The document discusses an automated online system for extracting, purifying, and analyzing plant hormones called auxins. It uses solid phase extraction coupled with ultra-high performance liquid chromatography and tandem mass spectrometry. The system allows extraction of samples using up to 50 μL of solvent compared to only 5 μL for older methods. It completes a full extraction, purification and analysis cycle in only 7 minutes, much faster than the over 30 minutes required by older methods. The automated high-throughput online system provides more accurate and reproducible results while being less time-consuming than previous manual methods.

1. Presented to:
Dr. Syed Hammad Raza
Presented by:
Muhammad Rehan Khalid
Roll No:
2220892 (Reg: 2018-GCUF-02371)
Class:
M. Phil. Botany (1st Semester)

2. Phytohormone:
Plant hormones (phytohormones)
are chemicals produced by plants
that regulate.
Death
Growth
Longevity
Reproduction
Development
Example:
Auxin Gibberellin Cytokinin
Ethylene Abscisic Acid Jasmonates
Brassinosteroids Salicylic Acid Strigolactones

3. Extraction
Purification
Analysis
“is defined as the
separation of some
Components from
crude solution of
interest”
“is defined as the
removal of Impurities
from the extracted
solution”
“is defined as the
detection and
Identification of
compound. It is used to
identify that, compound
extracted is of our
interest or not”
Terms Difference:

4. Some Important Terms:
Sorbents:
“Sorbents are insoluble materials or mixtures of materials used to
recover liquids through the mechanism of absorption or adsorption or
both”
Diff Between “Hormone” and “Vitamin”
Hormone
Produced both in animals
and plants
No catalytical activity.
Site of production in plants
are Roots, Shoots apex and
leaf.
Vitamins
Synthesized mostly in plant
body but transferred to
animal by food.
Concept of Essential and Non-
Essential Vitamin Exists in
animals
Have catalytic activity. Also
causes disease due to
deficiency.

5. The extraction, purification and analysis was very arduous process in past. But now a
days different techniques like “Solid Phase Extraction” and HPLC are done by
machines. The results are accurate and less time consuming.
As SPE (Solid Phase Extraction) is most effective with HPLC so I selected the newer
but on other hand an Online automatic system for doing all the process, rather than
doing manually.
“An automatic versatile system integrating solid-phase extraction with ultra-high
performance liquid chromatography–tandem mass spectrometry using a dual-
dilution strategy for direct analysis of auxins in plant extracts”(Zhong et al., 2014).
It was an online system. The online system was converted from the Shimadzu LC–
MS/MS system

6. Different Techniques:
Extraction
VPE (Vapor phase extraction)
VMAE (Vacuum microwave-assisted extraction)
SPE or SPME ( Solid Phase Extraction)
Purification
LLE (Liquid-liquid extraction)
SPE ( Solid Phase Extraction)
DLLME (Dispersive Liquid –Liquid Microextraction
VPE ( Vapor Phase Extraction)
Immuno-Extraction
dCPE (Dual Cloud Point Extraction
HF-LLLME (Hollow-Fiber Based- Liquid Liquid-Liquid Microextraction
Analysis
HPLC (High performance Liquid Chromatography)
UPLC (Ultra Performance-LC)
HPLC-FLD(Fluorescence Detector)
LC-MS, GC-MS, 2D-HPLC

7. Apparatus:
A LCMS-8040 tandem mass spectrometer (Shimadzu, Shimadzu, Kyoto, Japan) was
equipped with an
Electrospray ionization (ESI) interface
The LC system consisted of a CBM-20A controller
Two LC-30AD pumps (Pump A&B, which could stand ultra-high pressure up to 130 MPa)
Two LC-20AD pumps (Pump C&D, which could only stand normal pressure up to 40 MPa)
Two vacuum degassers
An autosampler
SPD-M20A photodiode array (PDA) detector.

8. Procedure:
1) First is dilution and extraction:
Sample solution was injected by autosampler and was transferred into Mixer 1 by Pump D. The
sample solution was efficiently diluted in Mixer 1 and then went through the SPE column, on which
the analytes of small molecular weight were adsorbed while the matrixes with larger molecular weight
were excluded.
2) Second is Desorption and Capture:
After Valve 1 was switched, the desorption solvent flowed through the SPE column and carried the
analytes to Loop 1, at a certain time, the desorbed analytes was “captured” in Loop 1.
3) Third is the second dilution, column-head stacking and UHPLC separation:
After Valve 2 was switched, the desorbed analytes in Loop 1 was driven into Mixer 2 by an organic
phase from Pump A and mixed with the aqueous phase from Pump B. Because the ratio of the organic
phase to the aqueous phase was set at 1:9 (v/v), the analytes were diluted in 90% (v/v) aqueous phase
in Mixer 2 and introduced to the UHPLC column for the following separation.

9. Pump
4 (A)
Pump
3 (B)
Pump
2 (C)
Pump
1 (D)
Auto Sampler
Mixer 1
Mixer 2
Valve 1
Valve 2
In Line Filter
Valve 3
Extraction Column
T Join
ANALYSIS
PURIFICATION
EXTRACTION
Pump
1 (D)
UHPLC Column
PDA-MS
Loop-1
Loop-2

10. Pump
4 (A)
Pump
3 (B)
Pump
2 (C)
Pump
1 (D)
Auto Sampler
Mixer 1
Mixer 2
Valve 1
Valve 2
In Line Filter
Valve 3
Extraction Column
T Join
ANALYSIS
PURIFICATION
EXTRACTION
Pump
2 (C)
Pump
3 (B)
UHPLC Column
PDA-MS
Loop-1
Loop-2

11. Pump
4 (A)
Pump
3 (B)
Pump
2 (C)
Pump
1 (D)
Auto Sampler
Mixer 1
Mixer 2
Valve 1
Valve 2
In Line Filter
Valve 3
Extraction Column
T Join
ANALYSIS
PURIFICATION
EXTRACTION
Pump
3 (B)
Pump
4 (A)
UHPLC Column
PDA-MS
Loop-1
Loop-2

12. Pump
4 (A)
Pump
3 (B)
Pump
2 (C)
Pump
1 (D)
Auto Sampler
Mixer 1
Mixer 2
Valve 1
Valve 2
In Line Filter
Valve 3
Extraction Column
T Join
ANALYSIS
PURIFICATION
EXTRACTION
Pump
1 (D)
Pump
2 (C)
Pump
3 (B)
Pump
3 (B)
Pump
4 (A)
UHPLC Column
PDA-MS
Loop-1
Loop-2

13. Comparison with old system:
Old system can endure only 5 µL of Solvent. But Online System can endure
up to 50 µL.
Old Method took more than 30 min. But the online New System took only 7
min per complete cycle.
This can be repeated again and again. We can perform 6 cycle as compared to
the old method.

14. Result