This ppt is based on the analysis techniques of macromolecules.

2. ADVANCED TECHNIQUES IN
ANALYSIS OF ORGANIC
COMPOUNDS
SUBMITTED BY
UPASANA
MOHAPATRA
PALB 6290
SUBMITTED TO
PROF.DR.K. M. HARINIKUMAR
ADVANCED CENTRE FOR PLANT
BIOTECGHNOLOGY
UAS,GKVK,BENGALURU

3. Flow of Presentation
Summary
References
Research Paper
Description about different techniques
HPLC-NMR
Techniques used for analysis
Organic Molecules
Need of analysis of Organic Molecules

4. ORGANIC MOLECULE
• The molecules of life which are
built of chains of carbon atoms
that are often quite long.
• Four main groups of organic
molecules that combine to build
cells and their parts-
carbohydrates, proteins, lipids,
and nucleic acids.

5. NEED OF ORGANIC COMPOUND
ANALYSIS
We do analysis of organic compounds
1. To measure their concentrations in any living
organisms.
2. To identify the compounds present in microorganisms
3. To identify the waste pollutants pressent in water
sample.
4. To identify the secondary metabolites present in the
medicinal plants.
5. To identify the volatile compounds present in the
aromatic plants.

6. TECHNIQUES USED FOR ANALYSIS
• Elemental Analysis
• Electrochemistry
• X-ray Diffraction
Microarray
• Biological Techniques For Chemical Analysis
• Chromatographic Techniques
• Chromatography-Spectrometer
• HPLC-NMR

7. ELEMENTAL ANALYSIS
For a lot of organic compounds having very
similar properties, elemental identification is
relatively simple.
• Flame test
• Photometry and spectrophotometry
• Atomic emission/absorption spectroscopy
• X-ray fluorescence analysis

8. ELECTROCHEMISTRY
• The techniques are applicable for identification
of electrochemically reducible (e.g., nitro,
nitroso, and azo) compounds and oxidizable
(aromatic amines, phenols) compounds.
• Voltammetric peak potentials are quantities
measured for identification.
• Two types of electrochemical devices, sensors
and selective HPLC detectors are used for
advanced chemical analysis.

9. X-RAY DIFFRACTION
• For structure determination of organic solids
and identification of crystalline phases.
• Eg.
Identification of minerals in geological
samples

10. MICROARRAY
They are very suitable for:
• Purposes of detection and screening
• Field and industrial analysis
• Beginning analysts
• Qualitative analysis

11. Biological Techniques for Chemical
Analysis
• Developing biosensor techniques combined
with electrochemical devices can be used for
screening of some chemicals.
• Methods based on bioassays, e.g.,ones using
enzymes, are specific to certain substances and
sensitive, i.e., suitable for qualitative
confirmation

12. CHROMATOGRAPHIC TECHNIQUESCHROMATOGRAPHIC
TECHNIQUES

13. GC
PRINCIPLE
separation of a gaseous
mixture into individual
compounds on passing a
gas flow through a thin
glass column, the inner
walls of which are coated
with a special nonvolatile
liquid
MEASURE
Time,
relative
time, Index
of retention
DETECTOR
MS,flame
ionization,
atomic
emission
APPLIC
ATION
Gas,
volatile,
and semi-
volatile
compoun
ds

14. LC
PRINCIPLE
Separation of a liquid
mixture into individual
components on passing a
liquid through a relatively
thin steel column packed
with particles or aporous
layer of stationary phase
MEASURE
Time,
relative
time, Index
of retention
DETECTOR
MS,flame
ionization,
atomic
emission
APPLICA
TION
Non
volatile
compound
including
almost all
biochemic
als

15. TLC(THIN LAYER
CHROMATOGRAPHY)
PRINCIPLE
• Separation
technique for a
liquid mixture
somewhat
resembling LC,
where the
stationary
phase is a layer
of solid
particles spread
on a flat plat
MEASURE
• Retention
factor
DETECTOR
• Visual,
detection
reactions,
• fluorescence,
UV–Vis,
• densitometer
APPLICATION
• Non-volatile
compounds

16. CAPILLARY
ELECTROPHORESIS
PRINCIPLE
• In the simplest
form,
separation of
dissolved
ionizable
compounds in
silica capillary,
due to
migration of
their ions on
application of
high electric
field
MEASURE
• Migration time
DETECTOR
• UV and also
fluorescence,
• MS
APPLICATION
• Nucleic acids
and
• nucleotides,
• pharmaceutical
s,
• proteins,ioniza
ble compounds

17. UV-VIS
PRINCIPLE
Measurements of light
absorption at different
wavelengths in ultraviolet and
visible part of the spectrum
due to electronicexcitation
APPLICATION
Detection for
HPLC
MOLECULAR SPECTROMETRY

18. IR
PRINCIPLE
• Absorption
measurement of IR
radiation
(wavenumbers from
13,000 to 10 cm–
1,wavelengths from
0.78 to1,000 mm) due
to vibration excitation
APPLICATION
• Structure elucidation
(determination of
functionalgroups),Qua
litative analysis II
(polymers,plastics,
resins,food, and so
on)
LIMITATIONS
• Relatively low
sensitivity(1–10 mg is
commonly needed for
Spectral recordingb);
low compatibility of
IR detector with GC
and especially LC

19. NMR
PRINCIPLE
• Absorption of
radiation in the
radiofrequency range
of the electromagnetic
spectrum(hundreds of
MHz) due to changes
in the spin states of
the atom nucleus
APPLICATION
• Structure elucidation
(determination of
functionalgroups),Qua
litative analysis II
(polymers,plastics,
resins,food, and so
on)
LIMITATIONS
• Relatively low
sensitivity
• slow progress in LC–
NMR

20. MS
PRINCIPLE
• Measurement of mass
(up to 106 Da) and
amount of ions(down
to a few counts)
• generated from
atoms/molecules of a
substance
APPLICATION
• All kinds of chemical
analysis
LIMITATIONS
• Lower applicability in
direct analysis of
unpolar highmolecular
compounds

21. Chromatography and spectrometry
Instrument Application Comment
Gas chromatograph–
mass
spectrometers
Volatile and semivolatile
organic
compounds
Rather simple, bench top,
inexpensive instruments
Liquid chromatograph
low resolution mass
spectrometers
Non-volatile lowmolecular
organic
compounds
Increasing role of tandem
instruments, i.e. ion traps
Liquid chromatograph–
high resolution
mass
spectrometers
Non-volatile organic
compoundsincluding
highmolecular
biocompounds,Proteomics
Expensive instruments:
time-offlight,
Orbitrap,ESI
Mass spectrometers for
nonvolatile
compounds without
chromatography
Bio compounds,
proteomics,
polymers
Sample as a thin surface
layer of organic compound
in matrix
(MALDI)

22. GCMS
LCMS

23. Flow chart for treating gas, liquid, and solid samples in chromatographic and
mass spectrometric analysis

24. HPLC-NMR
• HPLC principles to high-resolution NMR spectrometers
offers a powerful tool for analyzing and characterizing
complex chemical mixtures.
• The HPLC performs the separation of a complex
mixture and the NMR spectrometer takes a spectrum of
each separated component to identify its structure
molecular weight.

25. 25

26. The coupling of two instruments to make a new
technique
• More capabilities than either instrument alone.
• HPLC-NMR is made possible with a specially
designed flow probe.
• Complex mixtures of unknown alkaloids extracted
from plants have been separated and their
structures completely characterized by HPLC-NMR
using a variety of 2D NMR.
26

28. Eastern blot
(Detection of carbohydrate epitopes on proteins or lipids)
• The eastern blot is a biochemical technique used to analyse
protein post translational modifications (PTM) such as lipids,
phosphomoieties and glycoconjugates.
• Eastern blotting can be considered an extension of the
biochemical technique of Western blotting
• It is most often used to detect carbohydrate epitopes

29. Continued…
1. Multiple techniques have been used in Eastern blotting,
2. Example: use proteins blotted from SDS-PAGE gel on
to nitrocellulose membrane.
3. Transferred proteins are analyzed for post-translational
modifications using probes that may
detect lipids, carbohydrate, phosphorylation or any other
protein modification.

32. Results

35. RESULTS
Content of caryophyllene (I, μg/g)
in the different samples
Area of the peaks of the terpenes
identified

37. Conclusions
• Analytical techniques in particular molecular
spectrometry providing more information, are
preferred for identification.
• Chromatographyin combination with
spectrometric techniques sharply increases
possibilities and trueness of identification.
• Mass spectrometry is superior to other spectral
techniques in such features as sensitivity,
selectivity, generation possibility of molecular
mass/formula, and combinability with
chromatography

38. REFERENCES
• Chapter 2 Techniques and Methods of Identification
• Chapter A3,METHODS FOR ANALYSIS OF ORGANIC SUBSTANCES IN
WATER By Donald F. Goerlitz and Eugene Brown Book 5 LABORATORY
• Chapter 8 - ADVANCED INSTRUMENTAL ANALYSIS
• Power point prsentation by Prof Dr. K.M.Harinikumar
• Highperformance liquid chromatography analysis of plant saponins:An update
2005-2010 Jagmohan S. Negi, Pramod Singh, Geeta Joshi Nee Pant, and M. S. M.
Rawat Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal)
Herbal Research and Development Institute, Gopeshwar (Chamoli), Uttarakhand,
India
• Characterization of the Volatile Components of Cannabis Preparations by Solid-
Phase Microextraction Coupled to Headspace-Gas Chromatography with Mass
Detector (SPME-HSGC/MS)Sebastiano Arnoldi, Gabriella Roda*, Eleonora
Casagni, Lucia Dell’Acqua, Michele Dei Cas, Fiorenza Fare, Chiara Rusconi,
Giacomo LucaVisconti and Veniero GambaroDepartment of Pharmaceutical
Sciences, University of Milan, Via Mangiagalli 25, 20133, Milan, Italy