The Column Instrumentral Innovations 2013

4 December 2013 Volume 9 Issue 22
Cover Story
2 Instrumental Innovations
A selective review of companies who launched products in 2013.
Features
26 Q&A: Miniaturizing Military Detectors
Scientists at Sandia National Laboratories are thinking smaller in the development
of detectors for military use — from the detection of explosives and chemical
weapons to humans.
Scientist Ron Manginell from the laboratories spoke to Bethany Degg of
The Column about the on-going HPLC GC
research in this area. MS SPE
28 Diisobutyl Columns Reduce Solvent Consumption and Offer
powered by Rapid HPLC–MS Analysis of Plasma Oxycodone and its Metabolites
Linda L Risler1 and Anne E Mack2, 1Fred Hutchinson Cancer Research Centre,
2Agilent Technologies, Inc.
LIVE
Oxycodone and its metabolites were analysed by high performance liquid
2 014 chromatography–mass spectrometry (HPLC–MS) using a diisobutyl stable

ing i n Fundamentals of HPLC - Jan 2014
com
bond column. This article discusses the results.
32 A Mixed-Mode Stationary Phase for the Structural Analysis of
Fundamentals of LCMS - March 2014
Labelled N-Glycans using LC–MS–MS
Udayanath Aich, Julian Saba, Xiaodong Liu, Srinivasa Rao, and Chris Pohl,
Fundamentals of GC - May 2014
Thermo Fisher Scientifc.
This article describes the analysis of fluorescently labelled N-glycans released
HPLC Method Development - July 2014
from proteins by liquid chromatography–mass spectrometry (LC–MS).
Regulars
GC Method Development - Sept 2014
on the web 2014
18 News
LCMS Method Development - Nov 2014
Text to come, text to come, text to come, text to come, text to come, text to
come, text to come, text to come, text to come, text to come, text to come.
21 Tips & Tricks: Column Selection for an Appropriate Molar

Mass Separation Range
Daniela Held, PSS Polymer Standards Service GmbH, Mainz, Germany.
Shoulders in gel permeation/size-exclusion chromatograms (GPC/SEC) can be a
Instructor led training without leaving your desk result of sample characteristics or down to the wrong choice of columns or
column combinations. Proper selection helps to measure true results.
To fnd out more visit:
Headline_ headline_ 36 CHROMacademy
http://www.chromacademy.com/2014 Update on what’s new on the professional site for chromatographers.
Subhead_ subhead_ subhead_ subhead 37 Training Courses and Events
39 Staff
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4 December 2013 Volume 9 Issue 22
Cover Story
2 Instrumental Innovations
Features
26 Q&A: Miniaturizing Military Detectors
Scientists at Sandia National Laboratories are thinking smaller in the development
of detectors for military use — from the detection of explosives and chemical
weapons to humans. Scientist Ron Manginell from the laboratories spoke to
Bethany Degg of The Column about the on-going research in this area.
28 Diisobutyl Columns Reduce Solvent Consumption and Offer

Rapid HPLC–MS Analysis of Plasma Oxycodone and its Metabolites
Linda L. Risler1 and Anne E. Mack2, 1Fred Hutchinson Cancer Research Centre,
2Agilent Technologies, Inc.
Oxycodone and its metabolites were analyzed by high performance liquid
chromatography–mass spectrometry (HPLC–MS) using a diisobutyl stable
bond column. This article discusses the results.
32 A Mixed-Mode Stationary Phase for the Structural Analysis of

Labelled N-Glycans Using LC–MS–MS
Udayanath Aich, Julian Saba, Xiaodong Liu, Srinivasa Rao, and Chris Pohl,
Thermo Fisher Scientifc
This article describes the analysis of fluorescently labelled N-glycans released
from proteins by liquid chromatography–mass spectrometry (LC–MS).
Regulars
18 News
Analysis of cannabinoids, mummified snacks, an immune system hijack, and the
latest company and research news in brief are all featured.
21 Tips & Tricks GPC/SEC: Select the Right Columns for Your
Molar Mass Range
Daniela Held, PSS Polymer Standards Service GmbH
Shoulders in gel permeation/size-exclusion chromatograms (GPC/SEC) can be a
result of sample characteristics or down to the wrong choice of columns or
column combinations. Proper selection helps to measure true results.
Instrumental Innovations 36 CHROMacademy
Update on what’s new on the professional site for chromatographers.
Selected highlights of 2013 product launches 37 Training Courses and Events
39 Staff
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Encapsulated bonding technology

ACT has developed “encapsulated
bonding technology”, which is
reported to increase the ligand
Mass spectrometer for in vitro use coverage of silica surfaces to eliminate
AB Sciex introduced its first series of CE-marked mass spectrometers and kits designed the effect of unbonded silanol
specifically for in vitro diagnostic use*. The AB Sciex API 3200MD and 3200MD QTRAP groups in liquid chromatography (LC)
CE-IVD LC–MS–MS systems have been developed for clinicians and researchers in hospital separations. The higher ligand coverage
laboratories performing analyses of trace level multiple compounds in human samples for is reported to improve inertness,
diagnostic purposes. The company has also recently announced a series of CE-marked chromatographic performance, and
reagent kits for diagnostic testing. Mass spectrometry (MS) has the potential to improve stability. The resultant ACE SuperC18
the sensitivity and accuracy of testing in UHPLC/HPLC columns are inert and
clinical diagnostics, while also reducing costs stable across an extended pH range
compared to other technologies. These (pH 1.5 to 11.5), allowing exploitation
instruments are reported to bring the power, of beneficial selectivity changes at low,
reliability, and speed of MS in a robust, intermediate, and high pH. The columns can be used with both methanol and acetonitrile
off-the-shelf, and easy-to-use format. The mobile phases and can be rapidly equilibrated without memory effects. Designed for
SCIEX IVD-MS kits, coupled with the 3200MD use with LC–MS compatible buffers, they reportedly give ultra-low bleed for improved
CE-IVD instrumentation, are reported to offer LC–MS compatibility. Columns of 2-, 3-, and 5-µm particle sizes are supplied with dual
complete application-specific solutions to compatible UHPLC/HPLC Excel hardware and are stable up to pressures of 1000 bar
European hospitals and clinical laboratories or 15,000 psi. A 10-µm particle size is also available. The wide range of preparative
to improve diagnostics. column dimensions allows reproducible scale-up from analytical dimensions, allowing
increased loading capacity at elevated pH. The columns are reportedly ideal for method
* Only available in some
development.
European countries.
www.absciex.com www.ace-hplc.com
2 Instrumental Innovations 18
8 News 2
21 Tips and Tricks: GPC/SEC 26 Q&A: Manginell
2 28 Risler and Mack 32 Pohl et al. 36 CHROMacademy 37 Training & Events 39 Staff
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The Column www.chromatographyonline.com Instrumental Innovations
Flow path inertness products

As regulatory agencies drive detection limits lower for increasingly active and more
complex samples, chemists cannot afford adsorption caused by flow path activity. This
is particularly critical for food, environmental, and forensic sample matrices. To address
this need, Agilent laid the groundwork for flow path inertness in 2008 by introducing
Agilent J&W Ultra Inert GC columns — reported
to deliver consistent inertness and exceptionally
low column bleed.
Since then Agilent has expanded the range of
Ultra Inert liners and added inert fittings, ferrules,
and supplies. By minimizing activity along every
step of the GC and GC–MS flow path, the flow
path solutions are reported to improve system
performance, give better results, and allow the
processing of more samples without unplanned maintenance and recalibration. The
complete Inert Flow Path includes: Ultra Inert liners, Inert Flow Path split/splitless inlet,
Ultra Inert gold seals, Inert MS source, Inert CFT devices, UltiMetal plus Flexible Metal
ferrules, Gas Clean purifier, and Agilent J&W Ultra Inert GC column.
www.agilent.com
DAWN HELEOS®. The most advanced Optilab T-rEX®. The refractometer with ViscoStar®. The viscometer with Eclipse. The ultimate system for the DynaPro® Plate Reader II. Automated dynamic
multi-angle light scattering instruments for the greatest sensitivity and range. unparalleled signal-to-noise, stable separation of macromolecules and light scattering for proteins and nanoparticles in
absolute macromolecular characterization. baselines and a 21st-century interface. nanoparticles in solution. 96 or 384 or 1536 well plates, and now with an
on-board camera!.
©2012 Wyatt Technology. DAWN, Optilab, DynaPro and ViscoStar are registered trademarks, and the Eclipse is a trademark of Wyatt Technology Corporation.
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Triple quadrupole liquid HPLC system

chromatography mass Many scientists performing high performance
liquid chromatography (HPLC) require the most
spectrometer innovative instrumentation to accommodate a
Winner of three awards in summer 2013, the EVOQ wide range of current and future applications.
triple quadrupole liquid chromatography mass Many HPLC instruments have a vast array
spectrometer (LC–MS TQ) from Bruker reportedly offers of functions to meet these requirements;
a comprehensive solution for routine laboratories, however, with this comes additional pricing
enabling them to reliably quantify thousands of samples costs and system complexity. Furthermore, in
in the fastest sample to report time possible. The company has also added the EVOQ Elite some applications, technological developments are not required and can be detrimental
ER to the range in 2013, providing high sensitivity and specificity needed for proteomics to use. According to Cecil Instruments, Merit HPLC systems are low-cost and easy to use,
quantitation. The models are used in laboratories in a wide range of application areas, ideal for applications such as multi-compound screening, initial method development,
including food safety, environmental testing, petrochemicals, and drug testing. production situations, process development, and teaching. The systems are available in
The instruments feature ultrahigh pressure liquid chromatography (UHPLC) pump isocratic and binary modular configurations. The company report that in initial method
technology that gives short run-to-run cycle times and reproducible separations; as development an ultra-fast scanning UV–vis WaveQuest detector, instead of a diode array
well as several innovations in the ion source to maximize sensitivity and robustness. detector, may be used to check that all known components of the injected solution
Vacuum-Insulated Probe Heated Electrospray (VIP-HESI) technology preserves and ionizes are eluted with appropriate baseline resolution. Each system offers ultra-low drift and
thermally fragile molecules, and “Active Exhaust” entrains sprayed gases in the source for durable modules, with full PC control. This control is provided by software on-board the
high sensitivity, reducing chemical noise and cleaning. In addition, the EVOQ Elite ER is instrument modules, so there is virtually no software to learn to master.
reported to offer an extended mass range of 2,000 m/z. www.cecilinstruments.com/merit-hplc.html
Novel PACER software delivers exception-based data review, reportedly increasing
efficiency and accelerating sample-to-report times without compromising on data quality.
Dash Reporting software gives customized reporting to all users, with an intuitive system
which allows control of all report elements. The innovations behind the EVOQ and the
new EVOQ Elite ER reportedly deliver a complete platform for routine laboratory testing.
www.bruker.com
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Robotic platform
With the PAL RTC (Robotic Tool Change) robotic platform from CTC Analytics, sample
preparation is no longer the bottleneck. In seconds the RTC changes automatically
between different syringes (1 μL to 10 mL), liquid injection, headspace, and solid-phase
microextraction (SPME) methods without the need for manual intervention. This boosts
productivity and expands the application range. The optional vortex mixer, incubator, and
different syringes can automate most sample preparation steps. Liquid-liquid extraction,
derivatization, standard addition, and dilution reportedly become smooth and traceable.
PAL Sample Control
software controls the RTC.
With a few clicks you can
import or generate sample lists
and start the data acquisition.
Or you can quickly set up a
new workflow to eliminate
tedious manual operations.
PAL Sample Control interfaces
with most chromatographic
systems.
www.palsystem.com
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Syringe filters GC–MS headspace sensitivity

Whatman disposable syringe filters from GE Healthcare can be categorized into different The enrichment technique hot injection and trapping (HIT) for gas chromatography
families. Each family is optimized for specific applications or filtration requirements. For coupled to mass spectrometry (GC–MS) combines analyte trapping from several
example, Roby can be utilized for automated filtration systems and Whatman GD/X for headspace injections for each run, thereby offering extra concentrating power. The
hard-to-filter samples. Within each syringe filter family there are options for different technique can be performed with a MultiPurpose Sampler (MPS) instrument and Thermal
diameters, pore sizes, membrane types, and sterility. Whatman disposable syringe filters Desorption Unit (TDU) combined with a Cooled Injection System (CIS) PTV-type inlet, all
are designed to provide fast and efficient filtration of aqueous and organic solutions. available from Gerstel. The system is reportedly easily switched between standard and
The syringe filters are sealed without glue to maintain the purity of the filtered sample. HIT operation by specifying the number of injections in the Maestro software. HIT can
The filters can be used in numerous pharmaceutical, environmental, biotechnology, food be performed under integrated control with the GC–MS system or independently using
and beverage, and agricultural applications. Maestro software. The performance of the HIT technique has been demonstrated for
www.gelifesciences.com/LaboratoryFiltration flavour and off-flavour compounds in drinking water and beverages, resulting in good
linearity and repeatability and very low limits of detection (LOD).
www.gerstel.com
S/SL Inlet
Injection Volume: 1 mL
Split 1/10
(conventional HS)
CIS4 Initial Temp.: -50 ºC

Injection Volume: 1 mL
(HIT-HS)
CIS4 Initial Temp.: 10 ºC

Injection Volume: 2.5 mL x4
(HIT-HS)
Figure 1: Total ion chromatograms of canned coffee by SHS analysis.

(a) Conventional SHS with S/SL inlet (injection volume 1 mL, Split 1/10);
(b) HIT-HS with CIS 4 Initial Temp. -50 ºC (injection volume 1 mL, Split 1/1);
Whatman and Whatman GD/X are trademarks of GE Healthcare companies. (c) HIT-HS with CIS 4 Initial Temp. 10 ºC (injection volume 2.5 mL ×4, Split 1/1).
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Fingertight UHPLC fittings

With the increasingly popular transition to UHPLC technologies, there remains a demand
for fittings that can be “finger-tightened” into place. This year, IDEX Health & Science
has expanded its growing line of reusable fittings for UHPLC, adding the new VHP-3200
fitting to its portfolio. Senior technical specialist, John Batts, shared his thoughts on this
new fitting: “One of the major benefits of UHPLC is speed. There is a greater demand
for convenience without sacrificing quality. To that end, we
developed the VHP-3200. We focused on engineering
features into the fitting that brought tangible value
to those using them in the laboratory. To start with,
the fitting’s patented design allows it to function as
a ‘one-piece’ fitting. It can be hand-tightened without
tools and still hold to typical UHPLC pressures, while
allowing the use of a wrench for those extra-high
pressure applications. This fitting can also be
reused multiple times – with the same tube or
with different tubes in the same or different
receiving ports, even in higher-temperature
applications and aggressive chemical
environments.” He added: “These
features help facilitate more rapid column
change-overs, less system down-time,
and therefore higher productivity per system.”
www.idex-hs.com
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Light scattering detector Thermal desorper

Multi-angle light scattering (MALS) detectors are widely used in gel permeation/size-exclusion In 2013 Markes International launched the next generation of the TT24-7 thermal desorber
chromatography (GPC/SEC) within biopharmaceutical research. The Viscotek SEC–MALS for continuous monitoring of trace-level volatile and semi-volatile organic vapours in air
20 detector from Malvern Instruments measures absolute molecular weight across a or gas by gas chromatography (GC) or direct read-out instruments. According to Matt
range of macromolecular Bates, thermal desorption product manager at Markes, the TT24-7 Series 2 desorber uses
applications and provides a reciprocating twin-trap system to capture airborne volatiles to avoid breaks in sampling.
good radius of gyration He said: “The inert, uniformly-heated flow
(Rg) measurements for path makes it compatible with compounds
molecules larger than ranging in volatility from C2 through to C40,
10–15 nm. The system including reactive species.” He added that
reportedly gives a good the design of the instrument and the cold
data fit for molecular traps allows sampling flows up to 800 mL/
weight and size min with quantitative trapping, which he
extrapolation, improving says provides outstanding levels of sensitivity.
accuracy even at high The same flexibility, he points out, also
molecular weights. Its makes it possible to achieve cycle times as
vertical flow cell with low as 3–5 min, for situations where a rapid
radial optics can increase response to raised levels of toxic airborne
sensitivity and accuracy for low angle detection and allows the use of different mobile compounds is needed.
phases without the need to alter the optical properties of the detector. These and other innovations, Bates says,
www.malvern.com/secmalslaunch make the TT24-7 Series 2 suitable for a range
of challenging air monitoring applications,
including atmospheric chemistry research, continuous monitoring of controlled industrial
environments, counter-terrorism (NRT detection of chemical warfare agents), and
continuous monitoring of changing odour profiles.
www.markes.com
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Purification software THE VERY BEST FOR INSTRUMENTAL TLC

Millitrack Compliance from Merck Millipore is a novel e-solution designed to allow
regulatory compliance and optimization for Milli-Q Advantage and Milli-Q Integral DUE TO SOPHISTICATED INSTRUMENTS AND INNOVATIVE
laboratory water purification system users. Jean Mahooti, responsible for Merck SOFTWARE THIN-LAYER CHROMATOGRAPHY HAS BECOME
Millipore Lab Water MORE POWERFUL THAN EVER BEFORE.
Compliance services said:
“This e-solution enables DOCUMENTATION AND QUANTITATIVE EVALUATION HYPHENATING TLC WITH MS
EVALUATION WITH TLC VISUALIZER WITH TLC SCANNER USING THE TLC-MS INTERFACE
laboratories to achieve
good archiving practices
and ultimately offers full
compliance for users’
water systems.” He added:
“The built-in Millitrack
Compliance e-solution is
the optimal solution for
companies who want to
move towards a zero-paper
environment. In addition, Millitrack Compliance was designed with remote monitoring CAMAG DEVELOPS AND MANUFACTURES A COMPLETE SUITE OF DEDICATED INSTRUMENTS FOR ALL
STEPS OF THE TLC PROCESS:
and advanced diagnostic capabilities to facilitate work in the laboratory and boost
SAMPLE APPLICATION
lab productivity, allowing laboratories to save time and increase productivity while DETECTION
minimizing downtime.” CHROMATOGRAM DEVELOPMENT
EVALUATION
www.merckmillipore.com/millitrackcompliance DERIVATIZATION
DOCUMENTATION
WORLD LEADER IN
PLANAR CHROMATOGRAPHY WWW.CAMAG.COM
WWW.CAMAG.COM
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Ion chromatography system Modular gas generators

Metrohm has recently improved the operation of the Professional IC Vario Series. A Peak Scientific has released the Peak Precision Series,
liquid handling station enables a number of sample preparation techniques such as a group of compact modular gas generator systems
dilution, partial loop injection, pick-up techniques, and preconcentration. Although each that allow the separation and analysis of multiple
instrument consists of different units, they are very compact with short connections and gas types and complex mixtures with high accuracy.
visible flow paths. The new suppressor set-up is reportedly more environmentally friendly; The laboratory is a unique environment demanding
all suppressor types are compatible with the same holder and can be easily exchanged. precision, accuracy, reliability, and design. The Precision
The suppressor is said to be robust and does not require any exchange of spare parts. Series is reported to maintain gas flow with accuracy
The module for eluent production handles up to four solutions. A water purification and reliability. The systems incorporate a space-saving
system from ELGA can be stackable design and can fit neatly into confined
connected for ultrapure spaces. Looking to the future as natural resources run
water supply. This low and costs rise, the system is able to accommodate
contamination-free, helium to hydrogen conversion. The modular design
stable system runs reportedly allows for different combinations to suit
automatically and offers single gas chromatography (GC) and multiple GC
flexibility combined with applications. The system eliminates the inconvenience
user-friendly operation of gas cylinders — no more changing over, no more
and reliability. supply issues, and no safety concerns. As a result, the
www.metrohm.com range is very low maintenance and provides long-term

cost stability.
www.peakscientific.com/peak-precision
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GC–MS–MS system Core–shell particle columns

PerkinElmer has introduced the AxION iQT GC–MS–MS system which has the identification In 2013, Phenomenex continued to expand the line of sealable high performance liquid
capabilities of a QTOF and the quantitation capabilities of a triple quadrupole in one chromatography/ultrahigh pressure liquid chromatography (HPLC/UHPLC) Kinetex
instrument, according to the company. It has minimal core–shell particle columns,
setup requirements and is reported to be able to measure the company’s most important
up to 500 compounds per second. The system delivers innovation in recent years. A
automated method development tools to users, includes new addition to the line is a
advanced software, and is reportedly easy to use and high-powered 1.3-μm particle,
maintain. The company state that it is compatible with the smallest diameter in the
a wide array of gas chromatography (GC) instruments, Kinetex family. With high
enabling rapid and efficient ion transmission and providing manufacturing quality and
the quantitative dynamic range and low level sensitivity a tight specification range,
required for most complex matrices. The system offers Kinetex 1.3-μm columns are
a broad array of user-interchangeable ion sources reported to give a performance
(dual-filament electron ionization [EI], chemical approximately 50% greater
ionization [CI], or cold EI). It can also than fully porous 1.7-μm
characterize and quantify compounds particles, according to the
for a variety of applications, including company. This efficiency
the analysis of pesticides in food samples allows greater chromatographic
to ensure food safety and authenticity, resolution, performance, and productivity. The Kinetex core–shell family provides
quantitation of non-derivatized steroids, separation solutions for a wide range chromatographic applications and offers easy
cholesterol and therapeutic drug monitoring method transferability from UHPLC to HPLC to preparative-scale LC.
in research applications, analysis of drugs in www.phenomenex.com
forensic toxicology, and thorough analysis
of chemical compositions.
www.perkinelmer.com/outsidethebox
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Automated soluble fraction system MALS technology

Polymer Char recently introduced the CRYSTEX QC instrument for soluble fraction Postnova has introduced the MaxMALS Multi-Angle Light Scattering (MALS) technology for
determination in quality control laboratories at polypropylene manufacturing plants on the absolute molar mass, particle size, and structural characterization of proteins, biopolymers,
to the market. Traditionally, this test is performed through gravimetric and wet chemistry macromolecules, and particles, using a maximum number of 21 detection angles. This
methods which are time-consuming and have safety implications for the operator because technology is incorporated into the company’s new line of multi-angle light scattering
they require manual detector systems and reportedly
solvent handling at overcomes the traditional limits of
high temperature. The currently available multi-angle light
system fully automates scattering set-ups, such as insufficient
temperature rising number of detection angles and a
elution fractionation — more or less — complete lack of
to obtain the soluble low detection angles. This has been
fraction in resins that a significant problem for correct
is reported to be safer and reproducible characterization of
and simpler to use over large biomolecules, polymers, and
traditional methods. particles, as the information for molar
Samples do not have mass and size is directly derived from these low angles. The total number of angles and
to be weighed and all the availability of low angles are absolutely crucial for precise molar mass and particle size
solvent handling is automatic throughout the analysis. High repeatibility is obtained as measurement. Up to now only instruments with a smaller number of angles, such as 3°,
the instrument allows the use of up to 4 g of polymer. Furthermore, it is reported that 7°, 8°, or 18° in the best case, have been available. Typically, the lowest angles available
precise quantification is delivered through a built-in infrared detector that also measures in MALS are located at 35° for most commercial instruments and around 20° for others.
ethylene content. An integrated viscometer provides intrinsic viscosity in the same MaxMALS provides a maximum number of 21 detection angles and a complete series of
analytical process that lasts in total approximately 2 h. low angles located at 7°, 12°, 20°, 28°, and 36°. MaxMALS reportedly offers the highest
www.polymerchar.com number of total and lowest detection angles available on the market and the most precise
MALS instrument for molar mass and size determination.
www.postnova.com
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Deep well plates Light scattering detector

Porvair Sciences has developed and introduced 96-well round deep well plates that deliver a The SLD7000B from PSS is a seven-angle multi-angle light scattering detector that can be
full 2.00 mL liquid capacity for each well in an automation compatible SLAS/ANSI footprint, operated with any gel permeation/size-exclusion chromatography (GPC/SEC) system or
according to the company. The plates measure 45 mm in height and are compatible as a stand-alone instrument in batch-mode to measure absolute molar masses and sizes
with microplate stackers, of macromolecules in
washers, and automated solution. A metal-free
equipment. The plate design version has recently
incorporates features that been released
prevent “locking” when that is reported to
stacked and enable easy enable operation
heat sealing. Manufactured for applications at
under class 10,000 extreme pH values
conditions from ultra-pure and with high salt
grade polypropylene, the concentrations. It
plates are certified as combines the cell
RNase/DNase free, and do design of the SLD7000
not contain measurable contaminants, according to the company. Plates are also available with the robustness against corrosion of the PSS BioSECcurity SEC line. The detector
irradiated for sterile biological applications. A matching thermoplastic elastomer “cap measures the scattering intensity directly in the cell, therefore avoiding problematic
mat” is reported to provide easy friction sealing for transport, shipping, and short-term interfaces and the need for angular correction with the solvent. The detector can be
storage. For longer periods, the plate can be heat sealed to provide peelable, pierceable, used in the pH range 1–13 and is ideal for protein molar mass, purity, and aggregation
and re-sealable storage options. analysis in combination with a concentration detector (UV, RI). The wetted parts are
www.porvair-sciences.com PEEK (cell, tubing, frits).
www.pss-polymer.com
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GC filter status indicator LC–MS instrument

The Super Clean Gas Filter Electronic Indicator from SGT Shimadzu has released the LCMS–8050 triple quadrupole LC–MS–MS system, which
displays real-time filter status, rather than traditional is reported to feature extremely fast data acquisition rates. The system is reported to
last-minute colour change indication. It has an provide attogram-level (10 -18 g) sensitivity as the result of two features: The UFsweeper III
audio-visual early warning system, and can be linked to a collision cell which enhances collision-induced dissociation (CID) efficiency by optimizing
virtual indicator platform, or be configured to a preferred the collision cell pressure; and a newly designed heated electrospray ionization (ESI) source
replacement schedule. It has three operating modes: which improves desolvation and enhances ionization efficiency by adding a heated gas
Factory, manual, and virtual indicator. The factory mode combined with the nebulizer gas. The instrument’s newly engineered high voltage power
is based on default replacement advice of 12 months supply enables a maximum scan rate of 30,000 u/sec and a 5 msec polarity switching time.
and no configuration. The manual mode is reportedly It is reported that 1000 events with up to 32 channels can be included for a maximum
useful for users with a replacement schedule in place that of 32,000 multiple reaction monitoring (MRMs) for each analysis. The “plug-in & play”
deviates from the standard manufacturer’s replacement ion source is reported to enable an easy transition from ESI to atmospheric-pressure
advice and want to use the device for updates on status. chemical ionization (APCI) or to dual ionization mode (DUIS). The LabSolutions LCMS
The virtual indicator mode is an advanced operation mode Version 5.60 software integrates high performance liquid chromatography (HPLC) and
that calculates replacement-advice tailored to specific gas mass spectrometry (MS) control. An automated MRM optimization routine is reported to
configuration and instrument usage. provide unattended optimization of multiple compounds.
www.virtualindicator.com www.shimadzu.eu
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Complex biological sample analysis Size-exclusion chromatography

The Thermo Scientific Orbitrap Fusion Tribrid LC–MS instrument was introduced in In 2013, Tosoh Bioscience presented a new high temperature gel permeation/size-exclusion
June 2013 for the analysis of complex biological samples. The instrument is reported to chromatography (GPC/SEC) system to their SEC portfolio, the HLC-8321GPC/HT (HT
improve the results of tandem mass tag (TMT) experiments by increasing the depth and EcoSEC System). It is reported to provide stable thermostatization up to 220 °C for up to
quality of data compared to previous tools. This can solve many throughput challenges eight columns, autosampler, pumps, and an extremely stable refractive index detector
by enabling mass spectrometer users to determine relative quantification of proteins with an independent temperature control. The optional DF-8321 sample processing
in multiple samples simultaneously. The instrument combines three mass analyzers — unit is able to process up to 24 samples at temperature programmes ranging from
quadrupole, Orbitrap, 40–220 °C. In addition,
and linear ion trap the TSKgel SW series of
— into a unique aqueous HP-SEC columns
“tribrid” architecture. was expanded by three
The three different new ultrahigh-pressure
mass analyzers are liquid chromatography
reported to work (UHPLC) columns tailored
together to improve to different aspects
analytical performance of antibody analysis:
and enable new According to the company
experimental methods. The quadrupole mass analyzer selects precursors at isolation the TSKgel SuperSW mAb
widths down to 0.4 amu for excellent sensitivity and selectivity. The ultrahigh-field HTP enables easy transfer
Orbitrap offers resolution greater than 450,000 and scan rates up to 15 Hz. The ion of high performance liquid chromatography (HPLC) methods to fast UHPLC analysis;
routing multipole followed by dual-pressure linear ion trap is reported to provide MSn the TSKgel SuperSW mAb HR provides high resolution for the analysis of fragments,
higher energy collision dissociation (HCD), collision-induced dissociation (CID), and monomers, and aggregates in one run; and the TSKgel UltraSW Aggregate covers a
electron-transfer dissociation (ETD) fragmentation, as well as fast, sensitive mass analysis range of higher molecular weights, ideal for separating antibody dimers and higher
with scan rates up to 20 Hz. aggregates.
www.thermofisher.com www.tosohbioscience.de
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Mass detector for chromatography UHPLC RI detector

This year, Waters introduced the The Optilab UT-rEX from Wyatt is a refractive index (RI) detector specifically designed
Acquity QDa detector, which is for use with ultrahigh-pressure liquid chromatography (UHPLC) systems using columns
reported to bring high-quality, mass packed with small beads. Using a combination of miniaturized components, semiconductor
spectral data to chromatographic photodiode technology, and proprietary computer algorithms, the detector is reported
separations. A synergistic component to improve sensitivity, stability, and temperature range. As a universal detector, it can be
of a chromatographic system, it is a operated either stand-alone
small and easy-to-use mass detector — as the sole on-line detector
that brings mass spectrometry to downstream of a UHPLC
chromatography in a way that column — or in combination
gives analytical scientists access to with another detector, such as
mass spectral data regardless of UV–vis absorption.
their previous experience with mass The temperature regulation
spectrometry. The detector fully automates sample analysis and eliminates sample-specific capabilities of the detector
adjustments for certainty in sample results. Robust and reliable enough for routine are reported to allow control
use, the detector generates the mass spectral data expected of a single quadrupole of the flow path temperature
mass spectrometer in a mass detector no bigger than a familiar photodiode array (PDA) above and below ambient.
detector. By simplifying laboratory workflows, the detector is reported to increase The absolute refractive index
productivity by eliminating the need to run additional assays or turn to time-consuming measurement of the solution is reported to improve resolution, reduce sample and
alternative techniques to establish the levels and identity of specific sample compounds. solvent usage, and save time. In addition, the detector is linear over a wide range and
www.waters.com/separate is sensitive to samples that are not UV-active, while the dead volume for the instrument
is <1.5 μL. The small flow cell and proprietary temperature regulation enable stable RI
baselines and RIS signals, further enhancing the detector’s sensitivity.
www.wyatt.com
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The Column www.chromatographyonline.com Advertisements
PHARMACEUTICAL/DRUG DISCOVERY
Sophisticated Antibody Analysis by GPC/SEC with RALS
Monoclonal antibodies (mABs) are increasingly growing in importance

for the diagnosis and therapy of various diseases, including cancer and
Refractive index Multimers
autoimmune and infammatory disorders. One essential parameter to UV at 214 nm mABs
Detector signal (V)

125 Light scattering at 488 nm
defne their quality is the content of aggregates (dimers, trimers, and 2.0
FAST AND
Detector response (%)

higher aggregates). These aggregates can be formed during processing 100
1.5
and purifcation or are the result of long-term storage. As a result of 1.0
aggregation, antibodies lose their pharmaceutical effcacy and can 75

0.5
facilitate an immunology response.
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Antibody fragments which lack the Fc region can be used for the Dimers 10.0 12.5 15.0
Fragments
treatment of diseases. They can also be the result of degradation of full 25
length antibodies. Therefore, a GPC method, which offers the opportunity

0
to analyse antibodies and their aggregates, as well as antibody fragments 16 17 18 19 20 21 22 23 24 25
simultaneously, with superior resolution and high sensitivity is invaluable. Elution volume (mL)
GPC/SEC Solutions and Expert Support Experimental

Figure 2: Sensitive analysis of antibody aggregates. The light
scattering signal for the dimer is relatively high compared to that of
the mABs because of molar mass dependency and provides improved
GPC/SEC analysis was performed on a PSS SECcurity GPC system,
Macromolecular characterization from the experts equipped with a PSS SECcurity SLD1000 light scattering detector, using sensitivity for the detection of high aggregates (inset).
the following conditions:
Columns: PSS PROTEEMA, 5 µm, 2 × 300 Å (8 × Results
300 mm each) +precolumn Figure 1 shows an overlay of elugrams obtained for a full length antibody
GPC/SEC is our passion. We at PSS are fully dedicated to the advance-
Solvent: 100 mM sodium phosphate pH 6.7 + 0.25 M NaCl and antibody fragments analysed on a single set of columns.
ment of macromolecular liquid chromatography by means of developing
Flow rate: 1.0 mL/min All three detector signals for the analysis of a monoclonal antibody are
true solutions and providing competent and personal support.
Temperature: 25 °C shown in Figure 2. The light scattering signal shows improved sensitivity
Based on excellent products and the latest findings in material science, Detection: Refractive index (RI), ultraviolet (UV) at for high aggregates compared to the other signals.
we create easy-to-use and powerful solutions for QC and R&D. From a λ = 214 nm, PSS SLD1000 (right-angle light
single molar mass reference material to turn-key systems for GPC/SEC scattering [RALS]) at λ = 488 nm Conclusion
multi detection with light scattering, viscometry, mass spectrometry or Calibration: Light scattering The GPC/SEC method including UV, RI, and RALS can be used for
fully compliant GPC/SEC for the pharmaceutical industry: PSS offers all Injected mass: 60–80 µg the simultaneous determination of aggregate content of monoclonal
products and services for successful macromolecular analysis and expert Data acquisition, antibodies as well as antibody fragments. The column combination
support by GPC/SEC enthusiasts! calibration, and covers the separation range for all three types and provides a high
evaluation: PSS WinGPC UniChrom 8.1 resolution for the determination of the dimer content. Because of its
Discover what‘s new in macromolecular characterization and work
molecular weight dependency, the PSS SLD1000 RALS detector
with our specialists on your application challenges.
offers high sensitivity for very small quantities of high aggregates and
100 Multimers mABs also allows the determination of the absolute molecular weight of the
antibodies. In addition, it has a unique feature for a light scattering
80
detector as the wavelength can be altered to increase the sensitivity.
Detector signal (V)

60
Fragments
40 Low-molecular-weight
impurities
20
Dimers
0 Fragments
www.pss-polymer.com 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0
PSS Polymer Standards Service GmbH
Elution volume (mL)
contact: info@polymer.de In der Dalheimer Wiese 5, D-55120 Mainz, Germany
Figure 1: Separation range of the column combination. The red
Tel: +49 6131 962390 fax: +49 6131 962390 11
curve shows the UV signal of a full length antibody and its dimers
plotted against the elution volume. The blue curve is the elugram of E-mail: info@pss-polymer.com
Driving GPC/SEC forward antibody fragments and their high level aggregates. Website: www.pss-polymer.com
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Hijacking the Immune System
Staphylococcus aureus (S. aureus) evades clearance by the immune system by hijacking the very traps it
produces, transforming these same traps into a toxic compound according to a paper published in Science.1
Scientists from the University of Chicago (Chicago, USA) used high performance liquid chromatography
coupled to mass spectrometry (HPLC–MS) to identify the toxin as 2’-deoxyadenosine (dAdo).
Bacterial invasion of tissue triggers an array of processes as part of the immune response, in an attempt
Designer Cannabinoids
to first contain and then destroy an infection. S. aureus can permanently colonize the human body without
The variance in the toxic effects of designer cannabis drugs
causing harmful effects, usually on the skin and within the nose causing boils or abscesses. However,
marketed throughout Europe and the USA as “K2” or “Spice”
if it enters the blood stream it can result in blood poisoning, endocarditis, and meningitis among other
could be the result of stereoselective metabolism of enantiomers by
lung and liver enzymes, according to a study published in Analytical conditions.
Chemistry. Chiral liquid chromatography–tandem mass spectrometry S. aureus tissue infections are characterized by abscesses formed by immune cells cornering off the
(LC–MS–MS) was applied to the analysis of JWH–018 and infection for clearance by a specific type of cell known as a macrophage — so-called “big eaters” that
AM2201 metabolites in human urine. JWH–018 and AM2201 are roam the body clearing away infections. The traps are released by neutrophils (neutrophil extraceullar traps
metabolized by lung and liver enzymes to generate three primary [NETs]) in an attempt to contain the infection until the arrival of the macrophage. In the case of S. aureus
chiral metabolites — (ω)-carboxyl, (ω)-monohydroxyl, and (ω-1)- however, there is no macrophage activity and the infection remains uncleared.
monohydroxyl metabolites that have a high affinity for the human To investigate this mystery, S. aureus was co-cultured alongside immune cells, including neutrophils
cannabinoid type-1 receptor (CB1R). Solid-phase extraction with and macrophages. When NET production was activated, the macrophage began to die suggesting
chiral LC–MS–MS determined specific excretion patterns associated the production of a toxin by the bacterium. The team performed HPLC–MS to identify this toxin as
Photo Credit: SCIEPRO/Getty Images

with both JWH–018 and AM2201 compound metabolism. The 2’-deoxyadenosine (dAdo). Combining these results with data from genetic experiments using mutant
authors suggest the method could be used in future clinical studies. strains of S. aureus, the team deduced that the bacterium converted NETs to dAdo
— A.L. Patton et al., Anal. Chem. 85, 9390–9399 (2013). that is toxic to macrophages.
Olaf Schneewind, senior author of the paper, said: “These bacteria have endowed themselves with
Mummified Snacks weapons to not only anticipate every immune defense, but turn these immune
The ancient Egyptians embalmed meat mummies as an afterlife defenses against the host as well.” He added: “Sooner or later almost every human
snack in the same way as their dead, according to the findings of gets some form of S. aureus infection. Our work describes for the first time the
a gas chromatography–mass spectrometry (GC–MS) analysis. The mechanism that these bacteria use to exclude
team from the University of Bristol (Bristol, UK) and the American macrophages from infected sites.”
University in Cairo (Cairo, Egypt) collaborated to analyze the So what does this mean for drug development? Theoretically, scientists
chemical compositions of samples and tissues from meat mummies could target this mechanism of evasion using therapeutic proteins to block
excavated by the Cairo Museum and the British Museum. Of bacterial enzymes involved in the transformation of NETs by S.
particular interest, the team found evidence of the use of Pistacia aureus. This is promising but requires a lot more work. — B.D.
resin on a beef rib mummy, a substance rarely even seen in the
embalming of humans. — K.A. Clark, S. Ikram, and R.P. Evershed, PNAS Reference
Early Edition, DOI: 10.1073/pnas.1315160110 (2013). 1. V. Thammavongsa et al., Science 342, 863–866 (2013).
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Gulf of Mexico Research Initiative Grant

Competition
Early Use of Chili in Mexico
The Gulf of Mexico Research Initiative (GoMRI)
has announced plans to invest $105 million Residues of chili pepper (Capsicum) have been found on pottery dating back 2000 years in southern
between 2015 and 2017 to support research Mexico, according to a study published in PLOS ONE.1 The New World Archeological Foundation (NWAF) first
consortia investigating the effect and impacts excavated the pottery (thought to date from 400 BCE–300 CE) from Chiapa de Corzo between 1955 and 1963.
of hydrocarbon release into the environment. Ultrahigh pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS–MS) was performed
The funding will be awarded to four or on residues collected from the pottery. Extracts were collected by lightly scraping the interior of 13 pieces of
more institutions over two years, under the pottery with sandpaper, which were subsequently analyzed. Capsicum residues were identified in five out of the
programme known as RFP–IV. 13 pieces.
gulfresearchinitiative.org There has long been a connection between the fields of analytical chemistry and archeology leading to the
Biosimilar Characterization Laboratory development of molecular archeology, specifically when investigating the diet of ancient peoples. Lead author
Opens in Austria Terry Powis told The Column: “I was asked by a Mexican colleague, Emiliano Gallaga, one of my co-authors,
The Christian Doppler Laboratory for Biosimilar about two years ago to look for cacao in pottery vessels from the site of Chiapa de Corzo in the State of Chiapas.
Characterization has opened at the Paris-Lodron Although our focus was primarily to look for cacao in 2000-year-old pottery vessels from the site, we were also
University of Salzburg (Salzburg, Austria). The interested in looking for any additives or flavourings that may have been included in these particular drinks.”
laboratory was established to develop and The discovery of chili residues was an unexpected one, according to Powis. The vessels had been selected as cacao
transfer to practice novel and more efficient residues had been identified in similar artefacts in other areas. He said: “During the mass spectroscopy analysis we
methods to characterize the active ingredients were completely surprised at the fact that no cacao was present in any of the pots tested. In fact, chili was present,
in protein-based medicines. Scientists from the although we have no way of knowing at present whether the amount is strong or weak, just that it is present.”
University of Salzburg, Sandoz Pharmaceuticals, As an archeologist, the primary goal of Powis was to identify the exact species of chili pepper used but, as there
and Thermo Fisher Scientific are collaborating in are five different species, they were not able to determine this. He told The Column that this is a future path, and
these efforts. that they are attempting to conduct DNA analysis of Theobroma cacoa on archeological samples to link residues
www.thermoscientific.com to different modern species now in existence in different areas of the New World.
PerkinElmer Opens Center for Innovation When discussing the importance of the work, Powis said: “Because of our findings we can now pursue many
in Massachusetts, USA different cultural questions about how the Mixe-Zoque people used chili peppers in their daily social lives.
PerkinElmer has opened a new Center for We will also be able to move beyond its culinary use (but we still have a way to go) to include how they
Innovation in Hopkinton, Massachusetts, USA. used it in pharmaceutical and ritual contexts as well.” — B.D.
The facility features laboratory space where
engineers, scientists, and customers can meet Reference
Photo Credit: jklr/Getty Images

and discuss developments to benefit the life 1. T.G. Powis, E.M. Gallaga, R. Lesure, R. Lopez Bravo, L. Grivetti, et al,
sciences market. PLoS ONE 8(11), e79013 (2013). Bethany Degg, Assistant Editor, bdegg@advanstar.com
www.perkinelmer.com www.chromatographyonline.com
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Peaks of the Week News In Brief

We have a rich range of informative and practical content across our print and digital portfolio that we tweet, share, and
open for discussion. Get on-line today and join the discussions! Some of the highlights: Studies of Autism
Chromatographic methods are used in studies of
• What’s the Problem with the LLOQ? — A Case Study: Liked and shared by Twitter and Facebook followers. bit. autism to identify novel diagnostic biomarkers.
ly/1b5PjUc Zurawicz and colleagues have reviewed the
methods available in the journal Biomedical
• John Dolan: Dr. Troubleshooting: “John Dolan column in LCGC is a read reference for all users of liquid chromatography.
Chromatography, presenting a theoretical
John is a true master in this regard. In this 30th anniversary of this column I hope he can continue to contribute for many
introduction as well as example applications.
years to the resolution of LC troubleshooting.” — bit.ly/17DPZKp DOI: 10.1002/bmc.2911
• Professor Barry L. Karger: Scientist, Mentor, and Innovator: In graduate school, do you think that your thesis
advisor should also be your mentor? “No. If you know why you are going to graduate school, you can focus on your Graphene Oxide Adsorbent
studies & your research...” “While I have not been in graduate school since the previous millennium I think lack of mentorship Graphene oxide (GO) has been proposed
is the biggest failing for US post-graduate education. But what faculty member is qualifed to mentor advanced degree as an effective adsorbent of afotoxins prior
chemists for anything other than academic positions? Most graduates move into industrial positions and a career academic has to HPLC analysis. The different parameters
little of frst-hand practical utility to share. — Comments from LinkedIn. bit.ly/Ic7c7k affecting extraction were investigated, and
the method was applied to the determination
Like us Join us Follow Us of afotoxin levels in peanuts. DOI:1016/j.
chroma.2013.10.006
Online Highlights Plastic Pollution

A study published in the journal Scientifc Reports
suggests that chemicals from plastic debris
could impact fsh health. Microscopic plastic
Editors’ Series: Fundamentals, Optimization, You might have missed... debris found worldwide can be ingested and
and Practical Aspects of UHPLC: Series 1: bioaccumulated by fsh resulting in liver toxicity.
Fundamentals, Optimization, and Practical • Green Foodomics: Just another buzzword or a DOI: 10.1038/srep03263
Aspects of UHPLC new direction in food analysis? bit.ly/19nBA4h
The first in a three-part series on UHPLC to address • Practical Chemometrics for Profiling
Capillary LC and Nano LC in Food
and explain fundamental aspects of UHPLC when Contaminants: Q&A with LCGC Europe EAB
Luigi Mondello and colleagues have reviewed
preparing to apply the technique in the laboratory member. bit.ly/1hdpsfu
and compared the general principles of two
in practice. • An Insight into Analytical Quality by Design
bit.ly/168SWVL in the Pharmaceutical Industry: bit.ly/15Dp8fa miniaturized techniques, capillary LC and nano
LC, to conventional LC in relation to food
analysis. DOI: 10.1016/j.trac.2013.05.021
OnDemand Webcast Interviews
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Tips & Tricks GPC/SEC: Select the Right Columns
for Your Molar Mass Range
Daniela Held, PSS Polymer Standards Service GmbH, Mainz, Germany.
Shoulders in gel permeation/size-exclusion chromatograms (GPC/SEC) can be a result of sample characteristics or down to the wrong choice of
columns or column combinations. Proper selection helps to measure true results.
Resolution, molar mass separation range, solvent effcient separation over a large molar mass Selecting the Upper Molar Mass
consumption, and analysis time are key factors separation range is required. For this, columns Separation Limit
that have to be balanced when performing of the same dimensions and particle size (but When selecting a column or combination of
gel permeation chromatography/size-exclusion different porosity) are often coupled in series. columns for GPC/SEC, users often only know the
chromatography (GPC/SEC). Depending on This is a very successful approach but has some approximate average molar mass (average weight
the application and environment, whether potential pitfalls such as mismatch, which can molecular [Mw]). However, wrong column choices
routine quality control (QC) or research and lead to artifacts in chromatograms and the can be made if the breadth of the molar mass
development (R&D), one of these parameters resulting molar mass distributions. distribution (related to the polydispersity index
can be dominant and will affect GPC/SEC [PDI]) of the sample is not considered.
column selection. Column Types For example: If a typical sample PDI value for
The concept of specifc resolution has In general, linear, mixed-bed, or multipore synthetic polymers, produced by free-radical
been developed for GPC/SEC to understand columns have a broad pore size distribution polymerization, is two, the molar mass range
and compare resolution and to determine and cover a wide molar mass range with a covered can be very broad. In this case, users
the infuence of the molar mass accuracy.1 constant resolution. Single porosity columns should select an upper molar mass limit that is
Inspection of the calibration curve of a GPC/SEC with a narrow pore size distribution cover a 10× higher than the Mw of the highest samples
column can give a good indication of resolution. smaller molar mass separation range but with to be analyzed. Figure 1 shows the molar mass
The slope of the calibration curve directly a high resolution. Resolution can be increased distribution for a European reference material,
indicates resolution; the fatter the slope, the for all types of columns by adding a column a poly(styrene) of Mw = 181,200 Da and
higher the resolution.2 of the same type and porosity. The separation PDI = 2.26. Although the average molar mass is
Good resolution (a fat slope) can only be range of single porosity columns can be relatively low for this polymer, molar masses up
achieved for a limited molar mass separation expanded by adding a column of the same to 2 million Dalton are present.
Photo Credit: Daniel Kulinski/Getty Images
range when one column is used, which is type with different, correctly matched porosity. Figure 2 shows an example of when a fraction
acceptable for protein (or antibody) and The differences between linear, mixed-bed, of the sample molar masses is higher than the
oligomer analysis. However, when analyzing multipore columns, and single porosity columns exclusion limit (the upper molar mass separation
synthetic polymers or natural macromolecules was covered in detail in a past instalment of Tips limit). It shows the refractive index (RI) detector
with a broad molar mass distribution, an & Tricks.3 signal (green) overlaid with the corresponding
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The Column www.chromatographyonline.com Tips and Tricks: GPC/SEC
Figure 1: Many synthetic and natural macromolecules have a broad molar mass distribution
and therefore a higher polydispersity index (PDI). This example shows the molar mass range
covered for an poly(styrene) European reference material with a PDI around two and a
medium-molecular-weight.
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An artifcial shoulder appears in the Selecting the Right Combination
chromatogram at approximately 6 mL It is not possible to combine linear or mixed
elution volume when the exclusion limit bed columns and single porosity columns in
has been reached. This is not related to all combinations — if the porosities and the CLICK HERE
sample characteristics, but is the result of the calibration curve slopes do not match for each TO FIND OUT MORE AT
resolution change indicated by the change of of the columns combined, chromatographic www.malvern.com/morechoice
Material relationships
the slope of the calibration curve. artifacts (such as shoulders) can arise giving
This problem can be solved by adding a erroneous results.
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Figure 2: An upper molar mass separation limit that is too low can lead to artifcial
shoulders in the chromatogram. Choosing the proper separation range helps to solve this
problem.
Figure 3 shows an analysis of three different Porosity mismatch is not always readily
Dextran samples on two analytical GPC/SEC visible in the calibration curve. Figure 4 shows
columns with different porosities. One of the a calibration curve where a very small porosity
samples, Dextran 40 (red), seems to be bimodal and a very large porosity column have been
as a shoulder is present. However, this shoulder combined. The resulting calibration curve
is not part of the sample and cannot be seen has two regions with very different slopes
on a single column. This shoulder is a result of a and an infection point can be identifed.
mismatch of the porosities. This combination would defnitely produce
Subsequent calculation of the raw data chromatographic artifacts.
erroneously creates a shoulder in the molar mass On the other hand, the calibration curve
distribution of this sample. Furthermore, small can look as would be expected but there
changes in the column manufacturing process could still be a mismatch problem. Such a
can shift or eliminate the shoulder making it mismatch can only be detected by probing
appear to be a real property of the sample. the region of the infection point using
Long-term reproducible results based on such a reference materials with a broad molar mass
setup are therefore extremely hard to achieve. distribution.3
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Figure 3: Chromatograms of three Dextrans with broad molar mass distributions and different 13th International Symposium on Hyphenated Techniques in Chromatography and
molar mass averages. The red trace seems to be bimodal, but this is a chromatographic Separation Technology (HTC-13)
artifact resultant of porosity mismatch of the two combined columns.
The 13th International Symposium on
Hyphenated Techniques in Chromatography
and Separation Technology (HTC-13) will be
held at the Old Saint John Conference
Center, Bruges, Belgium, from January 29-31
2014. The three-day event will feature
recent findings from leading academic and
industrial experts on chromatographic and
mass spectrometric techniques covering
fundamental aspects, instrumental developments and state-of-the-art applications. A number of key
researchers in the field have been invited including: Alois Jungbauer, Peter Schoenmakers, Hian-Kee Lee,
Tadeusz Gorecki, Michal Holcapek, Ralf Zimmermann, Fred Regnier, Bruno Le Bizec, Gabriel Vivo-
Truyols, Philip Marriott, Luigi Mondello, Janusz Pawliszyn, Valérie Pichon, Bart Devreese, Gert Desmet,
Robert Shellie, Davy Guillarme, Hans-Gerd Janssen, Yvan vander Heyden, Emily Hilder, Frank David,
Hernan Cortes and Tuulia Hyötyläinen.
The HTC-13 programme features state-of-the-art overview lectures, tutorials, keynote and oral
presentations encompassing fundamental chromatographic developments and application areas. An
emphasis will be placed on oil and petrochemicals during sessions organized by the Royal Society of
Chemistry (RSC). Other areas of interest cover computational chromatography and life sciences, new
data-treatment methods, metabolomics, green hyphenated chromatography, natural products and
hyphenated electro-driven systems.
Instrument and equipment vendors will display their latest systems in the large exhibition space which is
integrated with catering and presentations of shortlisted posters.
Several awards will be presented including the Lifetime Achievement Award, sponsored by LC GC
Europe, honoring a scientist who has been distinguished by outstanding achievements in the field of
hyphenated techniques in chromatography and for distinguished service to the international
Up to now, it is not possible to overcome • Ensure that the upper molar mass separation chromatographic community, and the HTC-award, sponsored by Elsevier Science for the most innovative
these problems with advanced calibration limit is suffcient taking the PDI of your samples paper or poster. The most innovative poster will also be awarded a specific poster award by the
scientific advisory committee.
data ftting even if absolute detection, such as in account. Verify that all parts of the sample To encourage scientific exchange and friendship building, the scientific program will be topped off by a
on-line laser light scattering, is used. Molar mass elute in a region with suffcient separation rich social programme consisting of a welcome party, reception at the historical Town Hall, beer
degustation evening, symposium dinner and a farewell cocktail reception.
distributions can still show artifcial shoulders if by overlaying the calibration curve with the Short courses will be organized on January 28 on capillary electrophoresis, gas chromatography and
the porosities do not match. A better solution is sample. high-performance liquid chromatography troubleshooting and advanced mass spectrometry. HTC is also
organizing a job market. Scientists seeking a job that involves hyphenated systems (participants and
to optimize the separation by adding correctly • Avoid combining linear columns of different non-participants of the meeting) will be given facilities to meet potential employers.
matched columns. types; linear columns with single porosity The 3rd International Symposium on Hyphenated Techniques for Sample Preparation (HTSP-3) will be
held alongside HTC-13 on January 28-29 2014. The HTSP meeting is a strong programme in its own right,
columns (for example, linear + 100 Å); and with a number of experts from around the world as invited speakers. Participants of HTSP will be able to
catch a glimpse of some of the HTC-13 lectures and posters - and vice versa.
Summary single porosity columns with non-matching
There are some general rules that are more or porosities — or only do so following the Tel: +32 92649606 Fax: +32 92649606
E-mail: info@htc-conference.org
less true for all columns, independent of the manufacturer’s recommendation or by doing Website: http://www.htc-conference.org
manufacturer: careful mismatch tests.
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Figure 4: Calibration curve of two columns with non-matching porosities. The curve has two
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• Investigate unexpected shoulders carefully. References

Review the calibration curve with respect to 1. W.W. Yau, J.J. Kirkland, D.D. Bly, and H.J. Stoklosa,
changes in slope and use reference materials Journal of Chromotography A 125(1), 219–230
with a broad molar mass distribution to (1976).
John Hinshaw, renowned Specific topics covered include:
detect hidden porosity mismatch. Please 2. D. Held, The Column 7(2), 14–16 (2011). expert in gas chromatography ➤ Peak Problems: How to handle partially resolved
note that porosity mismatch can even occur 3. T. Hofe, The Column 4(4), 20–23 (2008). and the longtime author of or distorted peaks that yield poor quantitation
within one column, for example, if the the “GC Connections” column ➤ System Operation: The steps to follow for
in LCGC, provides invaluable restoring an idle GC column to operating
manufacturer blends materials in a linear or Daniela Held studied polymer chemistry in condition
advice about how to handle
mixed column to cover a wider molar mass Mainz (Germany) and is working in the PSS ➤ Air Leaks: What happens when air leaks into the
some of the most common
range. software and instrument department. She is also carrier-gas line, and what to do about it
difficulties faced by users of
responsible for education and customer training. ➤ Preventive Maintenance: How to avoid crises
gas chromatography as well through periodic maintenance of your GC system
In conclusion, applying these three basic rules as with best practices to avoid
➤ Upgrading GC: Guidelines for upgrading your
will help to establish GPC/SEC methods that problems in the first place. GC laboratory to use high speed GC and generate
E-mail: Dheld@pss-polymer.com your own gases
can be used over a long time period with high Website: www.pss-polymer.com
reproducibility.
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Q&A: Miniaturizing Military Detectors
Scientists at Sandia National Laboratories are thinking smaller in the development of detectors for military use — from the
detection of explosives and chemical weapons to humans. Scientist Ron Manginell from the laboratories spoke to
Bethany Degg of The Column about the on-going research in this area.
Q. You work for the Microsystems-Enabled Q. Sandia released details of MicroChem

Detection Department at Sandia National Lab — a detector for the military — in the
Laboratories, part of a network of US late 1990s. What was unique about the
National Security Laboratories — what is technology that you developed?
the purpose of this laboratory? A: The MicroChemLab system was very unique
A: Sandia’s primary missions are in nuclear in its system approach. While many groups
weapons and national security. have developed interesting detectors, the
MicroChemLab miniaturized laboratory system
Q. What are the main research interests of approaches to chemical detection. This includes
your department? inventing the world’s frst microfabricated
A: My department is primarily concerned preconcentrators, implementing the frst
with developing miniature, portable miniature gas chromatographs, and sensitive
detection systems for chemical, biological, microdetectors. Each of these elements had
and physical detection. Our main thrust chemical selectivity built in, allowing sensitive
for many years has been developing and feld analysis to take place in minutes, rather
“felding” handheld chemical detection than hours, and usability by lay persons.
systems for chemical agents and industrial
uses. Q. What are the applications of the
detector now?
Q. How did you become an inventor A: The system is still used for detecting chemical
working for Sandia? agents, but other targets have been added
A: I began working at Sandia as a graduate over time, including toxic industrial chemicals,
student intern, then as a postdoctoral pharmaceutical solvents, natural gas, and so
Photo Credit: Courtesy of the Author
appointee, and fnally as a full-time on. Recently, our development of a miniature

employee. The work in our area at Sandia pulsed-discharge ionization detector (mini-PDID)
has always been centred on research and has allowed us to detect a much broader
development, with a strong emphasis on range of compounds, including unique volatiles
new inventions. emitted by bacteria.
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The Column www.chromatographyonline.com Q&A: Manginell
Q. Why is gas chromatography (GC) your ionization potential, or the willingness for a A: Our research now is focused on New Strategies to Improve
method of choice when developing sensors chemical to give up an electron and be ionized. implementing our miniature GC-based Your CE-SDS Results:
for the detection of VOCs? Detection is electronic, meaning it is fast and systems with detectors like the mini-PDID and
A Data-driven Perspective
A: Even the best detectors, such as the sensitive. miniature MS. We are looking at these for a ON-DEMAND WEBCAST
gold-standard laboratory-grade mass variety of chemicals, including those produced Register Free at
spectrometers, utilize GC. GC separates out a Q. What are the advantages of your by bacteria in their metabolism. www.chromatographyonline.com/newstrategies
mixture of chemicals so that they are presented prototype technology compared with EVENT OVERVIEW:
Capillary SDS gel electrophoresis (CE-SDS) has been widely
to a detector, like the mass spectrometer, in current “e-nose” technology? Dr Ron Manginell is a principal member of adopted by the biopharmaceutical industry for automation
of therapeutic protein analysis. In this webcast, the speaker
a time series. This simplifes the job of the A: The bare response of our detectors are at the technical staff in the MicroSystems-Enabled will demonstrate the major challenges of CE-SDS sample
preparation with real-life data and examples. The speaker will
detector, as it only has to detect one compound least a factor of 10 better than typical e-nose Detection Department at Sandia National also share proven strategies to:
at a time if the GC separation is good. Our systems, but probably a factor of 100 in many Laboratories. He has worked at Sandia since n Achieve reproducible peak areas and migration times
n Minimize user-to-user sample preparation variability
systems use GC for the same reason, to cases. Further, we can detect not only patterns 1995 on microsensors, microfabrication, n Improve workfow efciency through automated sample
simplify the detector’s job. In addition, the of response, as e-noses do, but also specifc and microanalytical systems and has over 20 preparation
Who Should Attend:
time at which compounds exit a GC can be chemicals, thereby imparting greater chemical patents. In the last 15 years he has focused on
Anyone using CE-SDS, SDS-PAGE or similar technology for
used in their identifcation. Many groups claim specifcity. microanalytical systems for the detection of protein purity analysis, including:
n n
to have the latest and greatest detectors. But chemical agents, explosives, toxic chemicals, Biopharmaceutical
scientist and other
Lab Technicians,
lab managers,
like gold-standard MS, if they do not have a Q. What are the current applications and biological compounds. Ron has also analytical scientists and lab directors
specializing in the n Principal Investigators
separation column in front of them, they are that you have tested? Could it be used in developed microfuidics and control systems development and or
n Research scientists
production of monoclonal
n Biopharma QC managers
very likely to be confused in the real world. the feld to test for the use of chemical for biological applications, including cell, antibody therapeutics
n Research Engineers and and laboratory managers
weapons? antibody, DNA and protein capture, and development engineers
Q. Your team is currently working on A: Absolutely. That was the original intent and sensing systems. In 1997 Ron received a PhD Key Learning Objectives:
the development of a miniature pulsed- the chemical target for which most of our test in physics with honours from The University of n Pros and Cons of
CE-SDS technology Presenter
discharge ionization detector (mini-PDID). data was obtained. New Mexico (Albuquerque, New Mexico, USA) n Understand Kevin Overstreet
challenges related
What are the key features of this detector? for research performed at Sandia National to manual sample
Chemist, Bioproduct
Pharmaceutical Development
preparation Eli Lilly and Company
A: This detector is small, and has the ability to Q. What further developments are Laboratories; he received a B.S. degree in
n When to consider
detect organic compounds at parts-per-billion required in order for it to be put into physics with honours from the Rochester automated sample Moderator
preparation vs. Laura Bush
levels or lower. It generates intense ultraviolet action? Where do you see your future Institute of Technology (Rochester, New York, manual sample Editorial Director,
preparation LCGC
(UV) light internally that can ionize any chemical research taking you? USA) in 1991.
except neon and helium. This opens up a very Sponsored by Presented by
broad range of chemical analyses. The ionization To reach Ron Manginell contact Sue Holmes, Sandia Media and Communications,
energy can also be tuned downwards if desired at sholmes@sandia.gov For questions, contact Kristen Moore at kmoore@advanstar.com
to create chemical selectivity based on chemical
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Diisobutyl Columns Reduce Solvent Consumption
and Offer Rapid HPLC–MS Analysis of Plasma
Oxycodone and its Metabolites
Linda L. Risler1 and Anne E. Mack2, 1 Fred Hutchinson Cancer Research Centre, Washington, USA, 2 Agilent Technologies, Inc., California, USA.
Oxycodone and its metabolites (noroxycodone, oxymorphone,

and noroxymorphone) were analyzed by high performance liquid
chromatography–mass spectrometry (HPLC–MS) using a diisobutyl stable
bond column. Opioid compounds were isolated from human plasma
samples using solid-phase extraction (SPE) prior to HPLC–MS. The
proposed method is highly linear (R 2 >0.9900) and reproducible (<10%
difference between duplicates) for all compounds; and also reduces
analytical turnaround time and reduces solvent consumption.
Oxycodone is a semi-synthetic opioid the metabolic scheme of oxycodone.

synthesized from poppy-derived thebaine. Oxymorphone is approved for use as an
It was developed in Germany in 1916 to analgesic in its own right, but like oxycodone,
replace the only narcotic analgesic available it was also reformulated in 2011 owing to
at the time: Diacetylmorphine (heroin).1 high rates of abuse.6,7 Substance abuse of
Today, oxycodone is a schedule II drug in drugs such as oxycodone and oxymorphone
the USA2 and a class A drug in the UK3 makes accurate identifcation and
indicated for the relief of moderate to severe quantifcation of these compounds and their
pain. Although it has proven medical uses, multiple metabolites a clinical priority.
the high abuse potential of oxycodone led In recent years, high performance liquid
to the reformulation of extended release chromatography–mass spectrometry
formulations in an attempt to reduce abuse (HPLC–MS) (or LC–MS–MS) has become
Photo Credit: LAGUNA DESIGN/Getty Images
through non-oral routes.4 increasingly important in the feld of clinical

Oxycodone is metabolized by the and forensic toxicology. It has enabled the
cytochrome P450 enzyme system (CYP2D6 analysis of aqueous matrices, as well as
and CYP3A4 isoforms) to noroxycodone, hydrophilic, thermolabile, and non-volatile
oxymorphone, and noroxymorphone (a analytes that were not suffciently covered
secondary metabolite).5 Figure 1 shows by the established gold standard technique
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The Column www.chromatographyonline.com Risler and Mack
Figure 1: Oxycodone and its metabolic products.
gas chromatography–mass spectrometry require preparation prior to injection onto

(GC–MS).8 an HPLC column. In spite of clean-up with
Multi-analyte procedures that allow techniques such as SPE, matrix effects can either
simultaneous analysis of multiple drugs from reduce analyte response or enhance it. Both can
a drug class, or several closely related drug compromise quantifcation accuracy and ion
classes, are often used in clinical and forensic suppression can lead to false negative results.8
toxicology. These procedures help to save time, Chromatographic column and stationary
limit the number of methods that need to be phase selection can help to overcome the
established for a broad spectrum of analytes to challenges of multi-residue analysis in complex
be covered, and reduce the resources required matrices. For example, HPLC using stationary
during method development.8 However, phases with sub-2 µm particle sizes can
complex samples such as human plasma usually enhance separation power and reduce run
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Figure 2: Extracted ion chromatogram of a human plasma sample spiked with oxycodone,
noroxycodone, d6-oxycodone.
time; mixed mode column sorbents may offer a isopropanol, and ammonium hydroxide were
balance between extraction of as many analytes purchased (Fisher Scientifc) and Boric acid
as possible while also minimizing sample (JT Baker). Standard solutions of oxycodone,
matrix interference.8 This study describes the noroxycodone, oxymorphone (all 1 mg/
successful application of a reversed phase HPLC mL), and noroxymorphone (0.1 mg/mL) in
using a stable bond column for separation of methanol (Cerilliant) were used.
oxycodone and its metabolites from human A pooled internal standard was prepared
plasma extracts prior to MS identifcation. by mixing 25 µL aliquots of oxycodone,
noroxycodone, and noroxymorphone with
Methods 2.5 µL of oxymorphone and 25 mL methanol.
Reagents: Acetonitrile, ammonium Sample Preparation: Matrix samples were
acetate, methanol, methylene chloride, prepared by spiking 1 mL of clean human
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plasma with different concentrations of the gas temperature and fow rate: 350 °C at 40 ng/mL d6-oxycodone (an internal standard) In addition, such systems may aid
pooled internal standard. 12 L/min. and then extracted by SPE. Despite plasma being development of optimized drug formulations
Mixed-mode SPE extraction of metabolites a complex sample matrix, chromatograms were and help to monitor compliance with pain
is summarized as follows: Results and Discussion well resolved for each of the fve compounds. medication where urine analysis is not
• Sorbent material: Octyl (C8) and This study describes the successful This method employed demonstrated possible.
benzenesulphonic acid (SCX). chromatographic separation of oxycodone and high linearity, sensitivity, and reproducibility.
• Pre-condition: 2 mL methanol, then 2 mL its metabolites in human plasma using reversed Calibration curve coeffcients of determination References
deionized (DI) water. phase HPLC on a stable bond column, followed (r2) were ≥0.99000 over the concentration 1. E. Kalso, J. Pain Symptom Manage. 29(Suppl 5),
• Load: 1 mL spiked plasma sample + 1.5 mL by MS identifcation. Rapid, multi-compound range of 2–50 ng/mL for oxycodone S47–56 (2005).
borate buffer (pH 8.9). analysis within a single analytical run is highly and noroxycodone, and 0.2–5 ng/mL for 2. United States Congress: Controlled Substances Act
• Wash: 2 mL DI, 1 mL 10 mM ammonium valuable in the forensic determination of oxymorphone and noroxymorphone. Limits 1970. United States of America.
acetate (pH 4), 2 mL methanol. opioid compounds in human plasma. Optimal of detection were: 0.5 ng/mL for oxycodone, 3. HM Government: Misuse of Drugs Act 1971. United
• Elute: 3 mL methylene chloride/isopropanol/ chromatographic separation of compounds 1 ng/nL for noroxycodone, and 0.2 ng/mL for Kingdom.
ammonium hydroxide (80:20:2). in a complex sample matrix such as human both oxymorphone and noroxymorphone. 4. FDA news release: “FDA approves abuse-deterrent
• Samples were then air-dried at 60 °C plasma will depend on column and stationary Inter-sample variability of the method was labelling for reformulated Oxyontin” 16 April 2013.
and the remaining pellets reconstituted in phase selection. Compared with end-capped low, with less than 10% difference between 5. B. Lalovic et al. Drug Metab Dispos. 32, 447–454
60 µL 10 mM ammonium acetate (pH 4)/ stationary phases, non-end capped stationary duplicate samples over the aforementioned (2004).
acetonitrile (95:5). phase allow chromatographic separation of a concentration range for limits of detection. 6. Press release: “Endo announces FDA approval of a
mixture of compounds with varying polarity new formulation of Opana ER designed to be crush
HPLC–MS Analysis: HPLC separation was because of additional interactions with exposed Conclusion resistant” Endo Pharmaceuticals, 12 December 2011.
performed on an 1100 series HPLC system silanol groups. These interactions can be Oxycodone and its metabolites were 7. S.F. Butler et al. J. Pain. 14(4), 351–358 (2013).
(Agilent); Chromatographic column: 2.1 optimized by altering mobile phase conditions. successfully extracted from human plasma, then 8. F.T. Peters, Clin Biochem. 44, 54–65 (2011).
mm × 50 mm, 1.8-µm Zorbax Narrow Bore The small 1.8-µm particle size allowed for separated using reversed-phase HPLC prior to
RRHT StableBond SB-C18 (Agilent); Mobile superior resolution and effciency over 3.5-µm MS identifcation over a linear concentration Linda L. Risler is a researcher at the Fred
phase A: 20mM ammonium acetate, pH 4.0; or 5-µm particles. In addition, use of a short range. The separation technique employed Hutchinson Cancer Research Centre, Seattle,
mobile phase B: Acetonitrile. Mobile phase column length allowed a lowered analytical allowed for rapid analytical turnaround, clear USA.
fow rate: 0.300mL/min; Gradient: Hold 5% turnaround time, while the small internal chromatographic peak resolution, and reduced Anne E. Mack is an LC applications chemist
B: 2.33 min. Increase B from 5–20%: diameter reduced solvent consumption. solvent consumption This method demonstrated at Agilent Technologies, Inc.
2.33–4.33 min. Stop: 6 min. Post time: Figure 2 shows the extracted ion good linearity, sensitivity, and reproducibility.
4 min; Column compartment temperature: chromatograms (EIC) of a human plasma sample These advantages may make the method useful E-mail: anne_brooks@agilent.com
30 °C; MS identifcation: Electrospray spiked with 50 ng/mL each of oxycodone and for routine multi-compound forensic analysis of Website: www.agilent.com
ionization; Polarity: Positive; Spray chamber noroxycodone, 5 ng/mL noroxymoprhone and opioids in human plasma.
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A Mixed-Mode Stationary Phase for the Structural
Analysis of Labelled N-Glycans Using LC–MS–MS
Udayanath Aich,1 Julian Saba,2 Xiaodong Liu,1 Srinivasa Rao,1 Yury Agroskin,1 and Chris Pohl,1 1Thermo Fisher Scientifc, Sunnyvale,
California, USA, 2Thermo Fisher Scientifc, Mississuaga, Ontario, Canada
This article describes the analysis of fuorescently labelled N-glycans released from proteins by liquid chromatography–mass spectrometry (LC–MS). The
column for this separation used mixed-mode surface chemistry that combines weak anion-exchange and hydrophilic interaction liquid chromatography
(HILIC) retention mechanisms for high-resolution and high-throughput analysis of glycans. This combination of modes offers unique selectivity that
provides separation of glycans based on charge, size, and polarity. MS and MS–MS analyses were performed using a hybrid quadrupole-orbitrap-based
mass spectrometer in negative ion mode to provide detailed structural information of N-glycans released from proteins.
Glycans are involved in a wide range of The structures of glycans are highly
biological and physiological processes, diverse, complex, and heterogeneous
including recognition and regulatory because of post-translational
functions, cellular communication, gene modifications. This makes it challenging
expression, cellular immunity, growth, to comprehensively characterize
and development.1 They are commonly glycan profiles and to determine
investigated in therapeutic protein their structures. 3 To understand the
drug development because there is detailed structure of glycans using
strong evidence that bioactivity and liquid chromatography tandem
effcacy are affected by glycosylation. 2 mass spectrometry (LC–MS–MS), it
Both the structure and types of is essential to separate all isomeric,
glycans attached to the proteins charge, and branching glycan
Photo Credit: Science Photo Library - SCIEPRO/Getty Images
are usually examined. For these variations.

reasons, understanding, measuring, Liquid chromatography (LC) coupled
and controlling glycosylation in to mass spectrometry (MS) has
glycoprotein-based drugs, glycan emerged as one of the most powerful
content of glycoprotein products, and tools for the structural characterization
thorough characterization of biosimilars of glycans. Various modes of high
has become increasingly important. performance liquid chromatography
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The Column www.chromatographyonline.com Pohl et al.
Table 1: Structural characterization of glycans present in each peak by the separation of 2AB-labelled N-glycans from bovine fetuin. (HPLC) separation have been developed for
the analysis of glycans, including normal
phase (NP) or hydrophilic interaction
(HILIC),4 ion-exchange (IEX), and
reversed-phase.5 Porous graphitized carbon
LC columns that work based on a reverse
phase mechanism have also been used for
the LC–MS analysis of native unlabelled
N- and O-glycans.6 Because glycans are
highly hydrophilic and polar substances,
HILIC amide columns are extensively used
for glycan analysis as compared to reverse
phase chromatography.7 The separation
columns are typically amide, amine, or
zwitterionic-based packing materials. The
HILIC amide columns 8 separate glycans
by hydrogen bonding, resulting in a size
and composition-based separation. HILIC
amide columns are particularly useful for
the separation of 2AB or 2AA labelled
N-glycans released from antibodies, for
example MAb, in which the majority of
glycans are neutral (0 charge). However,
HILIC amide columns don’t provide good
separation of highly charged state (≥-2)
sialylated N-glycans because glycans of
different charge states are intermingled in
the separation envelope. Highly sialylated
glycans also retain strongly in HILIC
columns and that leads to unsatisfactory
separation. In addition, the inherited strong
ionic interactions between multiply charged
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glycan profile without the changes to Hypercarb cartridge (Thermo Scientific).

Figure 1: LC–MS analysis of 2AB-labelled N-glycans from bovine fetuin. the glycan profiles that labelling reaction with the help of a 24-port solid-phase
can introduce. Standardized MS data extraction (SPE) vacuum manifold (Thermo
acquisition method and data analysis Scientific) under vacuum. The released
software are essential to identify known glycans were conjugated with a 2-amino
and unknown N-glycan structures present benzamide (2AB) (Fisher Scientific) label
in proteins. LC–MS–MS analysis of glycans group.9
requires the processing of large set of data. 2. 2AB labelled N-glycans from fetuin
The incorporation of appropriate software (5,000 pmol) were dissolved in deionized
alleviates this issue, which enables the water (25 μL) in an autosampler vial
development of a true high-throughput (Thermo Fisher Scientific) (volume 250 μL).
workflow. 3. Acetonitrile (75 μL) was added to the
In this article, we highlight the structural same vial and mixed until uniform.
analysis of 2-aminobenzamide (2AB) Separation Conditions: Column:
labelled N-glycans from bovine fetuin 2.1 mm × 150 mm, 1.9 μm GlycanPac
using LC–MS. An example of a step-by- AXH-1 (Thermo Scientific); Mobile phase
step method for the release, labelling, A: Acetonitrile–water (80:20) (v/v). Mobile
separation, and structural elucidation phase B: Ammonium formate (80 mM,
of native and labelled N-glycans from pH 4.4). Column temperature: 30 °C.
glycans and the stationary phase often mechanisms. The WAX functionality proteins by LC–MS–MS technology is also Sample volume: 1 μL.
requires a high eluent buffer concentration provides retention and selectivity for presented. Gradient:
for HILIC amide column, adversely affecting negatively charged glycans while the Time %A %B Flow Rate Curve
MS sensitivity. HILIC mode facilitates the separation of Experimental Details (min) (mL/min)
This article will describe the use of a glycans according to their hydrophilicity Buffer Preparation: Ammonium formate -10 97.5 2.5 0.4 5
high-performance HPLC/ultrahigh-pressure and size. As a result, the column provides (80 mM, pH 4.4): Dissolve 5.08 ± 0.05 g of 0 97.5 2.5 0.4 5
liquid chromatography (UHPLC) column unparalleled separation capabilities for ammonium formate (crystal) and 0.60 g of 30 87.5 12.5 0.4 5
specifically designed for structural analysis highly charge native and 2AB or 2AA formic acid in 999.6 g of deionized water. 35 75.0 25.0 0.4 5
of glycans, either labelled or native, by labelled N-glycans. Furthermore, analyzing Sonicate the resulting solution for 5 min. 40 62.5 37.5 0.4 5
LC-fluorescence or LC–MS methods. The unlabelled glycans eliminates the extra Sample Preparation: MS Conditions: MS instrument: Q
column is based on innovative mixed-mode reaction step and time-consuming cleanup 1. Native N-glycans were released from Exactive hybrid quadrupole-Orbitrap MS;
surface chemistry combining both weak methods during labelling. Characterization glycoproteins with PNGase F enzyme (New Ionization mode: Negative ion mode; MS
anion-exchange (WAX) and HILIC retention of native glycans also retains the original England Biolabs) and purified with a 6 mL scan range: 380–2000 m/z; Resolution:
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70,000; AGC target: 1 × 106; Max IT: chromatogram (TIC) is shown in Figure 1. in Demonstrating Biosimilarity to a Reference 9. J.C. Bigge, T.P. Patel, J.A. Bruce, P.N. Goulding,
60 ms; dd-MS2 resolution: 17,500; For structural elucidation, data dependant Product, Draft Guidance; U.S. Department S.M. Charles, and R.B. Parekh, Anal. Biochem.
MS–MS AGC target: 2 × 105; MS–MS MS–MS spectra were acquired on all of Health and Human Services Food 230(2), 229–238 (1995).
max IT: 1000 ms; Isolation window: 2 m/z; precursor ions (z ≤ 2) and software was and Drug Administration, February 2012 10. A. Apte and N.S. Meitei, Methods Mol. Biol.
Dynamic exclusion: 90 s. used for data analysis.10,11 The detailed [On-line] www.fda.gov/downloads/Drugs/ 600, 269–81 (2010).
Data Processing and Software: structural information obtained from the GuidanceComplianceRegulatoryInformation/ 11. J. Saba, A. Apte, N.S. Meitei, and R. Viner,
Chromatographic software: ChromQuest MS–MS data (Table 1) further validated Guidances/UCM291128.pdf (accessed 18 Application Note 516: Automated Glycan
Chromatography Data System version the ability of the stationary phase to January 2013). Structural Isomer Differentiation Using
5.0 (Thermo Fisher Scientific); MS data separate glycans based on charge, size, 4. Thomas L. Chester, Analytical Chemistry 85(2), SimGlycan Software.
acquisition: Thermo Scientific Xcalibur isomers, and polarity. These results also 579–589 (2013).
software version 2.2 SP1.48; MS–MS data confirmed that the column would be ideal 5. (a) L. R. Ruhaak et al., Anal Bioanal Chem Udayananth Aich is a senior chemist
analysis: SimGlycan software (Premier for MS use. 397(8), 3457–3481 (2010). (b) Xiaoyu Chen and at Thermo Fisher Scientific, working in
Biosoft). Gregory C. Flynn, Anal Bioanal Chem 370(2), glycomics and glycoproteomics.
Conclusion 47–161 (2007). Julian Saba is a senior scientist, Life
Results The mixed-mode surface chemistry of 6. Pia H. Jensen, Niclas G. Karlsson, Daniel Science Mass Spectrometry at Thermo
Figure 1 shows the separation of neutral the column used here, combining weak Kolarich, and Nicolle H. Packer, Nature Protocols Fisher Scientific.
and acidic 2AB labelled N-glycans from anion-exchange and HILIC retention 7, 1299–1310 (2012). Xiaodong Liu is a manager, Column
bovine fetuin. The glycan elution profile mechanisms, is a high-performance, 7. (a) S. Hunter Walker, Brandon C. Carlisle, and Research and Development at Thermo
consists of a series of peaks grouped silica-based HPLC column for simultaneous David C. Muddiman, Analytical Chemistry Fisher Scientific.
into several clusters. The neutral glycans separation of glycans by charge, size, and 84(19), 8198–8206 (2012). (b) Michael Melmer Srinivasa Rao is an associate principal
elute first, followed by monosialylated, polarity. This mixed-mode approach offered et al., Analytical and Bioanalytical Chemistry research scientist, Thermo Fisher Scientific.
disialylated, trisialylated, tetrasialylated, unique selectivity and excellent resolution 398(2), 905–914 (2010). Yuri Agroskin is a director, research and
and, finally, pentasialylated species. of glycans released from bovine fetuin. 8. Thermo Scientifc Accucore 150 Amide HILIC development, Thermo Fisher Scientific
Analytes in each cluster represent glycans application note on “Analysis of Human IgG Dionex Products.
of the same charge. Within each cluster, References Glycans on a Solid Core Amide HILIC Stationary Chris Pohl is VP, Chromatography
glycans with the same charge are further 1. A. Varki, Glycobiology 3(2), 97–130 (1993). Phase”. Chemistry, Thermo Fisher Scientific.
separated according to their size and 2. C.R. Bertozzi, H.H. Freeze, A. Varki, and J.D. Esko,
polarity by HILIC interaction. Glycans in Biotechnology and the Pharmaceutical
The 2AB labelled N-glycans from Industry, Essentials of Glycobiology (2nd edition Cold
bovine fetuin were separated based on Spring Harbor Laboratory Press, Cold Spring Harbor,
E-mail: udayananth.aich@thermofisher.com
the separation conditions using a two New York, USA, 2009) Chapter 51. Website: www.thermofisher.com/glycanpac
eluent system and analysed. The total ion 3. Guidance for Industry, Scientifc Considerations
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4 December 2013 Volume 9 Issue 22

2 014 chromatography–mass spectrometry (HPLC–MS) using a diisobutyl stable

21 Tips & Tricks: Column Selection for an Appropriate Molar

28 Diisobutyl Columns Reduce Solvent Consumption and Offer

32 A Mixed-Mode Stationary Phase for the Structural Analysis of

Encapsulated bonding technology

compared to other technologies. These (pH 1.5 to 11.5), allowing exploitation

Flow path inertness products

Triple quadrupole liquid HPLC system

Syringe ﬁlters GC–MS headspace sensitivity

CIS4 Initial Temp.: -50 ºC

CIS4 Initial Temp.: 10 ºC

Figure 1: Total ion chromatograms of canned coffee by SHS analysis.

Fingertight UHPLC ﬁttings

Light scattering detector Thermal desorper

Puriﬁcation software THE VERY BEST FOR INSTRUMENTAL TLC

Ion chromatography system Modular gas generators

www.metrohm.com range is very low maintenance and provides long-term

GC–MS–MS system Core–shell particle columns

Automated soluble fraction system MALS technology

Deep well plates Light scattering detector

GC ﬁlter status indicator LC–MS instrument

Complex biological sample analysis Size-exclusion chromatography

Mass detector for chromatography UHPLC RI detector

Sophisticated Antibody Analysis by GPC/SEC with RALS

Monoclonal antibodies (mABs) are increasingly growing in importance

Detector signal (V)

Detector response (%)

and purifcation or are the result of long-term storage. As a result of 1.0

aggregation, antibodies lose their pharmaceutical effcacy and can 75

facilitate an immunology response.

length antibodies. Therefore, a GPC method, which offers the opportunity

GPC/SEC Solutions and Expert Support Experimental

Detector signal (V)

Photo Credit: SCIEPRO/Getty Images

Gulf of Mexico Research Initiative Grant

Photo Credit: jklr/Getty Images

Peaks of the Week News In Brief

Online Highlights Plastic Pollution

MORE ANGLES MORE DATA MORE CHOICE

Five Keys to Successful GC Methods

• Investigate unexpected shoulders carefully. References

Q. You work for the Microsystems-Enabled Q. Sandia released details of MicroChem

appointee, and fnally as a full-time on. Recently, our development of a miniature

Oxycodone and its metabolites (noroxycodone, oxymorphone,

Oxycodone is a semi-synthetic opioid the metabolic scheme of oxycodone.

through non-oral routes.4 increasingly important in the feld of clinical

Figure 1: Oxycodone and its metabolic products.

gas chromatography–mass spectrometry require preparation prior to injection onto

are usually examined. For these variations.

glycan profile without the changes to Hypercarb cartridge (Thermo Scientific).

CHROMacademy is announcing the launch of the interactive

Building on the success of our ground-breaking HPLC

Step 2. Select from nearly 50 different instrument symptoms which deal

With its ease of use, self explanatory graphics and a huge

27 April–1 May 2014 Fundamentals of GC - May 2014

ljones@advanstar.com kmosford@advanstar.com Chief Financial Officer

Executive Vice-President, Business Systems

Executive Vice-President, Human Resources

Photo Credit: Jonathan Kitchen/Getty Images

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