Sample Prep for Chromatographic Analysis of Difficult Matrixes
Summary
Significance of the Topic
Reliable sample preparation is essential for chromatographic analysis of challenging matrices such as biological fluids, foods and environmental samples. Effective cleanup and enrichment steps directly impact sensitivity, reproducibility and accuracy, particularly when target analytes are present at trace or ultra-trace levels. Innovations in extraction and cleanup methods reduce matrix interferences, shorten analysis time and minimize solvent consumption, meeting the needs of modern high-throughput laboratories.
Objectives and Study Overview
This work provides an overview of advanced sample preparation strategies designed for difficult matrices. Key goals are to:
- Present solid phase microextraction (SPME) developments including fiber coatings, mechanisms and applications.
- Describe high-specificity molecularly imprinted polymer (MIP)-based SPE for target analytes in complex foods and biological fluids.
- Summarize dispersive SPE (QuEChERS) for multi-residue pesticide analysis.
- Illustrate silver-ion SPE for fatty acid methyl ester (FAME) cis/trans fractionation.
- Highlight carbonaceous adsorbents for polar analytes and flash chromatography for purification of synthetic samples.
Methodology and Instrumentation
Several techniques and instrumentation platforms are covered:
- SPME fibers with varied coatings (PDMS, polyacrylate, PDMS/DVB, Carboxen) interfaced to GC and GC-MS, including manual and automated autosampler holders.
- SupelMIP SPE cartridges and 96-well plates targeting antibiotics, β-agonists, nitrosamines and pesticides.
- Dispersive SPE using salt-out and sorbent mixes for QuEChERS extraction of >100 pesticides in food matrices.
- Discovery Ag-Ion SPE cartridges for selective retention and stepwise elution of cis/trans and degree-of-unsaturation FAMEs.
- Spherical and irregular silica and C18 media for flash chromatography on VersaFlash systems.
Main Results and Discussion
SPME demonstrations show low-ppt detection of odor-causing compounds (e.g., IPMP, IBMP, MIB, geosmin) with linear calibration (r2>0.99). Fiber type, coating polarity and thickness govern capacity, selectivity and equilibrium time. MIP-SPE effectively cleans honey and milk, sharply reducing matrix background compared to liquid-liquid extraction. QuEChERS dSPE achieves rapid multiresidue pesticide cleanup with minimal solvent. Ag-ion SPE fractionates cis and trans FAMEs, improving peak resolution in GC analysis of edible oils and snack samples. ENVI-Carb SPE isolates highly polar contaminants like acrylamide and 1,4-dioxane from water at low-ng/mL levels. Flash cartridges with spherical particles deliver tighter bands and faster separations than irregular media.
Benefits and Practical Applications
These sample preparation approaches offer:
- Increased sensitivity and selectivity for trace analytes.
- Reduced analysis time, solvent use and labor through solvent-free or minimal-solvent methods.
- High robustness and reproducibility via automated workflows and well-characterized sorbents.
- Adaptability across diverse matrices including water, biological fluids, foods and synthetic reaction mixtures.
Future Trends and Opportunities
Emerging directions include in vivo and biocompatible SPME probes for real-time monitoring, integration of ambient ionization sources (DESI-SPME), further miniaturization and multiplexed MIP arrays for high-throughput screening. Advances in sorbent nanomaterials and machine-learning optimization promise enhanced selectivity and faster method development.
Conclusion
Innovations in sample preparation are critical for reliable chromatographic analysis of difficult matrices. Techniques such as SPME, MIP-SPE, QuEChERS, silver-ion fractionation, carbonaceous cleanup and flash chromatography collectively enable sensitive, selective and efficient workflows that meet the demands of modern analytical laboratories.
Used Instrumentation
- Gas chromatography with FID and mass spectrometric detection.
- Automated SPME autosamplers (e.g., Gerstel MPS, CTC Combi PAL).
- SupelMIP SPE cartridges and 96-well plates.
- Dispersion SPE kits for QuEChERS extraction.
- Discovery Ag-Ion SPE cartridges for FAME purification.
- VersaFlash flash chromatography systems with spherical silica and C18 media.
Content was automatically generated from an orignal PDF document using AI and may contain inaccuracies.
Sample Prep for Chromatographic
Analysis of Difficult Matrixes
2
Real World & Real Samples
0
2
4
6
Time (min)
Urine Sample without sample prep
0
2
4
6
Time (min)
Urine Sample with sample prep
3
Sources of Chromatographic Errors
4
Time Spend on Analytical Process
5
Sample Prep Innovations
•
Solid Phase Microextraction (SPME)
•
High specificity SPE
•
Dispersive SPE
•
Silver Ion SPE for FAMEs
•
Carbonaceous adsorbents
•
Flash chromatography
6
Solid Phase Microextraction (SPME)
• “Sample Prep Made Easy”
• Enrichment technique mainly for trace analysis
• Developed in collaboration with Janusz Pawliszyn, Univ. of Waterloo
• Unique and proprietary to Supelco
Users are...
•
GC and GC-MS analysts (HPLC & LC-MS)
•
Analyzing compounds in gases, liquids or
solids.
Interested in...
•
Sample enrichment
•
Solventless extraction
•
Using existing GC & HPLC systems
•
Economical sample prep
•
Reducing lab animal sacrifice
Users can expect...
•
Highly consistent, quantifiable results from
low concentrations of analytes
Users are...
•
GC and GC-MS analysts (HPLC & LC-MS)
•
Analyzing compounds in gases, liquids or
solids.
Interested in...
•
Sample enrichment
•
Solventless extraction
•
Using existing GC & HPLC systems
•
Economical sample prep
•
Reducing lab animal sacrifice
Users can expect...
•
Highly consistent, quantifiable results from
low concentrations of analytes
7
6.00
7.00
8.00
9.00
10.00 11.00 12.00 13.00 14.00 15.00
1
2
3
4
5
1. 2-Isopropyl-3-methoxypyrazine (IPMP)
2. 2-Isobutyl-3-methoxypyrazine (IBMP)
3. 2- Methylisoborneol (MIB)
4. 2,4,6-Trichloroanisole (I.S. 8ppt)
5. (±) Geosmin
1. 2-Isopropyl-3-methoxypyrazine (IPMP)
2. 2-Isobutyl-3-methoxypyrazine (IBMP)
3. 2- Methylisoborneol (MIB)
4. 2,4,6-Trichloroanisole (I.S. 8ppt)
5. (±) Geosmin
Odor-Causing Compounds in Water at
2 ppt (GC/MS)
Sensitive
8
Linearity of Odor-Causing Compounds from
Water at ppt Levels (SPME-GC/MS)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
0
2
4
6
8
10
12
part per trillion
IPMP r2=0.9900, yint=+0.015
IBMP r2=0.9959, yint=+0.028
MIB r2=0.9983, yint=0.021
Geosmin r2=0.9988, yint= -0.071
Quantitative
9
SPME Overview
• Solvent-free extraction technique for
nearly any sample or matrix
• Alternative to head-space GC and solid
phase extraction (SPE) techniques
• Directly interfaced with GC analysis
• Non-destructive to sample
• Reusable (100+ times)
• Inexpensive
• Fast
Manual SPME holder
and inlet guide.
Assembled SPME
fiber and holder
with fiber immersed
in a liquid sample.
11
SPME Fiber Coating: The Business
End
•An equilibrium is set up between analytes dissolved in the sample
(solution or gas phase) and in the liquid coating on the fiber.
•The fiber coating consists of:
•
GC-type phases
•
Particles
Enlargement of
the SPME fiber
coating
12
Distribution Constant
•Concentration of analyte in stationary phase compared to
concentration of analyte in solution:
K = ns/V1C2°
K = Distribution constant
ns = Moles of analyte in stationary phase
V1 = Volume of stationary phase
C2° = Final analyte concentration in water
13
Analyte
Adsorbed
Silica Rod
Liquid Polymer
Aqueous
Solution
Vial
Time
Adsorption Mechanism for SPME
14
Absorbent vs. Adsorbent Fibers
Absorbent-type fibers
(Film-type fibers)
Analytes are extracted by partitioning
•
Liquid phase
•
Retains by thickness of coating
Analytes do not compete for sites
Fibers can have high capacity
Adsorbent-type fibers
(Particle-type fibers)
Physically traps or interacts with
analytes
•
Porous particles
•
High surface area
Analytes may compete for sites
Fibers have limited capacity
15
Types of SPME Fiber Coatings
Coating
Type
Polarity
7 µm Polydimethylsiloxane (PDMS)
Absorbent
Nonpolar
30 µm PDMS
Absorbent
Nonpolar
100 µm PDMS
Absorbent
Nonpolar
85 µm Polyacrylate (PA)
Absorbent
Polar
60 µm PEG (Carbowax)
Absorbent
Polar
Coating
Type
Polarity
85 µm Carboxen-PDMS
Adsorbent
Bipolar
65 µm PDMS-DVB
Adsorbent
Bipolar
55 µm/30 µm DVB/Carboxen-PDMS
Adsorbent
Bipolar
15 µm Carbopack Z-PDMS
Adsorbent
Bipolar
Particles – Adsorption:
Films – Absorption:
16
PDMS-DVB Fiber SEM
• Cross section of the PDMS-DVB fiber. The center is a fused silica core,
surrounded by a Stableflex core. The 3-5µm DVB particles are
suspended in PDMS and layered over the cores. 275x magnification.
Photomicrograph of SPME fiber provided by Prof. Dan Armstrong, U. Texas Arlington
17
PDMS-Carboxen Fiber SEM
• 3000X magnification of the Carboxen PDMS coating. The 3-5µm
Carboxen-PDMS particles are suspended in PDMS.
Photomicrograph of SPME fiber provided by Prof. Dan Armstrong, U. Texas Arlington
18
97-0340
Carboxen™ Particle – Volume
Contribution
Contribution of pore types to total
Carboxen pore volume:
micropores (2-20Å) = 0.29 mL/g
mesopores (20-500Å) = 0.26 mL/g
macropores (>500Å) = 0.23 mL/g
Macropore
Mesopore
Micropore
19
Physical Properties of Divinylbenzene
and Carboxen 1006
Surface Area
Porosity (mL/g)*
Material (m2/g) macro meso micro
Divinylbenzene
750
0.58
0.85
0.11
Carboxen™ 1006
720
0.23
0.26
0.29
*Macropore = >500Å
Mesopore = 20-500Å
Micropore = 2-20Å
20
Comparison of SPME Fibers for the
Extraction of Small Hydrocarbons
Analyte
100µm PDMS
PDMS/DVB
Carboxen/PDMS
Ethane
0
0
750
Propane
0
0
20000
Butane
0
340
72100
Pentane
230
2150
108000
Hexane
460
9280
105000
(Analytes at 1 ppm in air, extracted for 10 min.)
(Absolute area responses)
Absorbent
Adsorbent
21
Molecular Weight Range for SPME
Fibers
0
150
300
450
Molecular Weight Range
7µm PDMS
30µm PDMS
100µm PDMS
DVB
DVB-Carboxen
Carboxen
22
Area Response vs. Fiber Type
Acenaphthene
MW 154
Decachlorobiphenyl
MW 502
Chrysene
MW 228
1.E+07
7.E+06
9.E+06
5.E+03
2.E+06
1.E+06
0.0E+00
2.0E+06
4.0E+06
6.0E+06
8.0E+06
1.0E+07
1.2E+07
30µm PDMS
Polyacrylate
PDMS-DVB
Carboxen-PDMS
23
Effects of Fiber Polarity & Coating
Thickness
• Fiber Polarity
•
Analyte selectivity
•
Better recovery of polar analytes
•
PEG
•
Polyacrylate
• Coating Thickness
•
Analyte selectivity
•
Extraction time
•
Sample capacity
•
Desorption time and carryover
24
Effects of Phase Coating Thickness of
PDMS on Analyte Recovery Relative to
Chrysene*
Analyte
%Relative Recovery
100µm
30µm
7µm
Benzene
2
1
<1
Toluene
5
1
<1
Naphthalene
13
4
1
Phenanthrene
37
27
16
Anthracene
49
38
32
Pyrene
69
54
47
Benzo(a)anthracene
105
91
96
Chrysene
100
100
100
Benzo(a)pyrene
119
127
131
Indeno(1,2,3-cd)pyrene
61
140
148
Benzo(g,h,i)perylene
61
117
122
*Absolute response of chrysene set to 100%
25
Factors Affecting Extraction Recovery
•Salts and pH
•Headspace vs. direct extraction
•Inlet liner volume
•Stirring (sample) & agitation (fiber)
•
Increases precision
•
Reduces time to reach equilibrium
•
Must be consistent for all analyses
•
Required for analytes with high
distribution constants
•
Sonication may increase temperature
26
Effects of Salt and pH
Salt usually increases analyte uptake
Use 25-30% NaCl to salt-out samples
Salt is not necessary for large non-polar analytes, such as PAHs
and large hydrocarbons, and may reduce recovery
Lower pH to extract acidic compounds
Raise pH to extract basic compounds
Beware of stability of analytes at different pH levels
27
No Salt
No Salt
Salt
Salt
Neutral
pH = 2
Neutral
pH = 2
Phenol
810
1003
6425
6150
Methylphenol
761
882
5485
7434
2-Nitrophenol
422
474
311
2315
2,4-Dimethylphenol
1344
1476
15000
20710
2,4-Dichlorophenol
5396
8138
19803
61664
2,4,5-Trichlorophenol
3115
11097
24270
96333
2,4-Dinitrophenol
0
11
765
1182
4-Nitrophenol
626
730
6536
11438
2,3,4,6-Tetrachlorophenol
3108
27683
33938
70440
2-Methyl-4,6-dinitrophenol
55
47
920
1685
Pentachlorophenol
2305
40582
22056
143905
The Effect of Salt and pH on Extraction of
Phenols by SPME
28
Headspace vs. Direct Immersion
•Analytical
considerations:
•
Volatility of sample
•
Extraction time
concerns
•
Sample matrix
•
Selectivity of analytes
Liquid or solid sample
Liquid sample
Headspace
extraction
Direct immersion
extraction
29
0.75mm ID Inlet Liner
50ppb
Standard Splitless Liner,
2mm ID
50ppb
94-0040
Inlet Liner Volume: Comparison for
Analysis of Gaseous VOCs by SPME
1
4
5
3
2
3
4
5
794-0050, 0051
Min
2
6
1
4
5
3
2
2
3
4
5
Min
6
1. Chlormethane
2. Vinyl chloride
3. Bromomethane
4. Chloroethane
5. Freon 11
30
SPME Automation
•Compatible with common GC
autosamplers (Gerstel MPS,
CTC Combi PAL, etc.)
•Improves reproducibility by
automating important
variables:
•
Heating
•
Agitation
•
Equilibration time
•
SPME automation video (~2 mins.)
31
794-0862
0
4
8
Min
12
1. Solvent
2. Internal standard
3. cis-Menthone
4. trans-Menthone
5. Menthol
1
3
4
5
2
SPME Fiber: 100µm PDMS
Sample: 4g peppermint cookie bar
Extraction: headspace, 1 min, 45°C
Desorption: 5 min at 250°C
Column: PTE™-5, 30m x 0.25mm ID,
0.25µm film
Detector:FID, 250°C
Injector: Splitless (3 min), 250°C
Peppermint Oil in Chocolate Cookie Bar
32
Milk Sample Off-Flavors by SPME-GC/MS
98-0385
Chromatogram provided by Ray Marsili, Dean Foods
Technical Center, Rockford, IL, USA.
G00507, 508
1
3
2
IS
4
6
10
15
20
Min
25
30
35
5
IS
1. Acetone
2. 2-Butanone
3. 3-Methylpentane
4. Pentanal
5. Dimethyldisulfide
6. Hexanal
IS. 4-Methyl-2-pentanone
Prior to Exposure to
Sunlight
After 1-Hour Exposure to
Sunlight
SPME Fiber: 75 µm PDMS/Carboxen
Sample: 3g of 2% milk + 10µL internal
standard solution, (20µg/mL 4-methyl-2-
pentanone) (9mL GC vial)
Column: Supel-Q™ PLOT, 30m x 0.32mm ID
Det.: GC/MS ion trap, m/z = 33-300
33
Residual Solvents in Commercial Ibuprofen
Brand “A”
Brand “B”
2
4
6
8
10
12
Time (min)
0.00E+00
2.00E+06
4.00E+06
3
1
2
4
7
8
9
12
5
2
4
6
8
10
12
Time (min)
0.00E+00
2.00E+06
4.00E+06
9
1
2
4
5
6
7
8
10 11
12
1.
Acetaldehyde
2.
Ethanol
3.
Acetonitrile
4.
Acetone
5.
2-Propanol
6.
2-Methylpentane
7.
3-Methyl pentane
8.
Hexane
9.
Ethyl acetate
10. 2,2-Dimethylpentane
11. 2,4-Dimethylpentane
12. Methylcyclopentane
34
10ppb Nitrosamines in Water: SPME-
GC/MS
96-0142
Chromatogram courtesy of J. Clark, Liggett Group, Inc.
1. Nitrosodimethylamine
2. Nitrosodiethylamine
3. Nitrosomethylethylamine
4. Nitrosodipropylamine
5. Nitrosopiperidine
6. Nitrosodibutylamine
7. Nitrosodiphenylamine
1
2
3
4
5
6
7
Min
4
6
8
1
0
1
2
1
4
1
6
1
8
2
0
2
2
Sample: analytes in (water + 25% KCl, pH 10)
SPME Fiber: 65µm PDMS-DVB
Extraction: immersion, 15 min (rapid stirring)
Desorption: 270°C, 1 min
Column: PTA-5 (amine deactivated,
30m x 0.32mm ID, 0.5µm film)
Oven: 50°C (1 min) to 250°C at 10°C/min,
hold 2 min
Carrier: helium, 30cm/sec
Det.: GC/MS (quadrupole, SIM)
Inj.: splitless, 250°C (0.75mm ID liner)
35
New Development: Biocompatible Fiber
Pipette Tips for Solvent Extraction
36
Single Use Biocompatible Fiber Probes
for in vivo Analysis
37
Comparison of SPME in-vivo PK Study of
Carbamazepine from Mice Whole Blood to
Extracts of Plasma Removed from Mice
Slide Courtesy of Ines de Lannoy-NoAb BioDiscoveries
SPME 1 mouse
Terminal blood draw
Plasma from 3 mice
0
60
120
180
240
300
1
10
100
1000
10000
Time (min)
CB
Z C
oncentration (ng/m
L)
38
Courtesy of
Joseph Kennedy
of Prosolia
SPME fiber Holder with Automated
DESI-1D Source
39
Solid Phase Microextraction (SPME)
Products
•
Fibers
•
Holders
•
Manual
•
For autosamplers
•
Accessories
•
Instructions
•
Applications on CD
sigma-aldrich.com/spme
40
Sample Prep Innovations
•
Solid Phase Microextraction (SPME)
•
High specificity SPE (SupelMIPs)
•
Dispersive SPE
•
Silver Ion SPE for FAMEs
•
Carbonaceous adsorbents
•
Flash chromatography
Users are...
•
Analytical chemists (LC, LC-MS, GC...)
Interested in...
•
Very selective extraction
•
Analysis at extremely low concentrations
(ppb, ppt)
•
Increasing specificity of sample prep from
complex matrixes
Users can expect...
•
More rigorous washing to remove matrix
•
Detect at lower levels
Users are...
•
Analytical chemists (LC, LC-MS, GC...)
Interested in...
•
Very selective extraction
•
Analysis at extremely low concentrations
(ppb, ppt)
•
Increasing specificity of sample prep from
complex matrixes
Users can expect...
•
More rigorous washing to remove matrix
•
Detect at lower levels
41
High Specificity Sample Prep
•
The specific innovation we will describe:
•
SupelMIP Molecularly Imprinted Polymers
•- SPE tubes
•- 96-well plates
42
The Molecular Imprinting Process
•
Molecularly imprinted polymers (MIPs) are polymers that have been
prepared by polymerizing either pre-formed or self-assembled monomer-
template complexes together with a cross-linking monomer. After removal
of the template molecule, a polymer with binding sites for the template is
obtained.
43
The MIP Binding Site
•Graphical
representation of
the MIP binding
site, which
contains a cavity
of the right size
and attractive
molecular
features that can
bind to the target
molecule(s).
44
SupelMIP Chloramphenicol: Analysis in
Honey
• Chloramphenicol is an antibiotic that is monitored in honey.
Background from honey sample cleaned
by SupelMIP SPE and LLE for
Chloramphenicol analysis
0.E+00
1.E+08
2.E+08
3.E+08
0
1
2
3
4
5
Time (min)
T
IC
150-
500 M/
Z
MIP
LLE
Position of
chloramphenicol peak
-1
0
1
2
3
4
5
6
-1
0
1
2
3
4
5
6
theoretical concentration (ng/mL)
ex
p
er
im
e
n
tal
co
n
ce
n
tr
at
io
n
(
n
g
/m
L
)
standard
MIP
LLE
generic
polymer
Comparison of ion suppression effect
between different clean-up methods for
honey. Samples were post-spiked with CAP
prior to analysis.
45
Overview of a Typical SupelMIP SPE
Procedure
•Very simple methods.
•Full protocols are
included with each MIP
product.
•Protocols may require
optimization depending
on the sample matrix.
47
SupelMIP Products
PAHs in edible oils
Nitroimidazoles in milk, eggs and other foods
Nonsteroidal anti-inflammatory drugs (
NSAIDS) in
wastewater and other matrices
Fluoroquinolones in bovine kidney, honey and
milk
Amphetamines and related compounds in urine
Chloramphenicol in plasma, urine, milk, honey
and shrimp
NNAL - nitroso compound in urine
TSNAs - tobacco specific nitrosamines in urine
and tobacco
β
-agonists and β-blockers in tissue, urine and
wastewater
Clenbuterol in urine
Triazines in water
Riboflavin in milk
sigma-aldrich.com/supelmip
48
Topics: Sample Prep Innovations
•
SPME (solid phase microextraction)
•
High specificity SPE (SupelMIP)
•
Dispersive SPE
•
Silver Ion SPE for FAMEs
•
Carbonaceous adsorbents
•
Flash chromatography
Users are...
•
Food safety analysts
Interested in...
•
Multi-residue pesticide analysis in food
and agricultural products
Users can expect...
•
Quick, easy, inexpensive extraction
method
Users are...
•
Food safety analysts
Interested in...
•
Multi-residue pesticide analysis in food
and agricultural products
Users can expect...
•
Quick, easy, inexpensive extraction
method
49
Dispersive SPE (dSPE or QuEChERS)
Multiresidue Pesticide Method
•Multi-residue (100’s) pesticide analysis
•Retains/removes key interferences in food
samples
•Analytes are un-retained
•
Quick (~30 min./6 samples)
•
Easy (no laborious steps)
•
Cheap
•
Effective (wide scope, low consumption)
•
Rugged (minimal sources of errors)
•
Safe (solvents and techniques)
50
Dispersive SPE Procedure
Procedure:
1. Food initially extracted with aq. miscible
solvent (e.g. ACN)
2. High amounts of salts (NaCl, Mg-sulfate)
and buffering agents added to induce
phase separation and stabilize
acid/base labile pesticides
3. Shake/centrifuge. Isolate aliquot of sup
for SPE clean-up.
4. Transfer supernatant to centrifuge tube.
Add bulk SPE phase(s) and salts.
Shake/vortex. Centrifuge and analyze
supernatant.
Standard dSPE product line configured for:
• CEN Standard Method EN – 15662
• AOAC Method 2007.01
Full details of the simple protocol is included with the product.
51
GC-MS of Pesticides from Oranges
Following Extraction with dSPE
G003590
52
Dispersive SPE Products
• Centrifuge tubes containing pre-
determined amounts of salts and
SPE sorbents to support the most
common method configurations
used today
sigma-aldrich.com/spe
Also available:
• Sample packs
• Custom tubes and packing materials
53
Topics: Sample Prep Innovations
•
SPME (solid phase microextraction)
•
High specificity SPE (SupelMIP)
•
Dispersive SPE
•
Silver Ion SPE for FAMEs (Discovery Ag-Ion)
•
Carbonaceous adsorbents
•
Flash chromatography
Users are...
•
Food analysts
Interested in...
•
Measuring cis/trans fats or degree of
unsaturation
Users can expect...
•
To fractionate FAME samples prior to GC
analysis, simplifying analytical
chromatography and improving method
accuracy
Users are...
•
Food analysts
Interested in...
•
Measuring cis/trans fats or degree of
unsaturation
Users can expect...
•
To fractionate FAME samples prior to GC
analysis, simplifying analytical
chromatography and improving method
accuracy
54
Discovery Ag-Ion SPE for FAME
Fractionation
• Silver ion anchored onto SCX SPE support
• Ag+ forms a charge transfer complex with unsaturated FAME double
bond
•
Ag+ = electron acceptor; double bond = electron donor
• Cis configuration offers greater steric accessibility = stronger retention
• Strength of interaction increases with no. of double bonds
55
Overview Discovery Ag-Ion SPE
Procedure
1. 1) Fatty acids (FA) extracted from food sample
2. 2) FA converted to FAMEs using BF3
3. 3) FAMEs are extracted into hexane
4. 4) Hexane sample applied to Discovery Ag-Ion
SPE cartridge
5. 5) FAMEs separated using different mixtures of
hexane:acetone to extract from cartridge
•
Increasing % acetone disrupts retention of
strongly retained FAMEs (cis and higher
number of double bonds)
6. 6) Fractions analyzed by GC
56
Cis/Trans Fractionation of FAMEs from
Potato Chips with and without Ag-Ion
SPE
57
Discovery Ag-Ion SPE Products
http://tinyurl.com/agionspe
58
Topics: Sample Prep Innovations
•
SPME (solid phase microextraction)
•
High specificity SPE (SupelMIP)
•
Dispersive SPE
•
Silver Ion SPE for FAMEs
•
Carbonaceous adsorbents (ENVI-Carbs)
•
Flash chromatography
Users are...
•
Analytical chemists, HPLC, GC doing
sample prep
Interested in...
•
Extraction of highly polar compounds
from water samples, and many others...
Users can expect...
•
High extraction efficiency
Users are...
•
Analytical chemists, HPLC, GC doing
sample prep
Interested in...
•
Extraction of highly polar compounds
from water samples, and many others...
Users can expect...
•
High extraction efficiency
59
Structural Classification of Carbons
60
Carbon Sorbents for Sample Prep
• Packed SPE tubes:
•
Supelclean™ ENVI-Carb PLUS – spherical carbon molecular sieve for
extraction of highly polar compounds from water samples
•
Supelclean ENVI-Carb-II/PSA SPE – multilayer SPE tubes for multiresidue
pesticide analysis in foods
•
Supelclean ENVI-Carb-II SPE – isolation/removal of pigments (e.g., chlorophyll
and carotenoids) and sterols commonly present in fruits, vegetables, and other
natural products
•
Supelclean ENVI-Carb-II/SAX/PSA SPE – additional ion exchange capability
•
Supelclean PSA SPE – polymerically bonded, ethylenediamine-N-propyl phase
that contains both primary and secondary amines
Dual Layer Supelclean ENVI-Carb-II/PSA SPE Tube
•
http://tinyurl.com/carbonspe
61
Supelclean ENVI-Carb PLUS
Examples of polar compounds:
Acephate
log P -0.85
Acrylamide
log P -0.67
1,4-Dioxane
log P -0.27
Oxamyl
log P -1.2
Spherical Carbon Molecular Sieve
• Extraction of highly polar
compounds from water samples
• > 70% Abs Recovery from 0.5 L
drinking water (1-100 ng/mL)
Procedure:
1. Condition w/ 10 mL MeOH & 10
mL DI water
2. Load up to 1 L sample
3. Reverse tube & elute w/ 4-5 mL
MeOH in opposite direction
62
GC-MS Analysis of 1,4-dioxane in water extracted using
ENVI-Carb Plus
6
8
10
12
14
16
18
20
22
24
Time (min)
1
2
3
• THF-d8 (Int. Std.)
• 1,4-dioxane-d8 (surrogate)
• 1,4-dioxane
Column: SPB-624, 30 m x 0.25 mm I.D. ,1.4 µm
Oven: 30 °C (1 min.), 7 °C/min. to 90 °C, 20 °C/min. to 200 °C (3 min.)
Inj: 200 °C
Carrier: helium, 1 mL/min constant flow
Injection: 2 µL, splitless
MS interface: 220 °C
Scan range: SIM
63
Topics: Sample Prep Innovations
•
SPME (solid phase microextraction)
•
High specificity SPE (SupelMIP)
•
Dispersive SPE
•
Silver Ion SPE for FAMEs
•
Carbonaceous adsorbents (ENVI-Carbs)
•
Flash Chromatography Users are...
•
Synthetic, organic chemists
•
Medicinal chemists
Interested in...
•
Purification of relatively large samples
from reaction mixtures or other samples
Users can expect...
•
Fast, simple, inexpensive purifications
•
High N (spherical particles)
Users are...
•
Synthetic, organic chemists
•
Medicinal chemists
Interested in...
•
Purification of relatively large samples
from reaction mixtures or other samples
Users can expect...
•
Fast, simple, inexpensive purifications
•
High N (spherical particles)
64
• Higher efficiency of spherical
particles translates to
narrower bands for more
concentrated fractions and
faster isolations.
• samples: 5-hydroxy-DL-tryptophan
and DL-tryptophan
• cartridges: 53 mm x 23 mm I.D.
• mobile phase: methanol:water (90:10)
detection: UV 254 nm
• flow rate: 20 mL/min.
Spherical particles
Irregular particles
80 mL
25 mL
Fraction 1 Volume
260 mL
400 mL
Irregular silica
40 mL
120 mL
Spherical silica
Fraction 2 Volume
Total Volume
Particle Type
Performance of Spherical vs.
Irregular Silicas in Flash
Application
65
VersaFlash Support Literature
& Products
• Silica & C18 Cartridges
•
Particle size options
•
Cartridge size options
•
Cartridges can be coupled
•
Reversible
•
Compatible with other systems
• All system components
66
Summary
•Solid Phase Microextraction (SPME) http://www.sigma-aldrich.com/spme
•High specificity SPE (SupelMIPs) http://www.sigma-aldrich.com/supelmip
•Dispersive SPE for pesticide extraction http://www.sigma-aldrich.com/spe
•Silver Ion SPE for FAMEs (Discovery Ag-Ion SPE)
http://tinyurl.com/agionspe
•Carbon adsorbents for polar compounds (ENVI-Carbs)
http://tinyurl.com/carbonspe
•Flash chromatography http://www.sigma-aldrich.com/versaflash
67
Acknowledgements/Collaborators
Prof. Janusz Pawliszyn, U. Waterloo, Canada
Ines de Lannoy, NoAb Biodiscovery (in vivo applications)
Joseph Kennedy, Prosolia (DESI)
Supelco and Fluka R&D Teams
For more information on the subjects presented
here, please contact [email protected] or
your regional sales team.
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