This document describes a study that developed an online automatic system integrating solid-phase extraction with ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) for direct analysis of auxins in plant extracts. The system allowed for dilution and extraction of samples using solid phase extraction, desorption and capture of analytes in a loop, and a second dilution before UHPLC separation and MS/MS analysis. This provided accurate, fast results for extracting, purifying, and analyzing phytohormones like auxins from plant samples.

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. 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

5. 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.

6. 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.