Sample Prep for Chromatographic Analysis of Difficult Matrixes
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
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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%