it is an instrumental problem.
2- Chemical
Some kinds of molecules have the tendency to form dimers or oligomers , and these
have a different behavior from the monomer.
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Instrumental Analysis
Sheet#7
Dr.Emad Hamdan Done by:Noor Aswad
The problem here is whatever it is a monomer or oligomer or dimer, still it is a function
of concentration.
Definitely, we all know that the dimer and
oligomer and monomer all have different
absorptivities, and this will make the different
concentrations to give different absorbencies
(±deviations).
Typical molecules that have such chemistry:
planar molecules like naphthalene.
Naphthalene present as monomers (separate molecules) at low concentration (diluted). As we
increase the concentration they tend to stack through bi- interaction.
Atomic absorption spectroscopy
I°
As in UV, Here we’ll measure absorbance, and that means that we have log It, and automatically
we want a light source. However, the nature of light source here is different from that of UV.
In UV we were using molecules, but here we are measuring the absorbance of atoms. The
analyte here is a metal (cation like Zn, Ni,..). We make solution of these metals (e.g. Ni in certain
type of chromium, lead in sea water, etc).
This technique is characterized by its high selectivity (unlike UV) and sensitivity (measure
these metals of very low concentrations), but if its principle is similar to UV, then why it has a
higher sensitivity? In UV we had a band spectrum (because of the associated vibrational and
rotational excitation), but the atomic spectrum is a linear spectrum (because we don’t have
molecules to have such vibrational excitations).
Take Ni spectrum as example, the number of lines
represents the number of possible electronic
transitions (i.e. the transition to E1 or E2 or E3..).
Height of the line represents the possibility of the
excitation (transition) process. (How much atoms tend
to undergo this transition, 20% of the atoms, 50%, etc.)
So, we measure at the wavelength that has highest
sensitivity (highest line).
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