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Chromatographic technique


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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).

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Chromatographic technique

  2. 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.
  3. 3. Chromatogram
  4. 4. Chromatography (Common terms)Solute Solvent Stationary phase Analyte Mobile phase
  5. 5. Chromatography (Common terms) contd. Effluent Chromatogram Immobilized Chromatograph phaseBonded Retentionphase time
  6. 6. Chromatography Preparative AnalyticalSeparate the components Operates with smallerof a mixture for further use amounts of material and(And is thus a form of seeks to measure the relativepurification) proportions of analytes in a mixture.
  7. 7. 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).
  8. 8. Chromatographic TechniquesTechniques by Techniques by Techniques bychromatographic physical state of separationbed shape mobile phase mechanism IonColumn GC exchange Affinity Paper LC Size exclusion
  10. 10. 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)
  11. 11. LATEST TECHNIQUES HPLC- High Pressure Liquid Chromatography LC/MS- Liquid Chromatography/Mass Spectrometry GC/MS- Gas Chromatography/Mass Spectrometry
  12. 12. HPLC
  13. 13. 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.
  14. 14. HPLC Overview Polychrom Computer (Diode Array) Detector Workstation Variable Solvent UV/Vis DetectorHPLC Solvent Delivery System Reservoirs Rheodyne Injector HPLC Column
  15. 15. %A %B %C Flow Rate Pressure to column {H2O} {MeOH} (mL/min) (atmos.) load Ready inject Rheodyne InjectorVarian 9010 Solvent Delivery System to injector through pump Column through C pulse dampener A B from solvent to Ternary Pump reservoir detector
  16. 16. 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.
  17. 17. LC/GC- MS
  18. 18. GC-MS
  19. 19. 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.
  20. 20. Mass Spectrometry
  21. 21. Reports On Different Types Of Estimation Of Plant Hormones
  22. 22. Indole-3-acetic Acid Levels of Plant Tissue as Determinedby a new High Performance Liquid ChromatographicMethod (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.
  23. 23. A Rapid Method for the Extraction and Analysis of AbscisicAcid 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.
  24. 24. Headspace Gas Chromatographic Determination ofEthylene Oxide in Air (Binetti et al, 1986) (
  25. 25.  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.
  26. 26. 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.
  27. 27. 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
  28. 28. Recoveries of standard IAA and ABA
  29. 29. 2-D HPLC set up
  30. 30. 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,
  31. 31. 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
  32. 32.  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.
  33. 33. 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.
  34. 34. 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
  35. 35. Introduction In this study the pH and polarity of the mobile phase were takeninto consideration to optimize the mobile phase for thechromatographic separation of 3 important plant hormones: (ABA),(IAA) and (GA3). GA3, IAA and ABA contain carboxylic groups and their retentiondepends on the percentage of ionized and non-ionized species. The optimum pH of the mobile phase should be taken into accountto study the influence of pH on retention in LC.
  36. 36. 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.
  37. 37. 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.
  38. 38. Chromatogram of the plant hormones