CHROMATOGRAPHIC TECHNIQUES FOR THE ESTIMATION OF PLANT

HORMONES

Chithra Rajagopal

INTRODUCTIONINTRODUCTION

ChromatographyChromatography : Greek word : Greek word chromachroma [color] + [color] + grafeingrafein [to [to write]write]

The collective term for a family of laboratory techniques for the The collective term for a family of laboratory techniques for the separation of mixtures.separation of mixtures.

Russian botanist Mikhail Semyonovich Tsvet -invented the first Russian botanist Mikhail Semyonovich Tsvet -invented the first chromatography technique in 1900 during his research on chromatography technique in 1900 during his research on chlorophyll. chlorophyll.

He used a liquid-adsorption column containing calcium He used a liquid-adsorption column containing calcium carbonate to separate plant pigments.carbonate to separate plant pigments.

Chromatogram

Chromatography (Common terms)

Solute Solvent Stationary phase Analyte Mobile phase

Chromatography (Common terms) contd.

Bonded phase

Chromatogram Chromatograph

EffluentImmobilized phase

Retention time

Chromatography

Preparative Analytical

Separate the components of a mixture for further use (And is thus a form of purification)

Operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture.

Chromatography theory

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

Chromatographic Techniques

Techniques by chromatographic bed shape

Techniques by physical state of mobile phase

Techniques by separation mechanism

Column

PaperGC

LC

Ion exchange

Size exclusion

Affinity

Major Plant Hormones

AUXINS CYTOKININS GIBBERLLINS ABA ETHYLENE

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)(Hedden,1993)

LATEST TECHNIQUES

HPLC- High Pressure Liquid ChromatographyHPLC- High Pressure Liquid Chromatography

LC/MS- Liquid Chromatography/Mass SpectrometryLC/MS- Liquid Chromatography/Mass Spectrometry

GC/MS- Gas Chromatography/Mass SpectrometryGC/MS- Gas Chromatography/Mass Spectrometry

HPLC

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.

SolventDelivery System

VariableUV/Vis Detector

HPLC SolventReservoirs

HPLCColumn

RheodyneInjector

Polychrom(Diode Array) Detector

ComputerWorkstation

HPLC Overview

Varian 9010 Solvent Delivery System

Rheodyne Injector

%A %B %C Flow Rate Pressure{H2O} {MeOH} (mL/min) (atmos.)

Ready

Ternary Pump

A

C

B

from solvent reservoir

Colum

n

to detector

to column

throughpulse

dampener

to injector

through pump

load

inject

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.

LC/GC- MSLC/GC- MS

LC/GC- MS

GC-MS

The molecules take different amounts of time (retention time) to The molecules take different amounts of time (retention time) to come out of the gas chromatographcome out of the gas chromatograph

Allows the mass spectrometer downstream to capture, ionize, Allows the mass spectrometer downstream to capture, ionize, and detect the molecules separately.and detect the molecules separately.

The mass spectrometer breaks each molecule into ionized The mass spectrometer breaks each molecule into ionized fragments and detecting the fragments using their mass to fragments and detecting the fragments using their mass to charge ratio.charge ratio.

Working

Mass Spectrometry

Reports On Different Types Of Estimation Of Plant Hormones

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.

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.

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

Add 10 ml di-methyl acetamide(DMA)Add 10 ml di-methyl acetamide(DMA) Keep for 1 hrKeep for 1 hr Submit to GC determinationSubmit 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.

IntroductionIntroduction

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.

Materials and methods usedMaterials 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

Recoveries of standard IAA and ABARecoveries of standard IAA and ABA

2-D HPLC set up2-D HPLC set up

Results and discussionResults 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,

Contd.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

IntroductionIntroduction

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.

LC-MS OptimizationLC-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.

Results and DiscussionResults 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

IntroductionIntroduction

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.

Chromatographic procedureChromatographic 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.

Results and DiscussionResults 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.

Chromatogram of the plant hormonesChromatogram of the plant hormones

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

GC-MS ANIMATION