LC GC June 2015

Volume 33 Number 6, 373–440
Volume 33 Number 6 June 2015

www.chromatographyonline.com
LCGC NORTH AMERICA
LC–MS Analysis of
Peptide Mixtures
in Proteomics
Studies
The Current State
of Superficially
Porous Particles
Single-Cell MS and MS
Imaging Using a Novel
Sampling Device
High-Throughput LC
and SFC Purification
June 2015
in Drug Discovery
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® Contents Volume 33 Number 6

JUNE 2015
Columns
386 COLUMN wATCH

Current State of Superficially Porous Particle
Technology in Liquid Chromatography
David S. Bell
Superficially porous particles continue to expand into new realms,
through new surface chemistries and particle sizes.
396 LC TROUBLEsHOOTING
Volume 33 Number 6 June 2015
www.chromatographyonline.com
Internal Standard Calibration Problems

John W. Dolan
LC–MS Analysis of Readers’ questions regarding problems related to internal standard
Peptide Mixtures
in Proteomics calibration of liquid chromatography methods are addressed.
Studies
The Current State
of Superficially
Porous Particles
402 PERsPECTIVEs IN MOdERN HPLC
Single-Cell MS and MS
Separation Science in Drug Development,
Imaging Using a Novel
Sampling Device Part I: High-Throughput Purification
High-Throughput LC
and SFC Purification
Mengling Wong, Brent Murphy, Joseph H. Pease, and Michael W. Dong
in Drug Discovery
An overview of modern practices of high-throughput purification
to support small-molecule drug discovery
Cover photography by XXXXXXX,
CoverXXXXXXX,
illustrationXXXXXX
courtesy of
Ella Marushchenko 414 Ms–THE PRACTICAL ART
Cover materials
Column courtesy
photography of
by Joe Single-Cell MS and High-Spatial-Resolution MS Imaging
XXXXX, XXXXXXX, XXXXXX
Zugcic of Joe Zugcic Photography Under Ambient Conditions Using a Novel Sampling Device
Column courtesy of Ning Pan, Wei Rao, and Zhibo Yang
Showa Denko America/Shodex A novel device can probe extremely small targets to sample and ionize
molecular species, facilitating single-cell MS and MS imaging.
DEPARTMENTS
Peaks of Interest . . . . . . . . . 384 438 THE EssENTIALs
Products & Resources. . . . . . . . . 431 GC Temperature Programming—10 Things
Calendar . . . . . . . . . . . . . . . . .435 You Absolutely Need to Know
Short Courses . . . . . . . . . . . 436 Learn about the critical aspects of GC temperature programming
Ad Index . . . . . . . . . . . . . . . . .437 and improve your method development and implementation.
WEB SEMINARS Peer-reviewed ArtiCle

maximizing Analysis efficiency 420 Analysis of Peptide Mixtures for Proteomics Research
through new GC–ms Approaches
Dominic Roberts
Using LC–ESI-MS with a Simple Microgradient Device
Thermo Fisher Scientific René Lenobel, Helena Rěhulková, Marek Šebela, Vojtečh Franc,
www.chromatographyonline.com/ Vladislav Kahle, Dana Moravcová, and Pavel Rěhulka
LCGCwebseminars
This setup is an inexpensive alternative for the separation of
both simple and complex peptide mixtures before MS analysis.
NEW ON
FeAtured interview
Torsten C. Schmidt on
An in-tube extraction (ITEX) method
for headspace sampling of beer
429 Caroline West, LCGC’s 2015 Emerging Leader Award
prior to GC–MS Winner, Focuses on the Fundamentals of Selectivity
www.chromatographyonline.com/ West recently spoke to LCGC about her scientific beginnings
lcgc-tv-library
and her recent work in HPLC, SFC, and HILIC.
Like LCGC on Facebook:
www.facebook.com/lcgcmagazine
LCGC North America (ISSN 1527-5949 print) (ISSN 1939-1889 digital) is published monthly with 1 additional issue in August as Buyers Guide by UBM Life Sciences,
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PHARMACEUTICAL n
HEALTH SCIENCES n
FOOD n
ENVIRONMENTAL n
CHEMICAL MATERIALS
©2015 Waters Corporation. Waters, ACQUITY QDa and The Science of What’s Possible are registered trademarks of Waters Corporation.
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Editorial Advisory Board

Kevin D. Altria GlaxoSmithKline, Ware, United Kingdom Ira S. Krull Professor Emeritus, Department of Chemistry and
Jared L. Anderson The University of Toledo, Toledo, Ohio Chemical Biology, Northeastern University, Boston, Massachusetts
Daniel W. Armstrong University of Texas, Arlington, Texas Ronald E. Majors LCGC columnist and analytical
consultant, West Chester, Pennsylvania
Michael P. Balogh Waters Corp., Milford, Massachusetts
R.D. McDowall McDowall Consulting, Bromley, United Kingdom
Brian A. Bidlingmeyer Agilent Technologies, Wilmington, Delaware
Michael D. McGinley Phenomenex, Inc., Torrance, California
Dennis D. Blevins Agilent Technologies, Wilmington, Delaware
Victoria A. McGuffin Department of Chemistry, Michigan
Peter Carr Department of Chemistry, University
State University, East Lansing, Michigan
of Minnesota, Minneapolis, Minnesota
Mary Ellen McNally E.I. du Pont de Nemours
Jean-Pierre Chervet Antec Leyden, Zoeterwoude, The Netherlands
& Co., Wilmington, Delaware
André de Villiers Stellenbosch University, Stellenbosch, South Africa
Imre Molnár Molnar Research Institute, Berlin, Germany
John W. Dolan LC Resources, Lafayette, California
Glenn I. Ouchi Brego Research, San Jose, California
Michael W. Dong LCGC columnist, San Francisco, California
Colin Poole Department of Chemistry, Wayne
Roy Eksteen Sigma-Aldrich/Supelco, Bellefonte, Pennsylvania State University, Detroit, Michigan
Anthony F. Fell School of Pharmacy, University of Douglas E. Raynie Department of Chemistry and Biochemistry,
Bradford, Bradford, United Kingdom South Dakota State University, Brookings, South Dakota
Francesco Gasparrini Dipartimento di Studi di Chimica e Tecnologia delle Fred E. Regnier Department of Chemistry, Purdue
Sostanze Biologicamente Attive, Università “La Sapienza,” Rome, Italy University, West Lafayette, Indiana
Joseph L. Glajch Momenta Pharmaceuticals, Cambridge, Massachusetts Pat Sandra Research Institute for Chromatography, Kortrijk, Belgium
Davy Guillarme University of Geneva, University Peter Schoenmakers Department of Chemical Engineering,
of Lausanne, Geneva, Switzerland University of Amsterdam, Amsterdam, The Netherlands
Richard Hartwick PharmAssist Analytical Laboratory, Kevin Schug University of Texas, Arlington, Texas
Inc., South New Berlin, New York
Dwight Stoll Gustavus Adolphus College, St. Peter, Minnesota
Milton T.W. Hearn Center for Bioprocess Technology,
Michael E. Swartz Boston Analytical, Salem, New Hampshire
Monash University, Clayton, Victoria, Australia
Caroline West University of Orléans, France
Emily Hilder University of Tasmania, Hobart, Tasmania, Australia
Thomas Wheat Waters Corporation, Milford, Massachusetts
John V. Hinshaw BPL Global, Ltd., Hillsboro, Oregon
Kiyokatsu Jinno School of Materials Science, Toyohashi
University of Technology, Toyohashi, Japan Consulting Editors: Jason Anspach, Phenomenex, Inc.; David Henderson,
Trinity College; Tom Jupille, LC Resources; Sam Margolis, The National Institute
of Standards and Technology; Joy R. Miksic, Bioanalytical Solutions LLC
The Fused-Core®
Advantage for Bioseparations
Faster Separations of mAbs, ADCs, Glycans, Proteins & Peptides
BIOshell™ columns are the most recent innovation in Fused-Core particle

technology: high efficiency reversed-phase columns for protein and peptide
separations and specifically engineered HILIC columns for the separation of
glycans. BIOshell columns can be operated in HPLC or UHPLC instrumentation
equipped with a mass spectrometer or any other detector.
To request an evaluation column, visit

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LLC, registered in the US and other countries. BIOshell and Solutions within are trademarks of Sigma-Aldrich Co. LLC.
Fused-Core is a registered trademark of Advanced Materials Technology, Inc. 83221
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PEAKS of Interest
Gyula Vigh Receives Arnold O.
Beckman Award for Separation Techniques
Gyula Vigh, Emeritus Professor of Chemistry and inaugural
New videos from LCGC
holder of the Gradipore Chair in Separation Science at Texas
A&M University (College Station, Texas) has been awarded RUDOLF KRSKA ON MYCOTOXIN
the Arnold O. Beckman Award for Outstanding Scientific ANALYSIS USING LC–MS-MS
Mycotoxins are an important group of
Achievements in the field of electrodriven separation tech- secondary metabolites produced by
niques. The award is sponsored by Sciex Separations, a divi- fungi that can cause disease in humans
sion of AB Sciex (Framingham, Massachusetts). and animals. Rudolf Krska from BOKU,
Vigh’s research focuses on both the theoretical and prac- IFA-Tulln, in Vienna, Austria, describes why he uses LC–
MS-MS in his analysis of mycotoxins and offers advice to
tical aspects of high performance chromatographic and separation scientists thinking about using the technique
electrophoretic separation methods. He joined Texas A&M in their research.
University in 1985 where he served two terms as Chairman
of the Analytical Division in the Department of Chemistry. In Other recent LCGC TV interviews include:
2001, he was appointed to Texas A&M University’s Gradipore
• Torsten C. Schmidt on novel extraction methods for GC
Chair in Separation Science. He has graduated 34 PhD stu-
dents, published 178 papers, and obtained five US patents. • Kevin Schug with tips for analyzing
three types of biological fluids.
In 2011, he received the Hungarian Society for Separation
Sciences (HSSS) Halász Medal Award recognizing research
excellence in the separation sciences. He retired in 2013. The
Visit http://www.learnpharmascience.com/lcgc/index.php
award was presented on April 28, 2015, at the 31st Interna- to see these videos and more.
tional Symposium on Microscale Bioseparations in Shanghai.
VWR Acquires Hichrom

VWR, a global distributor of analytical products head-
quartered in Radnor, Pennsylvania, has announced its
acquisition of the analytical instrumentation manufac-
IS THERE AN APP FOR THAT?
HIGHLIGHTS OF APPS FOR SEPARATION SCIENCE
turer Hichrom Limited, based in Berkshire, UK. 4G 100% 11:59 AM

“Known for its quality, excellence and technical exper-
tise, Hichrom is an investment that will help expand our APP NAME: HPLC Troubleshooting
portfolio and manufacturing capabilities to a specialized OFFERED BY: BoBo Labs
and growing industry,” said Peter Schuele, senior vice
president and president, EMEA and Asia-Pacific Lab and
PLATFORMS: Android (requires 2.3.3 and up)
Distribution Services. “Together with Hichrom, we are WHAT IT DOES: The HPLC Troubleshooting
excited to deliver the tools needed to enable science in app is designed to help beginner HPLC users with
HPLC troubleshooting. HPLC troubleshooting sec-
the largest product segment in the analytical instruments
tions include troubleshooting HPLC pressure is-
industry.” sues; troubleshooting peak issues like split peaks
and no peaks; HPLC retention time fluctuation; and
Markes International HPLC chromatography issues.
Wins Queen’s Award for Enterprise COST: Free
Markes International (Llantrisant, UK) has been awarded a
2015 Queen’s Award for Enterprise in International Trade
decreed by Her Majesty the Queen of Great Britain, on
the recommendation of the Prime Minister, David Cam-
eron. The awards are announced each year on the queen’s
birthday, April 21.
Founded in 1997, Markes International has shown sus-
tained growth in exports that, according to the company,
account for over 85% of total turnover. Alun Cole, Man-
aging Director of Markes International, said “It is an hon-
our to be recognized by Her Majesty the Queen for the
strength of our export business, and I believe it reflects
many years of hard work and dedication from everyone
at Markes.” ◾
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“So you’re sure this protein has a molecular weight of 150,000?”
“According to our assumptions, that’s correct.”
; ; ;
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COLUMN WATCH
Current State of Superficially
Porous Particle Technology in
Liquid Chromatography
A
The use of superficially major goal in high perfor- Fused-Core particles) exhibit a solid
porous particles in the mance liquid chromatography core surrounded by a thin layer of
manufacture of high (HPLC) over the past decade porous material. The SPP architec-
has been to develop particle technolo- ture was initially designed to improve
performance liquid
gies that provide enhanced separation mass transfer kinetics over fully
chromatography (HPLC) efficiency. The trend began with the porous particles (FPPs); however,
columns has become introduction of sub-2-µm particles further research has revealed that
prominent in recent years. along with advances in HPLC instru- the increased efficiencies observed in
mentation. Modern superficially small-molecule applications is largely
Over the course of the
porous particles (SPPs) for use with because of improvements in both
past decade most major small-molecule separations were intro- eddy diffusion (the A term in the
manufacturers have built duced in 2006 by Advanced Materials van Deemter equation) and longitu-
column lines around the Technologies. The advantage of SPPs dinal diffusion (the B term) (4). The
was that the technology provided improvement in eddy dispersion has
technology. At the recent
nearly similar efficiencies to the sub- been attributed to the inherent low
Pittcon conference in New 2-µm particles, but at a lower cost of particle size distribution resulting
Orleans, Louisiana, a large back pressure. The lower back pressure from the SPP construction process
number of oral and poster then allowed the technology to be used and on the roughness of the SPP
across a wider array of HPLC systems. surface (5). These features result in a
presentations centered on
Since its introduction, SPP tech- higher packing density than observed
the current research and nology has been embraced by most with FPP phases. The presence of the
advances using superficially major column manufacturers and solid core has a positive impact on
porous particles. This HPLC practitioners. At Pittcon 2015 the longitudinal diffusion and has
in New Orleans, Louisiana, it was been shown to reduce the longitudi-
installment aims to provide
clear from the number of presenta- nal diffusion by approximately 30%
some highlights of these tions that active research and devel- over fully porous particles (6).
recent trends. opment is continuing in this realm.
A few major trends were observed: Recent Trends in Superficially
• expansion of particle sizes and pore Porous Particle Design
geometries, Expansion of Particle
• additional chemical surface modi- Sizes and Pore Geometries
fications, and The initial offering from Advanced
• application beyond small molecule Materials Technology in 2006 con-
reversed-phase analyses. sisted of a 1.7-µm solid core and a
This installment is intended to 0.5-µm outer shell with a pore size
discuss recent observed trends in SPP of 90 Å (see Figure 1). Since that
development. For more background time most column manufactur-
on the technology, readers are ers have adopted some form of SPP
referred to several reviews published technology. As demonstrated in Fig-
in recent years (1–3). ure 2, SPPs at 2.7 µm can provide
David S. Bell is the chromatographic efficiencies nearly
guest author this month.
Ronald E. Majors Superficially Porous Particles similar to smaller sub-2-µm FPPs,
is the editor of Column Superficially porous particles (also but with about half the back-pressure
Watch. known as solid core, core–shell, or burden. A listing of some of the typical
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Joanna Simpson
comfortable with on the
spot sampling at home
Dried blood spot (DBS) sampling is an emerging technology in the clinical and pharmaceutical laboratory,
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brands and their SPP offerings is

provided in Table I. It is clear from
this list that SPP introductions have
grown to cover a large array of par-
Porous layer
ticle sizes and surface chemistries.
Several of the manufacturers in
Table I have introduced 2.0-µm or
sub-2-µm versions of the SPP tech-
nology. The smaller particles offer
Solid core 1.7 μm 2.7 μm increased efficiency over 2.7-µm and
larger particles and are generally
used with ultrahigh-pressure liquid
chromatography (UHPLC) instru-
mentation. The same trends in lower
optimum plate heights and f latter C
0.5 μm terms with smaller overall particle
size are observed with SPPs as previ-
ously was achieved with fully porous
Figure 1: Representation of superficially porous particle technology showing the particles. Some of these manufactur-
general architecture of modern superficially porous particles as introduced by ers have also introduced larger (4–5
Kirkland (Advanced Materials Technology) in 2006. (Adapted with permission from
www.hplc.eu/halo.htm.)
µm) shell particles. The larger par-
ticle sizes provide practitioners with
even less of a back-pressure burden
than the 2.7-µm particles and allow
t0 = 0.058 min easier transfer of methods based on
(a)
common 5-µm fully porous particles.
Studies have shown that the larger
200
sized SPP technology still provides
Absorbance (mAU)
tr = 0.407 min improved efficiency and f latter C

150 N = 5514 terms than similarly sized fully
Log (t0/N) = -3.20
porous particles (7,8). Figure 3 shows
100 a comparison of the van Deemter
plots (H versus u 0) for 5-µm SPPs
50 and 5-µm TPPs.
Particles in the range of 2.5 µm
0 appear to be most often used for
0 0.2 0.4 0.6 0.8
initial method development because
Time (min) typical back pressures allow them
(b)
t0 = 0.060 min to be utilized on both UHPLC and
250 HPLC systems. The smaller par-
ticle sizes tend to be used in high-
throughput environments where
200
tr = 0.474 min speed of analysis is important. Larger
Absorbance (mAU)
N = 6194 particle sizes have found use in rou-

150 Log (t0/N) = -3.23 tine testing laboratories where HPLC
systems continue to dominate. It is
100 unclear if even larger or even smaller
SPP versions will find utility, but it
50 is expected that further developments
within the current range of 1.3–5 µm
0 0.2 0.4 0.6 0.8 particle sizes will continue.
Time (min)
Additional Chemical
Figure 2: Ultrafast separations obtained using (a) superficially porous and (b) totally Surface Modifications
porous particle 50-mm columns. (a) Column: 50 mm × 2.1 mm, 2.7-µm Poroshell 120 It is also apparent from Table I that
EC-C18; mobile phase: 56:44 acetonitrile–water; flow rate: 1.94 mL/min; temperature:
40 °C; pressure: 549 bar. (b) Column: 50 mm × 2.1 mm, 1.8-µm Zorbax RRHD Eclipse
a wide variety of surface chemistries
Plus C18; mobile phase: 59:41 acetonitrile–water; flow rate: 1.82 mL/min; temperature: are now available that use the SPP
40 °C; pressure: 1003 bar. (Courtesy of Agilent Technologies.) technology. Because resolution is
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www.chromatographyonline.com JUNE 2015 LCGC NORTH AMERICA VOLUME 33 NUMBER 6 389
dependent on efficiency and selectiv- colleagues (16), large-molecule sepa- stricted access to the internal volume
ity, both the particle design (effi- rations suffer from potential issues for compounds up to about 5 kDa.
ciency) and the surface chemistry because of adsorption, secondary inter- Particles designed with 160-Å pores
(selectivity) are significant toward actions, low diffusion coefficients, and provide access to compounds up to
achieving separation (9). Classical poor kinetics. The same features of about 15 kDa, and those with 400-Å
surface chemistries such as C18-, SPP phases that have proven effective pores provide access for structures up
C8-, and phenyl-modified surfaces in small-molecule separations are also to around 500 kDa. Particles with
have been commercially available applicable to large-molecule separa- pore sizes around 160 Å are generally
since the inception of SPPs. More tions. To allow unrestricted access into used for peptides and small proteins,
unique surface chemistries, such as the pores for these large molecules, whereas larger proteins require further
polar phases suitable for hydrophilic- wider-pore structures are required. increases in average pore size (17).
interaction chromatography (HILIC) According to Schuster and colleagues One characteristic difference
(10) and biphenyl phases continue (21), 90 Å pore structures allow unre- between the newly developed wide
to be commercialized that broaden
selectivity options. Several presenta-
tions demonstrating that this trend is
continuing were presented at Pittcon
2015. For example, Dan Armstrong’s NEW VERSION
group from the University of Texas at
Arlington reported the development
of several HILIC phases bonded to
SPP surfaces (11). The same group
also demonstrated improved effi-
ciency for chiral separations when
chiral selectors are applied to the SPP
technology (12,13). Figure 4 shows
a comparison between cyclofructan-
based chiral stationary phases (CSPs)
bonded to totally porous and superfi-
cially porous particles. The improved
efficiency of SPP technology results
in increased resolution. The group
also demonstrated very fast chiral
separations using a number of dif-
ferent brush-type chiral station-
ary phases (CSPs) bonded to shell
particles. Other examples include
separate reports from Phenomenex
and Agilent representatives on the
development of high-pH-stable SPP-
based phases (14,15). The broadening
of selectivity on SPP architecture
through additional surface chemis-
tries is expected to continue in the
foreseeable future.
Application Beyond Small- Over 100 new features & Over 500,000 registered users
improvements in Origin 2015! worldwide in:
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Another recent trend has been the FOR A FREE 60-DAY EVALUATION, 120+ Fortune Global 500
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large-molecule separations. The ini- AND ENTER CODE: 2187 ◾ 3,000+ Government Agencies
tial SPP offerings were geared toward & Research Labs
small-molecule separations with par-
ticle pore sizes around 100 Å. Table
II lists manufacturers and selected
stationary-phase characteristics of 20+ years serving the scientific
recently developed larger-pore SPP & engineering community
columns. According to Fekete and
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Table I: Listing of some typical brands and their small molecule SPP offerings
Manufacturer Brand Pore Available Chemistries Particle

Size (Å) Size (μm)
Advanced Chromatography
ACE UltraCore 90 SuperC18, SuperPhenylHexyl (pH stable) 2.5, 5
Technologies
Advanced Materials Technology Halo 90 C18, C8, Phenyl-Hexyl, PFP, ES-CN, HILIC, Penta-HILIC 5.0
Advanced Materials Technology Halo 90 C18, C8, Phenyl-Hexyl, RP-Amide, PFP, ES-CN, HILIC, Penta-HILIC 2.7
Advanced Materials Technology Halo 90 C18, PFP 2
SB-C18, SB-C8, SB-Aq, EC-C18, EC-C8, Phenyl-Hexyl, EC-CN,

Agilent Technologies Poroshell 120 Bonus-RP, Peptide-Mapping, HILIC, PFP, HPH-C18, HPH-C8 4
(high pH phases)
Agilent Technologies Poroshell 120 C18, C8, Phenyl-Hexyl, PFP, and HILIC 2.7
ChromaNik SunShell 90 C18, C8, PFP, Phenyl-Hexyl, HILIC-Amide 2.6
Flare Diamond
Diamond Analytics 120 C18, C18MM (mixed mode), C18+, HILIC 3.6
Coreshell
Fortis SpeedCore 80 C18, Diphenyl, PFP, HILIC 2.6
Knauer Blueshell 80 C18, C18A, PFP, Phenyl-Hexyl, HILIC 2.6
Knauer Blueshell Classic 80 C18, C8 4.5
Macherey-Nagel Nucleoshell 90 RP-18, RP-18 Plus, Phenyl-Hexyl, PFP, HILIC 2.7
Macherey-Nagel Nucleoshell 90 RPC18, RP-18 Plus 5
Nacalai Tesque Cosmocore 90 C18, Cholesterol 2.6
PerkinElmer Brownlee SPP 90 C18, C8, Phenyl-Hexyl, RP-amide, PFP, ES-CN, HILIC 2.7
Phenomenex Kinetex 100 C18, XB-C18, EVO-C18, C8, Phenyl-Hexyl, Biphenyl, PFP, HILIC 5.0
Phenomenex Kinetex 100 C18, XB-C18, EVO-C18, C8, Phenyl-Hexyl, Biphenyl, PFP, HILIC 1.7, 2.6
Phenomenex Kinetex 100 C18 1.3
Restek Raptor 90 C18, ARC-C18, Biphenyl 2.7, 5
Supelco Ascentis Express 90 C18, C8, Phenyl-Hexyl, RP-Amide, F5, ES-CN, HILIC, OH5 2.7, 5.0
Supelco Ascentis Express 90 C18, F5, HILIC, OH5, ES-CN, C8, Phenyl-Hexyl 2.0
C18, C8, RP-MS, aQ, PhenylHexyl, Phenyl X, PFP, Polar Pre-

Thermo Fisher Scientifc Accucore 80 2.6
mium (RP-Amide), HILIC and HILIC Urea.
1.4 (C18
Thermo Fisher Scientifc Accucore XL 80 C18, C8
only), 4.0
Waters Cortecs 90 C18, HILIC 1.6, 2.7
YMC Meteoric Core 80 C18, C8 2.7
pore SPP packings and the initial shell thickness layers for efficiency recently (18,20–22). Figure 5 shows a
small-molecule particles lies in the in protein separations. The thinner comparison of a wide-pore sub-2-µm
shell thickness. The porous layer the porous layer, the more the effi- fully porous column and a 3.6-µm
used in particles developed for large- ciency increased; however, thinner wide-pore SPP phase for the analysis
molecule separations are generally layers also result in lower loading of an intact monoclonal antibody,
thinner than SPPs first introduced capacity, thus some compromise is rituximab. It is apparent from these
for small molecule separations. This typically arrived at (19). Demonstra- traces that both particle technologies
is because of the relatively slow dif- tions of effective separations of intact provide excellent peaks shape and
fusion of large molecules in and out proteins and monoclonal antibod- selectivity for this 150-kDa antibody,
of the porous layer. Schuster and ies (mAbs) along with performance although in this case the FPP pro-
colleagues (18) compared several evaluations have been published vides slightly better resolution. The
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Table II: Commercially available superficially porous particle columns with larger pore size structures
Manufacturer Brand Average Pore Available Chemistries Particle Size Outer Shell
Size Thickness
Advanced Materials Technologies Halo 160 C18, CN 2.7 0.5
Advanced Materials Technologies Halo 160 C18, CN 4.6 0.6
Advanced Materials Technologies Halo 400 C4, C18 3.4 0.2
Agilent Technologies AdvanceBio 450 C4, C8, Diphenyl 3.5 NA
Agilent Technologies Poroshell 300 300 C3, C8, C18, Extend-C18 5 0.25
ChromaNik SunShell 160 C18, RP-Aqua 2.6 0.5
ChromaNik SunShell 300 C4, C8, C18 2.6 0.5
Diamond Analytics Flare C18 Wide Pore 250 C18 3.6 0.1
PerkinElmer Brownlee Brownlee SPP 160 C18 2.7 NA
Phenomenex Aeris WIDEPORE 200 C4,C8, C18 3.6 0.2
Supelco BIOshell 160 C18, CN 2.7 0.5
Supelco BIOshell 160 C18, CN 4.6 0.6
Supelco BIOshell 400 C4, C18 3.4 0.2
Thermo Fisher Scientifc Accucore 150 150 C4, C18, Amide HILIC 2.6 0.5
YMC Meteoric Core 160 C18 2.7 NA
NA = information not available
O
25
F3C–S CN
(a) H2N N
N
Cl Cl
20
Fully porous Rs = 1.2
CF3
H (µm)
(b)
15 Hmin = 9.9 μm
Rs = 1.7
10
Hmin = 7.2 μm
(c) Rs = 2.1
Superfcally porous
5 0 1 2 3 4 5 6 7 8 9
Time (min)
0 2 4 6 8
u0 (mm/s)
Figure 4: Comparison of totally porous particles and

Figure 3: Efficiency comparison between fully porous and superficially porous particles in chiral chromatography.
superficially porous 5-μm particles. Column dimensions: Columns: Cyclofructan 6 (CF6-P) bonded to (a) 5-µm fully
250 mm × 4.6 mm, 5 µm; mobile phase: acetonitrile–water; porous particles, (b) 3-µm fully porous particles, and (c) 2.7-
test compound: benzophenone; k ’ = 6.2 (isoelutropic µm superficially porous particles; column dimensions: 150
conditions). (Data courtesy of Dr. Ken Broeckhoven, Vrije mm × 4.6 mm; mobile phase: heptane–ethanol, 92:8 for the
Universiteit Brussel.) fully porous particles and 95:5 for the superficially porous
particles; analyte: fipronil. (Adapted with permission from
reference 13.)
same wide-pore SPP phase is used to separate fragments
of a digested mAb, bevacizimab, in Figure 6. The effi- land, in commemorating his receipt of the LCGC Life-
ciency of the SPP particle results in excellent separation time Achievement in Chromatography Award, presented
of fragments in both the Fc and Fab regions. a talk entitled “Tools to Improve Protein Separations”
Activity around large-molecule SPP developments was that focused on recent advances in SPP technology from
also apparent at the recent Pittcon meeting. J.J. Kirk- Advanced Materials Technologies (23). As with small-
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25.0
C18 - 1.7 μm
1.7 μm 20.0
TPP 5 μm
15.0
H (μm)
3.2 μm solid core 0.2 μm porous
TTP 3.5 μm
10.0
C18 - 3.6 μm SPP
3.6 μm
SPP 2.6 μm
5.0 TPP 1.7 μm
TPP 1.6 μm
0 1 2 3 4 5 6
Time (min) 0.0
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
Figure 5: Comparative analysis of intact mAb rituximab u (mm/s)
(IgG1) u sing sub -2- µm tot ally porou s and 3.6 - µm
superficially porous phases. Column dimensions: 150 mm
× 2.1 mm; temperature: 80 °C (C18, 1.7-µm, 300-Å) and 90 Figure 7: Comparison of superficially porous and totally
°C (C18, 3.6-µm SPP, wide-pore). (Data courtesy of Davy porous par ticle per formance in SFC . Mobile phase:
Guillarme, University of Geneva.) 9 8:2 c arb o n dioxid e – m e than ol; m o d el co m p oun d:
b u t ylp a ra b e n . (Da t a co u r te s y of Dav y G uillarm e ,
University of Geneva.)
Fab 2015, in London, UK, Davy Guillarme presented com-

10 pelling evidence that SPP particles provide enhanced
800
700 FC efficiency over traditional particles in SFC (24). Figure
600 7 shows a comparison of FPP and SPP phases based on
4 11
500
400 the van Deemter relationship in SFC. As with HPLC and
300
200 UHPLC, SPP technology affords increased efficiency
12 13
100 1 23 56 7 8 9 over similarly sized particles in SFC. The alternative
0
selectivity, as demonstrated in Figure 8 using SPP tech-
2 3 4 5 6
Time (min) nology bonded with different polar phases, indicates that
the trend of broadening selectivity through modifica-
Figure 6: Separation of digested mAb (bevacizumab Fc
and Fab fragments). Column: 150 mm × 2.1 mm, 3.6-µm
tions of surface chemistry is spreading to the alternative
widepore SPP, C18; mobile-phase A: 0.1% trifluoroacetic techniques as well. Several recent publications support
acid in water; mobile-phase B: 0.1% trifluoroacetic acid in SPP use in SFC (25–28).
acetonitrile; gradient: 30 – 40% B over 11 min; flow rate:
0.35 mL/min; temperature: 90 °C; injected volume: 0.5 µL;
detection: fluorescence (excitation at 280 nm, emission at
Conclusion
360 nm). (Data courtesy of Davy Guillarme, University of HPLC columns packed with SPPs are rapidly becoming a
Geneva.) preferred tool for separations method development in the
modern analytical laboratory. SPPs continue to expand
molecule SPP packings, it is expected that continuous in their traditional realm of small-molecule separations
developments in surface chemistry aimed at improving through the introduction of alternative surface chem-
large-molecule separations will continue to emerge. istries and a widened assortment of particle sizes. SPPs
In addition to expansion of SPP technology to large- with wider pore structures have recently been made avail-
molecule reversed-phase separations, exploration of SPPs able that allow large molecules such as proteins and large
for use in ion chromatography and supercritical f luid peptides to be effectively separated. The particle technol-
chromatography (SFC) have surfaced. For instance, at ogy also continues to expand into additional separation
the Emerging Separations Technologies Symposium techniques such as SFC. Further developments in surface
organized by The Chromatographic Society and the chemistry modifications and fundamental investigations
Separation Science Group of the Analytical Division into particle architecture promise even further extension
of the Royal Society of Chemistry held on March 26, of the SPP platform utility.
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0.10 15 Silica
Absorbance (mAU) 23 6 11 16
10 7 14
1 9 13 5
4 8 12 17
0.05
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Time (min)
0.10 3+7
Diol
Absorbance (mAU)
6
4
0.05 11 13
10 15
2 8 5 14 16
1 9 12 17
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Time (min)
5+12 2-EP
0.10
6 7 4
9 11
Absorbance (mAU)
3 15 13
2 10 14
0.05 1 8 16
17
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Time (min)
0.10
Absorbance (mAU)
4-EP
6 7
3+4 15
0.05 9
8 10 11 5 14 13 16
1 2 12 17
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
Time (min)
Figure 8: Alternative selectivity for acidic and neutral compounds using various polar SPP columns in SFC. Silica column
(Sunshell): 5 –20% methanol in 4 min; diol column (Sunshell), 2-EP column and 4-EP column: 5 –25% methanol in 5.33
min; flow rate: 2.5 mL /min; back pressure: 150 bar; temperature: 40 °C; injected volume: 1 µL; detection: UV absorbance
at 220 nm. Peaks: 1 = ibuprofen, 2 = flurbiprofen, 3 = naproxen, 4 = ketoprofen, 5 = paracetamol, 6 = caffeine, 7 =
etophylline, 8 = thymine, 9 = uracil, 10 = warfarin, 11 = hydrocortisone, 12 = prednisolone, 13 = sulfamethoxazole, 14
= sulfadimethoxine, 15 = sulfamethazine, 16 = sulfaquinoxaline, 17 = sulfamethizole. (Adapted with permission from
reference 23.)
Acknowledgments Dr. Zachary Breitbach of the Uni- M.A. Mart’n, Trends Anal. Chem. 64,
The author would like to acknowl- versity of Texas at Arlington, and 17Ð28 (2015).
edge the following reviewers for Dr. Ron Majors of LCGC. (2) S. Fekete, E. Ol‡h, and J. Fekete, J.
their substantial suggestions to Chromatogr. A 1228, 57Ð71 (2012).
improve this work. Dr. Davy Guil- References (3) F. Gritti and G. Guiochon, J. Chro-
larme of the University of Geneva, (1) V. Gonz‡ lez-Ruiz, A .I. Olives, a nd matogr. A 1228, 2Ð19 (2012).
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(4) S. Fekete and D. Guillarme, LCGC North Am. 32(6), 420–433 This month’s guest coauthor:
(2014).
(5) G. Guiochon and F. Gritti, J. Chromatogr. A 1218, 1915–1938 David S. Bell is a manager in pharmaceuti-
cal and bioanalytical research at Sigma-Aldrich/
(2011). Supelco. With a B.S. degree from SUNY Platts-
(6) S. Deridder and G. Desmet, J. Chromatogr. A 1218, 46–56 (2011). burgh and a PhD in Analytical Chemistry from The
(7) K. Broeckhoven, D. Cabooter, and G. Desmet, J. Pharm. Anal. Pennsylvania State University, Dave spent the first
3, 313–323 (2013). decade of his career within the pharmaceutical
industry performing analytical method develop-
(8) G. Kahsay, K. Broeck hoven, E. Adams, G. Desmet, and D. ment using various forms of chromatography
Cabooter, Talanta 122, 122–129 (2014). and electrophoresis. During the past 15 years, working directly in the
(9) C.R. Aurand, H. Cramer, J. McKenzie, and D.S. Bell, LCGC chromatography industry, Dave has focused his efforts on the design,
North Am. 32(9), 704–717 (2014). development, and application of stationary phases for use in HPLC and
hyphenated techniques. In his current role at Supelco, Dr. Bell’s main
(10) D.S. Bell, LCGC North Am. 33(2), 90 –101 (2015). focus has been to research, publish, and present on the topic of molecu-
(11) M.D. Dolzan, D.A. Spudeit, Z.S. Breitbach, W.E. Barber, G.A. lar interactions that contribute to retention and selectivity in an array of
Micke, and D.W. Armstrong, J. Chromatogr. A 1365, 124–130 chromatographic processes. Direct correspondence to: dave.bell@sial.com
(2014).
(12) D.A. Spudeit, M.D. Dolzan, Z.S. Breitbach, W.E. Barber, G.A. Editor of Column Watch:
Micke, and D.W. Armstrong, J. Chromatogr. A 1363, 89–95 (2014).
(13) D.C. Patel, Z.S. Breitbach, M.F. Wahab, C.L. Barhate, and
Ronald E. Majors
is the editor of “Column Watch,” an analytical
D.W. Armstrong, Anal. Chem. in press (2015). consultant, and a member of LCGC’s editorial
(14) L.Y. Loo, L. Abadilla, M. Chitty, I. Rustamov, T. Tran, and advisory board. Direct correspondence about this
T. Farkas, “The Benef its of an Optimized and Robust High column to lcgcedit@lcgcmag.com
pH Stable Core-Shell Stationary Phase for the Analysis and
Purification of Basic Analytes,” Presented at Pittcon 2015, New
Orleans, Louisiana, 2015.
(15) W. Long, A.E . Mack, J. Link, and X. Wang, “Examining
Orthogonal Separations in Superficially Porous Particles: Maxi-
For more information on this topic,
mizing Resolution Through the Use of Bonding Chemistries
please visit www.chromatographyonline.com/column-column-watch
and New High pH Stable Columns,” Presented at Pittcon 2015,
New Orleans, Louisiana, 2015.
(16) S. Fekete, J. Schappler, J.-L. Veuthey, and D. Guillarme, Trends
Anal. Chem. 63, 2–13 (2014).
CHIRAL
(17) D.S. Bell, H.K. Brandes, and D. Wallworth, in Chromatography
Today 8(1), 4–8 (2015).
(18) S.A. Schuster, B.M. Wagner, B.E. Boyes, and J.J. Kirkland, J.
Chromatogr. A 1315, 118–126 (2013).
SCREENING
(19) S. Fekete, J.L. Veuthey, and D. Guillarme, J. Pharm. Biomed.
Anal. 69, 9–27 (2012).
(20) S. Fekete, R. Berky, J. Fekete, J.L. Veuthey, and D. Guillarme,
AND
J. Chromatogr. A 1236, 177–188 (2012).
(21) S. Fekete, S. Rudaz, J. Fekete, and D. Guillarme, J. Pharm. OPTIMIZATION SERVICES
Biomed. Anal. 70, 158–168 (2012).
(22) B.M. Wagner, S.A. Schuster, B.E. Boyes, and J.J. Kirkland, J. • FREE Screenings
Chromatogr. A 1264, 22–30 (2012).
• Over 90 % Hit Ratio
(23) J.J. K irk land, S.A. Schuster, B.M. Wagner, and B.E.Boyes,
• Quick Turnaround Time
“Tools to Improve Protein Separations,” Presented at Pittcon
• Scale-Up to Purification
2015, New Orleans, Louisiana, 2015.
(24) D. Guillarme, “The use of modern supercritical f luid chroma-
tography in pharmaceutical analysis,” Presented at Emerging
Separations Technologies, RSC Burlington House, Piccadilly
SUBMIT YOUR PROJECT TODAY IN < 5 MINUTES!
London, UK, 2015. WWW.PHENOMENEX.COM/CHIRALSCREENING
(25) A.G.G. Perrenoud, W.P. Farrell, C.M. Aurigemma, N.C. Auri-
gemma, S. Fekete, and D. Guillarme, J. Chromatogr. A 1360,
275–287 (2014).
(26) S. Delahaye, K. Broeckhoven, G. Desmet, and F. Lynen, Talanta
116, 1105–1112 (2013).
PA30110215_us_1
(27) A. Grand-Guillaume Perrenoud, J.-L. Veuthey, and D. Guil-

larme, Trends in Anal. Chem. 63, 44–54 (2014).
(28) E. Lesellier, J. Chromatogr. A 1228, 89–98 (2012).
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LC TROUBLESHOOTING
Internal Standard
Calibration Problems
I
Readers’ questions have recently had several email sample and the response (usually peak
regarding problems related inquiries from readers regarding area) is recorded. For calibration, a
to internal standard calibration issues for liquid chro- plot of X = concentration versus Y =
matography (LC) methods. I try to area is made, and the equation for
calibration of liquid
reply directly to the queries as quickly the regression line is used to deter-
chromatography methods as possible so that the readers send- mine the concentration of unknown
are addressed. ing the questions can get back up and samples. The same volume of the
running, and I collect the questions unknown sample is injected, and the
until there are enough on a particular area response is used to determine the
topic to share in LCGC. Last month concentration of the injected sample.
(1) we looked at one of these inqui- Corrections are then made for any
ries in a case study format. For this concentration changes during sample
month’s “LC Troubleshooting” dis- preparation, and the final sample con-
cussion, I will look at two additional centration is reported.
questions that center on issues relat- Internal standardization follows
ing to calibration using the internal a process that is similar to external
standard technique. standardization, except another com-
pound, the internal standard (IS) is
Over-Curve Samples with added at the same concentration in
Internal Standardization every sample and calibration standard
The first question comes from a early in the sample preparation pro-
reader who finds that occasional cess. For example, a 100-µL aliquot
samples occur at concentrations that of sample might be mixed with 10 µL
exceed the range of the calibration of IS, then processed. The calibration
curve. The question regards how to curve is constructed as a plot of X =
dilute the samples so that they can ratio of the concentration of analyte
be analyzed at concentrations within to concentration of internal standard
the calibration range. The problem versus Y = ratio of area of analyte
is complicated by the fact that the to IS. For application, the ratio of
method uses the internal standard analyte to IS area is determined for
method for calibration. unknowns and the equation for the
regression line then allows determina-
External Versus tion of sample concentration. The IS
Internal Standardization method is especially useful when the
Before we get into the problem itself, sample preparation process has many
let’s review the difference between steps or it is likely that volumetric
external standardization and internal losses might take place. This com-
standardization. With external stan- monly occurs with biological samples,
dardization, calibration samples (cali- such as plasma, where sample prepa-
brators) are made at concentrations ration may involve several transfer
covering the desired calibration range, steps, evaporation to dryness, recon-
such as 1, 2, 5, 10, 20, 50, 100, 500, stitution in a new solvent, and so
John W. Dolan and 1000 ng/mL. A fixed volume (for forth. Any physical losses in sample
LC Troubleshooting Editor example, 10 µL) is injected for each are compensated for by tracking
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the ratio of the analyte to IS, rather over-curve samples could be diluted up to 10-fold was permissible for over-
than the absolute area of the analyte, by twofold (or 10-fold, or whatever curve samples.
because the analyte–IS ratio should had been demonstrated) to obtain
stay the same, even with sample vol- accurate results. Documentation is Vital
ume changes. The internal standard method Whenever you devise a method for
doesn’t deal as simply with over-curve diluting over-curve samples, whether
Over-Curve Samples samples. If we use the same example external or internal standardization is
Now, what happens if the analyte as for the external standardization used, you need to be careful to docu-
concentration is greater than the above, the problem is quickly appar- ment it properly. At least three steps
upper end of the calibration curve ent. Diluting the ~1500-ng/mL sam- need to be documented:
(we’ll refer to these as “over-curve” ple by a factor of two does not change • Demonstrate that the process
samples)? It is not a good analytical the analyte to IS ratio at all! That is, works, using blank matrix spiked
practice to extrapolate the calibration when the sample is diluted twofold, at the appropriate levels. This
curve above the upper end or below both the analyte and IS peaks halve should be part of your validation
the lower end of the actual calibra- in size, but the ratio doesn’t change report.
tor concentrations. This is because and therefore the sample would still • Include written instructions
it may not be a valid conclusion that be over-curve. This is the reason the on how to deal with over-curve
the analyte response is the same out- IS was added in the first place—to samples (“say what you’re going
side the test range as inside it. For compensate for unintended or uncon- to do”).
example, many detectors will show trolled sample loss or dilution—and • Record how you followed the
a nonlinear drop-off in response at now it seems to defeat us. We’ll have directions when samples were
high concentrations; at low concentra- to try a different approach. diluted (“do what you said you
tions, nonlinear changes in response One simple way to handle the over- were going to do”).
may be seen because of absorptive curve problem with the IS method is to One last item needs to be docu-
losses or other factors. Unfortunately, dilute the sample before adding the IS. mented, which is how to deal with
such changes in response seldom are For example, if an over-curve sample multiple analyses of the same sample,
predictable, so the risk of reporting is found or is anticipated, the sample because usually you don’t want to
a faulty analysis result is large if the could be diluted twofold with blank report a value that is known to be
concentration lies outside the calibra- matrix before adding IS. Alternatively, or could potentially be wrong. The
tion range. twice the concentration of IS could be acceptable procedure is usually
For external standardization, the added to the undiluted sample. Either recorded as part of a standard operat-
solution to over-curve samples is technique would effectively dilute the ing procedure (SOP) or as part of the
quite simple—just dilute the sample analyte-to-IS ratio twofold and bring method document. For example, if
until its concentration is within the it back into the calibration range for the initial analysis of the sample gave
calibration range. For example, let’s the ~1500-ng/mL example used here. over-curve results and the sample was
consider a calibration curve that cov- As with the external standard method, diluted 10-fold and reanalyzed, the
ers 1–1000 ng/mL, as in the example the effectiveness of sample dilution data table might include two entries.
above, and a sample that is estimated would have to be demonstrated as The one for the initial analysis might
to contain ~1500 ng/mL based on an part of the validation process, and the list “o/c” (over-curve) instead of an
extrapolated curve. We could dilute analytical method would need to be assay value and the reanalysis might
this sample by a factor of two with written to allow this procedure. For include the assayed value with a
injection solvent and reinject the example, when our laboratory was footnote that the sample was diluted
sample. If it now assays as 725 ng/ analyzing plasma samples using inter- before analysis. This documentation
mL, we would correct for the dilu- nal standardization, for validation we acknowledges that the sample was
tion factor and report 1450 ng/mL as often would prepare plasma spiked at analyzed twice and that the over-
the sample concentration. Of course, five and 10 times the concentration of curve result should be ignored.
this could not be done without proper approximately 80% of the upper point
supporting evidence that dilution on the calibration curve. These samples Practical Application
was an acceptable solution to such were frozen to mimic normal sample Finally, how would you implement
problems. You could show this dur- handling, then thawed and diluted dilution of IS-calibrated samples on
ing the validation process by prepar- five- or 10-fold with blank plasma a practical basis? If you are analyz-
ing test samples at known over-curve and treated as normal samples. If the ing random-concentration samples
concentrations, then diluting them diluted samples then gave acceptable and the over-curve problem occurs
after sample preparation and show- assay results (following correction for only occasionally, such as ≤10% of
ing that you obtained the appropriate dilution), we had demonstrated that the time, it may be most efficient
results. This information would be dilution was a valid way to analyze to analyze all the samples normally.
included in the validation report and such over-curve samples. Our methods The over-curve samples could then
the method would be written so that were then written so that any dilution be diluted and reanalyzed with the

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next batch of samples. If, on the other assume a known volume. Because samples, calibration standards, and
hand, over-curve samples were com- the sample comes to the laboratory control samples would all have to be
mon half the time, for example, it already in a container, we must assume treated in the same manner, and the
might be worthwhile to prepare every that adsorption on the container has IS calibration technique should allow
sample in the normal manner and already occurred. This means that we acceptable quantification of drug con-
in the diluted form. Your analytical need to determine the volume of the centrations. Of course, the success of
method would then describe how to original sample, because the adsorptive this procedure could not be assumed,
handle the results. For example, if the loss, and thus the amount of analyte but would require validation through
normal dilution samples are within desorbed with additives, will be related demonstration of acceptable recoveries
range, their values would be reported to the sample volume. from blank matrix spiked with known
and their diluted versions would be There are at least three ways to concentrations of drug.
ignored; the over-curve samples would determine the sample volume, and
instead report the diluted values and none is perfect. One could use a gradu- Conclusions
ignore the normal dilution ones. This, ated pipette to withdraw all the sample Internal standardization is the most
of course, would mean that every from the sample tube and measure it in common standardization technique
sample would have to be injected the pipette. This would work, but the in some laboratories, such as those
twice, adding time and expense to pipette would then need to be rinsed analyzing drugs in biological matrices.
the analysis process. Other analytical with surfactant to release any adsorbed For analyses that require little sample
strategies may be more appropriate. sample. Another option would be to manipulation, such as simple dissolu-
Economics, run times, and other fac- pour the sample from the original con- tion or dilution of samples before injec-
tors will help you decide which tech- tainer into a calibrated tube for further tion, external standardization often is
nique is best for your laboratory. sample pretreatment. This would add preferred. Most of us tend to work in a
Other alternatives to avoid the the cost of a calibrated tube to the sam- laboratory where one or the other stan-
problem of over-curve samples may be ple pretreatment. Still another option dardization technique is used almost
possible. If the detector is sufficiently would be to pour the sample into a exclusively. If this is your situation, be
sensitive at low concentrations, it may tared tube and determine the volume especially careful if you switch stan-
be better to dilute all samples and, if by weight or pour it into an empty dardization techniques, because prac-
necessary, extend the lower end of the tube and weigh the original container tices that aren’t problematic with one
calibration curve so that over-curve to determine the volume technique may create problems when
samples are effectively eliminated. Or After the sample volume had been the other calibration scheme is used.
if the detector has acceptable response determined, the surfactant-additive
characteristics above the range of the mixture could be added. I would favor References
calibration curve, extend the calibra- spiking this mixture with the internal (1) J.W. Dolan, LCGC North Am. 33(5), 312–
tion curve to sufficiently high con- standard, then adding an aliquot in 316 (2015).
centrations so that over-curve samples proportion to the measured sample (2) United States Food and Drug Administra-
are rarely, if ever, encountered. volume. The walls of the original con- tion, Guidance for Industry: Bioanalytical
tainer (as well as any pipettes or other Method Validation (FDA, Rockville, Mary-
Accounting for Dilution surfaces that contacted the sample) land, 2001).
with Internal Standardization would need to be rinsed with this IS
I had a related question from a reader mixture and then transferred to the John W. Dolan
“LC Troubleshooting”
who is analyzing drugs in urine or holding tube to remove any adsorbed Editor John Dolan has
cerebral spinal fluid (CSF) as a matrix analyte from its walls and to mix with been writing “LC Trou-
using an internal standardization the sample. This step would surely bleshooting” for LCGC
for more than 30 years.
scheme. The problem with the target add uncertainty to the measurements One of the industry’s
drugs is that they stick on container because of the approximation of the most respected profes-
walls, pipette tips, and other surfaces. original volume. With bioanalytical sionals, John is currently
To counteract adsorption, surfactants samples such as these, however, the the Vice President of and a principal instruc-
tor for LC Resources in Lafayette, California.
and other additives are mixed into analysis guidelines from the United He is also a member of LCGC’s editorial advi-
study samples, calibration standards, States Food and Drug Administra- sory board. Direct correspondence about
and control samples. The question is tion (2) allow uncertainty (%RSD) this column via e-mail to
how to report the concentration of in precision and accuracy of ±15% John.Dolan@LCResources.com
drug in the original samples. at all levels above the lower limit of
This problem would be fairly simple quantification (LLOQ) and ±20% at
if the same volume of each sample the LLOQ. It is unlikely that these
existed, but I suspect that is not the additional transfer and measurement For more information on this topic, please
case. CSF is probably collected in steps would degrade the precision and visit www.chromatographyonline.com/
column-lc-troubleshooting
a syringe and an arbitrary volume accuracy beyond the limits if the rest of
of urine is obtained, so we cannot the method is performing well. Study
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402 LCGC NORTH AMERICA VOLUME 33 NUMBER 6 June 2015 www.chromatographyonline.com
PERSPECTIVES IN MODERN HPLC
Separation Science in Drug

Development, Part I: High-
This installment provides
an overview of modern
Throughput Purification
practices of high-through-
put purification to support
small-molecule drug dis-
covery. It describes the use
of reversed-phase liquid
D rug development is a highly
complex, expensive, and multi-
disciplinary process (1,2). The
modern molecular approach to new drug
development generally starts with an
fication and analysis (characterization);
and compound management for weighing,
creating stock solutions, and storing the
compounds for future testing. Assays that
are performed by multiple groups include
chromatography (LC) and understanding of the pathophysiology of physicochemical property screening, bio-
supercritical fluid chroma- a disease and a molecular target, followed logical testing, and drug metabolism and
tography (SFC) for purify- by the synthesis of molecules that bind to pharmacokinetics. Assay results are used
ing diverse samples in a the target (hits and leads), which are sub- to generate the next hypothesis–com-
sequently optimized by medicinal chem- pound design cycle in an iterative fashion.
centralized laboratory set-
ists (3) into a development candidate. The In this installment, we describe the
ting. A case study is used to drug candidate is then scaled up by pro- modern practice and recent advances
illustrate the principles and cess chemists, characterized by analytical in the high-throughput purification of
rationales for selecting oper- chemists, and formulated by pharmaceuti- compounds of interest (COI) in this
ating parameters for these cal scientists. If safety and efficacy profiles drug discovery cycle. The goal of a high-
applications. This is the first of the drug candidate are acceptable, it throughput purification laboratory is to
may eventually become a new commercial provide purified materials in sufficient
installment of four articles
drug product after a stringent regulatory quantity and quality (chemical and chiral
on “Separation Science approval process. Analytical chemistry, purity) to support candidate characteriza-
in Drug Development” to particularly the separation science, plays tion and further biochemical assays. The
describe the modern prac- an important role throughout this long next installment will focus on the high-
tice of separation science in and arduous process (4,5). throughput characterization process to
supporting small-molecule Because the lifetimes of patents for new generate quality control and analytical
drug molecules are relatively short (for data for internal submissions to “com-
drug discovery and develop-
example, 20 years in the United States), pound management.”
ment. The complete series the speed in selecting the “right” candi- Additional details of the typical pro-
will be published through- dates and the subsequent reduction in cesses and Genentech’s sample workflow
out the year in LCGC North “time to market” are important factors that in a small-molecule discovery chemistry
America. can impact the survival of the pharmaceu- organization are summarized in Figure
tical company. In early discovery, it is vital 2. The analytical chemistry functions of
to minimize the time between compound purification and analysis (shown in blue
design and assay results, which are sub- boxes) are often handled by a centralized
sequently used to drive the next round of analytical group, although many of these
Icon image: Joe Zugcic, Joe Zugcic Photography
compound design in an iterative process tasks can be done with either open-access
called the “drug discovery cycle” (shown instrumentation or can be outsourced to
in Figure 1). external contract research organizations
Mengling Wong, Brent
In the drug discovery cycle, medicinal (CROs). A compound management group
Murphy, and Joseph H.
and computational chemists are typically is often responsible for archiving the entire
Pease are the guest coauthors
involved in hypothesis generation and portfolio of compounds and distributing
of this installment. Michael
compound design. Medicinal chemists are them for testing, typically as stock solu-
W. Dong is the editor of
Perspectives in Modern HPLC and responsible for the synthesis of test com- tions (for example, 10 mM in dimethyl-
a coauthor of this installment. pounds; analytical chemists for their puri- sulfoxide [DMSO]).
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Post-registration
Design Synthesis
Pre-registration
QC of DMSO Physicochemical properties
Open access stock solutions pKa
LogP
LogD
Structure
Kinetic solubility
elucidation
Thermodynamic solubility
Purifcation Crystal form
Assay and Purifcation Compound
analysis management
Compound testing
Registration QC
In vivo PK
Compound In vitro ADME
management Formulation
Registration Biopharmacology
Figure 1: The drug discovery cycle. CRO and Commercial HT screening
compounds
A challenge in a centralized analytical Figure 2: Sample workflow and processes in a small molecule discovery chemistry
laboratory such as ours is the diversity organization. The light blue boxes represent the functions likely to be performed by
a centralized analytical chemistry group.
of compounds received for purification.
Although most samples are drug leads or
active pharmaceutical ingredients, they
can also be synthetic starting materials,
intermediates, small polar fragments (<150 Chemist sample submission in
molecular weight [MW]), or linkers used electronic lab notebook
with antibody–drug conjugates (>1500
MW). Another challenge is that many
Initial screenings by reversed-phase
compounds have one or more chiral cen- UHPLC–UV using a broad gradient with 1
ters, which complicate the purification high and low pH
strategy as high chiral purities are prereq-

Sample recovery:
uisites for ensuring the accuracy of assay Reversed-phase HPLC 2 centrifugal evaporation,
purifcation using a Triage lyophilization,
results. Samples are submitted to the puri- narrow gradient range
rotary evaporation
fication laboratory as crude mixtures in
vials, flasks, or multiwell plates from par- QC collected fractions 3
allel chemistry efforts. The goal is to gen-
erate a pure powder of sufficient quality
(typically >90% pure with no single impu- Sample recovery:
rity >5%) for compound management.
“Genevac”, lyophilization, 4
rotary evaporator
Having an analytical chemistry group in
close proximity to medicinal chemists is
an important benefit for shortening the HT characterization:
purity and structure
drug discovery cycle through quick com- identifcation
munication and sample transfers.
Compound registration
High-Throughput Purification
Strategy and Workflow
Figure 3: The high-throughput purification workflow in a centralized analytical
Figure 3 shows a schematic diagram of laboratory supporting discovery chemistry in a pharmaceutical company. The four
the purification workflow used in our typical separations steps in the workflow are as follows: initial reversed-phase LC–m
laboratory that may exemplify processes –MS screen; reversed-phase LC or SFC preparative purification; quality control of
used in other centralized groups (6). Four collected fractions; and sample recovery.
major steps are used: initial screening;
purification by reversed-phase liquid nal sample and the amount of purified ally preferred because of its capacity for
chromatography (LC) or supercritical material needed. Many samples from handling compounds of a wide polarity
fluid chromatography (SFC); quality medicinal chemists are crude mixtures range (5). If separation by reversed-phase
control (QC) of collected fractions; and containing starting materials, by-products, LC is not successful, then achiral SFC
sample recovery. Achiral compounds are and residual catalysts, which can interfere may be the next screening step (7). In gen-
separated by reversed-phase LC or SFC with the isolation process. Removing these eral, SFC is preferred as the final purifica-
depending on the purity of the origi- impurities by reversed-phase LC is gener- tion step when larger amounts of COI are
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www.chromatographyonline.com June 2015 LCGC NORTH AMERICA VOLUME 33 NUMBER 6 405
required (such as >300 mg) or for those (8–10). Using either of these mobile- sub-2-μm particles (when available), with
that are unstable or insoluble in the pres- phase conditions, we usually are able to a flow rate of 1.5 mL/ min, a run time
ence of water. obtain baseline resolution for most sam- of 1.5 min and a 5–60% B gradient. For
ples. Trifluoroacetic acid, triethylamine, chiral SFC, we screen six 50 mm × 4.6
Rapid Initial Screening and the ammonium salts of formate, mm columns packed with 3-μm particles,
Using Reversed-Phase LC acetate, or carbonate are less desirable for with a flow rate of 4 mL/min, a run time
All purifications in our laboratory start volatility and recovery reasons (5,8). An of 2.5 min, and a 5–60% B gradient.
with an initial ultrahigh-pressure LC example of the initial screening step is We selected a combination of screening
(UHPLC) screening step. Exceptions are shown later in the case study. phases that appears to work well for our
COI that are unstable in water or single type of compounds based on an internal
compounds requiring immediate chiral Rapid Achiral and study (14). This method development
purity assessment where SFC is preferred. Chiral Screening Using SFC process usually allows scale-up to pre-
We typically screen using reversed-phase For achiral SFC separations, normal phase parative SFC using isocratic or gradient
LC–ultraviolet (UV)–mass spectrometry columns packed with silica, cyano, diol, conditions. An example of this screening
(MS) with fast, broad gradients using a and 2-ethylpyridine bonded phases are process is discussed later in the case study.
short sub-2-μm C18 column with both the most popular, though reversed-phase
acidic and basic mobile phases as the columns such as C18 have found some Preparative Reversed-Phase LC
weak solvents (mobile-phase A) (4,5). success (11,12). For chiral separations, The goal of preparative chromatography,
These 2-min screening methods allow columns packed with derivatized polysac- whether by LC or SFC, is to obtain puri-
quick assessment of sample purity, peak charides (coated or immobilized) on silica fied materials of sufficient quality (purity,
confirmation (MS), and subsequent support or Pirkle-type chiral stationary form) and quantity (yield) in a reason-
selection of optimal preparative reversed- phases are the most common (4,12,13). able time (for example, 10–20 min).
phase LC parameters. Our default Our approach to achiral and chiral Contrary to the predominance of flash
mobile-phase additives are 0.1% formic SFC method development is to screen the chromatography by normal-phase LC in
acid and 0.1% ammonia for high MS compounds rapidly using short gradients. organic chemistry laboratories, reversed-
sensitivity and low volatility for ease of For achiral SFC, we typically screen six phase LC is commonly used in our labo-
elimination in the sample recovery step 50 mm × 3.0 mm columns packed with ratory because of its higher reproducibility
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Table I: Comparison of techniques used in high-throughput purification

Reversed-Phase LC SFC Flash
Primary purpose • Initial screen and method • Chiral purification • Preworkup before submission
development • Achiral purification if reversed-phase LC to purification laboratory
• Purification of crude attempts fail
samples • Large quantity >300 mg because of ease
of recovery
Types of samples • “Dirty” samples with many • Chiral separation • Starting materials, intermedi-
components • Achiral separation of closely-eluting ates, or very crude mixtures
impurity
Pros • Versatile with high resolu- • Speed • Loading capacity is high
tion and selectivity • Chiral method development • Dry or wet load possible
• Automated for multiple • Less solvent usage, less waste • Low equipment cost and ease
sample purification of use
Cons • Difficult to remove mobile • Compounds insoluble in carbon dioxide • Toxic solvents
phase lead to precipitation at the injector • Low resolution and
• Not applicable to com- • Higher initial investment reproducibility
pounds that are unstable
in water with additives
Instrumentation • HPLC and UHPLC: • SFC analytical and prep with PDA, MS, • Low or medium pressure sys-
Preparative LC with PDA, ELSD, or polarimeter detection tem with on-line UV detection
MS, or ELSD
Typical analytical and • C18 • Achiral: Pyridyl amide, 2-ethylpyridine • Silica semipreparative columns
preparative phases • Chiral: Polysaccharide-based or cartridges packed with
larger particles
Typical mobile phases 0.1% formic acid or ammo- 0.1% ammonia or formic acid in metha- Heptane with alcohols or chlo-
nia in water with acetoni- nol, ethanol, isopropanol, or acetonitrile, roform
trile or methanol carbon dioxide
and resolution, MS-compatibility, and of the preparative equipment at hand. Col- fraction collection are UV, MS, (termed
high likelihood for recovery of desired umn lengths tend to range from 50 to 100 UV-directed or MS-directed purification,
materials in a solid powder form (4–6). mm for speedy separations, although longer respectively), and evaporative light scatter-
Preparative normal-phase LC is mostly column lengths may be needed to provide ing detection (ELSD). While UV-directed
supplanted by the “greener” SFC in cen- higher resolution. Common particle sizes systems are simple and less expensive, they
tralized laboratories because of the higher used are 5 and 10 μm for cost and pressure are limited to chromophoric compounds.
speed and faster sample recovery (7,13). considerations. Mobile phases (water and MS-directed systems are versatile and
Table I summarizes comparative charac- acetonitrile [or methanol]) with additives preferred for purification of compound
teristics and parameters used in reversed- (0.1% formic acid or ammonia) are similar library collections. ELSD-directed collec-
phase LC, SFC, and normal-phase LC. to those used in initial screening. Flow tions are typically used for purification
Since concepts and practices of prepara- rates are geometrically scaled to preparative of natural products or nonchromophoric
tive LC and SFC can be found in many column diameters (5). We typically use products. In our laboratory, we use both
books (6,7,15) and articles (16–18) in narrower gradient ranges to increase the UV- and MS-directed purification sys-
great detail, we will only summarize resolution around the COI. For instance, tems with automated fraction collection
those for high-throughput purification four standard ranges are typically used and subsequent QC. Fractions containing
applications. in our laboratory (5–50% B, 20–60% B, the COI are then recombined for sample
Columns packed with octadecylsilane 30–70% B, or 40–80% B) depending on recovery. This semiautomatic approach
(C18) bonded phase remain the universal the hydrophobicity of the COI (19), which allows us more flexibility to handle a vari-
columns of choice in reversed-phase LC can be estimated from initial screens. ety of complex samples and amounts.
because of their reliability and sample Diluents of strong solubilizing power
capacity (5). Newer packing materials such such as dimethylformamide or DMSO Preparative SFC
as hybrids (for example, Waters BEH or are typically used to dissolve the sample. The last decade witnessed significant
Phenomenex Gemini-NX) are popular Low-solubility compounds may require improvements in the reliability and per-
because of their compatibility with high- larger injection volumes or multiple injec- formance of SFC instrumentation. The
pH mobile phases, allowing improved tions. Sample concentrations are kept benefits of higher speed, resolution, and
retention and better peak shapes for basic high when possible to minimize injec- easier sample recovery using environmen-
pharmaceuticals (5). Column inner diam- tion volumes or the number of injections tally friendly solvents (carbon dioxide)
eters (21.2–30 mm i.d) are dictated by (15). Common detection modes where are well publicized and recognized (7,18).
sample amounts and the flow rate ranges signals can be used to trigger automated The principles, practices, and applications
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Affinity - Empty - ERLIC - Hydrophilic - IEX - RPC - SEC
Product Sample Packed Void Elution

Formats Capacity Volume Volume Volume
UltraMicroSpin™ Column 3-30µg 25µL 12µL 2-25µL
MicroSpin™/TIP Column 5-60µg 50µL 25µL 5-50µL
MacroSpin™ Column 30-300µg 180µL 90µL 25-180µL
96-Well Spin Plate 10-100µg 75µL 35µL 7-75µL
96-Well MACROspin Plate 25-250µg 200µL 100µL 25-200µL
Page*Eraser™ µFilter Tips 2-200µL 0.1µL
VOLUME EFFECTS ON SPE TIPS DISPLACEMENT CHROMATOGRAPHY VS.

Desorption Increases From Excessive Sample & Wash Volumes SELECTIVE ELUTION
VS.
MASS RELATIVE TO COLUMN SIZE MASS OVERLOADED SPE CARTRIDGE

Excess capacity; everything retained. Capacity limited; the most strongly-retained
Separation through selective elution. solutes displace those less strongly-retained.
Results in a separation with selective reten-
tion, displacement chromatography.
EXAMPLE: ERLIC-CAT chromatography
Column: TARGA® C18, 300µm x 30mm Trap

Sample Solvent: 0.1% FA water (volumes as shown). Trap Column in 0.1% FA water
Gradient: 98% water, 0.1% FA, 2% ACN to ACN, 0.1% FA
The Nest Group, Inc.
Not Just Columns
Not Just ... Answers!
Columns ... Answers!
45 Valley Road, Southborough, MA 01772 USA
TARGA® is a registered trademark of Higgins Analytical, Inc.
E-mail: sales@nestgrp.biz
UltraMicroSpin™, MicroSpin™, MACROspin™ are trademarks of Harvard Apparatus, Inc. For more Info: www.nestgrp.com/minispin.shtml
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Phospho-Peptide and Glyco-Peptide
micro-SPE Isolation and Desalting
Enhance Selectivity - Eliminate Sample Losses
ERLIC-WAX ISOLATION DISPLACEMENT CHROMATOGRAPHY VS.

MULTI PHOSPHORYLATED PEPTIDES SELECTIVE ELUTION
VS.
MASS RELATIVE TO COLUMN SIZE MASS OVERLOADED SPE CARTRIDGE

Excess capacity; everything retained. Capacity limited; the most strongly-retained
Separation through selective elution. solutes displace those less strongly-retained.
Results in a separation with selective reten-
tion, displacement chromatography.
EXAMPLE: ERLIC-CAT chromatography
Sample: Tryptic Digest

Column: ERLIC-WAX microSPE p/n: SEM HIL-DE
Load in 85% ACN, 0.1% FA. Elute 10% ACN pH 2, 100mM Na-methylphosphonate
Desalt on MACROspin TARGA ® C18 p/n: SMM SS18R
Analytical column: PolyWAX LP™ 4.6 x 100mm, 5µm, 300Å, p/n: P104WX0503
VOLUME EFFECTS ON SPE TIPS

Desorption Increases From Excessive Sample & Wash Volumes
A CHEMISTRY FOR EVERY COMPOUND

Affinity - Empty - ERLIC - Hydrophilic - IEX - RPC - SEC
Product Sample Packed Void Elution

Formats Capacity Volume Volume Volume
UltraMicroSpin™ Column 3-30µg 25µL 12µL 2-25µL
MicroSpin™/TIP Column 5-60µg 50µL 25µL 5-50µL
MacroSpin™ Column 30-300µg 180µL 90µL 25-180µL
96-Well Spin Plate 10-100µg 75µL 35µL 7-75µL
96-Well MACROspin Plate 25-250µg 200µL 100µL 25-200µL
Page*Eraser™ µFilter Tips 2-200µL 0.1µL
Column: TARGA® C18, 300µm x 30mm Trap

Sample Solvent: 0.1% FA water (volumes as shown). Trap Column in 0.1% FA water
Gradient: 98% water, 0.1% FA, 2% ACN to ACN, 0.1% FA
The Nest Group, Inc.

Not Just Columns ... Answers!
45 Valley Road, Southborough, MA 01772 USA
E-mail: sales@nestgrp.biz
TARGA® is a registered trademark of Higgins Analytical, Inc. For more Info: www.nestgrp.com/minispin.shtml
PolyWAX LP™ is a trademark of PolyLC, Inc.
UltraMicroSpin™, MicroSpin™, MACROspin™ are trademarks of Harvard Apparatus, Inc.
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weak mobile phase with increasing con-

centration of cosolvents such as methanol,
N W ethanol, or isopropanol. The cosolvents
1 1.12 1.19
5.0e-2
0.03
B,P1.03 are spiked with acidic or basic additives to
0.0
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 keep the analytes in non-ionized forms to
2 W 0.53 0.55N P 1.21 B
prevent sample adsorption on these polar
5.0e-1
1.43
0.0 stationary phases (4,13). Our laboratory
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
B pioneered the use of ammonia in place
3
Absorbance (AU)
1.43
2.0e-2
0.21
of triethylamine or diethylamine in SFC
0.0
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 (8), which yields similar separations and
5.0e-2
4 P 1.21 allows easier recovery of the COI as a free
0.0 base. Earlier concerns of reduced lifetime
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
of columns have not been observed.
5.0e-1 5 0.56 N
Sample precipitation in the injector,
0.0
which can cause equipment failure, is
6 0.53 W a real issue in preparative SFC. While
2.0e-1
0.0 a strong diluent such as DMSO can be
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
used for achiral SFC, we prefer to use
Time (min)
methanol as the diluent to match the
Figure 4: Chromatograms showing the results of step 1 of initial sample screening of
mobile phase most often used in SFC.
the case study sample. The sample consisted of bupivacaine (B), propranolol (P, COI), Sample concentrations are scaled back
naproxen (N), and warfarin (W), which were subjected to UHPLC–UV–MS screening 2–3-fold from those used in reversed-
using either acidic (chromatogram 1) or basic (chromatogram 2) mobile phases, A, phase LC to minimize potential prob-
with an acetonitrile gradient, B. UHPLC column: 30 mm × 2.1 mm, 1.7-µm Waters
Acquity BEH C18 (Waters) at 35 °C; mobile-phase A: 0.1% (v/v) formic acid or 0.1%
lems. Yields and sample throughput are
(v/v) ammonium hydroxide in water; mobile-phase B: acetonitrile; linear gradient: often increased with “stacked” injections
5–100% B in 1.5 min; flow rate: 0.8 mL/min; detection: UV absorbance at 254 nm; whereby injections are stacked to maxi-
temperature: 35 °C; injection volume: 1 μL at 0.1 mg/mL. The use of 0.1% formic acid mize efficiency (an injection mode in
mobile phase shows poor resolution of the four components, chromatogram 1. The
use of 0.1% ammonium hydroxide mobile phase shows excellent resolution of all four
which the next sample is injected before
components, chromatogram 2. Individual standards of bupivacaine, propranolol, the complete elution of the current sam-
naproxen, and warfarin analyzed under basic mobile phase conditions are shown in ple). Achiral and chiral method develop-
chromatograms 3–6, respectively. ment strategies in SFC and column selec-
tion are published elsewhere (21–23).
Post-Purification
80,000 QC Using UHPLC–UV–MS
75,000 Solvent
70,000 peak N W P In our laboratory, post purification QC
65,000
60,000
by UHPLC of the collected fractions is
Absorbance (AU)
55,000 performed concurrently during purifica-

50,000
45,000 tion or after the purification is complete.
40,000
35,000 Additional analysis of the fractions by
30,000
25,000
UHPLC under opposite pH mobile
20,000 phase conditions provides an orthogo-
15,000
10,000 B nal purity assessment and decreases the
5,000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617 1819 20
chances of missing coeluted impurities.
0 0.5 1 1.5 2 2.5 3 4.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
A more detailed description of high-
Time (min)
throughput structure confirmation and
quantitation will be discussed in the next
Figure 5: Reversed-phase LC–UV preparative chromatogram showing step 2a
purification of the sample to isolate the COI (propranolol) from other components. installment. Results from these quick
HPLC column: 50 mm × 30.0 mm, 5-µm Phenomenex Gemini-NX; mobile-phase A: QC analyses of the fractions collected
water with 0.1% ammonium hydroxide; mobile-phase B: acetonitrile; linear gradient: allow for a rational combination of all
2–80% B in 10 min; flow rate: 60 mL/min; detection: UV absorbance at 220 nm;
COI-containing fractions into one con-
temperature: ambient; injection volume: 1.4 mL containing the entire sample of 140 mg
in dimethylformamide. Fraction collection time points triggered by the UV signal are tainer for improving sample recovery and
labeled with vertical dashed lines: 0.5–1.25 min, the solvent peak (dimethylformamide); optimizing yield or purity.
2.5–3 min naproxen and warfarin; 6.4–7.2 min, propranolol; 7.7–7.9 min, bupivacaine.
Fractions 7–8 and 13–15 were pooled and analyzed. The isolated COI, propranolol, was
Sample Recovery
subjected to further SFC chiral purification to obtain pure enantiomers.
A vital step in purification of a COI is
of SFC are available in numerous books SFC operates in the normal-phase the removal of all solvents and addi-
and articles (7,17,18,20). mode with carbon dioxide serving as the tives to yield a pure powder that is
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exposure to organic solvents, if present in

large quantities. Rotary evaporators are
0.53
0.55
efficient for removing organic and aque-
6.0e-2 1.21
4.0e-2
Original sample P ous solvents, but do not usually lead to
W
2.0e-2 N
1.43
B dry powders. We use rotary evaporators
0.0 to reduce large volumes of solvents prior
Absorbance (AU)
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
0.56 to the final drying steps in a centrifugal
0.53
2.0 W
N evaporator or lyophilizer.
1.0
Fractions 7-9
0.0 A Case Study Illustrating
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
1.23 the Purification Strategy:
1.5
P Isolation of R- and S-Propranolol
1.0 Fractions 13–15
5.0e-1
To illustrate the workflow in high-
0.0 throughput purification as illustrated in
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Figure 3, we present here a case study
Time (min)
on the isolation of R- and S-propranolol
from a drug mixture. The sample was a
Figure 6: Chromatogram showing step 3a of the workflow in the analysis of
collected and pooled fractions using UHPLC (reversed-phase LC–UV–MS) with the mixture of commercial drugs consisting
initial screening conditions of Figure 4, chromatogram 2. UV detection was set to of 50 mg of racemic propranolol, 20 mg
220 nm and an injection volume of 1 μL was used. Chromatograms: upper, original of bupivacaine HCl, 20 mg of naproxen,
sample; middle, fractions 7–9; and lower, fractions 13–15 (containing the racemic COI,
and 50 mg of warfarin. This sample was
R- and S-propranolol).
used to simulate a more complex purifica-
tion scenario requiring a reversed-phase
LC cleanup step before chiral SFC to yield
0.05
purified enantiomers. The sample was dis-
0.00
AD solved in 1.4 mL of dimethylformamide
at 100 mg/mL. This sample solution was
0.02
0.00 Cel-1 further diluted 1000:1 in methanol to
Absorbance (AU)
-0.02
0.02 ~0.1 mg/mL for initial screening.
0.00 Cel-4
-0.02
0.05 Step 1: Initial
0.00
ID UHPLC–UV–MS Screening
0.50
The initial screening of the sample was
Whelk O-1 performed under typical high-through-
0.00
0.05
put screening UHPLC conditions with
AS
0.00 a broad gradient using a 30 mm × 2.1
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
mm i.d. sub-2-μm C18 column. Both
Time (min)
acidic and basic mobile phases A were
used consecutively in separate gradient
Figure 7: Chromatograms showing the chiral column screening for separating
R- and S-propranolol using an automated chiral SFC analytical screening system separations using a standard protocol
(Waters Acquity UPC 2). SFC conditions: columns: Chiralpak AD, Lux Cellulose-1, Lux as shown in Figure 4 (first and second
Cellulose-4, Chiralpak ID, Regis Whelk-O 1 (S,S), Chiralpak AS; all columns were 50 chromatograms). Note that basic mobile
mm × 4.6 mm, 3-µm particle size except for Whelk-O 1, which has a particle size of phase A yielded an acceptable separation
3.5 µm; column temperature: 40 °C; mobile-phase A: carbon dioxide; mobile-phase
B: 0.1% ammonium hydroxide in methanol; linear gradient: 5–60% B in 1.8 min; run of all four components. Under high-pH
time: 2.5 min; flow rate: 4 mL/min; back-pressure regulator: set to 120 bar; detection: conditions, the acidic naproxen and
UV absorbance at 220 nm; injection volume: 1 µL of 0.1 mg/mL solution. warfarin are ionized and have lower
retention, while the basic propranolol
amenable to weighing, distribution, Centrifugal evaporation systems are and bupivacaine are un-ionized and are
and long-term storage. Solvent removal popular because of their efficiency, pro- strongly retained (5). The initial screens
can be achieved by centrifugal evapo- grammability, and the convenience of provided quick assessments of sample
ration (that is, Genevac), lyophiliza- using the same vials from fraction collec- purity and impurity profile, which
tion (freeze-drying) (24,25), or rotary tion. These type of systems should not is useful information for devising a
evaporation. Occasionally, a residual be used for thermally labile or semivola- purification strategy. Results indicated
oil instead of a solid is obtained and tile compounds. Freeze dryers are best the need for a two-stage purification
must be submitted as such. Newer suited for removing aqueous solvents by process: reversed-phase LC to isolate
evaporation approaches using trapping sublimation at room temperature. The the COI from other components and a
columns may be able to address this downsides are longer evaporation time chiral SFC purification step to obtain
issue (26). and potential equipment damage from purified enantiomers.
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Step 2a: Preparative the appropriate fractions using UHPLC Preparative chiral SFC-UV purifica-
Reversed-Phase LC Purification conditions similar to those used in the tion was performed on a PIC-100 SFC
A preparative reversed-phase LC–UV initial screening (Figure 6). If the purity system using a 150 mm × 21.2 mm,
isolation step was used to isolate the and identity (%area at 220 nm and MS) 5-μm Phenomenex Lux Cellulose-1 col-
COI from the sample mixture. Figure were deemed acceptable, the pooled frac- umn (Figure 8) under optimized iso-
5 shows the chromatogram and oper- tions were evaporated to yield materials cratic conditions. An initial exploratory
ating details of a single injection (1.4 for chiral SFC. A total recovery weight of injection was made to identify the elu-
mL) of the entire sample dissolved in the R- and S-propranolol from this step tion time and loadability of the sample.
dimethylformamide. Note that while was 44.7 mg after rotary evaporation of To increase throughput, stacked injec-
narrower gradient ranges are typically fractions 13–15 shown in Figure 5. tions were used with a 2-min cycle
used in this step to maximize the res- time, leading to a total run time of 25
olution around the COI (13), this was Step 2b: Chiral SFC Purification min. Fractions containing methanolic
not the case for this study because the Chiral method development using solutions of each purified enantiomer
sample contains components of widely an automated column-mobile phase were transferred to round-bottomed
ranging hydrophobicity. Fraction col- screening system was performed flasks and rotary evaporated to remove
lection was triggered by the UV signal. (Waters Acquity UPC2 SFC system). the 120 and 170 mL of methanol con-
The collected fractions of interest The isolated propranolol racemic sam- taining the respective stereoisomers.
were pooled and subsequently ana- ple was dissolved in 2 mL of methanol Each enantiomer was then transferred
lyzed (QC) in step 3a. The column (22.4 mg/mL) and then further diluted to a pretared vial using a 2:3 (v/v)
dimensions (50 mm × 30.0 mm) were to 0.1 mg/mL. The diluted sample was mixture of water and acetonitrile for
dictated by the purification amount screened on six chiral columns under subsequent sample recovery to obtain
(140 mg) with flow rate (60 mL/min) standard gradient conditions with the solid powder.
scaled to the column size. results shown in Figure 7. This screen
was completed in less than 30 min and Step 4: Sample Recovery
Step 3a: QC of Collected Fractions led to the selection of Phenomenex The vials were frozen on dry ice and
This reversed-phase LC–UV–MS QC Lux Cellulose-1 column for the purifi- placed in a Genevac HT-4 evaporator.
step was used to confirm the identity of cation step. Lyophilization or freeze drying is also
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We are an independent, international, and diverse team of employee-owners not bound
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2520
Open
2000
Absorbance (AU)
1500
1000
500
Close
-100
0 2 4 6 8 10 12 14 16 18 20 22 24 25
Time (min)
Figure 8: Preparative SFC chromatograms of the final step 2b purification showing a

sequence of six consecutive staggered injections of the racemic propranolol sample isolated
in the final step 2a purification to yield purified enantiomers on a PIC-100 system. 44.7 mg
of the isolated propranolol was dissolved in 1.5 mL of methanol diluent and a ”scout’’
injection of 0.2 mL was made to identify retention times and loadability. Injections (0.2 mL)
of the sample were made every 2 min under optimized isocratic conditions. Preparative
SFC conditions: column: 150 mm × 21.2 mm, 5-µm Phenomenex Lux Cellulose-1; mobile-
phase A: carbon dioxide; mobile-phase B: 0.1% ammonium hydroxide in methanol;
RSA™ Glass isocratic run: 25% B for 25 min; flow rate: 70 mL/min; back-pressure regulator: set to 100
bar; temperature: 40 °C; detection: UV absorbance at 220 nm.
Why RSA
AutoSampler Vials? 2.00
1.50
• Least Reactive Vial 1.00
0.50
R-Propanolol
OPEN
0.00
on the Market
Absorbance (AU)
• Low Basic & Polar 1.00

S-Propanolol
0.50
Analyte Adsorption 0.00
• Consistent Vial to Vial 0.15

0.10
(R,S)-Propanolol
Performance 0.05
0.00
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Why MicroSolv Time (min)
Technology Corp? Figure 9: Chromatograms of the final step 3b of QC or purity analysis of R- and
S-propranolol using a Waters UPC2 analytical SFC system. A 0.1-mg/mL solution of each
• Technical Expertise enantiomer was prepared by weighing out a sample and dissolving it in methanol. SFC
conditions: column: 50 mm × 4.6 mm, 3-µm Phenomenex Lux Cellulose-1; mobile-phase
• Method Development A: carbon dioxide; mobile-phase B: 0.1% ammonium hydroxide in methanol; isocratic
run: 20% B for 2.5 min; flow rate: 4 mL/min; back-pressure regulator: set to 120 bar;
• Industry Innovation detection: UV absorbance at 220 nm; temperature: 40 °C; injection volume: 2 µL. The top
two chromatograms result from analyses of the isolated pure propranolol enantiomers
and the bottom chromatogram is from analysis of the original propranolol racemate.
LCMS Recommended possible, although centrifugal evapora- of 20% methanol with 0.1% ammonium
tion is typically preferred for quicker hydroxide on a Phenomenex Lux Cellu-
drying. The recovery for each enan- lose-1 column (Figure 9). Isolated samples
tiomer was 20.1 mg and 22.1 mg for were identified as R- or S-propranolol
peaks 1 and 2, respectively. respectively by retention time matching
with genuine reference standards.
www.RSA-Glass.com Step 3b: Final SFC QC
The enantiomeric purity was confirmed The Overall Purification Process
using a Waters Acquity UPC2 system and The entire purification process took two
an isocratic method with a mobile phase days with an overall recovery of >80%
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yielding a purity of COI that exceeded immobilized polysaccharides, and new Conclusion
our general criterion of >95% for both SFC bonded phases in our examples. The In this installment, we discussed modern
chemical and chiral purity. The next emerging dominance of superficially technologies and processes used in high-
step is high-throughput characteriza- porous particles (SPP) is likely to impact throughput purification that support
tion, which will be described in the next high-throughput screening method devel- small-molecule drug discovery. We also
installment of this series. This case study opment and sample analysis in the analyti- described the workflow taking place in
describes our purification processes and cal scale (31), although SPPs are less likely a centralized group using rapid initial
serves to illustrate our parameter selection to be implemented in preparative-scale screening, reversed-phase LC or SFC
rationales using chromatographic prin- separations because of cost factors. Newer purification, quality control analysis of
ciples to balance sample throughput with and more effective columns for SFC and resulting fractions, and sample recovery.
quality, scale, and other requirements. chiral separations are also expected to A case study, in which two purified enan-
continue to be active research areas in the tiomers of propranolol from a drug mix-
Status and Trends in foreseeable future. ture were isolated and purified, illustrated
High-Throughput Purification
The emergence of SFC as a preferred
technology for chiral analysis and purifi-
cation is perhaps the most notable trend
(7,8,20,27). Further development of newer
instruments combining supercritical fluid
extraction (SFE) with SFC may prove use-
ful for on-line analysis of labile samples P
GRZQWR
extracted from complex matrices (28). Polymicro P
Two-dimensional (2D) systems combin- Technologies™
ing two orthogonal separation modes (for
Tight-Tolerance
example, reversed-phase LC-SFC) may
also offer quicker and more automated
VS Capillary
approaches for samples from complex Tubing
matrices (29,30).
The adaptation of automation in solid
and liquid handling improves the reli-
ability and productivity of the purification
process (16,17). Automation would impact
most in areas such as fraction pooling
and QC or weighing of powder samples,
particularly in laboratories with more pre-
dictable workflow in handling library puri-
fication. In our laboratory, we use a semi- THE WORLD LEADER IN
automated approach for specific processes
CAPILLARY TUBING WHEN
in the workflow (for example, screening,
chiral method development, analysis of TOLERANCES MATTER
collected COI, and drying) to maintain ,PSURYHÁRZFRQWUROGHOLYHU\ HDVLO\LQWHUIDFHVZLWKH[LVWLQJ
flexibility to handle diverse sample types SUHFLVLRQGHDGYROXPHUHSHDWDELOLW\ FRQQHFWRUWHFKQRORJ\
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The steps involved from sample isola- 'UDZQIURPKLJKSXULW\V\QWKHWLF ÁXLGGHOLYHU\UDWHVDQGGHFUHDVHG
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is labor-intensive, time consuming, and FRQVLVWHQWO\VXSHULRUSHUIRUPDQFH DVQDQRÁRZOLTXLGFKURPDWRJUDSK\
somewhat unpredictable at times. We DQGRSWLPDOÁRZFRQWUROZLWK,' DQGPDVVVSHFWURVFRS\$EOHWREH
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vacuum evaporation to be the most use-
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ful. New approaches (such as Shimadzu’s 7HFKQRORJLHV96FDSLOODU\WXELQJ
Crude2Pure system) with automated
evaporation from trapping resins may offer
another viable alternative (26).
Finally, new advances in column
technologies will continue to impact this molex.com/lcgc/polymicrotubing.html
application area. We have described the

routine use of sub-2-μm particles, hybrids,
magenta
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the principles and rationales used to (14) C. Hamman, M. Wong, I. Aliagas, D.F.
achieve the end goal of delivering materi- Ortwine, J. Pease, D.E. Schmidt, and J.
als of sufficient quality and quantity in a Victorino, J. Chromatogr A. 1305, 310–319
reasonable time span. (2013).
(15) D.A. Wellings, A Practical Handbook of Pre-
Acknowledgments parative HPLC (Elsevier, Oxford, United
Get to Know The authors would like to thank Chris

Hamman, Dr. Kelly Zhang, and Snahel
Kingdom, 2006).
(16) W. Goetzinger, X. Zhang, G. Bi, M. Towle,
Patel of Genentech; Dr. Szabolcs Fekete
Metrohm of the University of Geneva; William
D. Cherrak, and J.N. Kyranos, Int. J. Mass
Spec. 238, 153–162. (2004).
Farrell and Christine Aurigemma of (17) M. Liu, K. Chen, D. Christian, T. Fatima,
Pfizer; Larry Miller of Amgen; Professor N. Pissarnitski, E. Streckfuss, C. Zhang, L.
Larry Taylor of Virginia Tech; and Dr. Xia, S. Borges, Z. Shi, P. Vachal, J. Tata, and
Tom Waeghe of MAC-MOD Analytical J. Athanasopoulos, ACS Comb. Science 14,
for their editorial inputs and suggestions. 51–59 (2012).
Titration
(18) L.T. Taylor, J. Supercritical Fluids 47, 566–
References 573 (2009).
(1) Drug Discovery and Development: Technol- (19) M.W. Dong, LCGC North Am. 31(8), 612–
ogy in Transition, 2nd Edition, R.G. Hill 621 (2013).
and H.P. Rang, Eds. (Churchill Livingston, (20) Supercritical Fluid Methods and Protocols
Ion Chromatography Edinburgh, Scotland, 2012). (Methods in Biotechnology), J.R. William
(2) Drug Discovery: Practices, Processes, and Per- and A.R. Clifford, Eds. (Humana Press,
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(3) T. Nograd and D.F. Weaver, Medicinal Argoullon, Current Topics in Med. Chem.
Electrochemistry Chemistry: A Molecular and Biochemical 12, 1250–1263 (2012).
Approach, 3rd Edition (Oxford University (22) N. Hicks, V.S. Sharp, and J. Stafford,
Press, New York, New York, 2005). LCGC North Am . 31(8), 622 – 628,
(4) Handbook of Pharmaceutical Analysis by (2013).
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Spectroscopy (Elsevier/Academic Press, Amsterdam, Gibbons, J. Chromatogr. A 1218(22), 3629–
Netherlands, 2005). 36 (2011).
(5) M.W. Dong, Modern HPLC for Practicing (24) Freeze-Drying/Lyophilization of Pharmaceu-
Scientists (Wiley, Hoboken, New Jersey, tical and Biological Products, 3rd Edition, L.
2006), Chapters 2, 4, and 8. Rey and J.C. May, Eds. (CRC Press, Boca
(6) D.B. Kassel, in HPLC for Pharmaceutical Raton, Florida, 2010).
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Eds. (Wiley, Hoboken, New Jersey, 2007), pean J. Pharm. and Biopharm. 85, 162–169
pp. 535–575. (2013).
(7) Supercritical Fluid Chromatography: Advances (26) M.W. Dong, LCGC North Am. 32(4), 270–
Laboratory Process and Applications in Pharmaceutical Analysis, 279, (2014).
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(8) C. Hamman, D.E. Schmidt, M. Wong, and (28) T. Uchikata, A. Matsubara, E. Fukusaki,
M. Hayes, J. Chromatogr. A 1218, 7886– and T. Bamba, J. Chromatogr. A 1260,
Find out more at
7894 (2011). 69–75 (2012).
www.metrohmusa.com/technology
(9) Z. Wu, W. Gao, M.A. Phelps, D. Wu, D.D. (29) C.J. Welch, M. Biba, J.R. Gouker, G. Kath,
Miller, and J.T. Dalton, Anal. Chem. 76, P. Augustine, and P. Hosek, Chirality 19,
839–847 (2004). 184–189 (2007).
(10) M.C. Garcia, J. Chromatogr B. 825(2), 111– (30) M. Ventura, W. Farrell, C. Aurigemma, K.
123 (2005). Tivel, M. Greig, J. Wheatley, A. Yanovsky,
(11) M. Ashraf- Khorassani, L.T. Taylor, and E. K.E. Milgram, D. Dalesandro, R. DeGuz-
Seest, J. Chromatogr. A 1229, 237–248 (2012). man, P. Tran, L. Nguyen, L. Chung, O.
www.metrohm.com (12) T. Berger and B. Berger, LCGC North Am. Gron, and C.A. Koch, J. Chromatogr. A
28(5), 344–357 (2010). 1036, 7–13 (2004).
(13) Preparative Enantioselective Chromatography, (31) S. Fekete, D. Guilla rme, a nd M.W.
G. Cox, Ed. (Wiley-Blackwell, Hoboken, Dong, LCGC North Am. 32(6), 420 –
New Jersey, 2005). 433 (2014).
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Joseph H. Pease leads the Analytical Chemistry Things that look clean...
& Purification Department in Small Molecule
Discovery Chemistry at Genentech, Inc. He earned
a BS degree in Chemistry from U.C. Davis and a PhD
in Biophysical Chemistry from U. C. Berkeley. Joe
joined Syntex in 1990 as a research scientist special-
izing in NMR and structure-based drug discovery.
After Roche acquired Syntex in 1994 to form Roche
Bioscience, he stayed with the company until its closure in 2010 and
then joined Genentech in 2011. During his career, Dr. Pease has held
leadership positions in discovery research, preclinical development, and
informatics.
Brent Murphy has a BS in Biochemistry and

Molecular Biology from U. C. Santa Cruz. He
is a senior research associate in the Analytical
Chemistry & Purification department in Small
Molecule Discovery Chemistry at Genentech, Inc.
Over his 13 years in the laboratory, he has gained Things that are guaranteed clean.
experience with a variety of projects from purifi-
cation of natural products and peptides to chiral
separations of small molecules. Current interests include large-scale
preparative SFC and SFE. His prior job experience was with the Lokey
Group at U. C. Santa Cruz, Theravance, and Amgen.
Mengling Wong earned a BS in Biochemistry

from U. C. Davis. She is a senior research associate
in the Analytical Chemistry & Purification depart-
ment in Small Molecule Discovery Chemistry at
Genentech, Inc. Over her 14 years at Genentech, she
has acquired analytical and purification skills in the
areas of peptides and small molecules. Her interests
are in SFC and HPLC technologies and their applica-
tions in separating early discovery chiral and achiral molecules. Prior to
Genentech, she was at Keystone Division of Biosource International as
a laboratory supervisor in the synthesis and separations of oligonucle-
otides.
Michael W. Dong is a principal in MWD

Consulting focusing on training and consulting
services in HPLC, UHPLC, pharmaceutical analysis,
and drug quality. He was formerly Senior Scientist
at Genentech, Small Molecule Analytical Chemistry
and QC, Research Director at Synomics Pharma,
Research Fellow at Purdue Pharma, and Senior Staff
Scientist at Applied Biosystems/PerkinElmer. He
holds a PhD in Analytical Chemistry from City University of New York,
and a certificate in Biotechnology at University of California, Santa Cruz. CLE
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MS – THE PRACTICAL ART

Single-Cell MS and High-
Spatial-Resolution MS
Imaging Under Ambient
Conditions Using a Novel
We have developed
a miniaturized,
Sampling Device
multifunctional device,
M
called the “Single-Probe,”
ass spectrometry (MS) is a cal research (1–3). The mechanisms of
that is capable of probing crucial analytical technique biological processes at the cellular level
small targets and of based on the measurement are important for understanding life. It is
sampling and ionizing of mass-to-charge ratios (m/z) of ions of well known that cells of the same tissue
interest. Traditionally, MS has been used can behave differently, even under the
molecular species. It has
to analyze gas-phase samples or samples same conditions. Yet the current methods
been coupled with a mass in liquid solution. More recently, com- for analyzing the chemical makeup of
spectrometer and applied bined with advances in sampling and bulk cellular composition (such as with
to a variety of applications, ionization methods, instrument automa- cell lysates) provide limited information
tion, and data processing, MS has been because the measured results are an aver-
including single-cell MS and
used to analyze a broad range of samples. age of all of the samples combined. In
MS imaging of biological Of particular interest is the application particular, any cell preparation method
tissues, and detection of of MS to emerging techniques such as is likely to alter a cell’s environment and
sulfated proteins. In this single-cell MS analysis and MS imaging structure, changing the chemical com-
of biological tissues. position of cellular content. MS has been
article, we describe the
We developed a novel apparatus, called used for single-cell analysis because of its
probe and summarize the “Single-Probe,” which is a miniatur- high sensitivity—it is capable of detect-
recent work applying it ized, multifunctional device. The name ing a broad range of molecular species.
to single-cell MS and MS “Single-Probe” reflects the unique fea- Single-cell MS constitutes a new field in
tures of this sampling device: integrated bioanalysis, and a variety of techniques to
imaging studies.
design, multiple functions, and versatile apply it have been developed. The major
applications. difference among various approaches to
The single-probe device comprises single-cell MS lie in their sampling and
several integrated, microminiaturized ionization methods. To date, various
components, and is capable of probing MS methods have been developed for
extremely small targets and sampling and single-cell analysis, including secondary-
ionizing molecular species. The probe ion mass spectrometry (SIMS) (4–6),
can be coupled with a mass spectrometer, matrix-assisted laser desorption–ioniza-
and in this way it has been applied to tion (MALDI) (4,7,8), laser desorption–
various applications including single-cell ionization mass spectrometry (LDI-MS)
MS, MS imaging of biological tissues, (9,10), capillary electrophoresis–elec-
and the detection of sulfated proteins. trospray ionization MS (CE–ESI-MS)
In this article, we describe the probe and (11–13), and live, single-cell MS (14–18).
summarize our recent work using it in Although these established single-cell
single-cell MS and MS imaging studies. MS techniques have served in a variety
Ning Pan, Wei Rao, of studies, their inherent limitations
and Zhibo Yang are MS Analysis of Live, Single Cells constrain their broader application. For
the guest authors of
this month’s installment.
Single-cell analysis is a new field that has example, SIMS provides high spatial reso-
Kate Yu is the editor of the potential to reshape approaches to lution, but it must be operated under high
MS—The Practical Art. biological and pharmaceutical bioanalyti- vacuum conditions. MALDI, currently the
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dominant technique because of its good on the nano-ESI emitter, because the extracellular culture medium or drug
resolution (19), requires time-consuming emitter is too small (<5 mm). The con- compounds. Placed on the Z-translation
sample preparation, and the required tinuous flow of sampling solvent provides stage, the cell-containing plate can be
matrix changes the conditions of bio- a consistent, fresh, liquid junction from lifted precisely, by 0.1-µm increments,
logical samples (20). Therefore, neither which to extract cellular compounds for and monitored by means of the micro-
SIMS nor MALDI-MS can be used for MS analysis in real-time. The single- scope. The penetration is confirmed
live cells. Single-cell MS analysis of liv- probe MS technique allows for in situ, visually using the appended microscope
ing cells has been reported using the live analysis of a single cell under ambi- and sideways camera. The insertion of
live single-cell MS technique (14–18), ent conditions, with little or no prior the probe is monitored by an immediate
in which a sharp nanoelectrospray ion- sample preparation required. change of mass spectrum over the back-
ization (nano-ESI) emitter penetrates Before the single-cell MS experiments, ground signal. A time delay, of 1 to 2 s, is
the cell membrane and extracts cellular the live cells are rinsed with phosphate- usually observed between the moment of
contents. The intracellular contents are buffered saline (PBS) to remove insertion and detection of the MS signal.
mixed with the ionization solvent in the
emitter and the mixture then is trans-
ferred to a mass spectrometer for analysis.
Because the sampling, cell-extraction
preparation, and measurement steps must www.sedere.com
be performed separately, this technique
Low Temperature
cannot be used for in situ or real-time Evaporative Light-Scattering Detectors
cellular analysis.
The single-probe device, which can be SEDEX with SAGA becomes unsaturable,
coupled with a mass spectrometer, can without impact on sensitivity
overcome the barriers to live, single-cell
analysis. The single-probe device com- SAGA Desactivated SAGA Activated
bines two capillaries into a laser-pulled,
dual-bore, quartz needle. One capillary
(the solvent-providing capillary) deliv-
ers the solvent to the tip of the device; a SEDEX
AUTOMATED SAGA is a smart
small liquid junction is formed between algorithm that
GAIN
two bores at the quartz needle, to pick up ADJUSTEMENT adapts gain setting
the component of the cell. The solution dynamically to avoid
signal saturation
containing the cellular content is drawn
by the other capillary (the nano-ESI
signal saturation
emitter) via capillary action, and then
is ionized for immediate MS analysis
(Figure 1a). With a diameter of 6–10
µm, the probe tip is small enough to be
inserted into an individual cell (Figure
1b). The entire single-cell MS experimen-
tal system includes an automated X,Y,Z-
translational stage system, two digital
light microscopes, and a Thermo LTQ
Orbitrap XL mass spectrometer.
During our experiment, the cell
sample was placed on the stage system,
which is controlled by LabView software,
developed by the Laskin group (21). Cell
HIGH PERFORMANCE AND HIGH THROUGHPUT QUALITY CONTROL AND EDUCATIONAL LABORATORIES
penetration is achieved by precisely lift-
ing the Z-stage using the microscopes as SEDEX 90LT SEDEX LC
the visual guide (Figure 1c). To ionize LT-ELSD™ LT-ELSD™
the sampled species for MS analysis, an
ionization voltage of approximately 3 kV
energizes the conductive union. The volt-
age charge is transmitted to the nano-ESI SENSITIVITY
emitter continuously through the solvent FLEXIBILITY
inside the single-probe device. The ion-
EXPERIENCE
ization voltage cannot be applied directly
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in the single cells (Figure 2b). In the

positive mode, we identified 18 differ-
ent lipid species; in the negative mode,
(a) Solvent-
providing inlet we detected adenosine phosphates
Solvent Analyte (AMP, ADP, and ATP). In the experi-
Nano-ESI (b)
emitter ments of cells treated with the antican-
cer drugs doxorubicin, paclitaxel, or
Mass OSW-1, we detected the corresponding
spectrometer drugs and their metabolites in indi-
vidual HeLa cells. In these experiments,
Dual-bore HeLa cells, under normal culture con-
Probe ditions, were dosed with compound for
tubing
trip
4 h at a series of concentrations: 10 nM,
1 mm Liquid surface 100 nM, 1 µM, and 10 µM. Untreated
junction HeLa cells served as controls. Ions for
Ionization voltage the drugs’ metabolite compounds were
(c)
Sample probe
Solvent
Nano-ESI not present in the extracellular PBS, but
the compounds were detected inside the
single cells.
Conductive union MS inlet
Syringe Though still in its infancy, single-cell
z MS analysis has already attracted a lot
y
Digital microscope of attention in the research community.
Sample x Its ability to analyze samples with a level
Stage system
of precision previously impossible offers
Figure 1: (a) Photograph of the constructed single-probe device, zoomed in on the the potential to substantially improve
region around the tip. (b) Schematic of the construction of the single-probe device, our understanding of many biological
showing the solvent-providing capillary, the dual-bore tubing with the pulled probe tip, systems. The single-probe device can,
and the nano-ESI emitter. (c) Diagram showing the setup for the operation of the single- potentially, provide a robust tool for
probe device for sample acquisition.
single-cell MS experiments. With further
improvements, it will allow better and
easier performance of single-cell analysis
for a wide range of biological samples.
782.5669:PC (34:1) +Na+
754.5355:PC (32:1) +Na+
(a) (b) 100
High-Resolution
798.5410:PC (34:1) +K+
820.5253:PC (36:4) +K+
Ambient MS Imaging
Relative abundance
872.5573:PC (40:6) +K+

772.5267:PC (32:0) +K+
848.5570:PC (38:4) +K+
HeLa cell Single-probe tip MS imaging is an emerging technology

that has attracted considerable inter-
est from researchers in many diverse
24 µm fields since its debut in 1997 (23). The
process of MS imaging involves the MS
100
investigation of the surface of a sample
50 µm
0
750 800 850 900
in a regular grid, with each individual
m/z
grid position producing an entire mass
spectrum. Compared with traditional
Figure 2: (a) Photograph of the tip of the single-probe device entering the target cell for
acquisition of MS spectra. (b) Typical single cell analysis spectrum showing some of the MS techniques capable of analyzing the
identified peaks of phosphatidylcholine (PC) species in the positive ion mode. chemical composition of samples, MS
imaging makes it possible to map the
Single-probe sampling continues until no few seconds, after which solvent flowing spatial distribution of species of inter-
cytoplasm-MS signals are observed. Then through the probe removes any debris. est on a sample’s surface. In particular,
the single-probe device is lifted, and the This unclogging technique increases the certain MS imaging techniques can
constant solvent flow flushes the device, robustness of the single-probe device and create MS images at subcellular resolu-
preventing any carryover effects. Each allows its reuse. If clogging cannot be tion. Such images provide a wealth of
cell measurement requires approximately overcome, then the single-probe device information about the distribution of
3 min, making this a high throughput can be quickly replaced with a fresh one. metabolites. These techniques can be
technique. If the probe becomes clogged, We used the single-probe MS tech- used in various applications such as bio-
a miniature coil can be placed around nique to successfully analyze indi- marker discovery (24), tissue-metabolite
the clogged parts (such as the probe tip vidual HeLa cells (22) (Figure 2a) and profiling (25), precision histological
or nano-ESI emitter) and heat them for a observed cellular metabolites and lipids identification (such as for cancer versus
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MS (TOF-SIMS) (28) MS imaging,

the samples require the application of
Inner Outer Cortex a matrix, the presence of a vacuum, or
(a) medulla medulla
both to detect surface metabolites. Both
of these technologies allow the imaging
of samples at very high spatial resolu-
tions. MALDI is capable of a resolution
of 5 µm and TOF-SIMS at a pixel size of
<1 µm. The nonambient nature of these
0.20 mm
techniques, however, can increase sample
1 mm analysis time during preparation. It may
(b) m/z 734.5700 [PC(32:0) +H]+ m/z 782.5696 [PC(34:1) +Na]+ m/z 814.5726 [PC(38:5) +Na]+ Max also cause changes to the sample that
could introduce artifacts into the resul-
tant MS image (29).
Ambient MS imaging is a relatively
recent advance. The technique involves
performing MS imaging in an ambient
0.20 mm
environment in which the sample has
undergone only minimal pretreatment
Min
(30). Thus, compared with nonambient
methods, ambient MS imaging allows
Figure 3: (a) Optical image of kidney section imaged using the single-probe device. (b) the sample—such as a tissue section
MS images of three phosphatidylcholine (PC) species showing their different distributions from a mouse brain or a tumor—to be
within the sample. The imaging resolution was at 8.5 µm.
analyzed in an environment approach-
normal cells) (26), and many others. tion involves the way in which samples ing that of its native state. The result
Broadly speaking, MS imaging tech- are prepared before analysis. In non- is fewer artifacts and the potential for
niques are split between two categories: ambient MS imaging, such as MALDI creating a better representation of the
nonambient and ambient. The distinc- (27) and time-of-flight secondary-ion sample in the MS image.
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The first ambient MS imaging tech- resolution of 8 ± 2 µm was achieved ther improve the manufacturing process
nique, desorption electrospray ionization using a rhodamine grid (37). The for the probe so that it can become more
(DESI) MS imaging, was described in small size of the single-probe device tip consistent and robust in operation and
2004 (31), and a proliferation of ambient (which can be as small as 6 µm) made user-friendly, allowing the device to be
MS imaging techniques followed (30). In this outcome possible. We then applied more readily used by a larger number of
DESI MS imaging, a spray of solvent is the device to the imaging of biological researchers. The single-probe device has
focused onto a sample and the analytes samples made by cryosection. That appli- the potential to become an effective tool
are desorbed from the sample’s surface by cation, however, presented a much bigger for elucidating analyte distributions with
means of a droplet-pickup mechanism. challenge. Cryosectioned biological tis- high-spatial-resolution ambient MS imag-
The analytes are then transmitted into sues are known to demonstrate a certain ing. We would like to apply this tech-
the mass spectrometer for analysis. Sur- amount of surface heterogeneity that can nique for investigation of an increasingly
face-extraction techniques such as liquid introduce inconsistencies during sur- sophisticated range of applications—
microjunction surface-sampling probe face microjunction formation (21). The imaging drug distributions within tissue
(LMJ-SSP) (32), liquid-extraction surface inconsistencies would create difficulties and biomarker discovery for cancers, to
analysis (LESA) (33), and nano-DESI during MS imaging that could affect the name but two—that may be of greater
(34) create a liquid-surface microjunction spatial resolution of the images produced. interest to the biological, pharmaceutical,
from which metabolites are extracted We were able to minimize this effect by and medical research communities.
during MS imaging. The techniques have creating thin sections (~12 µm) of mouse
demonstrated greater sensitivity than brain and kidney. In conjunction with References
that provided by DESI imaging (35). the custom-designed LabView control (1) H. Andersson and A. van den Berg, Curr.
Nevertheless, one of the biggest obstacles software for the XYZ stage, the thinness Opin. Biotech. 15, 44–49 (2004).
for ambient MSI techniques is the lack of the tissue sections allowed the single- (2) D.J. Wang and S. Bodovitz, Trends Biotech-
of spatial resolution. Though DESI MSI probe device to achieve a stable micro- nol. 28, 281–290 (2010).
is capable of a resolution of 35 µm (36), junction with a flat surface throughout (3) S.S. Rubakhin, E.V. Romanova, P. Nemes,
it is routinely performed at 100–200 µm the analysis. MS imaging of these tis- and J.V. Sweedler, Nature Methods 8, S20–
to maintain high sensitivity throughout sues were made with a Thermo LTQ S29 (2011).
the analysis. Both LMJ-SSP and LESA Orbitrap XL mass spectrometer, which (4) M.L. Pacholski and N. Winograd, Chem.
create relatively large liquid microjunc- provided high mass resolution at 60,000 Rev. 99, 2977–3006 (1999).
tions, with spatial resolutions exceeding m/Δm. Overall, we were able to achieve (5) K. Chughtai and R.M.A. Heeren, Chem.
500 µm. Of the aforementioned tech- a maximum lateral resolution of 8.5 µm Rev. 110, 3237–3277 (2010).
niques, nano-DESI MS imaging provides for an analysis of mouse kidney tissue, (6) E.J. Lanni, S.S. Rubakhin, and J.V. Sweedler,
the greatest spatial resolution, with MS a resolution that is among the highest J. Proteomics 75, 5036–5051 (2012).
images created at approximately 12 µm spatial resolutions reported for ambient (7) K.A.Z. Berry, J.A. Hankin, R.M. Barkley,
in lateral resolution. Yet such perfor- MS imaging techniques (Figure 3). Also, J.M. Spraggins, R.M. Caprioli, and R.C.
mance cannot compare with the spatial the high mass accuracy made it possible Murphy, Chem. Rev. 111, 6491–6512 (2011).
resolution of nonambient techniques to identify an entire host of metabolites, (8) A. Amantonico, J.Y. Oh, J. Sobek, M. Heine-
such as MALDI MS imaging, which are such as sphingomyelin (34:1) (725.5575 mann, and R. Zenobi, Angew Chem. Int.
capable of imaging precision at subcel- m/z, [M + Na]+), phosphatidylcholine Edit. 47, 5382–5385 (2008).
lular levels. (PC) (32:0) (734.5700 m/z, [M + H]+), (9) M.P. Greving, G.J. Patti, and G. Siuzdak,
We have applied the single-probe PC (34:1) (782.5696 m/z, [M + Na]+), Anal. Chem. 83, 2–7 (2011).
device to MS imaging and have been PC (36:4) (820.5256 m/z, [M + K]+), (10) P.L. Urban, T. Schmid, A. Amantonico,
able to improve the spatial resolution of and many others with a mass confidence and R. Zenobi, Anal. Chem. 83, 1843–1849
ambient MS imaging techniques. The below 4 ppm, which allows detailed dis- (2011).
single-probe device is readily adapted to tributions of metabolites for each of the (11) J.S. Mellors, K. Jorabchi, L.M. Smith, and
the surface microjunction mode of MS areas within the mouse kidney sample to J.M. Ramsey, Anal. Chem. 82, 967–973
imaging, so that a high degree of sensi- be elucidated. (2010).
tivity can be achieved during the analysis Accordingly, we expect to be able to (12) P. Nemes, A.M. Knolhoff, S.S. Rubakhin,
(37). The probe was modified to reduce further improve the spatial resolution of and J.V. Sweedler, Anal. Chem. 83, 6810–
the length of the emitter, minimizing the single-probe device to the extent that 6817 (2011).
the effect of carryover and thus improv- it may approach the resolution offered by (13) P. Nemes, S.S. Rubakhin, J.T. Aerts, and J.V.
ing the spatial resolution of the images nonambient techniques such as MALDI Sweedler, Nat. Protoc. 8, 783–799 (2013).
produced. One major advantage of the MS imaging. The enhanced resolution (14) Y. Fukano, N. Tsuyama, H. Mizuno, S.
single-probe device is that its integrated would allow subcellular levels of MS Date, M. Takano, and T. Masujima, Nano-
design allows for relatively easy setup imaging to be conducted in the ambi- medicine-UK 7, 1365–1374 (2012).
during MS image acquisition. ent environment, improving analysis (15) T. Masujima, Anal. Sci. 25, 953–960 (2009).
The single-probe device was used first time as well as reducing the possibility (16) H. Mizuno, N. Tsuyama, T. Harada, and T.
to create MS images of standardized of introducing artifacts during sample Masujima, J. Mass Spectrom. 43, 1692–1700
samples. From those images, a spatial preparation. We would also like to fur- (2008).

(17) N. Tsuyama, H. Mizuno, E. Tokunaga, and T. Masujima, Anal. Sci. Zhibo Yang, PhD, obtained his B.S. (1997)
24, 559Ð561 (2008). and M.S. (2000) degrees from the University of
Science and Technology of China. In 2005, he
(18) M.L. Tejedor, H. Mizuno, N. Tsuyama, T. Harada, and T. Masujima,
received his PhD in Physical Chemistry under
Anal. Chem. 84, 5221Ð5228 (2012). the supervision of Professor Mary T. Rodgers at
(19) A. Zavalin, J.H. Yang, A. Haase, A. Holle, and R. Caprioli, J. Am. Soc. Wayne State University. He has conducted post-
Mass Spectrom. 25, 1079Ð1082 (2014). doctoral research with Dr. Julia Laskin at Pacific
Northwest National Laboratory (2005–2008) and
(20) N. Tsuyama, H. Mizuno, and T. Masujima, Anal. Sci. 27, 163Ð170
Professor Veronica M. Bierbaum at the University
(2011). of Colorado, Boulder (2008–2012). In 2012, he joined the Department of
(21) I. Lanekoff, B.S. Heath, A. Liyu, M. Thomas, J.P. Carson, and J. Chemistry and Biochemistry at the University of Oklahoma as an assistant
Laskin, Anal. Chem. 84, 8351Ð8356 (2012). professor. His current research is focused on the development and applica-
tion of novel mass spectrometry techniques for bioanalysis. Dr. Yang was
(22) N. Pan, W. Rao, N.R. Kothapalli, R. Liu, A.W.G. Burgett, and Z. Yang,
the recipient of the 2014 ASMS Research Award.
Anal. Chem. 86, 9376Ð9380 (2014).
(23) R.M. Caprioli, T.B. Farmer, and J. Gile, Anal. Chem. 69, 4751Ð4760
Kate Yu
(1997).
“MS — The Practical Art” Editor Kate Yu
(24) K. Schwamborn, J. Proteomics 75, 4990Ð4998 (2012). joined Waters in Milford, Massachusetts,
(25) I. Lanekoff, M. Thomas, J.P. Carson, J.N. Smith, C. Timchalk, and J. in 1998. She has a wealth of experience in
Laskin, Anal. Chem. 85, 882Ð889 (2013). applying LC–MS technologies to various appli-
cation fields such as metabolite identification,
(26) S. Gerbig, O. Golf, J. Balog, J. Denes, Z. Baranyai, A. Zarand, E. Raso,
metabolomics, quantitative bioanalysis, natu-
J. Timar, and Z. Takats, Anal. Bioanal. Chem. 403, 2315Ð2325 (2012). ral products, and environmental applications.
(27) A. Rompp and B. Spengler, Histochem. Cell Biol. 139, 759Ð783 (2013). Direct correspondence about this column to
(28) J. Brison, M.A. Robinson, D.S.W. Benoit, S. Muramoto, P.S. Stayton, lcgcedit@lcgcmag.com
and D.G. Castner, Anal. Chem. 85, 10869Ð10877 (2013).
(29) J.C. Vickerman, Analyst 136, 2199Ð2217 (2011).
(30) C. Wu, A.L. Dill, L.S. Eberlin, R.G. Cooks, and D.R. Ifa, Mass Spec-
For more information on this topic, please visit
trom. Rev. 32(3), 218Ð243 (2012).
www.chromatographyonline.com/column-ms-practical-art
(31) Z. Takats, J.M. Wiseman, B. Gologan, and R.G. Cooks, Science 306,
471Ð473 (2004).
(32) G.J. Van Berkel, A.D. Sanchez, and J.M. Quirke, Anal. Chem. 74,
6216Ð6223 (2002).
(33) V. Kertesz and G.J. Van Berkel, J. Mass Spectrom. 45, 252Ð260 (2010).
(34) J. Laskin, B.S. Heath, P.J. Roach, L. Cazares, and O.J. Semmes, Anal.
Chem. 84, 141Ð148 (2012).
(35) W. Rao, A.D. Celiz, D.J. Scurr, M.R. Alexander, and D.A. Barrett, J. CHEERS TO 20 YEARS
Am. Soc. Mass Spectrom. 24, 1927Ð1936 (2013).
(36) D.I. Campbell, C.R. Ferreira, L.S. Eberlin, and R.G. Cooks, Anal. Bio-
anal. Chem. 404, 389Ð398 (2012).
(37) W. Rao, N. Pan, and Z. Yang, J. Am. Soc. Mass Spectrom. 26, 986Ð993
WWW.LABX.COM
(2015).
Ning Pan, PhD, earned her bachelor’s

degree in chemistry from Shandong Normal
University, in China, in 2007. In 2010 she earned
a master’s degree in organic chemistry from
Shandong Normal University and in 2013 she
earned a PhD in analytical chemistry from Shan-
dong Normal University and Ohio University.
She is currently a Postdoctoral Research Associ-
ate in Chemistry at the University of Oklahoma.
Her research focuses on single-cell mass spectrometry analysis.
Wei Rao, PhD, earned his bachelor’s degree

in genetics at the University of Cambridge, UK,
in 2005. After graduation he went on to com-
plete his PhD in proteomics under Professor R. The premier scientific website for buying and selling
E. Isaac in 2010 at the University of Leeds, UK. laboratory equipment and supplies for over 20 years.
His first postdoctoral position was with Profes-
WWW.LABX.COM
sor D.A. Barrett at the University of Nottingham
in the UK, looking at ambient surface MS imag-
ing of proteins. He is currently employed at the
University of Oklahoma under Dr. Zhibo Yang, working on high spatial
and mass ambient MS imaging using the single-probe device.
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Analysis of Peptide Mixtures for

Proteomics Research Using LC–ESI-MS
with a Simple Microgradient Device
This work presents an on-line combination of a simple microgradient

device for reversed-phase liquid chromatography (LC) and electrospray
ionization (ESI) tandem mass spectrometry (MS-MS). The formation
of an S-shaped gradient of the strong mobile phase (based on
acetonitrile) inside a microsyringe is described under the conditions
applied for capillary separation of proteomic samples and their
analysis using ESI-MS-MS on a hybrid instrument. The system was
successfully tested for a complex peptide mixture originating from
the digestion of the whole-cell lysate from Francisella tularensis
subsp. Schu S4 and it represents a cheap alternative for the separation
of both simple and complex peptide mixtures before MS analysis.
T here are many methods available ing, where intact proteins are separated
for studying biological systems that by 1D or 2D gel electrophoresis before
are commonly applied with the aim digestion and analysis (6). LC techniques
to describe their structure and function. hyphenated with mass spectrometry (MS)
Biological samples are usually very com- are already well-established in the pharma-
plex (for example, in the case of analyzing ceutical, biotechnological, nutrition and
proteomes) and thus the only possibility health care, and chemical industries (7).
to obtain valuable results often resides Peptides are separated either by LC or
in an efficient sample separation. So far, nanoflow LC (nanoLC) connected on-line
various separation techniques have been or off-line to electrospray ionization (ESI)
introduced in proteomics (1). Protein MS or off-line to MALDI MS (4,8,9).
separation by one-dimensional (1D) or This is applicable for complex peptide
two-dimensional (2D) gel electrophoresis mixtures in shotgun proteomics as well
(2) is usually followed by an enzymatic or as for digested protein fractions from 1D
chemical protein digestion. The obtained electrophoresis gel slices or less complex
peptide mixtures can be directly analyzed peptide mixtures coming from 2D gel
or further separated depending on the electrophoresis spots. Various commercial
researcher’s choice, needs, and experimen- 2D LC systems are available for separating
tal design. During the last few years, liquid peptides (10,11), which couple two mutu-
chromatography (LC) has had widespread ally orthogonal LC separation techniques.
use as a separation technique for proteins In a typical arrangement, the first dimen-
and peptides (3,4). In the shotgun pro- sion is based on a strong cation exchanger
teomics strategy, which involves the initial whereas the second dimension is run on
digestion of the whole protein sample, an a reversed-phase column. The separations
efficient chromatographic separation of may be run on sophisticated and expensive
peptides is required before recording tens commercial LC systems or on specialized
René Lenobel, Helena of thousands of tandem mass spectra (5). 1D LC systems designated for proteomics
Řehulková, Marek Šebela,
Vojtěch Franc, Vladislav This is in contrast with matrix-assisted (such as for the particular separation of
Kahle, Dana Moravcová, laser desorption–ionization time-of-flight peptides). Although LC peptide separa-
and Pavel Řehulka (MALDI-TOF) peptide mass fingerprint- tion is useful, it is also costly and time
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Latest Developments and Future Directions in Data Processing
& Analysis Software for LC–MS-MS and GC–MS-MS
LIVE WEBCAST
Wednesday, July 15, 2015 at 8 am PDT/ 11 am EDT/ 4 pm BST/ 5 pm CEST
Register free at www.chromatographyonline.com/lcgc/pesticide_residue_analysis_series
Series Part 4 Event Overview: Series Moderator

Safeguarding the environment and the global food supply requires con- Richard Fussell, Ph.D.
tinuous monitoring of more and more compounds. Pesticide residues Global Vertical Marketing Manager,
Food and Beverage, Chromatography
analysts are challenged to detect, identify, and quantify hundreds of & Mass Spectrometry Division
diferent pesticides with a fast turnaround time (often within 24 hours Thermo Fisher Scientifc
of receipt). Data processing and analysis can become a bottleneck. Presenter:
Method development, data analysis and processing can be simplifed Charles Yang
with the use of comprehensive compound databases, pre-determined Senior Marketing Specialist,
methodologies, and intuitive software. Environmental and Food Safety,
Thermo Fisher Scientifc
This webinar will provide pesticides residue analysts with valuable

information on software method development and data processing for LCGC Moderator:
the analysis of pesticide residues in food for both LC–MS and GC–MS. Laura Bush
Editorial Director
Technical experts will review the latest in software advances to help LCGC
with data interpretation and reporting. They will guide you through
industry-relevant trends and new techniques that allow you to see Key Learning Objectives:
more, do more, and be more productive. Example workfows will be
n Learn how to quickly set up
shown, along with data analysis using the latest in compound data-
acquisition methods with
bases and spectral libraries.
TraceFinder software
Who Should Attend: n Proceed to rapidly analyze data with
integrated software workfows
n Researchers and analysts in pesticide analysis
n Learn about the latest in
n Food scientists interested in learning the latest technologies for
comprehensive compound
sample preparation of food matrices
databases and libraries for triple-
n Anyone struggling with sample preparation challenges for pesticide
quadrupole and HRAM LC and GC
residue analysis in food mass spectrometry
Register free at www.chromatographyonline.com/lcgc/pesticide_residue_analysis_series

Sponsored by Part 1: Sample Prep Tips and Tricks Using QuEChERS and Accelerated Solvent Extraction
ON-DEMAND WEBCAST, originally aired March 24, 2015
Part 2: Workflow Guide for the use of LC-MS
ON-DEMAND WEBCAST, originally aired April 29, 2015
Part 3: Maximizing Analysis Efficiency through New GC-MS Approaches Register for
ON-DEMAND WEBCAST, originally aired June 17, 2015 the Pesticide
Presented by
Part 4: Latest Developments & Future Directions in Residue Analysis
Data Processing & Analysis Software for LC-MS/MS & GC-MS/MS
Wed., July 15, 2015 at 8 am PDT/ 11 am EDT/ 4 pm BST/ 5 pm CEST Webinar
Series
For questions, contact Kristen Moore at kmoore@advanstar.com
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consuming. The relatively low sensitivity

of conventional LC hampers its applica-
(a) bility for the analysis of biological samples,
7
2 such as peptides from protein digests in
4 5 6 proteomic research. This low sensitivity in
1 LC is because of the low levels of endoge-
3 3 3 nous proteins and the large dynamic range
of proteomes (12). Moreover, samples to
(b)
be analyzed are also frequently available in
tiny amounts (at the microliter level). For
2
that reason, LC systems have already been
miniaturized to achieve higher sensitiv-
ity namely in coupling with electrospray
1 3 mass spectrometers as detectors. Currently,
4 nanoLC separations are performed using
flow rates in the range of several hundreds
of nanoliters per minute (13).
A simple microgradient-generating
7 3
device has been introduced (14), where
5 an S-shaped acetonitrile gradient was eas-
6
ily formed in a microsyringe with a 250-
µm i.d stainless steel needle. The gradient
Figure 1: The microgradient device coupled to an ESI mass spectrometer: (a) a scheme emerged in the region where the needle
of the whole setup and (b) a photograph showing implementation of the device for spreads into the interior of the syringe.
nanoLC–MS. 1 = syringe infusion pump, 2 = microsyringe, 3 = microtight union, 4 =
injection capillary for sample loading, 5 = capillary column, 6 = nanospray needle, 7 =
Such a way of preparation provided a high
ESI-QTOF instrument. An arrow denotes the microtight union, which is disconnected reproducibility of retention times (rela-
for sample loading. tive standard deviation [RSD] of about
0.1%) and stability of the gradient inside
the syringe for several hours. This was
verified using electrochromatography on a
350 monolithic polyacrylic column and seven
(a) (b)
alkylphenones as a model mixture (14).
300 An off-line device for both preconcen-
(c) tration and separation of peptides (with
250 elution using a linear gradient of aceto-
Voltage (mV)
nitrile formed in a microsyringe followed

200 by MALDI-TOF MS-MS analysis) was
also reported (15). The peptide mixture
150 was separated using a reversed-phase
monolithic capillary column and the elu-
100 tion was performed by a gradient of ace-
tonitrile from 0% to 60% (v/v) in water
50 containing 0.1% (v/v) trifluoroacetic
acid. The main part of this system was
0 represented by a gastight 50-µL microsy-
0 20 40 60 80 100 120 140 160 180 200 ringe, where the acetonitrile gradient was
Time (min) formed because of a turbulent mixing of
the strong and weak mobile phases in
Figure 2: UV detection records of the gradient shape formed in the microsyringe of
the microgradient system. The mobile phases used were 0.1% (v/v) formic acid in water the high-dispersion region of the syringe.
(A), and 0.1% (v/v) formic acid in acetonitrile with the addition of 1% (v/v) acetone al- This region is formed by a steep exten-
lowing UV detection of the gradient shape at 275 nm (B). Three elution arrangements sion of the tube diameter between the
were used (the respective solutions are described in the order of their consecutive as-
needle end and glass syringe barrel. Frac-
piration into the microsyringe through the loading capillary (the order of elution was
thus reverse): (a) 3 µL of 60% (v/v) B and 3 µL of 2% (v/v) B were loaded only into the tions of the separated mixture were then
sample loading capillary—in fact, this represents an isocratic elution; (b) three repeti- directly spotted onto a MALDI target
tions (represented by solid, dotted, and dashed lines) of a two-step gradient elution that had been covered with α-cyano-
consisting of 12 µL of 60% (v/v) B, 22 µL of 2% (v/v) B, and 1 µL of blank (the same 2%
4-hydroxycinnamic acid as a matrix.
[v/v] B solution mimicking a sample) showing the reproducibility of gradient formation;
(c) a four-step gradient elution consisting of 12 µL of 60% (v/v) B, 8 µL of 40% (v/v) B, 8 Consequently, MALDI-TOF-TOF-MS
µL of 20% (v/v) B, 20 µL of 2% (v/v) B, and 1 µL of blank. measurements were done. The results
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HPLC GC MS SPE IR
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alternative and cheap gradient LC sepa-

ration system for MS analysis of moder-
ately complex mixtures that can be used
(a)
800,000 800,000
582.32
especially in the case when no suitable LC
600,000 system is available. The gradient was gen-
Intensity
722.32 740.40 erated in a microsyringe before the separa-
600,000 400,000
547.32 tion of peptides on a capillary column. A
Intensity
200,000
syringe infusion pump was used to deliver
400,000 0 the mobile phase. The system was first
6 8 10 12 14 16 18
Time (min) optimized for a tryptic digest of bovine
serum albumin (BSA) and then run with
200,000
a Francisella tularensis lysate. Its applica-
bility for survey analyses in proteomics
0 was confirmed.
6 8 10 12 14 16 18
(b) Time (min)
200,000 120,000
547.32 582.32 Experimental
Chemicals and Consumables
80,000 722.32
Intensity
120,000 740.40
Acetonitrile, formic acid, and water were of
40,000 LC–MS-grade quality, and acetone was of
Intensity
LC-grade quality (Sigma-Aldrich Chemie).

80,000 0 A standard tryptic digest of BSA was from
60 70 80 90 100 110 120 130
Time (min)
Bruker Daltonik. Reprosil-Pur C18-AQ
40,000 5-µm particles were obtained from Dr.
Maisch. PEEK microtight unions (5/16–
24, 360-µm o.d.) were obtained from
0
60 70 80 90 100 110 120 130 Upchurch Scientific and polyimide-coated
(c) Time (min) fused-silica capillaries (360-µm o.d., 200-
300,000
300,000 547.32 582.32 µm or 100-µm i.d.) were obtained from
200,000
Agilent Technologies.
Intensity
722.32
200,000 100,000 740.40

Preparation of Samples from
Intensity
Francisella tularensis subsp.

0
60 70 80 90 100 110 120 130 tularensis Strain Schu S4
Time (min)
100,000 Ready-to-analyze protein digests were

provided by Professor Lenka Hernychová
of the University of Defence in Hradec
Králové, Czech Republic. In brief, they
0
60 70 80 90 100 110 120 130 were prepared as follows: The whole cell
Time (min) lysate was obtained by disruption of har-
vested bacterial cells using a French press
Figure 3: Nanofow microgradient LC separation of tryptic digest of BSA (100 fmol). (19); proteins were then isolated from the
Column: 100 mm × 100 µm, 5-µm Reprosil-Pur C18-AQ (Dr. Maisch); mobile-phase A: lysate by a trichloroacetic acid–acetone
0.1% formic acid in water; mobile-phase B: 0.1% formic acid in acetonitrile. (a) Iso-
cratic elution—the gradient was aspirated as 25 µL of 60% (v/v) B plus 1 µL of sample; precipitation procedure (20) and finally
(b) two-step gradient elution—the gradient was aspirated as 12 µL of 60% (v/v) B, subjected to reduction, alkylation, and
22 µL of 2% (v/v) B plus 1 µL of sample; (c) four-step gradient elution—the gradient in-solution digestion by bovine trypsin at
was aspirated as 12 µL of 60% (v/v) B, 8 µL of 40% (v/v) B, 8 µL of 20% (v/v) B, 20 µL 37 °C overnight (21).
of 2% (v/v) B plus 1 µL of sample. The main panels show base peak chromatograms
(m/z range of 460–1200), and the insets show the corresponding extracted ion chro-
matograms of four selected precursors with m/z values of 722.32, 547.32, 582.32, Setup of the Microgradient Device
and 740.40 demonstrating the effciency of peptide separation under various modes. The chromatographic separation device
used in this work consisted of a KDS100
obtained after preconcentration and sepa- sensitivity of analyses. Recent studies have syringe infusion pump (KD Scientific,
ration were significantly better than those also demonstrated the applicability of the Holliston), a gastight 50-µL syringe (ILS
measured after a direct sample deposition system for an elegant analysis of protein Innovative Labor Systeme GmbH or SGE
(15). Despite its obvious simplicity, the post-translational modifications such as Analytical Science) and a capillary-based
system was sophisticated enough and effi- N- and O-glycosylations (16–18). flow system (Figure 1a). When mounted
cient in the separation of peptide mixtures The aim of this work was to introduce into the infusion pump, the microsyringe
from protein digests, which resulted in an a simple gradient device coupled on-line was connected to a capillary (250-mm
increased identification rate and higher to ESI-MS with the purpose to present an long, 360-µm o.d., 200-µm i.d.; loading
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Table I: An overview of identification results with tryptic digest of bovine then washed out with 60% (v/v) B. The
serum albumin using different elution modes microgradient was formed by spontaneous
Two-Step Four-Step mixing of mobile phases after a consecu-
Isocratic Elution
Gradient Elution Gradient Elution tive aspiration of their aliquots through
Number of queries 830 2000 3654 the loading capillary in the following
Mascot score 1426 2707 4260
order: 60% (v/v) B, 40% (v/v) B, 20%
(v/v) B, and 2% (v/v) B (see Results and
Sequence coverage (%) 29 45 52
Discussion section). Finally, the sample
Peptides signifcant 63 104 166
in 2% (v/v) B was aspirated into the load-
Sequences signifcant 15 24 33 ing capillary. After that, the microsyringe
became filled up with the microgradient
volume of 7.85 µL) through a microtight column was washed with acetonitrile and the sample was ready for separation
union. Another microtight union was used before use. initiated after repeated assembling of the
for connecting the loading capillary to a whole flow system. During separation, the
capillary column (100-mm long, 360-µm Chromatographic Separations flow rate was set at 250 nL/min.
o.d., 100-µm i.d.; containing C18 5-µm Mobile-phase A consisted of 0.1% (v/v)
particles). At this particular place, the formic acid in water, and mobile-phase B Modeling of the Gradient Profile
system was only disconnected when intro- was 0.1% (v/v) formic acid in acetonitrile. For visualization of the gradient profile in
ducing mobile phases into the syringe and The following mobile phases were used to microsyringe during modeled separation
individual samples into the 250-mm-long form the microgradient: 2%, 20%, 40%, runs omitting ESI-MS, mobile-phase B
capillary serving thus as a sampling loop. and 60% (v/v) B. Before the analyses, pep- was composed of 0.1% (v/v) formic acid in
At the opposite end, the capillary column tide samples were dissolved in 2% (v/v) B acetonitrile containing 1% acetone. The
was tightly connected to the nanospray in A. To introduce sample and prepare the addition of acetone allowed monitoring
needle of the ESI-MS instrument (Figure microgradient, the flow system was dis- the increasing content of acetonitrile in the
1b). The capillary separation column was connected between the capillary column mobile phase by UV spectrophotometry
packed with the C18 sorbent resuspended and loading capillary. The column was at a detection wavelength of 275 nm (14).
in methanol using a capillary loader tool wetted with 80% (v/v) B and equilibrated Sample loading was simulated by aspirat-
(The Loader Kits, Proxeon). The packed with 2% (v/v) B, and the microsyringe was ing a blank of 2% (v/v) B.
Therapeutic Protein Separations Using

Using Diamond-Based Wide-Pore HPLC Columns
ON-DEMAND WEBCAST (Originally aired May 27, 2015)
Register for free at www.chromatographyonline.com/lcgc/therapeutic
Who Should Attend: EVENT OVERVIEW:

■ Method developers for
Can new wide-pore HPLC column technology assist you in your therapeutic
UHPLC and HPLC methods protein separations? Dr. David Jensen will present research conducted by Diamond
in pharmaceutical, chemical, Analytics and Dr. Szabolcs Fekete, et. al. at the School of Pharmaceutical Sciences at
environmental, agrochemical, the University of Geneva on the performance of these columns for the separations
university and governmental of therapeutical proteins, including monoclonal antibodies (mAbs) and antibody-
laboratories
drug conjugates (ADCs).
■ Practitioners of liquid
chromatography and LC/MS Key Learning Objectives
■ Introduce diamond-based superfcially porous particles for characterization of
Presenter therapeutic proteins including mAbs and ADCs.
Dr. David Jensen ■ Compare current USP method for flgrastim—the active protein ingredient for the frst
Research and biosimilar approved by US FDA.
Development Scientist
■ Highlight the benefts of diamond-based wide-pore HPLC columns in a variety of protein
Diamond Analytics
separations.
Moderator
Sponsored by Presented by
Laura Bush For questions contact
Editorial Director Kristen Moore at
LCGC
kmoore@advanstar.com
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active exclusion after 5 spectra for next

18 s; excluded mass range of precursors:
50–350 amu and 1500–2200 amu. All
(a)
400,000 60 data were collected over the mass range
of 50–2200 amu with an acquisition time
50
of 500 ms for MS and 250–750 ms for
MS-MS according to precursor intensity.
300,000
Raw data were processed by DataAnaly-
40
% acentonitrile
sis v.4.2 SP4 (Bruker Daltonik) and saved
Intensity
as mgf files. Searches for protein identifi-

200,000 30 cation were done using the Mascot 2.4.1
search engine (in-house server; Matrix Sci-
20 ence) either against SwissProt 3.9.2014 or
a custom sequence database consisting of
100,000
Francisella tularensis subsp. tularensis strain
10
Schu S4 proteins (downloaded from the
UniProtKB database; 1528 entries). Each
0 0 search was performed with a mass toler-
60 70 80 90 100 110 120 130
Time (min)
ance for precursors and corresponding
(b)
fragment ions of ±50 ppm and ±0.05 Da,
400,000 60 respectively. Search parameters were as fol-
lows: Trypsin was set as a protease with
one missed cleavage allowed; carbamido-
50
methylation of cysteine was set as a fixed
300,000
modification and oxidation (M), acetyl
% acentonitrile
40 (protein N-term), Gln->pyroGlu (N-term
Intensity
Q), Glu->pyroGlu (N-term E), Pyro-carb-

200,000 30 amidomethyl (N-term C), variable modi-
fication; +2 and +3 was set as a peptide
20 charge; other settings — expectation value
for peptides: 0.01, significance threshold:
100,000
p < 0.05, false discovery rate: <1%.
10
Results and Discussion

0 0 This work describes a microgradient LC
60 70 80 90 100 110 120 130
device for proteomic analyses, which can
Time (min)
simply be used when coupled to a nano-
electrospray ion source on those occasions
Figure 4: Nanofow microgradient LC separation of Francisella tularensis subsp. tu-
larensis Schu S4 tryptic digest (2 µg). Column: 100 mm × 100 µm, 5-µm Reprosil-Pur
where no nanoLC system is available. The
C18-AQ (Dr. Maisch); mobile-phase A: 0.1% formic acid in water; mobile-phase B: 0.1% present system can be run at flow rates of
formic acid in acetonitrile. (a) Two-step gradient elution—the gradient was aspirated a few microliters down to several hundred
as 12 µL of 60% (v/v) B, 21 µL of 2% (v/v) B plus 2 µL of sample; (b) four-step gradient nanoliters per minute and thus it fits the
elution—the gradient was aspirated as 12 µL of 60% (v/v) B, 8 µL of 40% (v/v) B, 8 µL of
20% (v/v) B, 19 µL of 2% (v/v) B plus 2 µL of sample. The main panels show base peak
nanoESI setups commonly used in pro-
chromatograms (m/z range of 460–1200). The gradient profle is illustrated as a dashed teomic analyses.
line over the chromatograms with percentage of B shown on the secondary y-axis. Before chromatographic experiments,
the gradient shape in the microsyringe
MS and Data Processing QTofControl 3.2 software (Bruker Dal- was assessed by recording UV spectra of
for Identification of Proteins tonik) at the following settings: Source the eluate. Figure 2 shows a comparison
The instrument used was a maXis ultra- settings: capillary voltage: 4000 V; dry gas: of data acquired from three arrangements
high-resolution QTOF tandem mass 6 L/min, dry temperature: 160 °C. Tune and resulting in isocratic, two-step, and
spectrometer equipped with a nanoelec- page settings: ion funnel RF: 400 Vpp; four-step gradient elution modes. The
trospray ion source (Bruker Daltonik). multipole RF: 400 Vpp; quadrupole ion isocratic arrangement was set to aspirate
Measurements were run in a data-depen- energy: 5 eV; collision energy: 8 eV; col- the strong and weak mobile phases only
dent manner. From each survey scan, four lision RF: 1200 Vpp; ion cooler RF: 350 into the capillary. Almost no mixing of
precursors were selected automatically for Vpp; transfer time: 85 µs; pre-pulse stor- the mobile phases appeared in this case,
collision-induced dissociation (CID) frag- age: 7 µs. MS-MS settings: auto MS-MS: which comes out from a sharp increase
mentation and MS-MS spectra acquisi- on; 4 precursor ions; threshold for switch- in the concentration of acetonitrile-con-
tion. The whole system was controlled by ing from MS to MS-MS mode: 5000 cts; taining strong mobile phase as monitored
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by ultraviolet (UV) absorption (Figure 2, probability-based score as a confidence and ribosomal components (for example,
chromatogram 1). On the other hand, the measure of identification results. Thus, a rRNA), p-values of 9.3E-54 to 2.7E-17, by
aspiration of the strong and weak mobile significant improvement was observed gene ontology enrichment analysis using
phases into the microsyringe through not only when comparing the isocratic the Database for Annotation, Visualization
the capillary resulted in a mixing of both and two-step gradient arrangements, but and Integrated Discovery (DAVID) (26).
mobile phases and the UV trace of eluent namely when upgrading to the four-step This is not surprising as ribosomal pro-
showed a gradual increase in acetonitrile gradient setup. Table I summarizes data teins represent the most abundant proteins
concentration (Figure 2; chromatograms from the above separations and demon- in bacterial cytosol (27). The number of
2 and 3 depict a two-step and four-step strates the highest identification rate in that identifications achieved in this study is not
gradient, respectively). This result shows case when the four-step gradient was used comparable with those available from cur-
that the turbulent mixing, which appears (Mascot score = 4260, sequence coverage = rent state-of-art nanoLC–MS and MS-MS
inside the microsyringe, is important for 52%, number of sequences = 33). In addi- experiments with digested cell lysate protein
the acetonitrile gradient formation. As can tion, a complete list of peptides confirmed extracts, which appear at the level of thou-
be seen in the case of the two-step gradient from ESI MS-MS sequencing and data- sands. The reason resides in the maximum
(Figure 2, chromatogram 2), the gradient base search can be found in supplementary pressure limit of about 70 bar produced by
shape was reasonably reproducible for all tables (available at chromatographyonline. the gastight microsyringe when pushing the
three repetitions of manual loading and com/Rehulka-supplementary). These mobile phase down through the capillary
the use of four-step elution (Figure 2, chro- results clearly confirm the advantages of column. This precludes the possible use of
matogram 3) provided a less steep gradient application of the given microgradient longer columns with a narrower diameter
curve according to expectations. setup for proteomic experiments. and stationary phases made of smaller par-
All three elution modes were also exam- A tryptic digest made from the whole ticles. Both of these column characteristics
ined in experiments performed to separate cell lysate of Francisella tularensis subsp. significantly influence the separation effi-
peptides from a tryptic digest of BSA. The tularensis strain Schu S4 was chosen for ciency for very complex peptide mixtures.
reason was to optimize the whole instru- evaluation of the performance of the Nevertheless, quite long analysis times may
mental setup for more-complex samples. A microgradient separation with a complex be required for the instrumental approach
comparison of the obtained results is pre- peptide sample. F. tularensis is a patho- (28). Survey 2D gel electrophoresis–based
sented in Figure 3. The simple isocratic elu- genic Gram-negative bacterium that is studies, which do not use a sensitive fluo-
tion with 60% (v/v) acetonitrile led to a fast the causal agent of the serious infectious rescent labeling, provide “only” hundreds
elution of BSA tryptic peptides (a duration disease called tularemia (22). F. tularensis of protein spots. However, much higher
of about 4 min since the time when the subsp. tularensis is the most virulent form sample loads such as 200 µg are necessary
first BSA peptide appeared in the eluate). of the microbe (23). The molecular mech- in this case (29). To some extent, the use of
A low separation efficiency was observed anisms responsible for the pathogenicity the microgradient system for protein identi-
in this case, as it is presented in the inset are not completely understood (24). So far, fication could be competitive to minigel 2D
of Figure 3a, where extracted ion chro- no safe and efficient vaccine that would electrophoresis experiments because of the
matograms (XICs) of four selected peptide protect against multiple strains has been low sample consumption, easy applicability,
precursors of BSA are shown. The elution developed (25). In this context, identifying and time-saving advantage.
of these precursors occurred during a short immunogenic proteins in proteomic stud-
time window (within 2.5 min) and a sig- ies may represent a step forward. Concluding Remarks
nificant overlapping of signals (for example, An aliquot (2 µg) of the Francisella pro- This work shows a simple and inexpen-
those for the precursors at m/z 722.32 and tein digest was subjected to separation in sive alternative setup for nanoLC separa-
582.32) appeared. The two-step gradi- the microgradient chromatographic system tion coupled with ESI-MS and MS-MS
ent (2% and 60% [v/v] acetonitrile) pro- using both two-step and four-step gradi- analysis of peptide samples in proteomics
longed the duration up to 35 min. Selected ent options (Figure 4). The time periods of studies. The applicability of the micro-
monitored precursors were eluted within separation at the flow rate of 250 nL/min gradient chromatographic system was
about 19 min and were separated success- were predetermined by the total volume demonstrated with a complex peptide
fully (Figure 3b, inset) in this arrangement. of the aspirated microgradient-forming mixture originating from digested
Finally, the four-step gradient (2–20–40– solutions: 140 and 196 min, respectively. whole-cell lysate protein extract of the
60% [v/v] acetonitrile) achieved much bet- According to expectations, the four-step bacterium Francisella tularensis. When
ter separation: peptides were eluted in a gradient proved to be more efficient, which summarizing advantages of the opti-
time window of about 50 min and selected resulted in a higher number of identified mized microgradient system, we could
monitored precursors were nicely separated proteins when compared to the other elu- point out the following:
within about 26 min (Figure 3c, inset). tion mode. In total, there were 119 and 93 • It is simple to construct by connect-
Compared to isocratic elution, the results proteins, respectively, assigned to F. tular- ing a gastight microsyringe, capillary,
of MS analysis were improved in several ensis subsp. tularensis strain Schu S4 entries and capillary column to a nanoESI
parameters: number of acquired MS-MS in the SwissProt protein database under the ion source of an MS instrument
scans, number of significant peptide hits given search conditions (see supplementary and it is much cheaper for purchase
and sequences in the Mascot search, over- tables). A majority of the identified pro- compared to commercial separation
all protein sequence coverage, and Mascot teins were found associated with ribosomes systems.

• No splitting of the mobile phase flow is required and thus the (13) L. Rieux, E.-J. Sneekes, and R. Swart, LCGC North Am. 29, 926–935
solvent consumption is very low as a consequence. (2011).
• It is small in size and can be placed somewhere in the laboratory. (14) V. Kahle, M. Vázlerová, and T. Welsch, J. Chromatogr. A 990, 3–9 (2003).
• The reproducibility of the system is high—four selected peptide (15) D. Moravcová, V. Kahle, H. Řehulková, J. Chmelík, and P. Řehulka, J.
precursor ions from a BSA digest showed an RSD of total elu- Chromatogr. A 1216, 3629–3636 (2009).
tion volumes of less than 0.6% in four LC–MS separation runs (16) V. Franc, M. Šebela, P. Řehulka, R. Končitíková, R. Lenobel, C.
(in the case of an automated loading of gradient, the retention Madzak, and D. Kopečný, J. Proteomics 75, 4027–4037 (2012).
times are even more reproducible with an RSD of 0.1% [14]). (17) V. Franc, P. Řehulka, A. Padiglia, R. Medda, G. Floris, and M. Šebela,
• If contaminated, the system can be cleaned easily by simply Electrophoresis 34, 2357–2367 (2013).
washing the capillary and microsyringe. If necessary, the (18) V. Franc, P. Řehulka, M. Raus, J. Stulík, J. Novak, M.B. Renfrow, and
microsyringe or capillary can be replaced by cheap spare parts M. Šebela, J. Proteomics 92, 299–312 (2013).
after dismantling the system. (19) L. Balonová, L. Hernychová, B.F. Mann, M. Link, Z. Bílková, M.V.
Of course, there are also disadvantages. For example, it requires Novotný, and J. Stulík, J. Proteome Res. 9, 1995–2005 (2010).
a manual handling for sample injection and column washing; (20) M. Hubálek, L. Hernychová, M. Brychta, J. Lenčo, J. Zechovská, and J.
there is no software control; the shape of the gradient is not per- Stulík, Proteomics 4, 3048–3060 (2004).
fectly linear; there is a pressure limitation given by the syringe use (21) H.K. Hustoft, L. Reubsaet, T. Greibrokk, E. Lundanes, and H. Malerod,
(~70 bar in our case); and shorter columns with a lower back pres- J. Pharm. Biomed. Anal. 56, 1069–1078 (2011).
sure have to be used, which influences the separation efficiency. (22) A. Tärnvik and L. Berglund, Eur. Respir. J. 21, 361–373 (2003).
(23) J. Ellis, P.C.F. Oyston, M. Green, and R.W. Titball, Clin. Microbiol. Rev.
Acknowledgments 15, 631–646 (2002).
The financial support of grant No. NT13721-4/2012 of Min- (24) K. Konecna, L. Hernychova, M. Reichelova, J. Lenco, J. Klimentova,
istry of Health, Czech Republic, and a long-term organization J. Stulik, A. Macela, T. Alefantis, and V.G. DelVecchio, Proteomics 10,
development plan no. 1011 from the Faculty of Military Health 4501–4511 (2010).
Sciences, University of Defence, Czech Republic, are gratefully (25) D. Rockx-Brouwer, A. Chong, T.D. Wehrly, R. Child, D.D. Crane, J.
acknowledged. This project was also supported by the Ministry of Celli, and C.M. Bosio, PLOS One 7, e37752 (2012).
the Interior of the Czech Republic (Project No. VG20112015021) (26) D.W. Huang, B.T. Sherman, and R.A. Lempicki, Nature Protoc. 4,
and by the Academy of Sciences of the Czech Republic (Insti- 44–57 (2009).
tutional support RVO: 68081715). M.S. was supported by the (27) Y. Ishihama, T. Schmidt, J. Rappsilber, M. Mann, F.U. Hartl, M.J.
Education for Competitiveness Operational Programme project Kerner, and D. Frishman, BMC Genomics 9, 102 (2008).
CZ.1.07/2.3.00/30.0004 (POSTUP: Encouraging the creation (28) T. Köcher, P. Pichler, R. Swart, and K. Mechtler, Nat. Protoc. 7, 882–
of excellent research teams and intersectoral mobility at Palacký 890 (2012).
University in Olomouc) from the Ministry of Education, Youth (29) S.L. Hanna, N.E. Sherman, M.T. Kinter, and J.B. Goldberg, Microbiol-
and Sports of the Czech Republic. Professor Lenka Hernychová ogy 146, 2495–2508 (2000).
from Masaryk Memorial Cancer Institute in Brno, Czech Repub- René Lenobel, Marek Šebela, and Vojtěch
lic, is thanked for providing us with Francisella tularensis subsp. Franc are with the Department of Protein Biochemistry
tullarensis strain Schu S4 protein digest. and Proteomics at the Centre of the Region Haná for
Biotechnological and Agricultural Research, Faculty of
Science at Palacký University in Olomouc, Czech Republic.
References
Helena Řehulková and Pavel Řehulka are with the
(1) J.R. Yates, C.I. Ruse, and A. Nakorchevsky, Annu. Rev. Biomed. Eng. Department of Molecular Pathology and Biology, Faculty
11, 49–79 (2009). of Military Health Sciences at the University of Defence in
(2) F. Chevalier, Proteome Sci. 8, 23 (2010). Hradec Králové, Czech Republic. Vladislav Kahle and
(3) D.R. Mueller, H. Voshol, A. Waldt, B. Wiedmann, and J. Van Oostrum, Dana Moravcová are with the Institute of Analytical
Chemistry of the ASCR, v. v. i. in Brno, Czech Republic.
Subcell. Biochem. 43, 355–380 (2007).
Direct correspondence to: pavel.rehulka@unob.cz ◾
(4) G. Chen and B.N. Pramanik, Expert Rev. Proteomics 5, 435–444
(2008).
(5) Y. Zhang, B.R. Fonslow, B. Shan, M.-C. Baek, and J.R. Yates III, Chem.
Rev. 113, 2343–2394 (2013).
(6) B. Thiede, W. Höhenwarter, A. Krah, J. Mattow, M. Schmid, F.
Schmidt, and P.R. Jungblut, Methods 35, 237–247 (2005).
(7) P. Schoenmakers, Annu. Rev. Anal. Chem. 2, 333–357 (2009).
(8) Y. Ishihama, J. Chromatogr. A 1067, 73–83 (2005).
(9) J.Z. Bereszczak and F.L. Brancia, Comb. Chem. High Throughput Screen. S E P A R A T E S from the rest
12, 185–193 (2009).
(10) E. Nägele, M. Vollmer, P. Hörth, and C. Vad, Expert Rev. Proteomics  Better Prices & Selection
Sonntek, Inc.
1, 37–46 (2004).  Lab Solutions & Support INTERESTED? Ph:201-236-9300
(11) D.R. Stoll, X. Li, X. Wang, P.W. Carr, S.E.G. Porter, and S.C. Rutan, J.  Research lamps/HPLC sonntek@aol.com
 Instruments & Systems www.sonntek.com
Chromatogr A 1168, 3–43 (2007).
(12) P.R. Cutillas, Curr. Nanosci. 1, 65-71 (2005).
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Caroline West, LCGC’s 2015 Emerging

Leader Award Winner, Focuses on
the Fundamentals of Selectivity
Caroline West, the winner of the 2015 LCGC Emerging Leader in Chromatography award, has a
diverse set of scientific interests—in high performance liquid chromatography (HPLC), supercritical
fluid chromatography (SFC), hydrophilic-interaction chromatography (HILIC), and enantioselective
separations—but all are primarily focused on the fundamentals of chromatographic selectivity. West,
who is an associate professor of analytical chemistry at the University of Orleans in France, has
published approximately 50 papers on these topics, including an influential review on graphitic carbon
stationary phases, a significant paper that unravels retention mechanisms in the hydrophilic-interaction
chromatography (HILIC) mode, and an article on the development of a useful classification of stationary
phases for SFC. West recently spoke to LCGC about her scientific beginnings and recent work.
tionary phases get started? What were I believe that the most important
the biggest challenges in creating it, information we gained from this work is
and what benefits does this classifica- that the diversity of selectivities that are
tion bring to the field? available to the SFC chromatographer is
West: At the time I started my PhD the- extremely wide and strongly dependent
sis, in 2002, SFC was quite unpopular. on stationary phases. I hope it is helpful
I did not receive much encouragement to those who are now getting acquainted
to get involved in this field and rather with the technique, especially in the
was met with skepticism, which is a pity early steps of method development when
because SFC really needs more involve- a column must be selected. We keep on
ment from academics. I am very happy increasing the list of columns tested
to say that this is changing now, at least whenever interesting ones appear on
in Europe. Although much has been the market, especially focusing on high-
done in the past, it is clearly not suffi- performance stationary phases (such
cient yet because the way this technique as sub-2-µm particles and superficially
is practiced has changed a great deal over porous particles). I hope to make the
Where or how did your interest in chem- the years. The purpose of my PhD the- whole thing available on the internet in
istry and analytical chemistry begin? sis was to investigate the applicability of an open source format.
West: As a young student I was inter- porous graphitic carbon (PGC) station-
ested in forensics. But that was a long ary phases in SFC. I wanted to compare What prompted your research into
while ago, when “CSI” was not a TV PGC to other stationary phases that were the retention behavior of zwitter-
series and nobody knew much about it. more commonly used in SFC, and that ionic stationary phases for HILIC?
So it had the attraction of mystery to is how we ended up building a classifica- What effect has this research had for
me. All I knew was that chemistry, and tion of columns. The biggest challenge HILIC users?
analytical chemistry in particular, was was to design a column-testing procedure West: Zwitterionic stationary phases were
one way to get to forensics. Later, I did that would be adequate for a very large a pretext to investigate HILIC retention
spend some time in a forensics labora- range of columns, because we wanted to mechanisms. I had a feeling that SFC and
tory, working on the analysis of explo- include the most polar phases (like bare HILIC mechanisms had a lot in com-
sives residues, but I did not pursue that silica) down to the least polar phases mon (a combination of adsorption onto
field. Instead, I moved on to the funda- (like C18-bonded silica). All of them had a stationary phase and partition involving
mentals of chromatography. to be tested with the same analytes and a layer of adsorbed mobile-phase compo-
the same mobile-phase composition. Lin- nents), so I wanted to get a closer look at
You have done some significant ear solvation energy relationships (LSER) that. Also, the usual LSER methodol-
research in SFC. How did your proj- with Abraham descriptors proved to be ogy is intended for neutral species, while
ect on the classification of SFC sta- an efficient tool in this respect. HILIC required users to take account of
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ionizable species as well, so some method- impurity profiling of pharmaceutical have come to me with an idea and some
ological development was necessary there. compounds. While chiral SFC has financial support to carry on with their
So we developed a model to describe long been a favorite in pharmaceutical projects. I accept them when the idea is
HILIC retention mechanisms. My under- companies, especially for the purpose of interest to me and fits with the proj-
lying thought was that it would be great of purifying enantiomers at a low cost, ects of our laboratory, and when I know
to use this model for a classification of it is now being considered for achiral that I can provide access to a machine
HILIC systems. I did not have time to do analysis too. In this respect, there are and will be able to afford sufficient time
it myself, but I am glad that others (Wolf- still a number of points that need to to the student.
gang Lindner’s team) have used this idea. be explored in more detail, such as the
appropriate operating compositions to Have you faced any difficulties as
Can you tell us about your research in ensure both good chromatography and a young, female scientist based in
other areas of separation science such mass spectrometric detection, or the Europe?
as enantioselective separations? possibility to validate methods to meet West: I believe that my difficulties
West: Enantioselective separations have the requirements of quality control are those encountered by any young
kept me busy for the past five years. While laboratories. woman who wants to have children and
SFC is very successful at it, the general lack be involved in her job. You can do it,
of understanding of the enantioseparation You have many publications (~50 arti- but you definitely need some help at
processes is a topic of frustration for me. I cles and 1 book chapter) and you have home. I consider myself very lucky in
thought that LSER models again could a high h-index (16) for such a young the matter. Actually, all the students I
be helpful in this field. But here too, the researcher. Is it hard to find the bal- have supervised so far have been young
usual model is not sufficient because there ance between your research and the women. It was not something I chose;
is nothing in Abraham descriptors to take demands of teaching, supervising it is simply the result of a large propor-
account of the shape of the molecules. So PhD and master students, and giving tion of women in analytical sciences (as
I introduced two extra descriptors to take lectures at various universities and compared to other chemistry special-
account of flexibility and sphericity. They conferences? ties). They all have been brilliant, so I
proved to be rather good, we were able West: Teaching is somewhat refresh- hope they will not give up science.
to provide some accurate descriptions of ing because it forces me to keep up to
retention and separation mechanisms. An date with other analytical chemistry What do you plan to focus on next? Is
immediate application of this work is to areas that I do not practice in research. there one big problem in separation
compare enantioselective systems—chiral However, it also means that my research science that you really want to tackle?
stationary phases, effects of mobile-phase activities are strongly segmented, and West: I am interested in the limits of
composition, and a comparison of LC it is difficult to keep focused on your SFC for the analysis of polar molecules,
and SFC to facilitate method transfer. A projects when you are often interrupted. particularly biomolecules. While the way
future application could be to have some This lack of continuity is a great source to approach the technique has changed,
prediction of the chromatographic system of exasperation to me because it impairs I expect it will move even further to less
best suited to any required enantioresolu- productivity. The supervision of PhD carbon dioxide and more water in the
tion. We have had some success with that, and master students is also a highly mobile phase (that is the other end of the
but it still needs some improvement. demanding task if you really want to technique, usually called “enhanced-flu-
follow their progress and help them idity LC”). And next, when everybody
What research are you the most proud improve in their science, thus I prefer will have been converted to SFC, my job
of thus far? to limit their numbers. Students should there shall be finished. It will start to be
West: Self-satisfaction is not a familiar not suffer from my limited time allow- boring to me, so I will move to some-
feeling to me, as I am a real perfection- ance. This is a two-way commitment: thing very different.
ist. Do not misunderstand me: I do not They provide hands and brains, and I
consider perfectionism a good quality, must provide education, assistance, and This interview has been edited for
and it is probably getting worse with interest in their work. length and clarity. To read more about
time, now that I know that people may West visit: www.chromatographyonline.
read my papers! I can measure the prog- Do your students’ interests influence com/2015-lcgc-awards. For more inter-
ress I have made in my own understand- the research projects you select? views like this please visit chromatogra-
ing of chromatographic processes, and I West: To be honest, I should say that phyonline.com and look under the "Fea-
hope that the work we have published minor projects are brought up by stu- tured LCGC Interviews" section on our
has helped in appreciating the merits of dents, while major projects surge from homepage. ◾
unified SFC for what they really are. my own desire, but are sometimes influ-
enced by students. I have often noticed
What is your research group cur- that a certain degree of ignorance is an For more information on this topic,
rently focused on? advantage to propose innovative ideas please visit
West: We are busy developing high- because limited knowledge also implies www.chromatographyonline.com
performance achiral SFC methods for limited preconceptions. Some students
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Products & resources

Mass spectrometer Chiral columns
The Vion IMS Q-Tof mass Immobilized chiral
spectrometer from Waters is columns from Daicel are
designed for ion mobility mass designed with polymers
spectrometry analyses in research immobilized onto
and routine applications. According silica. According to the
to the company, the instrument company, the various
allows users to identify and selectors used in the
quantify analytes with fast method Chiralpak IA, IB, IC, ID,
development and high sample IE, and IF columns allow
throughput. the columns to separate
Waters Corporation, racemates of most pharmaceutical, agrochemical, and fragrance
Milford, MA. compounds.
www.waters.com/vion Chiral Technologies, Inc., West Chester, PA.
www.chiraltech.com
Air samplers GC–MS-MS system

Thermosorb-N air samplers from The Q Exactive GC hybrid quad-
Ellutia are designed to monitor rupole-Orbitrap GC–MS-MS
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workplace. According to the Scientific is designed to provide
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the air sampler, it captures any of samples in a single analy-
nitrosamines present so that sis for compound discovery,
they can be analyzed, and levels identification, and quantitation.
can be reported. According to the company,
Ellutia, the system combines high-
Charleston, SC. resolution accurate-mass mass
www.ellutia.com spectrometry with gas chromatography. Thermo Fisher Scientific,
San Jose, CA. www.thermoscientific.com/QExactiveGC
HOW TO IMPROVE THROUGHPUT

IN YOUR LC/GC LAB
ON-DEMAND WEBCAST Originally aired June 25, 2015
Register for free at http://www.chromatographyonline.com/lcgc/throughput Key Learning Objectives
EVENT OVERVIEW: ■ Demonstrate how one common interface
can increase productivity and simplify
This webcast is focused on how a laboratory using multi-vendor and/or multi-
workfows in the lab – even when running
technique chromatographic instrumentation can enhance their productivity and multiple chromatographic techniques
ensure data integrity and full traceability of results. Application and use scenarios ■ See how advanced, fully embedded
commonly encountered by food, environmental and industrial labs will be highlighted, calculation and reporting features can lower
all employing a common software solution to unify methods, raw data, metadata and risks by ensuring full and comprehensive
fnal results. This software solution has the following features and benefts: traceability given that data no longer need
to be exported or transcribed
■ Common, intuitive user interface experience across all chromatographic techniques ■ Demonstrate how a more sophisticated
■ Robust integration that yields highly reproducible results, even for complex algorithm can fully automate the processing
chromatograms in GC environmental analyses, without manual intervention of even complex chromatograms –
enhancing your lab’s productivity while
■ Powerful, customizable calculation and reporting capability which eliminates increasing reproducibility and consistency
having to export results to 3rd party tools. For example, reporting the purity of in results
incoming raw materials or comparing actual results to a products labeling claims.
Who Should Attend Presenter:

■ Analytical scientists, researchers, lab directors/managers/technicians that test Kyle Oberndorfer
organic samples across food, environmental and industrial end markets (and more) Liquid Chromatography Product Manager
PerkinElmer
■ Liquid and gas chromatographers that use instruments from multiple providers
Moderator:
For questions, contact Kristen Moore at kmoore@advanstar.com Kristen Moore, Multimedia Producer, LCGC
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Sample cleanup Autosampler vials

The EconoPrep sample cleanup MicroSolv’s reduced surface activity glass vials are
system from FMS is designed to designed to be less reactive to a sample without
combine manual sample preparation any coating or surface treatments. According to the
steps into one automated process. company, the autosampler vials are suitable for
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fractionation, and concentration for avoid sodium adducts from glass vials.
up to eight samples simultaneously MicroSolv,
in less than 1 h. Eatontown, NJ.
FMS, Inc., www.mtc-usa.com
Watertown, MA.
www.fms-inc.com
Sample cleanup system Sample preparation guide

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Optimize is designed for applications Phenomenex is designed to help users
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Certified reference materials kit Light-scattering detector

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multiresidue pesticide method, and supercritical fluid chromatography.
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transition tables, is available for Alfortville, France.
download for use with the kit. www.sedere.com
Restek Corporation,
Bellefonte, PA.
www.restek.com
SPE cartridges SPE columns

SupelIMIP solid-phase TARGA C18 UltraMicro Spin
extraction cartridges from solid-phase extraction columns
Supelco are designed for from The Nest Group are
the analysis of aminoglyco- designed in reversed-phase
side antibiotics in difficult solvent-resistant housings for
matrices such as meat, purification of small samples
honey, and cell culture. (3–30 µg). According to the
According to the company, company, desalting with its
the cartridges are based water-wettable columns allows
on molecularly imprinted loading in 100% water, improv-
polymer technology. ing recovery of polar peptide,
Supelco/Sigma-Aldrich, glycopetide, or glycan molecules before LC–MS.
Bellefonte, PA. The Nest Group,
www.sigma-aldrich.com/supelmip Southborough, MA.
www.nestgrp.com
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Mass spectrometer Sample preparation columns

The Orbitrap Fusion Lumos Clean Screen XCEL columns
Tribrid mass spectrometer from UCT are designed
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Scientific is designed throughput, reduced sample
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molecule quantitation According to the company,
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According to the company, step load, wash, and elute
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Thermo Fisher Scientific, UCT, LLC,
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www.thermoscientific.com www.unitedchem.com
Detector modules Water purification systems

UV–vis and fluorescence EMD Millipore’s AFS 40E, 80E, 120E,
detector modules from and 150E water purification systems are
D-Star are designed designed to provide clinical laboratories
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systems. According to the company, the systems provide users with
company, the modules are continuous real-time monitoring and
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laboratories, QC, and R&D. Billerica, MA.
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Instruments, Inc.,
Manassas, VA.
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UHPLC/HPLC Method Development for

Pharmaceutical-Related Substances
How to Translate Methods, Maximize Resolution, and Comply with USP <621>
ON-DEMAND WEBCAST (Originally aired May 26, 2015)
Register for free at http://www.chromatographyonline.com/lcgc/method
EVENT OVERVIEW: Who Should Attend?
The frst half of this webinar will focus on chromatographic selectivity and how it ■ Method developers for UHPLC and HPLC methods
can be exploited by adjusting key method parameters to maximize resolution for in pharmaceutical, chemical, clinical, environmental,
various analyte mixtures and classes. This will include optimizing analytical method agrichemical, university and governmental
development workfows to reduce development time, but maintain critical peak pair laboratories
resolution. This rationale will be applied to an HPLC / UHPLC method development ■ Anyone looking to move methods across diferent
case study for a pharmaceutical-related substances analysis. instruments, particle sizes and types
The second half of the webinar will review HPLC/UHPLC isocratic and gradient
method translation activities with example pharmaceutical assay and related Presenter:
substances methods. Highlights of the new USP <621> guidance regarding changes Alan P McKeown BSc, PhD, CChem, FRSC
to chromatographic methods will be included. Method translations between diferent Business Development Director
particle sizes and column geometries, including totally porous and solid-core Advanced Chromatography Technologies
particle platforms will be discussed, and diferences between these situations will be Ltd (ACT)
highlighted. Finally, maximizing resolution for complex natural product samples will
be illustrated using solid-core particle columns. Moderator:
Laura Bush
Key Learning Objectives: Editorial Director
■ Utilize chromatographic selectivity to maximize resolution LCGC
■ Optimize HPLC/UHPLC method development workfows For questions, contact Kristen Moore
■ Understand how to develop methods that comply with the new 2014 USP <621> at kmoore@advanstar.com
guidance
■ Translate isocratic and gradient methods efectively between totally porous and Presented by Sponsored by
solid-core particle columns
■ Leverage solid-core columns for ultrahigh resolution separations of natural
product samples
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Autosampler syringes Data analysis and graphing software

Autosampler syringes Origin and OriginPro 2015 data
for liquid and gas analysis and graphing software
chromatography from from OriginLab add more
Hamilton are designed than 100 new features and
specifically for CTC PAL improvements. According to
liquid chromatography the company, enhancements
autosampler systems. include collapsible menus,
According to the company, project file search for string,
the S-Line syringes complement its existing C-Line and X-Type thumbnail previews of graphs,
syringes, and are suitable for everyday use. and tooltips that display folder
Hamilton Company, or window comments in
Reno, NV. Project Explorer.
www.hamilton.com OriginLab,
Northampton, MA.
www.originlab.com
Combustion ion chromatograph LC system

Metrohm’s combustion PerkinElmer’s Altus UPLC system is
ion chromatograph (CIC) is designed for use in food, environmental,
designed to automate the and industrial laboratories for the
determination of halogens detection of adulterants, contaminants,
and sulfur. According to and pollutants. According to the company,
the company, the system’s the system is controlled through Waters’
autosampler can run both Empower 3 software and combines
solid and liquid samples, and advanced fluidics with hybrid particle
flame sensor technology is used to measure the light intensity from the columns.
pyrolysis oven during combustion. PerkinElmer, Inc.,
Metrohm USA, Waltham, MA.
Riverview, FL. www.perkinelmer.com
www.metrohmusa.com/CIC
Sample cleanup system Capillary tubing

Pickering’s DEXTech automated Tight Tolerance Vision
Sample-Clean system for Dioxin System flexible fused-silica
and PCB samples reportedly uses capillary tubing from Poly-
a multistage process that reduces micro Technologies, offered
the throughput to 96 min and the by Molex, is designed to pro-
solvent consumption to less than vide precise internal diam-
640 mL. According to the com- eter tolerances. According to
pany, the system was developed the company, the tubing is
in cooperation with the German drawn from high-purity syn-
governmental laboratory, Chemi- thetic fused-silica preforms
cal and Veterinary Analytical Insti- and has industry-standard
tute Münsterland-Emscher-Lippe outer diameter dimensions.
(CVUA MEL), and LCTech GmbH Polymicro Technologies,
Germany. Pickering Laboratories, Mountain View, CA. Phoenix, AZ.
www.pickeringlabs.com www.molex.com/polymicro/capillarytubing.html
2D-LC system SEC–MALS detector

Shimadzu’s Nexera-e comprehen- The Dawn Heleos-II mul-
sive two-dimensional liquid chro- tiangle light scattering
matograph is designed for use with (MALS) detector from Wyatt
complex sample matrices. The Technology is designed for
system reportedly incorporates the absolute molecular weight
company’s Nexera X2 photodiode- and size determinations of
array detector for sampling rates polymers and biopolymers
up to 200 Hz and can be used in solution. According to the
with its LCMS-80 triple quadrupole company, the detector may
LC–MS-MS system for ultrahigh- be connected in series to
speed data scanning. any chromatographic system
Shimadzu Scientific to determine absolute molar masses without the use of reference stan-
Instruments, dards or column calibration.
Columbia, MD. Wyatt Technology Corp., Santa Barbara, CA.
www.ssi.shimadzu.com www.wyatt.com
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CALENDAR
29–31 July 2015 20–21 October 2015
ISPPP 2015, 35th Symposium and Gulf Coast Conference 2015
Exhibit on the Separation and Houston, TX
Characterization of Biologically www.gulfcoastconference.com/index.cfm
Important Molecules
Philadelphia, PA 20–22 October 2015
www.isppp.org International Symposium on
GPC/SEC and Related Techniques
21–25 June 2015 17–19 August 2015 2015
42nd International Symposium Conference on Small Molecule Philadelphia, PA
on High Performance Liquid Science (CoSMos 2015) www.gpcevent.com
Phase Separations and Related San Diego, CA
Techniques (HPLC 2015) www.cosmoscience.org/blog/2015- 3–4 November 2015
Geneva, Switzerland cosmos/ 24th International Light
www.hplc2015-geneva.org Scattering Colloquium (ILSC)
19–20 August 2015 Santa Barbara, CA
22–24 June 2015 International Conference on www.wyatt.com/events/ilsc/ilsc-2015-
IFCC-EFLM European Congress of Environmental Forensics registration.html
Clinical Chemistry and Laboratory Putrajaya, Malaysia
Medicine www.ienforce.upm.edu.my/index.php 5 November 2015
Paris, France ILSC 2015 Focus Meeting
www.paris2015.org 23–28 August 2015 Santa Barbara, CA
35th International Symposium on http://www.wyatt.com/events/ilsc/ilsc-
28 June–1 July 2015 Halogenated Persistent Organic 2015-focus-meeting.html
RDPA 2015: Recent Pollutants (DIOXIN2015)
Developments in São Paulo, Brazil 3–6 November 2015
Pharmaceutical Analysis www.dioxin2015.org 7th International Symposium on
Perugia, Italy Recent Advances in
rdpa2015.chimfarm.unipg.it 6–10 September 2015 Food Analysis
Euroanalysis XVIII Prague, Czech Republic
30 June–3 July 2015 Bordeaux, France www.rafa2015.eu/RAFA_2015_1st_flyer.pdf
21st International Symposium www.euroanalysis2015.com
on Separation Sciences 12–15 November 2015
(ISSS 2015) 28–30 September 2015 GPSS: Gazi Pharma Symposium
Ljubljana, Slovenia 4th International Conference Series
www.isss2015.si on Forensic Research & Technology Antalya, Turkey
Atlanta, GA www.gpss2015.org
22–24 July 2015 forensicresearch.conferenceseries.com
SFC 2015 — 9th International 15–17 November 2015
Conference on Packed 5–7 October 2015 12th International Symposium
Column SFC International Symposium on on Persistent and Toxic
Philadelphia, PA Applied Chemistry (ISAC) Substances (ISPTS)
www.greenchemistrygroup.org/Registra- Bandung, Indonesia Riverside, CA
tion.html situs.opi.lipi.go.id/isac2015/ pts2015.ucr.edu
26–29 July 2015 16–20 October 2015 18–19 November 2015

PREP 2015: 28th International Native Mass Spectrometry-Based Petroleum, Refining, and
Symposium on Preparative and Structural Biology Environmental Monitoring
Process Chromatography Pacific Grove, CA Technologies (PEFTEC 2015)
Philadelphia, PA www.asms.org/conferences/asilomar-con- Antwerp, Belgium
www.prepsymposium.org ference/asilomar-conference-homepage www.peftec.com
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SHORT COURSES
Somerset, NJ Edison, NJ
easinc.org/wordpress/?page_id=941 proed.acs.org/course-catalog/courses/
how-to-develop-validated-hplc-methods-
HPLC rational-design-with-practical-statistics-
and-troubleshooting/
1–4 September 2015
High Performance Liquid 5 November 2015
Chromatography: Fundamentals, ILSC 2015 Focus Meeting
GC Troubleshooting, and Method Santa Barbara, CA
Development http://www.wyatt.com/events/ilsc/ilsc-
8–11 September 2015 Chicago, IL 2015-focus-meeting.html
Gas Chromatography: proed.acs.org/course-catalog/courses/
Fundamentals, Troubleshooting, high-performance-liquid-chromatography- Hyphenated
and Method Development fundamentals-troubleshooting-and-
Chicago, IL method-development/ 9 November 2015
proed.acs.org/course-catalog/courses/ Comprehensive Gas
gas-chromatography-fundamentals-trou- 14–15 October 2015 Chromatography-Mass
bleshooting-and-method-development How to Develop Validated Spectrometry Training
HPLC Methods: Rational Design Milton Keynes, UK
15–16 November 2015 with Practical Statistics and www.anthias.co.uk/training-courses/1day-
Practical Gas Chromatography Troubleshooting comprehensive-gcms-agilent-qqq
Hi-tech, Low-tech and No-tech

Preparing Samples for LC and LC-MS
ON-DEMAND WEBCAST
Register for free at http://www.chromatographyonline.com/lcgc/tech
EVENT OVERVIEW: Key Learning Objectives
Sample preparation for chromatographic analysis of small molecules such as drugs and ■ Key developments in three main
pesticides has evolved signifcantly over the past decade, becoming faster, more automated, approaches to sample preparation for
and capable of extracting analytes for analysis at part-per-trillion and lower levels. This the analysis of small molecules: SPE,
presentation will compare and contrast three approaches that have evolved most signifcantly. QuEChERS, and no sample prep.
Solid-phase extraction is a classical technique that has received a recent high-tech boost ■ Tips for choosing the best approach,
from automation and miniaturization. Over the past decade, the QuEChERS (Quick, Easy, depending on variables such as
Cheap, Efective, Rugged and Safe) approach provided a simple, low-tech alternative that has sample type, analytes, and desired
greatly impacted the analysis of pesticides and residues from numerous matrices, especially analytical performance, plus
foods. Finally, multidimensional detection has led to a no-tech sample preparation approach managerial variables including cost,
in LC-MS analysis: none. Especially in multi-residue drug analysis from biological samples, throughput and simplicity
methods involving no sample preparation at all are generating interest. The choice of ■ How these decisions play out in real-
approach depends on several chemical variables, including sample type, analytes, and desired life examples from the analysis of drug
analytical performance, plus managerial variables including cost, throughput and simplicity. and pesticide residues in biological
Using examples drawn from the analysis of drug and pesticide residues in biological samples, samples
these three approaches will be compared and contrasted with a view toward assisting
laboratories in choosing or improving their own methods and techniques for these often Presenter
difcult analyses. Nicholas H. Snow
Department of Chemistry and Biochemistry
Who Should Attend
Center for Academic Industry Partnership
■ Analysts working in analysis of small molecules using HPLC and LC-MS who wish to Seton Hall University
improve their sample preparation
■ Scientists working in felds such as pharmaceutical, biological, and environmental analysis Moderator
Laura Bush
For questions, contact Kristen Moore at kmoore@advanstar.com Editorial Director, Spectroscopy
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AD INDEX
ADVERTISER PAGE ADVERTISER PAGE
CASSS � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 376 Pickering Laboratories � � � � � � � � � � � � � � � � � � � � � � � � � � � 376

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A Faster, More Sensitive Sample Preparation

Solution for Bioanalytical Samples
Live Webcast: Tuesday, June 23, 2015 at 8:00am/ PDT/ 10:00am CDT/ 11:00am EDT
Register for free at http://www.chromatographyonline.com/lcgc/faster Analysts will learn how to…
■ Increase the sensitivity of their
EVENT OVERVIEW
downstream analysis
Bioanalysis poses many challenges due to the complexity of the matrices involved. These highly
complex samples require a sample preparation step to remove matrix interferences however ■ Process small or limited sample
sample volume and required sensitivity must be considered when choosing a sample preparation volumes without losing
technique. While there are many options for sample preparation of bioanalytical samples, most are sensitivity
not amenable to extremely small sample volumes such as 10 µL or they result in dilute eluents that ■ Analyze compounds that pose
require a timely dry down step to achieve the sensitivity needed for accurate analysis. challenges during a dry down
step such as peptides and
This webinar will introduce a faster, more sensitive thermolabile compounds
sample preparation solution for bioanalytical samples Presenter
■ Save time and labor costs
that will allow analysts to: Matt Brusius
Sample Preparation
■ Increase the sensitivity of their downstream analysis Product Manager
Phenomenex Sponsored by
■ Process small or limited sample volumes without
losing sensitivity Moderator
■ Save time and labor costs Kristen Moore
Presented by
■ Providing data from clinical, pharma, forensics. Multi Media Producer
LCGC
Who Should Attend
■ Analysts who are working with bioanalytical samples that would like to increase their
sensitivity, reduce processing time, avoid loss of analytes that pose challenges during a dry For questions, contact Kristen Moore at
down step, or process small or limited sample volumes. kmoore@advanstar.com
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THE ESSENTIALS
GC Temperature Programming—10
Things You Absolutely Need to Know
1. Temperature affects not only reten-
tion but also relative retention in gas
chromatography (GC) and therefore, Peak 13
tR: 23.39 min
when we change temperature, we Elution temp.: 264 °C
also change the selectivity of the Initial temp. 40 °C hold for 1 min
Peak 8,9
tR: 21.82 min
separation. This is true as we alter Ramp rate
Final temp.
10 °C/min to 300 °C
300 °C hold for 10 min
Elution temp.: 248 °C
Gradient time (tG) 26 min

the isothermal separation tempera- Column 30 m x 0.32 mm, 0.15-µm df DB5 Peak 1
Carrier gas Helium at 35 cm/s tR: 16.42 min
ture, but also as we change the slope Hold-up time (t0) 1.43 min Elution temp.: 194 °C Retention range:
6.97 min
of the temperature program gradient.
2. An increase of around 30 °C in oven 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0
temperature will reduce retention Time (min)
time by 50%.
Figure 1: Extract of chromatogram showing all pesticide analyte peaks eluted from a
3. Use the method shown in Figure 1 river water extract, analyzed under the screening conditions shown.
to “screen” samples.
4. If the peaks are eluted within a
“window” of less than 7 min (more
accurately t g /4) then isocratic anal- Time (min) Temperature Temperature
(°C) gradient
ysis may be possible (6.97 in our (°C/min)
0 149
example, so isothermal analysis may 8 203 6.75
10 203 0
be possible). 23 285 6.83
5. To approximate the required isother-
mal temperature for the separation,
calculate the temperature at which
the last analyte of interest is eluted 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0
and subtract 45 °C (239 °C in our
example). Isothermal separation at 249 °C,
6. To optimize the separation, alter the optimized empirically in steps of 10 °C
from 219 °C
isothermal temperature in steps of
10 °C, within a range of ±50 °C. If a
suitable separation is not obtained a
temperature gradient should be used.
2.0 3.0 4.0 5.0 6.0
7. For splitless injection, the initial Time (min)
oven temperature should be 20 °C

Figure 2: Optimum separations derived from a thermal gradient analysis (top) and an
below the boiling point of the sample isothermal analysis (bottom).
solvent and an initial hold time of
30 s should be used initially. For split initial isothermal hold unless the ini- elution temperature of the critical
injection, start with an oven tem- tial oven temperature is more than pair (203 °C to help separate peaks
perature 45 °C lower than the elution 30 °C below the boiling point of the 8 and 9 in our screening example).
temperature of the first peak from sample solvent. Empirically determine the length
the screening chromatogram (149 °C 8. The optimum ramp rate for any sep- required for the hold (start with a
in our exercise). For poorly resolved, aration can be estimated as 10 °C 1-min hold) and then resume the
early eluted peaks, decrease initial per hold-up time (7 °C/min in our gradient at the same slope as before.
temperature rather than adding an screening example). 10. Set the final temperature at 20 °C
9. If a suitable gradient slope cannot above the elution temperature of the
be obtained to separate compounds last analyte in the screen, but bear
eluted in the middle of the tempera- in mind that a higher temperature
ture gradient, insert a mid-ramp iso- “burn” period may be required to elute
thermal section at 45 °C below the high-boiling matrix components.
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GCMS-TQ8040 – Smart enough for everyday use in your laboratory
Smart Productivity
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Volume 33 Number 6, 373–440

Volume 33 Number 6 June 2015

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