Trends in Sample Preparation For Chromatography

Trends in Sample Preparation for
Chromatography
P
Presented
t d by
b
Ronald E
E. Majors
Agilent Technologies
Wilmington,
g
DE
USA
Sources of Error Generated During

Chromatographic
Analysis
Sources of Error Generated During
Chromatographic Analysis
Introduction
6.0% (6%)
Sample Introduction
Contamination
(4%) Sample
Contamination 4.0%
Integration 6.0%
Chromatography (7%)
Columns 11.0%
Columns (11%)
C
Chromatography
7.0%
Integration (6%)
Instrument
Instrument
(8%)
8.0%
Operator
19.0%
Operator (19%)
Calibration
(9%)
9 0%
Calibration 9.0%
30.0%
Sample
Processing(30%)
Sample
Processing
(R E Majors,
(R.E.
Majors LC/GC Magazine,
Magazine 2002)
Time Spent on Typical Chromatographic

Analysis
Time Spent on Typical
Chromatographic Analysis
Data Management 27.0%
Data Management (27%)
Collection
6.0% (6%)
Collection
Analysis 6.0%
Analysis (6%)
Sample Processing
61.0%
Sample Processing
(61%)
(R E Majors,
(R.E.
Majors LC/GC Magazine.
Magazine 2002)
Tribute to Academics Working Primarily

in the Area of Sample Preparation
K.-S. Boos, University of Munich, Germany
L.G. Blomberg,, Karlstad Univ., Sweden
U. Brinkmann (emeritus), H. Irth and H. Lingeman, Free Univ. of
Amsterdam, The Netherlands
M.F. Burke (emeritus), Univ. of Arizona, USA
J. Haginaka, Mukogawa Womens Univ., Japan
M.-C. Hennion, V. Pichon, ESPCI, France
J.A. Jonsson and L. Mathiasson (emeritus), Univ. Lund, Sweden
H K Lee
Lee, Univ.
Univ of Singapore,
Singapore Singapore
H.K.
J. Pawliszyn, Univ. of Waterloo, Canada
S. Petersen-Bjergaard and K.E. Rasmussen, U. Oslo, Norway
B. Sellergren, Tech. Univ. Dortmund, Germany
Countless others who have devoted research to sample prep
Outline of Sample
p Prep
p Presentation
Overview of Trends
Liquid-liquid extraction
Solid-phase extraction
Formats
F
Chemistries
Automation
Trends in Sample Preparation

TREND
Smaller samples
EXAMPLES
Life sciences, proteomics studies
encountered
IMPLICATION
Lower bed mass, less solvent, faster results,
less evaporation time in SPE
Simpler methods--Just
Protein crashing instead of SPE
Cuts down on # of sample prep steps (e.g.
enough sample prep
QuEChERS instead of lengthy multi-
less error (better data), faster results, higher
step processes
recovery)
Simple
Simple LLE
Continued growth of MS-MS for LC, CE and

GC applications
Greener approaches
Reduced use of organic solvent (e.g.
Better for environment, less exposure of
SPME, hot water extractions)
workers to toxic solvents, lower purchase and

disposal costs
High throughput
More selectivity
SPE pipette tips, 96-well plates
Seamless integration to analysis
On-line extraction
Favors different formats more attuned to
Multi-functional autosamplers
automation (e.g. pipette tips, 96-well plates)
Immunoaffinity sorbents
For use with less specific and less sensitive
MS-MS
MS (e.g. UV)
Molecularly imprinted polymers (MIPs) detectors than MS
Molecularly-imprinted
RAMs
Improved chemistries
Mixed mode sorbents
Higher capacity
Polymeric
P l
i SPE packings
ki
M
More
rugged
d phases
h
Time Consuming and Laborious LiquidLiquid Extraction

Typical Separatory Funnels
Manual labor
labor
vigorous shaking
TimeWaiting
for layers to
separate
New Formats for LLE

Microextractions
Liquid
q
p
phase microextraction ((LPME))
Single drop microextraction (SDME)
Dispersive liquid-liquid microextraction (DLLME )
Microextractions with addition of hollow fiber membranes

Supported
pp
liquid
q
membranes ((SLM))
Electromembrane extraction (EME)
Supported Liquid Extraction (SLE)
Schematic of a Single Drop LPME Apparatus
Stir bar
LPME Can Be Automated
Dispersive
p
LiquidLiquid
q
q
Microextraction
(DLLME)
Based upon a three
three-component
component solvent system
system.
Extraction vessel is usually a centrifuge tube
Mixture of immiscible organic
g
extraction solvent ((e.g.10s-L
g
of
tetrachloroethylene) and a dispersive solvent (e.g. 1-mL
acetone) injected rapidly into an aqueous solvent (~ 5 mL) with a
syringe.
Forms cloudy mixturedue to finely dispersed extraction
solvent droplets
Extraction is instantaneous; no shaking is needed
Mixture is centrifuged (e.g. 1.5 min at 6000 rpm) and extraction
solvent sedimentates to bottom of tube and is removed with
syringe
Extraction of Pesticides from Water

using DLLME: Extraction Solvent
Selection*
*S
S.S.
S Caldas,
Caldas F.P.
F P Costa,
Costa and E
E.G.
G Primel
Primel, XII Congresso Latino-Americano
Latino Americano de Chromatografia E
Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 2730, 2008, Poster Tu-145
Optimization of Volume of Extraction

Solvent in DMLLE of Pesticides in Water
Water*
*S.S. Caldas, F.P. Costa, and E.G. Primel, XII Congresso Latino-Americano de Chromatografia E
Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 2730,
27 30, 2008, Poster Tu-145
Tu 145
Investigation on the Type of Dispersion Solvent*
*S.S.
S.S. Caldas, F.P. Costa, and E.G. Primel, XII Congresso Latino
Latino-Americano
Americano de Chromatografia
E Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 2730, 2008, Poster Tu145
Overall Results of DLLME Study of

Pesticides from Water*
Water
The variables in method development included: choice of
dispersion solvent and its volume, choice of extraction solvent
and its volume, pH if necessary, and centrifuge speed
Volume of water: 5 mL containing phosphoric acid
acid, pH 2
Extraction Solvent: 60-L of carbon tetrachloride
Dispersive solvent: 2 mL of acetonitrile
For all three pesticides, the linear range was found to be 0.0011.0 mg/L and limit of quantitation (LOQ) was 0.02 mg/L.
Overall, the DLLME technique is simple, fast, provides good
recovery, is low cost, and provides good enrichment factors.
*S.S. Caldas, F.P. Costa, and E.G. Primel, XII Congresso Latino-Americano de Chromatografia E
Technicas Relacionadas (Colacro XII), Florianopolis, Brazil, October 2730, 2008, Poster Tu-145
Microscale Automation of Sample Prep

using Modern Autosampler (Agilent
7693A)
Example, LLE using 2-mL vial
Steps in Supported Liquid-Liquid Extraction
1) Apply aqueous sample so that it permeates no more than 75% of the bed height of the column
2) Wait 5-15
5 15 minutes
3) Apply a suitable water immiscible organic solvent and collect the effluent
(Courtesy of Biotage)
Product Examples: Varian Hydromax, Biotage Isolute HM-N, Mercks Extrelut
Salting Out LLE

Addition of an inorganic salt into a mixture of water and a watermiscible organic solvent causes a separation of the solvent from
th mixture
the
i t
and
d fformation
ti off two-phase
t
h
system
t
Sometimes referred to as salt-induced phase separation
g acetone,, MeOH,,
Occurs with manyy common solvents ((e.g.
EtOH, acetonitrile, etc.)
Salt and salt concentration causes different degree of phase
separation
Also occurs with saccharide addition (sugaring out!) and with
water-soluble polymers with salt addition.
Useful for polar (as well as non
non-polar)
polar) analytes and often used
for partitioning of metal chelates and other metal complexes
Recoveries of Nitroaromatics, Nitramines, and Nitrate

Esters from Water by Salting-Out
Salting Out LLE (%)
(%)**
**G.M. Nikolic, J.M. Perovic, R.S.

Nikolic, and M.M. Cakic, Physics,
Chemistry
y and Technology
gy 2 ((5),
) 293299 (2003).
Salting-out LLE of Pharmaceutical in

Biofluids with Acetonitrile*
Acetonitrile
Abbott Laboratories authors investigated automated salting-out
LLE for Aids drug Kaletra in human plasma; consists of two
compounds
d Rit
Ritonavir
i and
dL
Lopinavir
i
i
MgSO4 was used for salting out; ACN was organic solvent
g 96-well p
plate was used to develop
p GLP analytical
y
A single
methodaccuracy, precision, linearity, carryover, matrix effect,
recovery and selectivityin less than a day
LC-MS/MS
MS/MS was used for analysis
LC
Only 325-L/well (sample + salt solution + internal standard +
organic solvent) was used for entire assay
Recoveries were in range of 62
62-84%
84% across lots,
lots species
species, and
concentration levels;
% CV was 2.7-6.5% for Lopinavir and 3.3-6.5% for Ritonair
In recent publication, authors used NH4OAca MS friendly salt
as salting-out reagentfor hydrophobic drug candidate &
metabolite in human plasma
* J. Zhang et al, Biomed. Chromatogr. 23, 419-425 (2009)
QuEChERS* (Pronouced Catchers)

A Low Cost, Highly Effective Sample Preparation Technique for
Multiclass, Multiresidue Analytical Approach for Pesticides in Foods
extraction
clean-up
quantitation
confirmation
QuEChERS
method
GC-MS/MS
LC-MS/MS
Quick
Easy
Cheap
Ch
Effective
Rugged
Safe
*M. Anastassiades,, S. J. Lehotay,

y, D. Stajnbaher,
j
, F.J. Schenck,, Journal of AOAC
International (JAOAC) 86, p. 412-431, 2003
Flow Diagram of QuEChERS Process

Step 1
Weigh 10 g of sample into a 50-mL Centrifuge-Tube (1)
Step 2
Add 10-mL of Acetonitrile (2)
Extraction
Step 1
Shake vigorously 1 min (3)
Add 4
4-g off MgSO
M SO4 & 1
1-g off NaCl
N Cl (4)
Step 3
Shake vigorously 1 min (3)
Add ITSD Solution

S l ti (5)
Step 4
Shake 30 s and Centrifuge (3,6,7)
St 5
Step
T k aliquot
Take
li
t & add
dd M
MgSO
SO4 (and
( d sorbent)dSPE
b t) dSPE (8)
Shake 30 s and Centrifuge(9,10)
Step 6
[Add 0.1%
0 1% acetic acid and analyte
analyte protectants
protectants (11)]
Step 7
Analyze
y by
y GC-MSD or LC-MS/MS
Dispersive
SPE Step 2
(co rtes of Ste

(courtesy
Steve
e Lehota
Lehotay, USDA)
Pictorial Representation of the QuEChERS

Sample Prep Process
Pictorial Representation of the QuEChERS

Sample Prep Process
Step One: Salting-out LLE

Three Methods Currently Practiced:
1) Original unbuffered method
2) AOAC 2007.01 buffered method
(US)
3) EN15662 method (Europe)
4) All three methods in rest of world
Step 2:
Dispersive SPE Kits
for QuEChERS
QuEChERS Advantages
9 A batch of 6-12 extracts can be prepared in 3040 min by a single analyst with $1-3 of
disposable materials per sample and generate
<12
12 mL
L solvent
l
t waste.
t
9 Consistently high recoveries (mostly 90-110%

with
ith RSD
RSDs < 5%) off a wide
id range off GC
GC- and
d LCLC
amenable pesticides are achieved from many
matrices.
matrices
(courtesy
(cou
tesy of
o Steve
Ste e Lehotay,
e otay, USDA)
US )
Recoveries of 15 Pesticides in Different Matrices

120%
n=40
n=31
Rec
covery
100%
n=43
n=45
n=43
n=45
n=43
n=43
80%
60%
40%
20%
on
d
Al
m
M
ilk
e
Fo
lia
g
n
be
a
So
y
Pe
tF
oo
d
ry
D
or
n
O
at
s
lfa
s
Al
fa
M
ol
as
se
Si
la
ge
0%
No differences were found vs. matrix for individual pesticides

or concentration
(courtesy of Steve Lehotay, USDA)
Analysis
y
of 17 Representative
p
Pesticides in
Apple by QuEChERS (AOAC Buffered
method)) with the Detection by
y LC/MS/MS
17 Representative Pesticides Information

Pesticide
Category
Pesticide
Category
Pesticide
Category
Acephate
Organophosphate
Dichlorvos
Organophosphate
Thiabendazole
Benzimidazole
Carbaryl
Carbamate
Imidacloprid
Neonicotinoid
Carbendazim
Benzimidazole
Cyprodinil
Anilinopyrimidine
Penconazole
Diazinon
Organophosphate
Dichlofluanid
Sulphamid
Methamidophos Organophosphate
Thiophanatemethyl
Benzimidazole
Tolyfluanid
Sulphamide
Triazole
Ethoprophos
Organophosphate
Propoxur
Carbamate
Kresoxim methyl
Kresoxim-methyl
Strobilurin
Pymetrozine
Pyridine
Apple Matrix Blank
Chromatogram of Apple Sample Spiked with 5ng/g (5ppb)

Of 17 Pesticides
Apple AOAC Recovery and Repeatibility

Results ((n = 6))
Pesticides
Low QC (5ppb)
Mid QC (50ppb)
High QC (200ppb)
recovery
RSD % (n=6)
recovery
RSD % (n=6)
recovery
RSD % (n=6)
Methamidophos
77 57
77.57
5 03
5.03
91 79
91.79
2 75
2.75
91 22
91.22
5 54
5.54
Acephate
78.36
4.11
89.06
4.58
97.24
4.94
Pymetrozine
70.67
5.75
77.20
11.54
88.06
12.53
Carbendazim
Ca
be da
83.13
83
3
6.29
6
9
94.05
9
05
4.34
3
91.39
9
39
2.78
8
Imidacloprid
96.16
4.79
90.03
2.76
97.41
1.85
Thiabendazole
70.07
4.84
80.20
2.86
91.28
3.73
Dichlorvos
96.73
6.13
90.97
2.85
85.03
0.95 *
Propoxur
95.23
1.68
94.86
2.33
95.08
2.98
Thiophanate methyl
102.17
13.53
79.47
13.43
89.33
3.74
Carbaryl
94.56
3.56
99.94
2.44
104.48
2.05
Ethoprophos
100.88
2.56
97.93
2.15
92.71
2.09
Penconazole
110.97
2.80
96.93
1.79
102.62
2.63
Cyprodinil
98.90
4.01
93.48
1.54
95.08
2.02
Dichlorfluanid
77.38
9.84
76.29
11.98
89.72
8.71
Diazinon
133.53
0.74
116.50
1.83
108.14
2.26
Kresoxim methyl
97.13
2.28
90.31
2.03
97.25
2.00
Tolyfluanid
114.08
4.77
92.63
7.86
101.37
5.48
* n = 3.
-Lactam Analysis
y
in Beef Kidney
y
1 g sample in a 50 mL centrifuge
FEP tube tube
add internal standards (PENV,
(PENV CEFD)
add 2 mL water + 8 mL acetonitrile
vortex briefly, shake for 5 min
centrifuge for 5 min at 3450 rcf
supernatant + 500 mg C18 sorbent
mix for 30 s
evaporate 5 mL supernatant to 0.5 mL
filter 0.5 mL extract with the Mini-UniPrep
Mini
-UniPrepTMTM
LC-MS/MS analysis
Slide adapted from Kate Mastovska, USDA-ARS
QuEChERS for Acrylamide in Foods

1 g sample in a 50 mL FEP tube
add d3-acrylamide
acrylamide at 500 ng/g
add 5 mL hexane, vortex
add 10 mL water + 10 mL MeCN
+ 4 g MgSO4 + 0.5 g NaCl
shake vigorously for 1 min
discard the hexane layer
1 mL of the upper layer
+ 50 mg PSA + 150 mg MgSO
4
mix for 30 s
centrifuge
t if
for
f 1 min
i att 3450 rcff
Slide adapted from

Kate Mastovska,
USDA ARS
USDA-ARS
Extraction/Cleanup for
Acrylamide in Foods
Centrifuge Tube
discard ...... removal of fat
hexane layer
MeCN layer
matrix
take 1 mL aliquot for

dispersive SPE cleanup
water layer
excessive salts
Slide adapted from Kate Mastovska,

USDA-ARS
Status of QuEChERS
After AOAC International interlaboratory trials were
s ccessf l and the method is no
successful,
now AOAC
International Official Method 2007.01. In Europe,
Official EN1566.2
EN1566 2 has been published
published.
Many laboratories have implemented the method
successfully
f ll for
f 200-350
200 350 pesticides
ti id iin ffood
d and
d
lowered costs (4-fold faster, less labor, lower cost).
Commercial products for QuEChERS have been
introduced
The streamlining features of QuEChERS are being
used in more applications
pp
(acrylamide;
(
y
; vet. drugs).
g )
Most Popular SPE Formats: SPE

Cartridges and Disks
Typical SPE Disk Configuration
polypropylene
packing
frits
PTFE
Disk
or fiberglass
Pre-filter
(Optional)
Solid Phase Extraction Pipette Tip*
* Designed to work with x-y-z liquid handling

systems without needed for modification
*C
Can also
l b
be used
d manually
ll
* No vacuum manifold required
* Flow can be bi-directional
* Disposable, no carryover or cross-contamination
* Recommended for small samples (less than 100-uL)
* Examples:
Agilent Cleanup C18 Pipette Tips
Varian SPEC Plus PT
Millipore ZipTip
DPX Disposable Pipette Extraction
* Ansys SPEC Plus PT
Disposable Pipette Extraction (DPX)

Adsorbent sealed in pipette tip but loosely
packed
High efficiency extractions
Extractions are rapid (<3 minutes)
Extraction efficiency is flow rate
independent
Use less sorbent, so less solvent
is required
Minimal solvent waste generated
Readily automated
William E. Brewer, US patent 6,566,145 B2

(courtesy of DPX Labs)
Application number of PCT/US08/54584

41
Basics of DPX Extraction Technology
(courtesy of DPX Labs)
GC/MS Analysis of NIDA-5 Drugs of Abuse using DPX

P
Pre-extraction
t ti
(courtesy of W. E. Brewer, Clemson Veterinary Diagnostic Center and DPX Labs)

Industry Example - Gerstel Automated

SPE & DPX
CTC PAL base hardware

Gerstel
G
l custom SPE module
d l
LC (LC/MS), GC (GC/MS)
Maestro Software/interfaces
to ChemStation
Industry
I d t Standard
St d d SPE Format
F
t
44
MICRO EXTRACTION BY PACKED

SORBENT (MEPS)
Samples as small as 3.6-uL

Can be automated; SPE
steps and injection in
same device
(
(courtesy
t
off SGE)
Schematic Diagram
g
of 96-Well Extraction
Plate System
Extraction
plate
Polyethylene manifold lid
Polypropylene
manifold
To vacuum
base
pump
O i
O-ring
On / off valve
(in off position)
Collection plate
Vacuum Gauge
Needle valve
((courtesy
y of Agilent)
g
)
Comparison of Protein Precipitation and SPE

Cleanup of Plasma Using 96-Well Plate Format
Oasis uElution Plate
(courtesy of Waters)
LC/MS runs using water

water-ammonia
ammonia
Gradient on Xterra MS column
Chemistries of SPE
Advantages of Polymers in SPE

((relative to Silica-based))
Water-wettable-wont deactivate/dewet if dried out
Wide pH range
rangecan
can use more dramatic washing conditions for interference
removal and elutions
Sphericalhomogeneous packed bed and reproducible flows
High surface areasareas 600-800
600 800 m2/g; higher loading capacity; can reduce sorbent
bed volumes, less solvent, less sample
No acidic silanol sites
Higher retention of polar compounds due to frequent mixed mechanisms
(lipophilic-hydrophilic balanced); allows development of generic methods
Disadvantages of Polymers in SPE

Not as many phase chemistries
More expensive
Polymeric SPE Recovery Performance-Dry vs.

Wet Sorbent
140
120
100
80
60
DRY WET
DRY WET
DRY WET
DRY WET
DRY WET
DRY WET
DRY WET
40
20
0
acetaminophen
p
brompheniramine
p
propranolol
doxepin
p
mianserin
SampliQ OPT (Agilent Technologies)

fluoxetine
Dihydroxy
y
y
naphthalene
Selective Chemistries in SPE

Mixed
Mi d Mode
M d Phases
Ph
(both
(b th silicaili
& polymer-based)
l
b
d)
Phases that contain two (or more) types of functional groups to
allow multiple chemical interactions for selectivity and wettability
purposes
- SAX and PS-DVB
- SAX and SCX (removal of ions when interested in
neutral compounds)
- C18 and SCX
- C18 and SAX
-C
C18
8a
and
d WAX
- C8 and SCX (mainly for drugs of abuse)
- DVB and hydrophilic (drugs in biol. Fluids)
- PS-DVB
PS DVB and
dh
hydrophilic
d
hili (d
(drugs in
i biol.
bi l Fluids)
Fl id )
- RPC and Cyano (matrix removal, alternative to
protein ppt)
- DVB-amide and SCX
Immunoaffinity Phases for Sample

Preparation
High Abundance Proteins in Human Plasma

-1-antitrypsin
p 3.8%
Immunoglobulin G
16.6%
Immunoglobulin A 3.4%
Transferrin 3.3%
1DGE/2DGE
Haptoglobin 2.9%
Other 15%
Albumin
54.3%
Analysis and Identification

Protein Expression
Drug Targets
Disease Markers
Depletion of High Abundance Proteins in Human

Serum/Plasma
Anti-albumin resin
Affinity purified polyclonal
antibodies bound to resin.
Mixed resin bed for simultaneous
removal all six proteins
from serum
Currently up to 20 proteins can be
depleted
Robust chemistry
Anti-transferrin resin
Anti-haptoglobin resin
Anti--1-antitrypsin-resin
Anti-IgA resin
Anti-IgG-resin
Individual Ab materials are mixed in
selected percentages and packed into a
column format.
Agilent MARS Column

Multiple Affinity Removal System
H High-Abundant Proteins
(Albumin, IgG, IgA, Transferrin,
Haptoglobin, Antitrypsin)
L Low
Low-Abundant
Abundant Proteins
(Biomarkers for disease and
drug targets)
Column and optimized buffers are

used to remove
e o e the
t e top six
s most
ost
abundant proteins in human serum
and plasma samples.
Attach to HPLC instrument and pump
samples through - proteins of
y
interest are collected and analyzed.

H H
L HL L
H L H
LL H
H H LH
Crude
C
d Human
H
Serum
L Low
Low-Abundant
Abundant Proteins
drug targets)
Column and Optimized buffers are

used to remove
e o e the
t e top six
s most
ost
and plasma samples.
y

H H
L HL L
H L H
LL H
H H LH
Crude
C
d Human
H
Serum
LL
L LL LL
Low-Abundant
Proteins
Free from
Interferences
L Low
Low-Abundant
Abundant Proteins
drug targets)
Column and Optimized buffers are

used to remove
e o e the
t e top six
s most
ost
and plasma samples.
y
Immunoaffinity Column Elution Profile 50 mm column

Total column run cycle = 20.00 min, for
injection, elution, and regeneration (4.6 x
50 mm column).
l
)
Capacity =
15-20 L serum per injection
1.2 - 1.6 mg total serum proteins
Multiple Affinity Removal column is

reusable, protein binding capacity is
unchanged after 200 injections of serum
Comparison of Run #20 and Run #200
2500
Flow-through,
Low Abundant
Proteins
2000
1500
Bound,
B
d Hi
Highh
Abundant
Proteins
Injection
0.25 mL/min
1000
Elution
1 0 mL/min
1.0
500
1
2
3
4
5
6
Time
(min)
0.00
9.00
9.01
12.50
12.60
20 00
20.00
Flow
%B
rate
0.00
0.250
0.00
0.250
100.00 1.000
100.00 1.000
0.00
1.000
0 00
0.00
1 000
1.000
Re-equilibration
1 0 mL/min
1.0
End run
(20.0 min).
0
0
2.5
7.5
10
12.5
Retention Time (min)

Max.
Max
pressure
120 bar
120
120
120
120
120
15
17.5
Venture AF Immunoaffinity Column for On-Line

Aflatoxin Analysis
(courtesy of Grace Davison Discovery Sciences)

R t i t d Access
Restricted
A
Media
M di (RAM)
* Developed for analysis of low MW compounds
in complex matrices, especially drugs in biological
fluids.
* Macromolecular sample compounds have limited
y to sorption
p
sites of column p
packing
g and
accessibility
elute at column void volume; small molecules will
diffuse into matrix and interact with stationary phase.
* Can be used off-line, on-line via column switching,or
as an HPLC column alone.
Typical Structure of Restricted Access Medium
Use of Coupled Column RAMRPC System for Analysis of Drugs in Plasma

A.
B.
100-uL plasma
Column A: RAM Column

Mobile phase: water, 0.5 mL/min
C l
B:
B LiChrospher
LiCh
h 60 RP S
Select
l tB
BioTrap C8 Column
Mobile phase: Trichloroacetic acid,
Acetonitrile, 0.1%
Triethanolamine,
pH2, 1.0 mL/min
SPS C8
ISRP C8
Peaks:
1. Epirubicinol
2 Epirubicinal aglycone
2.
3. Epirubicin
4. Epriubicin aglycone
5. 7-deoxyepirubicinal aglycone
(compounds in 5.6-8.2 ng/mL range)
LiChrospher RP-4 ADS
A. Rudolphi and K.-S. Boos, LC/GC 15, 814-823 (1997).
Molecularly
y Imprinted
p
Polymers
y
((MIPs))
LC Chromatogram Showing Selective Cleanup of

Clenbuterol from Beef Liver using Molecularly
Imprinted SPE Phase*
H
O
Cl
H2N
H
N
C(CH3)3
Cl
Clenbuterol
a) Blank b) Spiked (1-ng/g) beef liver extract

*Ensing, Berggren, Majors, LCGC
Peaks
1) Clenbuterol
2) Bromoclenbuterol (template
bleeding)
Most Successful Story for Acceptance of New Sample

Prep Method: Janusz Pawliszyn
Pawliszyn* and coworkers
coworkers SPME
SPME GC
SPME-GC
SPME-LC
1000
2000
3000
Number of Publications
* University of Waterloo, Waterloo, ON, Canada
4000
Gerstels Twister for Stir-Bar Sorptive

Extraction (SBSE)
Recovery of Solutes as a Function of OctanolWater Partition Coefficients for SBSE and SPME
Volume of coated polydimethylsiloxane: SPME ~ 0.5-L

SBSE ~ 25-125-L (10-mm X 0.5-mm)
(Sandra and David, LCGC, 2003)
Thin Film Microextraction

1. Rotate thin-film as it
is inserted into the
li
liner
Stainless
Steel Wire
Thin-film
2cm
1cm
2. Place cap
onto liner
Gerstel
Twister
Desorption
Liner
3. Place
liner into
MPS2 Tray
Gerstel
GC Liner
with
Coiled
Thin-film
4. Exchange of liner between the tray and

the Twister Desorption Unit (TDU) via the
MPS2 autosampler arm
(courtesy of J. Pawliszyn, June, 2009)
How does SPME membrane

d i work?
device
k?
100 m PDMS fiber
Af: 10 mm2
Vf: 0.61mm3
1 cm x 1 cm PDMS membrane
Am:200 mm2
Vm : 2.55 mm3
Ratio of extraction p
phase volume ((Vm/Vf))=4.5
Ratio of extraction phase surface area (Am/Af)=20
Extraction Time Profiles of Fluoranthene by

Twister and Thin-film Coupled to a Drill
Extraction time profile of fluoranthene
E
Extracted
d amount/ ng
140 00
140.00
120.00
100 00
100.00
thin-film
extraction
t i t
twister
extraction
80.00
60.00
40.00
20.00
0.00
0
100
200
300
400
Extraction time/ min
500
(co rtes of JJ. Pa

(courtesy
Pawliszyn,
lis n JJune,
ne 2009)
Extraction Efficiency Comparison Between PDMS Fiber and PDMS

Membrane (Extraction of Ice Wine Volatile Constituents)
4.0E+07
3 5E+07
3.5E+07
area
a counts
3.0E+07
2.5E+07
PDMS membrane
2 0E+07
2.0E+07
1.5E+07
1.0E+07
5 0E 06
5.0E+06
PDMS-100 fiber
0.0E+00
0
10
15
20

25
30
RT (min)
35
40
45
50
55
60
SPME Multiwell Method

Well filled with
sample
Insert SPME fibre

for extraction
Agitate well until

equilibrium is reached
N2
Reconstitute and
Evaporate solvent
inject into GC or LC
Desorb in well filled

with solvent
Remove fibre
from well
Alternative Approaches to Stir Bar

Sorptive Extraction
a)
Magnet
PDMS
Glass
Glass
stopper
Conventional PDMS Twister

c)
b)
Magnet
Inner phase
PDMS
Dual-Phase Twister
Solvent
PDMS
tubing
Silicone-membrane
S
Sorptive
ti Extractor
E t t
(courtesy of Prof. Carlo Bicchi, Univ. of Torino, Italy, see May 2009 LCGC Magazine)
Summary and Future Directions

With tandem LC-MS and GC-MS systems, sample prep steps may be reduced
(e.g. QuEChERS, salting-out LLE)
New formats handle smaller samples for LLE and SPE and are amenable to
automation; chip-based SPE miniaturization is now available
Polymeric SPE sorbents bring many advantages including higher capacity, reduced
bed mass and more rugged performance
Specialty phases like mixed mode SPE, MIPs, immunoaffinity, RAMs can be used
to remove specific interferences or specific analytes
SPME and SBSE are into their next generation formats
Automation of many sample prep protocools is readily available; sample prep can
be integrated into analytical system
Acknowledgements
Gerstel, Dr. Ed. Pfannkoch

J. Pawliszyn,
y Univ. Waterloo,Canada
Agilent, Limian Zhou and Carol Ball Haney
USDA, Steve Lehotay and colleagues
O h
Orachem
Biotage
DPX Labs
SGE

Trends in Sample Preparation For Chromatography

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Trends in Sample Preparation For Chromatography

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Trends in Sample Preparation for

Sources of Error Generated During

Time Spent on Typical Chromatographic

Data Management 27.0%

Data Management (27%)

Tribute to Academics Working Primarily

Trends in Sample Preparation

Protein crashing instead of SPE

Cuts down on # of sample prep steps (e.g.

enough sample prep

QuEChERS instead of lengthy multi-

less error (better data), faster results, higher

Continued growth of MS-MS for LC, CE and

Reduced use of organic solvent (e.g.

Better for environment, less exposure of

SPME, hot water extractions)

workers to toxic solvents, lower purchase and

SPE pipette tips, 96-well plates

Seamless integration to analysis

Favors different formats more attuned to

automation (e.g. pipette tips, 96-well plates)

For use with less specific and less sensitive

Mixed mode sorbents

Time Consuming and Laborious LiquidLiquid Extraction

New Formats for LLE

Microextractions with addition of hollow fiber membranes

Supported Liquid Extraction (SLE)

Schematic of a Single Drop LPME Apparatus

LPME Can Be Automated

Extraction of Pesticides from Water

Optimization of Volume of Extraction

Investigation on the Type of Dispersion Solvent*

Overall Results of DLLME Study of

Microscale Automation of Sample Prep

Example, LLE using 2-mL vial

Steps in Supported Liquid-Liquid Extraction

Salting Out LLE

Recoveries of Nitroaromatics, Nitramines, and Nitrate

**G.M. Nikolic, J.M. Perovic, R.S.

Salting-out LLE of Pharmaceutical in

QuEChERS* (Pronouced Catchers)

*M. Anastassiades,, S. J. Lehotay,

Flow Diagram of QuEChERS Process

Weigh 10 g of sample into a 50-mL Centrifuge-Tube (1)

Add 10-mL of Acetonitrile (2)

Shake vigorously 1 min (3)

Shake vigorously 1 min (3)

Add ITSD Solution

Shake 30 s and Centrifuge (3,6,7)

(co rtes of Ste

Pictorial Representation of the QuEChERS

Pictorial Representation of the QuEChERS

Step One: Salting-out LLE

9 Consistently high recoveries (mostly 90-110%

Recoveries of 15 Pesticides in Different Matrices

No differences were found vs. matrix for individual pesticides

17 Representative Pesticides Information

Apple Matrix Blank

Chromatogram of Apple Sample Spiked with 5ng/g (5ppb)

Apple AOAC Recovery and Repeatibility

QuEChERS for Acrylamide in Foods

Slide adapted from

take 1 mL aliquot for