Plant hormones are of vital importance for normal functioning of plants that coordinate the growth and development of plants with response to the environment. Plant hormones are difficult to analyze because they occur in very low amounts in plant extracts which are very rich in interfering substances, especially secondary metabolites. To cope with this problem the plant extract must undergo several purification steps using unrelated separation mechanisms in order to increase orthogonality and purification efficiency (Dobrev et al., 2005). High performance liquid chromatography and Gas liquid chromatography are frequently used in the purification and quantification of plant hormones like Abscisic acid, Indole acetic acid etc. A method for estimation of Abscisic acid in Arabidopsis thaliana includes an extraction of plant tissues with acetone/water/acetic acid (80:19:1, v/v), evaporation of the extracts and finally injection into the liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) system in multiple reaction monitoring (MRM) mode (Carbonell and Jáuregui, 2005). A novel metabolic profiling approach to the analysis of acidic phytohormones and other metabolites based on a simplistic preparation scheme and analysis by chemical ionization-gas chromatography/mass spectrometry has also been developed (Schmelz et al., 2004). But Current metabolomic approaches are able to quantify highly abundant primary and secondary metabolites but do not perform well at detecting trace levels of phytohormones. Separate profiling methods, with comparatively more elaborate sample preparation procedures, are now making phytohormone profiles accessible using trace analysis chemical ionization GC/MS techniques. Using LC/MS detection, a significant phytohormone profiling advance was recently achieved (Chiwocha et al. 2003).

1. CHROMATOGRAPHIC TECHNIQUES
FOR THE ESTIMATION OF PLANT
HORMONES
Chithra Rajagopal

2. INTRODUCTION
 Chromatography : Greek word chroma [color] + grafein [to
write]
 The collective term for a family of laboratory techniques for the
separation of mixtures.
 Russian botanist Mikhail Semyonovich Tsvet -invented the first
chromatography technique in 1900 during his research on
chlorophyll.
 He used a liquid-adsorption column containing calcium
carbonate to separate plant pigments.

4. Chromatogram

5. Chromatography (Common terms)
Solute Solvent Stationary phase Analyte Mobile phase

6. Chromatography (Common terms) contd.
Effluent
Chromatogram Immobilized
Chromatograph phase
Bonded
Retention
phase
time

7. Chromatography
Preparative Analytical
Separate the components Operates with smaller
of a mixture for further use amounts of material and
(And is thus a form of seeks to measure the relative
purification) proportions of analytes in a
mixture.

8. Chromatography theory
 Components of a mixture may be interacting with the stationary
phase based on charge (ion-ion-interactions, ion-dipole-
interactions), van der Waals' forces, relative solubility or
adsorption (hydrophobic interactions, specific affinity).

9. Chromatographic Techniques
Techniques by Techniques by Techniques by
chromatographic physical state of separation
bed shape mobile phase mechanism
Ion
Column GC exchange Affinity
Paper LC
Size exclusion

10. Major Plant Hormones
AUXINS CYTOKININS GIBBERLLINS ABA ETHYLENE

12. PROBLEMS IN PLANT HORMONE ESTIMATION
 Efficiency in extraction of the plant tissue is considerably low.
 Although extractable hormones may be released from tissues
relatively quickly ,it is not possible to determine how much of the
hormone pool has been recovered.
(Sundberg,1990)
 Even for immunoassays, sample preparation accounts for a large
proportion of the time and effort expended in performing an
analysis.
(Hedden,1993)

13. LATEST TECHNIQUES
 HPLC- High Pressure Liquid Chromatography
 LC/MS- Liquid Chromatography/Mass Spectrometry
 GC/MS- Gas Chromatography/Mass Spectrometry

14. HPLC

15. Principle of HPLC
 Forces the analyte through a column of the stationary phase by
pumping a liquid (mobile phase) at high pressure through the
column.
 The sample to be analyzed is introduced in small volume to the
stream of mobile phase and is retarded by specific chemical or
physical interactions with the stationary phase as it traverses the
length of the column.
 The use of pressure increases the linear velocity giving the
components less time to diffuse within the column, leading to
improved resolution in the resulting chromatogram.
 Common solvents methanol and acetonitrile.

16. HPLC Overview
Polychrom Computer
(Diode Array) Detector Workstation
Variable
Solvent UV/Vis Detector
HPLC Solvent Delivery System
Reservoirs
Rheodyne
Injector
HPLC
Column

17. %A %B %C Flow Rate Pressure to column
{H2O} {MeOH} (mL/min) (atmos.)
load
Ready
inject
Rheodyne
Injector
Varian 9010 Solvent Delivery System to injector
through pump
Column
through C
pulse
dampener
A B
from solvent to
Ternary Pump reservoir detector

18. LC/GC- MS
LC/GC- MS
 Coupling of liquid chromatography (LC) or gas chromatography
(GC) separations to a mass spectrometer provides physical
separation of metabolites, introducing different compounds into
the mass spectrometer at different times.
 Separation of metabolites from interfering substances allows for
improved quantitative accuracy.
 Applications of GC/MS include drug detection, fire investigation,
environmental analysis, explosives investigation, proteomics and
identification of unknown samples.

19. LC/GC- MS

20. GC-MS

21. Working
 The molecules take different amounts of time (retention time) to
come out of the gas chromatograph
 Allows the mass spectrometer downstream to capture, ionize,
and detect the molecules separately.
 The mass spectrometer breaks each molecule into ionized
fragments and detecting the fragments using their mass to
charge ratio.

22. Mass Spectrometry

23. Reports On Different Types Of Estimation Of Plant
Hormones

24. Indole-3-acetic Acid Levels of Plant Tissue as Determined
by a new High Performance Liquid Chromatographic
Method (Philip et al, 1977)
 A method for the analysis of lndole-3-acetic acid (IAA) in plant
extracts based on high performance liquid chromatography
separation of IAA on a miroparticulate strong anion exchange
column
 And quantitation with two selective detectors: an electrochemical,
carbon paste amperometrc detector and/or a fluorescence
detector.

25. A Rapid Method for the Extraction and Analysis of Abscisic
Acid from Plant Tissue( Kerry et al, 1980)
 The method makes use of silica Sep-pak prepacked cartridges.
 The ABA extracts are loaded on to the Sep-pak cartridges which
are then washed with a series of solvents resulting in the removal
of pigments and other unwanted compounds.
 The ABA is then eluted from the cartridge and the levels of this
hormone are estimated by gas chromatography.

26. Headspace Gas Chromatographic Determination of
Ethylene Oxide in Air (Binetti et al, 1986)
(

27.  Add 10 ml di-methyl acetamide(DMA)
 Keep for 1 hr
 Submit to GC determination
 The peak areas in the headspace chromatograms of the
standard solutions are plotted against the corresponding ETO
concentration in order to obtain the calibration curve.

30. Introduction
 Common purification procedures such as column
chromatography, solid phase extraction (SPE), liquid–liquid
extraction, etc. are employed for plant hormone purification
– Require significant amounts of solvent, time and labor
 IAA and ABA exhibit many similar chemical properties
– Relatively hydrophobic compounds containing a carboxylic
group
– common chromatographic techniques very often end up in
the same fraction
 2D HPLC system obtain very pure separate fractions of IAA and
ABA and quantify these compounds with much higher reliability.

31. Materials and methods used
 Chemicals and materials:-
– Unlabelled IAA and ABA
– Radioactive IAA and ABA
– Deuterated ABA
– 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG, 97%)
– HPLC grade methanol and acetonitrile
– Formic acid and Ammonium hydroxide

32. Recoveries of standard IAA and ABA

33. 2-D HPLC set up

34. Results and discussion
– In the First dimension the sample was loaded into silica-
cyanopropyl column.
– When run in reversed-phase mode the polar sorbent of this
column allows the elution of IAA and ABA with relatively low
proportion of organic solvent.
– Low concentration of organic solvent in the segment applied
to the second dimension allowed concentrating IAA and ABA
on the more hydrophobic column (silica-C18) used in the
second dimension,

35. Contd.
 IAA and ABA were well retained and separated in the second
HPLC dimension with capacity factor higher than 2 and resolution
4.
 Relatively high throughput since the injection-to-injection cycle
time is less than 30 min
 The results show that quantification by 2D-HPLC with on-line UV
(ABA) and FLD (IAA) detection are statistically identical (with
95% confidence) to the ones measured by GC–MS

37.  Includes an extraction with acetone/water/acetic acid (80:19:1, v/
v), evaporation of the extracts and finally injection into the liquid
chromatography-electro spray ionization tandem mass
spectrometry (LC–ESI–MS–MS) system in multiple reaction
monitoring (MRM) mode.
 The objective of this work has been to show the applicability of
the method to quantify the endogenous content of ABA in
Arabidopsis thaliana leaves at three different degrees of water
stress.

38. LC-MS Optimization
 An LC column of 50 mm length used to analysis a high number of
samples.
 Gradient was done in such a way that ABA elutes at approx 7 min for
avoiding matrix interferences.
 A standard solution of ABA (1 ng µl-1 ) into the MS at 5µl min-1.
 MRM acquisition method was used for the quantification of extracts.

39. Results and Discussion
 The high specificity of the MRM acquisition mode allows us to
obtain clean chromatograms (1 peak)from non-purified crude
plant extracts, thus avoiding possible interferences to the
analysis.
 The main contribution of this method is its speed and simplicity,
allowing ABA to be determined in a few hours.
 solvents such as methanol/water/acetic acid (80:19:1, v/v) and
acetone/ water/acetic acid have given consistent results and
avoid the formation of ABA-Me

42. Introduction
 In this study the pH and polarity of the mobile phase were taken
into consideration to optimize the mobile phase for the
chromatographic separation of 3 important plant hormones: (ABA),
(IAA) and (GA3).
 GA3, IAA and ABA contain carboxylic groups and their retention
depends on the percentage of ionized and non-ionized species.
 The optimum pH of the mobile phase should be taken into account
to study the influence of pH on retention in LC.

43. Chromatographic procedure
 The mobile phases used:- Acetonitrile-water (26:74:30:70%; v/v)
 The chromatographic column equilibrated for each mobile condition
with a time limit of 30 min.
 Column temperature :- 250 C
 Separation through Isocratic elution with a flow rate of 0.8 mL/ min.
 The standard solution of individual acid prepared in the mobile
phase and chromatographed separately to determine the retention
time for each acid.
 OD was measured at 208, 265, 280 nm for GA3, ABA and IAA.

44. Results and Discussion
 The mobile phase was adjusted to different pH values in order to
select a suitable pH condition for chromatographic separation.
 Retention factor values, k, for the plant hormones studied were
determined in ACN-water mixtures at 26% and 30% (v/v) of
acetonitrile.
 Six pH values (4.0, 4.5, 5.0, 5.5, 6.0 and 7.0) were investigated
for the mobile phase.
 The GA3, ABA and IAA content of 2 plant samples were
determined in acetonitrile-water, 26% (v/v) containing 30 mM
phosphoric acid at pH 4.00.

45. Chromatogram of the plant hormones

46. GC-MS ANIMATION
http://www.shsu.edu/~chm_tgc/sounds/flashfiles/GC-MS.swf