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SOLID-PHASE EXTRACTION
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Objective:
Sample Preparation (GC HPLC)
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SPE Column
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Methodology of SPE
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Practical work flow
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4 Steps in SPE
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Conditioning of silica-based sorbents
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Case 1: when the analyte of interest is polar/ionic
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Case 2: when the analyte of interest is nonpolar
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Case 3: when the analyte of interest is a mix of polar
and nonpolar
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SPE Column accessories
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Types of sorbants (Packing materials)
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Stationary Phases
(Silicagel)
Reversed Phase
C18
C8
C4
Adsorption
Si-OH
Normal Phase
NH2
CN
C-OH(OH)
Mixed Phase
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Kinds of Exhange Resins
Anion Exchange
N+
NH2
Cation Exchange
C6H5-SO3H
COOH
SO3H
Biochromatography
WP PEI (NH2)
WP Butyl (C4)
WP CBX (COO)
Sephadex G-25
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Intensity of Interactions
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Reversed Phase Principle
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Mixed Mode Principle
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Polymer- Phase Principle
(-CH-CH2)n-
N-CH3
R
C=O
CH3
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Polymer- Phase Principle
(-CH-CH2)n-
N-CH3
R
C=O
CH3
SO3H or SO3H or CH2N+R3
CH2N+R3
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Experiment 1:
Rapid Extraction of natural dyes in beverages
Sample Preparation:
1 mL blackberry-juice is dissolved in 2 mL of distilled water.
Column Conditioning:
A 3 mL C18 (Baker: 7020-03) SPE cartridge is conditioned with 1 mL methanol followed
by one column volume of distilled water.
Sample Addition/Wash:
The prepared sample is aspirated through the column. A 5 mL distilled water wash is
used to remove sugars, sugar colouring and organic acids.
Elution:
The dye(s) [anthocyanines, flavonoids, tannins and/or alkaloids)] is (are) eluted with a
column volume of methanol. Sometimes propanol-2 will be more successful.
Analysis:
For detailed analysis- an absorption spectrum can be taken from the eluate.
- TLC-experiments can be done.
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Experiment:
Rapid Extraction of natural dyes in beverages: sample load
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Experiment 1:
Rapid Extraction of natural dyes in beverages: sample load
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Experiment 1:
Rapid Extraction of natural dyes in beverages: washing step
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Experiment 1:
Rapid Extraction of natural dyes in beverages: elution step
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Experiment 1:
Rapid Extraction of natural + synthetic dyes in beverages:
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Experiment 1:
Rapid Extraction of natural + synthetic dyes in beverages: sample load
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Experiment 1:
Rapid Extraction of natural + synthetic dyes in beverages: elution step
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Experiment 1:
Rapid Extraction of natural + synthetic dyes in beverages
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Application Database
Environmental
Water, Soil
Forensic/Clinical/Pharmaceutical
Serum, plasma, urine, blood, plant
extracts
Food/feed
Juice, grain, milk
Biological/biotech
Water, plasma, urine 36
Environmental
- PAH ‘s from water and soil
- PCB‘s from oil-
- Pesticides from water/soil-
- etc.
Food/Feed/Beverages
- Aflatoxine from corn meal
- Caffeine from de-caffeinated diet cola
- Vitamin E from juice
- etc.
Pharmaceutical/Clinical/
Biological/Forensic
- Benzodiazipines from serum
- Anabolic Steroids/Urine
- Aflatoxine from liver
- Marijuana in blood etc
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Comparison of LLE vs SPE Disk
LLE SPE disk
Uses 200 - 500 ml solvent Uses 2 - 20 ml solvent
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Problems with the LLE Procedure
Poor results
Forming of emulsions
Irreproducible extractions
Low recoveries 39
What are the Benefits of SPE?
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