about spectroscopy

1. APPLICATIONS OF
ABSORBANCE
SPECTROSCOPY IN NATURAL
PRODUCT RESEARCH
By Zartaj
240331

2. Absorbance Spectroscopy
 Absorbance spectroscopy is a
molecular spectroscopy method that uses the
wavelength-
dependent absorption characteristics of
materials to identify and quantify specific
substances. It ranges from Visible light region
to Ultraviolet region; 200-400nm. It is also
known as UV/Vis spectroscopy.

3. Natural Products
 A natural product is a chemical compound or
substance produced by a living organism or
which can be prepared by chemical synthesis
by life. For instance, biotic materials (wood,
silk), bio-based materials (bioplastics,
cornstarch), bodily fluids (milk, plant
exudates), and other natural materials (soil,
coal).

4. Applications in Natural Product
Analysis
 Absorption spectroscopy is an excellent
technique for following ligand-binding
reactions, enzyme catalysis and
conformational transitions in proteins and
nucleic acids.
 Here follows the broad significance of
absorption spectroscopy in the areas of natural
product research.

5. Protein Concentrations
 The concentrations of proteins or nucleic acids
in solution can be easily and accurately
determined by absorbance measurements
following the LAMBERT-BEER law;
 A= -log10(I/I0) OR A=ecl

6. Protein Concentrations
 Absorption coefficients of proteins
 Proteins usually show absorption maxima
between 275 and 280nm, caused by the
absorbance of the two aromatic amino acids
tryptophan and tyrosine and, to a small extent,
by the absorbance of cystine (disulfide bonds)

7. Protein Concentrations
 The absorbances of Trp and Tyr depend on
the microenvironment of their chromophores,
and they are slightly red-shifted when
transferred from a polar to a nonpolar
environment, such as in the interior of a
globular protein.

8. Protein Concentrations
 The absorption coefficienteof a protein can be
calculated in a simple fashion. First the
numbers of its Trp, Tyr and Cys disulfide
bonds (nTrp, nTyr and nSS, respectively) are
counted, and then e calculated by use of
lambert beer law and the values obtained are:
5500, 1490, and 125 L/molcm respectively.
These represent average values for the
chromophores in folded proteins.

9. Absorbance of Proteins
 The peptide groups of the protein main chain
absorb light in the ‘far-UV’ range (180–30nm).

10. Absorbance of Proteins
 The aromatic side chains of Tyr, Trp and
Phe also absorb light in this region and, in
addition, they absorb in the 240–300nm
region. This region is called the ‘near-UV’ or
the ‘aromatic’ region.
 Disulfide bonds that form between two
cysteine residues also show an absorbance
band near 260nm. Many cofactors absorb light
in the UVvis

11.  (NADH) and reduced flavin–adenine
dinucleotide (FADH2) show spectra in the
near-UV.
 Haem groups and copper-containing
cofactors absorb in the visible region.
Therefore haemoglobin is red and
plastocyanin is blue.

12. Concentrations of nucleic acids
 The concentrations of nucleic acids in solution
are routinely determined from their strong
absorbance at 260nm.
 Proteins absorb much more weakly than
nucleic acids. Contaminating proteins
therefore hardly affect the concentrations of
nucleic acids.

13. Absorbance of Nucleic Acids
 Nucleic acids show a strong absorbance in the
region of 240-275nm. It originates from the pi
to pi* transitions of the pyrimidine and purine
ring systems of the nucleobases.
 In native DNA the bases are stacked in the
hydrophobic core of the double helix and
accordingly their absorbance is considerably
decreased relative to the absorbance of single
stranded DNA.

14.  The decrease in absorbance upon base
stacking in the interior of DNA or RNA double
helices is called hypochromism. It provides a
very sensitive and convenient probe for
monitoring strand dissociation and unfolding
(‘melting’) of DNA double helices.

15. Applications in Enzyme Kinetics
 An enzyme catalyses the conversion of one or
several substrates to one or several products.
 The rate of the catalysed reaction or the
activity of the enzyme can be determined by
measuring either the decrease in substrate
concentration or the increase in product
concentration as a function of the reaction
time.

16.  When the substrate (S) and the product (P)
differ in absorbance, the progress of an
enzymatic reaction can be followed directly by
monitoring the change in absorbance as a
function of time.
 The absorbance changes are linearly related
with the changes in concentration (via the
Lambert–Beer relation)

17.  Therefore,the reaction rates can be
calculated from the absorbance data if
absorption coefficients of the reacting species
are known.
 NADH-linked enzyme reactions, such as
those catalysed by the lactate,
dehydrogenases provide excellent examples
for absorbance-based enzyme assays. NADH
shows an absorbance maximum near 340nm.

18. UV-Vis Spectrophotometric Methods for
Methotrexate Assay
 Methotrexate (MTX) is a well-known
anticancer drug used in the chemotherapy of
several malignant diseases, such as acute
lymphocytic leukemia, osteosarcoma, breast
and bladder cancers.

19. UV-Vis Spectrophotometric Methods for
Methotrexate Assay
 UV/VIS spectroscopy for prolonging the drug
delivery has been used. However, the
evaluation of the drug content is still a
challenge for researchers.
 An analytical curve is generated by plotting the
area under the curve or the absorbance for
UV-Vis spectrophotometric method.

20.  Any change in the absorbance intensity and
any bathochromic or hypsochromic shift are
investigated by analyzing the impurities curve
/peaks in the absorbance graph.
 If there are any impurities found by the shifts of
the spectrum, these are easily recognized and
reported.

22. The Use in Bioprocess and
Fermentation Monitoring
 A bioprocess is a specific process that uses
complete living cells or their components
(bacteria, enzymes) to obtain desired
products.
 Fermentation is a metabolic process that
produces chemical changes and the extraction
of energy from carbohydrates in the absence
of oxygen by Bacteria.

23.  There is an increasing need for real-time
analytical tools to monitor bioprocess and
fermentation in biological and food
applications.
 Spectroscopic sensors, when combined with
PAT, enable simultaneous, real-time
bioprocess monitoring of parameters like
biological, chemical, and physical variables
during the process.

24.  The development and implementation of these
spectroscopy methods in the field of food
analysis are based on the interactions
between matter and light that resulted in
absorption, emission, and scattering events
characteristic of the sample.

25.  Therefore, on the basis of the absorption
measurement, the presence and concentration
of analytes in the food matrix as a
consequence of its chemical and physical
properties can be determined and quantified.

26.  In bioprocess and fermentation monitoring,
optical density (OD) provides the most relevant
information to make the measurements. This
effect can be used to determine the
concentration of biomass in turbid samples.

27.  For example, in the wavelength range
between 350 and 400 nm, several researchers
have reported the usefulness of this range to
differentiate between viable and dead
microbial cells due to an increase in
absorption associated with microbial cell
contents and nucleic acids originating from the
damaged microbial cells.

28.  From the collected data, the information can
be visualized as a pattern that contains
information about a complex bioprocess—for
example, fermentation.

29. Phenolic Compounds
Evaluation
 Phenolic compounds play an important role in the
colour, flavour and “mouth feel” attributes of
wines. Consequently, the measurement of
phenolic compounds during fermentation is
important to better understand and control the
winemaking process . UV-Vis spectroscopy was
used to monitor the phenolic composition during
winemaking, where models used to predict
phenolic compounds were developed. The
accuracy and robustness of the calibration models
were evaluated using the slope and intercept,
interclass correlation coefficients and standard
error of measurement.

30. Conclusion
 The advent of new types of UV-Vis
instrumentation and sample presentation options
has aided in the development of new possibilities
to monitor many processes in biological samples.
The literature reports several considerable
successes to this end, particularly for the routine
screening of typical constituents, such as
anthocyanins, phenolics, sugars, and
antioxidants, and complex food matrices. The
challenges of isolating matrix interferences remain
but the incorporation of data mining and data
analysis techniques extends the possibilities of
using UV-Vis spectroscopy in natural products
analysis and quantification.