Sample Preparation

SOLID-PHASE EXTRACTION

Dr. Saleem A Bokhari

GCU Lahore
 Outline

 Principles of solid phase extraction

 Salient features and benefits
 Silica products and polymers
 Practical workflow
 Applications

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 Objective:
Sample Preparation (GC HPLC)

 we perform sample preparation to:

 remove interference from the sample

 concentrate analytes to improve detection
 more accurate results
 protect equipment to ensure proper maitenance

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SPE Column

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 Methodology of SPE

SPE is an extraction process whereby an aqueous sample is

filtered through a thin bed of sorbent particles, the analytes
of interest are removed from the liquid matrix, and
concentrated onto the sorbent.

Once concentrated, the analytes are removed by an eluting

solvent.

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Practical work flow

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4 Steps in SPE

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Conditioning of silica-based sorbents

Add an organic solvent to rinse and activate

the alkyl-chains (C18, C8 etc.)!

Don´t let the column run dry during conditioning!

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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

 Non polar: van der Waals ~20 KJ/mole

 Polar: Dipole / Dipole ~ 40 KJ/mole

Hydrogen bond ~40 KJ/mole

 Electrostatic: Ionic ~600 KJ/mole!

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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
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- Marijuana in blood etc

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 Comparison of LLE vs SPE Disk
LLE
 Uses 200 - 500 ml solvent
 Shaking / continuous process
 Forms emulsions
 Little selectivity
 Takes 1 - 2 hours / sample
SPE disk
 Uses 2 - 20 ml solvent
 Filtration process
 No emulsions formed
 Wide selectivity
(adsorbent)
 Takes 10 - 20 min. / sample
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Problems with the LLE Procedure

 Tedious and time-consuming

 Shaking and separation time
 Evaporation time

 Expensive-labor and materials

 Time factor
 Solvent cost and exposure
 Solvent disposal

 Poor results
 Forming of emulsions
 Irreproducible extractions
 Low recoveries 39
 What are the Benefits of SPE?

 SPE uses less solvent than LLE

 SPE is faster (at least 5 times)
 High capacity: Many samples at a time
 Total SPE costs are considerably less than LLE
 High selectivity: broad choice of bonded phases and solvents
 Automation much easier

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