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1158 LCGC NORTH AMERICA VOLUME 26 NUMBER 12 DECEMBER 2008 www.chromatographyonline.com
Ronald E. Majors
Column Watch Editor
SAMPLE PREP PERSPECTIVES
Practical Aspects of Solvent
Extraction
Columnist Ron Majors
discusses some of the
practical considerations in
the successful application
of the popular yet age-old
technique of solvent
extraction (also known as
liquidliquid extraction, or
LLE). After a brief review
of the basics, guidelines on
the selection of the
appropriate extraction
solvents and how to use
acidbase equilibria to
ensure efficient extractions
of ionic and ionizable
compounds are provided.
Problems in LLE and the
solutions to these
problems are highlighted.
A newer technique called
dispersive liquidliquid
microextraction is
introduced.
R
ecently, I had the opportunity to
participate in Colacro XII, a
chromatography symposium held
in a Latin America country every two
years. This year's meeting was held in
Florianopolous, Brazil, October 27-30.
During the symposium, I perused the
large number of applications posters for
the sample preparation techniques being
used. I was surprised to find many of the
investigations of such diverse matrices as
natural products, fruits and vegetables,
petroleum products, and body fluids
used solvent extraction (liquidliquid
extraction, or LLE) as the initial (or the
only) sample preparation technique. So,
I thought that I would devote an install-
ment of Sample Prep Perspectives to
this well used technique. In our last
comprehensive sample preparation sur-
vey, nearly 40% of the respondents
reported on the use of LLE as at least
one of their routine sample preparation
procedures (1).
Earlier, I wrote on the basics of LLE
providing some of the theory (which
will not be repeated here), examples of
classical LLE including continuous and
countercurrent chromatography, and
some of newer techniques of the time
(2). More recently, I covered approaches
for miniaturization of classical LLE (3).
In this installment, I will provide some
practical hints to those who are consid-
ering the use or already are using LLE in
hopes of improving your methodology.
Quick Review of LLE
LLE is performed using two immisci-
ble liquids and soluble samples. LLE is
useful for separating analytes from
interferences by partitioning the sam-
ple between these two immiscible liq-
uids or phases. Usually, one phase in
LLE will be aqueous (often the denser
or heavier phase) and the second phase
is an organic solvent (usually the
lighter phase). The more hydrophilic
compounds prefer the polar aqueous
phase, while more hydrophobic com-
pounds will be found mainly in the
organic solvent. Analytes extracted into
the organic phase are recovered easily
by evaporation of the solvent, while
analytes extracted into the aqueous
phase often can be injected directly
onto a reversed-phase high perform-
ance liquid chromatography (HPLC)
column. Obviously, when the analyte
of interest in found in the aqueous
phase, evaporation is a much slower
process. However, the following dis-
cussion assumes that an analyte of
interest is concentrated preferentially
into the organic phase, but similar
approaches are used when the analyte
is extracted into an aqueous phase.
The generic flow diagram of Figure 1
summarizes the steps involved in a typ-
ical LLE separation. Because extraction
is an equilibrium process with limited
efficiency, significant amounts of the
analyte can remain in both phases.
248 LCGC NORTH AMERICA VOLUME 25 NUMBER 3 MARCH 2007 www.chromatographyonline.com
Ronald E. Majors
Column Watch Editor
COLUMN WATCH COLUMN
New Chromatography
Columns and Accessories at
Pittcon 2007: Part I
This months installment
of Column Watch is the
first of a two-part series in
which Ron Majors examines
the trends and highlights in
columns and consumables
at Pittcon 2007. Here,
he discusses new HPLC
columns and packings,
including reversed-phase,
specialty, ion-exchange and
ion chromatography, size-
exclusion, preparative, and
specialty columns displayed
at Pittcon 2007.
ittcon 2007 the 58th Pitts-
burgh Conference on Analytical
Chemistry and Applied Spec-
troscopy returned to the
massive McCormick Place in Chicago,
Illinois, on February 25March 2, 2007.
This years event hosted more than 1000
instrument manufacturers and 1abora-
tory suppliers in more than 2500 booths.
In addition to attending the exposition,
the conferees listened to 1250 oral pre-
sentations, viewed more than 975 post-
ers, checked numerous company seminar
rooms, or attended one of 110 short
courses.
Undoubtedly, Pittcon still remains the
most important yearly international ana-
lytical exhibition where companies intro-
duce their latest instruments, instrument
accessories, columns, sample preparation
products, and other consumable prod-
ucts. Because many past attendees have
purchased one or more new products
within three months after attending the
show, most exhibitors attempt to maxi-
mize their booth traffic to meet as many
potential customers as possible.
The purpose of this report is to pro-
vide information about many of the new
separation consumables and accessory
products that were displayed at Pittcon
2007. In some cases, products that were
introduced during 2006 but after Pittcon
2006 (1,2) might be included for reasons
of completeness. The information is
based upon manufacturers responses to a
questionnaire mailed in December 2006.
Because of space limitations and the fact
that some manufacturers did not respond
to the questionnaire, this report cannot
be considered an exhaustive listing of
all new products that were introduced
in Chicago. However, over the years,
these Pittcon summaries have provided
a good source of information that would
be difficult for one individual to gather
during the four days of the exhibition. In
addition, the products introduced have
shown definite correlations to current
research, development, and application
activity in the separation sciences.
As in previous years, columns and
other products recommended by their
manufacturers primarily for biomolecule
separations or sample preparation are
denoted in the tables with the designa-
tion BIO. Some of these products can
be used for general high performance
liquid chromatography (HPLC) separa-
tions as well, but their main emphasis is
for biological samples. I will cite specific
information about bioapplications where
appropriate. Because the sub-2-m
HPLC columns are still quite new and
require special care in their use, I have
classified them as specialty columns or in
a new series if an entire family was intro-
duced at Pittcon.
In this months coverage, I will
describe new introductions in the areas
of reversed-phase, normal- and bonded-
phase, ion-exchange and ion, size-exclu-
sion (SEC), large- and preparative-scale,
and specialty chromatography columns.
Next month, I will look at gas chroma-
tography (GC) products, sample prepara-
tion products, and hardware, accessories
and kits for chromatography and sample
preparation.
Trends and Highlights
General: This year, I observed that there
P
SAMPLE PREP PERSPECTIVES
Electromembrane Extraction:
The Use of Electrical Potential
for Isolation of Charged
Substances from Biological
Matrices
R
ecently, efforts have been
devoted to the miniaturiza-
tion of existing liquidliquid
extraction methods to make them more
versatile and powerful. Reducing the
use of hazardous organic solvents due
to environmental and cost concerns,
time reduction, ease of automation, pos-
sible online coupling, high-throughput
capability, and the small amounts of
matrix available are major incentives
that have motivated scientists working
towards miniaturization. In bioanalysis,
which in this context is defined as the
analysis of small drug molecules and
their metabolites in biological samples,
the complexity of the matrices requires
selective and specific sample prepara-
tion methods to isolate the analytes of
interest. Macromolecules, salts, cellular
material, fat, or lipids in biological
matrices such as urine, plasma, whole
blood, and breast milk can disturb
the separation and data analysis steps.
In addition, the analytes of interest
can often exist in low concentrations
(pg/mLg/mL). Therefore, a sample
preparation method with high degree of
selectivity and enrichment is crucial for
a successful analysis.
With these considerations kept in
mind, a totally new approach to sample
preparation was proposed in 2006 (1).
The innovative concept, called electro-
membrane extraction (EME), combined
the technical setup for hollow-fiber liq-
uid-phase microextraction (HF-LPME)
(2,3) with known principles for elec-
troextraction (410). This combination
Astrid Gjelstad
This minireview of a
new sample preparation
technique called
electromembrane
extraction demonstrates
how the combination of
an electroextraction with
hollow-fiber liquid-phase
microextraction can lead
to a very selective, rapid
sample preparation method
for the extraction of
charged substances from
complex matrices such as
plasma, breast milk, and
urine. Several important
parameters for successful
extraction are presented
and application examples
of various analytematrix
combinations are tabulated.
Ronald E. Majors
Sample Prep Perspectives Editor
offers a highly selective sample prepara-
tion method using simple equipment
and gains a high degree of enrichment
within a short period of time.
The EME method extracts charged
substances from a small sample volume
through a thin membrane of organic
solvent immobilized in the wall of a
hollow fiber and into a receiver solution
inside the lumen of the hollow fiber.
This extraction process is forced by an
applied potential difference across the
membrane, and this combination of
well-known liquidliquid extraction
processes with electrokinetic migration
yields a rapid and selective sample prep-
aration method for ionic substances.
EME has shown to be compatible with
a wide range of biological matrices
for example, plasma, whole blood,
urine, and breast milk preparing
clean extracts in a short period of time
with simple and inexpensive equipment.
This installment of Sample Prep
Perspectives is a minireview of the
EME technique and shows the investi-
gation of several parameters that affect
recovery of charged analytes. In addi-
tion, a number of application examples
will illustrate its potential for the suc-
cessful extraction of drugs from com-
plex matrices.
Experimental
The technical setup for the equipment
used in EME is based upon earlier
experience with HF-LPME and is
shown in Figure 1a. The hollow fiber
used is made of porous polypropylene,
92 LCGC NORTH AMERICA VOLUME 28 NUMBER 2 FEBRUARY 2010 www.chromatographyonline.com
Volume 29 Number 1 January 2011
www.chromatographyonline.com
LCMS Analysis
of Herbicides in
Lake Water
Whole Blood
Sampling Techniques
Hydrogen Carrier Gas
Considerations
Reversed-Phase LC
Solvent-Strength Selectivity
Ronald E. Majors
Sample Prep Perspectives Editor
SAMPLE PREP PERSPECTIVES SAMPLE
436 LCGC NORTH AMERICA VOLUME 25 NUMBER 5 MAY 2007 www.chromatographyonline.com
QuEChERS A New Sample
Preparation Technique for
Multiresidue Analysis of
Pesticides in Foods and
Agricultural Samples
esticide residue analysis of food
and environmental samples has
been performed for over 40
years. Most pesticide methods
are not oriented toward measuring a
single pesticide. Instead, residue-moni-
toring laboratories are geared to perform
multiclass, multiresidue methods to
detect a wide variety (in the hundreds)
of pesticides potentially in the sample.
Because of the wide range of chemical
properties of pesticides (including acidic,
basic, and neutral) and the wide variety
of matrices (polar, nonpolar, fatty, waxy,
and so forth), the sample initially must
be cleaned up using a compatible sample
preparation technique before injection
into the chromatographic system. Ideally,
a multiresidue method should be fast
and easy to perform, require a minimum
amount of chemicals (especially solvents),
provide a certain degree of selectivity,
and still cover this wide array of ana-
lytematrix pairs. Although many sample
preparation protocols involve lengthy
multistep procedures, if the number of
steps can be minimized by use of a sim-
ple sample preparation procedure, repro-
ducibility (precision) and accuracy can be
improved and time can be saved.
For many years, the standard approach
for pesticides multiresidue methods for
foods and agricultural products involved
the use of organic solvent extraction
(usually acetone), followed by water dilu-
tion and partitioning into a nonpolar
solvent (such as methylene chloride and
petroleum ether). This approach worked
fine for nonpolar pesticides but certain
polar compounds such as organophos-
phorus insecticides and several modern
pesticides were partially lost. More
sophisticated approaches were needed
and analysts began to experiment with
different organic solvents for the initial
extractions. Next, the addition of vari-
ous salts to the mixtures was found to
affect recovery by changing solvent
polarity and this became the fashion-
able approach (salting out effect). Some
of these methods became the standard
official multiresidue methods used in
government, contract, and testing labs
today.
With environmental and health con-
cerns about the use of chlorinated sol-
vents, workers investigated various non-
halogen-containing solvents and solvent
mixtures such as ethyl acetate, acetoni-
trile, and cyclohexaneethyl acetate for
extraction. For various reasons, none of
these procedures gave an entirely satisfac-
tory set of results, particularly in the area
of extraction efficiency for wide ranges
of pesticides. Extraction solvent compat-
ibility with the analytical technique, be it
high performance liquid chromatography
(HPLC) or gas chromatography (GC)
with and without mass spectrometry
(MS) detection was another complicating
problem.
Therefore, new extraction techniques
This months installment of
Sample Prep Perspectives
describes a new extraction
technique called QuEChERS
(standing for quick, easy,
cheap, effective, and
safe and is pronounced
catchers) for the sample
preparation of pesticides
in foods and agricultural
samples. The technique
uses simple glassware, a
minimal amount of organic
solvent, and various
salt/buffer additives to
partition analytes into an
organic phase for cleanup
by dispersive solid-phase
extraction (d-SPE). The
technique provides good
recoveries, is reproducible,
and costs less than other
sample preparation
approaches. The technique
is being adopted by many
laboratories worldwide.
It has the potential for
applications outside of the
pesticide in foods area.
P
364 LCGC NORTH AMERICA VOLUME 31 NUMBER 5 MAY 2013 www.chromatographyonline.com
COLUMN WATCH
Column Watch comprises
Part II of our yearly
report on new products
introduced at Pittcon. Gas
chromatography columns,
sample preparation
products, and hardware,
accessories, and small
tabletop instruments
mainly for sample
preparation are covered.
New Chromatography
Pittcon 2013, Part II
T
he Pittcon 2013 Conference and
Expo (less formally known as the
64th Pittsburgh Conference on Ana-
lytical Chemistry and Applied Spectros-
copy) was held for the first time in the city
of Philadelphia, Pennsylvania, on March
1721, 2013. This years event hosted over
1011 instrument manufacturers and labo-
ratory suppliers in more than 1925 booths.
the conferees listened to more than 2200
technical presentations (orals, posters,
workshops, invited and contributed talks,
and award symposia), checked on numer-
ous company seminar rooms, or attended
one of 100 short courses.
Although the attendance was slightly up
this year compared to last year, the average
attendance had been shrinking over the
past several years. Still, Pittcon remains
one of the most important international
analytical exhibitions where companies
introduce their latest instruments, instru-
ment accessories, software, columns, and
sample preparation and other consumable
products. Several notable companies were
missing again this year, but enough were
present such that visitors had plenty of
opportunities to investigate new (and old)
technologies.
products that were displayed at Pittcon
2012 (1,2) may be included for reasons of
completeness. The information is based on
manufacturers responses to a questionnaire
mailed in early 2013. Because of space lim-
itations and the fact that some manufactur-
ers did not respond to the questionnaire,
this report cannot be considered a com-
prehensive listing of all new products that
were introduced in Philadelphia. However,
over the years, these Pittcon introduction
summaries have provided a good source of
information that would be difficult for one
individual to gather during the four days
of the exhibition. In addition, the products
introduced have shown definite correla-
tions to current research, development,
and application activity in the separation
sciences.
As in previous years, columns and other
products recommended by their manufac-
turers primarily for biomolecule separations
or sample preparation are denoted in the
tables with the designation BIO. Some of
these products may be used for general
(HPLC) separations as well, but their main
emphasis is for biological samples.
In part I of this coverage (3), I described
new column introductions in the areas of
HPLC, reversed-phase LC, normal- and
bonded-phase LC, hydrophilic interac-
tion liquid chromatography (HILIC),
supercritical fluid chromatography (SFC),
ion-exchange and ion chromatography,
size-exclusion chromatography (SEC), and
specialty chromatography. In this install-
ment, I will look at gas chromatography
(GC) columns, sample preparation prod-
ucts, and hardware, accessories, and small
tabletop instruments, mainly for sample
preparation.
General: This year, I observed that there
was a continuing trend in high-efficiency
chromatography driven by dramatic
increases in chromatographic efficiency,
especially in HPLC or ultrahigh-pressure
liquid chromatography (UHPLC). In addi-
tion, the introduction of more products
with inert surfaces driven by instrumental
increases in sensitivity especially through
Ronald E. Majors
Column Watch Editor
Column Watch features Ron
Majors yearly report on
new products introduced
at Pittcon. This years event
returned to Chicago, IL.
Amid a worldwide financial
crisis, the attendance
was redued compared to
recent years but Pittcon
still showcased the lastest
separations technology.
This month columns are
highlighted, covering all the
modes of HPLC and liquid
phase separations. Next
month, GC columns, sample
prep, and chromatographic
accessories will be covered.
New Chromatography
Pittcon 2009: Part I
P
ittcon 2009, the 60th
Pittsburgh Conference on
Analytical Chemistry and
Applied Spectroscopy, was held in the
massive McCormick Place in Chicago,
Illinois, on March 813, 2009, the sixth
time the Conference has been held there.
This years event hosted nearly 1000
instrument manufacturers and labora-
tory suppliers in more than 2200 booths.
the conferees listened to 2500 technical
presentations, checked numerous com-
pany seminar rooms, or attended one or
more of 120 short courses.
Undoubtedly, Pittcon remains the
most important yearly international
analytical exhibition, where compa-
nies introduce their latest instruments,
instrument accessories, software, col-
umns, sample preparation equipment,
and other consumable products. Because
many past attendees have purchased
one or more new products within three
months after attending the show, most
exhibitors attempt to maximize their
booth traffic to meet as many potential
customers as possible.
products that will be displayed at Pittcon
2008 (1,2) might be included for rea-
sons of completeness. The information
is based upon manufacturers responses
to a questionnaire mailed in December
2008. Because of space limitations and
the fact that some manufacturers did
not respond to the questionnaire, this
report cannot be considered an exhaus-
tive listing of all new products that were
introduced in Chicago. However, over
the years, these Pittcon introduction
summaries have provided a good source
Ronald E. Majors
Column Watch Editor
of information that would be difficult
for one individual to gather during the
four days of the exhibition. In addition,
the products introduced have shown
definite correlations to current research,
development, and application activity in
the separation sciences.
As in previous years, columns and
other products recommended by their
manufacturers primarily for biomolecule
separations or sample preparation are
denoted in the tables with the designa-
tion BIO. Some of these products can be
used for general high performance liquid
chromatography (HPLC) separations as
well, but their main emphasis is for bio-
logical samples.
In this months coverage, I will
describe new introductions in the areas
of HPLC: reversed-phase, normal- and
bonded-phase, ion-exchange and ion,
size-exclusion, and large- and prepara-
tive-scale chromatography; specialty
chromatography columns; and thin-
layer chromatography (TLC). Next
month, I will look at gas chromatogra-
phy (GC) columns, sample preparation
products, and hardware, accessories
and kits for chromatography, and
sample preparation.
General: This year, I observed that
there was a continued trend in high-
throughput techniques, especially in the
HPLC and sample preparation areas.
More devices and instrumentation that
support automation made their appear-
ance, and autosamplers with sample
preparation functionality have been
reappearing. Many application-specific
columns for HPLC and GC and various
devices for solid-phase extraction (SPE)
and sample preparation were intro-
duced. Again at Pittcon, a considerable
number of new accessories, hardware,
COLUMN WATCH
206 LCGC NORTH AMERICA VOLUME 27 NUMBER 3 MARCH 2009 www.chromatographyonline.com
Quantification of
Capsaicinoids in Hot
Sauces Using LCMS with
Monolithic Silica Capillaries
Trends in Sample Preparation
Intuition and LC Gradient Elution
Volume 31 Number 3 March 2013
RON MAJORS
A TRIBUTE TO THE MAN WHO
HAS BEEN CHROMATOGRAPHYS
MASTER COMMUNICATOR
FOR MORE THAN FOUR DECADES
Volume 31 Number 6 June 2013
ES263121_LCGC0613_CV1.pgs 06.03.2013 19:11 ADV
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ES257427_LCGC0613_437_FP.pgs 05.29.2013 02:12 ADV
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438 LCGC NORTH AMERICA VOLUME 31 NUMBER 6 JUNE 2013 www.chromatographyonline.com
CONTENTS
LCGC North America (ISSN 1527-5949 print) (ISSN 1939-1889 digital) is published monthly with 1 additional issue in August as Buyers Guide
by Advanstar Communications Inc., 131 West First Street, Duluth, MN 55802-2065, and is distributed free of charge to users and specifiers
of chromatographic equipment in the United States and Canada. Single copies (prepaid only, including postage and handling): $15.50 in the
United States, $17.50 in all other countries; back issues: $23 in the United States, $27 in all other countries. LCGC is available on a paid sub-
scription basis to nonqualified readers in the United States and its possessions at the rate of: 1 year (13 issues), $74.95; 2 years (26 issues),
$134.50; in Canada and Mexico: 1 year (13 issues), $95; 2 years (26 issues), $150; in all other countries: 1 year (13 issues), $140; 2 years (26
issues), $250. Periodicals postage paid at Duluth, MN 55806 and at additional mailing of fices. POSTMASTER: Please send address changes
to LCGC, P.O. Box 6168, Duluth, MN 55806- 6168. PUBLICATIONS MAIL AGREEMENT NO. 40612608, Return Undeliverable Canadian Ad-
dresses to: Pitney Bowes, P. O. Box 25542, London, ON N6C 6B2, CANADA Canadian GST number: R-124213133RT001. Printed in the USA.
COLUMNS
448 COLUMN WATCH
The Inert Flow Path Story for GC and GCMS:
Eliminating the Weakest Links
Mitch Hastings and Ronald E. Majors
For the next improvements, we must look beyond the column.
456 LC TROUBLESHOOTING
Gradient Elution, Part IV: Dwell-Volume Problems
John W. Dolan
How can the gradient dwell volume impact results?
464 MS THE PRACTICAL ART
HydrogenDeuterium Exchange Mass Spectrometry:
An Emerging Biophysical Tool for Probing Protein
Behavior and Higher-Order Structure
Joomi Ahn and St. John Skilton
HDX-MS as a means for performing routine biophysical analysis
472 PERSPECTIVES IN MODERN HPLC
The Essence of Modern HPLC: Advantages, Limitations,
Fundamentals, and Opportunities
Michael W. Dong
The fundamental concepts of HPLC are re-examined.
506 THE ESSENTIALS
Troubleshooting GC Retention-Time, Efficiency, and
Peak-Shape Problems
What problems in chromatogram may reveal, and what to do
ARTICLES
480 A Tribute to Ron Majors
Laura Bush
For more than 40 years, he has been a master communicator. Thats
just one reason he has so many fans, and friends, around the world.
494 HPLC Analysis of Very Polar Compounds in Bioanalysis
Joe Pesek answers questions about IEX, HILIC, polar-embedded, and
polar-endcapped columns, and aqueous normal phase chromatography.
PEER-REVIEWED ARTICLE
486 Morphological Comparison of Silica-Based Monolithic and
Particulate Beds by Confocal Laser Scanning Microscopy
Stefan Bruns, Alexandra Hltzel, and Ulrich Tallarek
This analysis offers insight for optimizing the structure of monoliths.
ON THE WEB
WEB SEMINARS
Tips and Tricks on Screening and
Conrmatory Methods for Resi-
dues and Contaminants in Foods
Dr. Je Layne, Phenomenex
Multi-Residue Pesticide Analysis
in Herbal Products Using
Accelerated Solvent Extraction
with Triple Quadrupole
David Steiniger and Aaron Kettle,
Thermo Fisher Scientic
Editors Series: A Generic HPLC-
UV Platform Method for Cleaning
Verication
Michael W. Dong, PhD, Genentech
Simply Intelligent:
Chromatography Data System
Software Goes Mass Spec
Darren Barrington-Light and Frank
Tontala, Thermo Fisher Scientic
chromatographyonline.com/WebSeminar
Join the LCGC Group on Facebook:
www.facebook.com/lcgcmagazine
Join the LCGC Group on Twitter:
https://twitter.com/LC_GC
Join the LCGC Group on LinkedIn
http://linkd.in/LCGCgroup
olume umber
une
Cover design by Dan Ward.
DEPARTMENTS
Peaks of Interest . . . . . . . . . . 446
Product Resources . . . . . . . . . 498
Ad Index . . . . . . . . . . . . . . . . . 504
ES262370_LCGC0613_438.pgs 06.01.2013 02:13 ADV
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2013 Waters Corporation. Waters and The Science of Whats Possible are trademarks of Waters Corporation.
Pharmaceutical & Life Sciences
|
Food
|
Environmental
|
Clinical
|
Chemical Materials
FOR CONSTANT
INNOVATION IN DMPK,
PARTNERSHIP IS
ELEMENTAL.
ES257449_LCGC0613_439_FP.pgs 05.29.2013 02:12 ADV
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What if you could purify exactly how you want?
What if you could trust your system 100 percent?
What if you could meet all your purication challenges?
Micrograms to kilograms.
Highest purity with maximum recovery.
Save time and cut costs.
Agilent solutions can help you do just that.
Instruments. Columns. Software. Services.
Everything you need to purify your way:
www.agilent.com/chem/purifyyourway
PURIFY
YOUR WAY
Analytical Semipreparative Preparative Pilot
g mg g kg
Agilent Technologies, Inc. 2013
ES257455_LCGC0613_440_FP.pgs 05.29.2013 02:13 ADV
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Using Focused Gradients on a Combined Analytical/Preparative HPLC
System to Optimize the Scale-up Process from 4.6 to 50 mm Columns
Andreas Tei, Pierre Penduff, Ron Guilliet, Helmut Schulenberg-Schell, Agilent Technologies, Inc.
5301 Stevens Creek Blvd., Santa Clara, CA 95051 | (800) 227-9770 (Directory) | www.agilent.com/chem/purifyyourway
ADVERTORIAL
This Application Note describes an optimized purication
process
[2,3,4]
using of focused gradients
[5]
after scale-up
from a 4.6 to a 50 mm id column.
Direct scale-up from analytical ow rates of 1.5 mL/min to
preparative ow rates of 177 mL/min on a combined system in
gradient mode is a tremendous challenge. Solvent delivery must
be accurate with low dwell volume

to perform well at analytical
ow rates
[1]
. Further, the UV detector must have a wide dynamic
range to cope with the vastly different compound concentrations.
Experimental
[6]

Agilent SD-1 Binary Purication System comprising, 410 Autosampler,
325 UV/VIS Dual Wavelength Detector, 4 x 0.15 mm ow cell, 440
Fraction Collector, manual injection valve with 10 mL loop, manual
2-position/6-port valve, OpenLAB ChemStation CDS C.01.03
Calibration mixture: acetamidophen (8,3%), propylparaben (29,9%),
butylparabene (29.9%), valerophenone (11.9%) and octanophenone
(19.9%) in DMSO. Total concentration 250 mg/mL
Purication sample: salycilic acid (35.5%) and ethylparaben (64.5%)
in DMSO. Total concentration 469 mg/mL
Eluent A: Water + 0.05 % TFA, Eluent B: Acetonitrile + 0.05 % TFA
Analytical column: Agilent Prep-C18 Scalar, 4.6 x 250 mm, 10 m
Preparative column: Agilent Load & Lock column, 50 x 243 mm,
packed with 300 g of Agilent Prep-C18 , 10 m, at 1500 PSI
Generic gradient conditions on the 4.6 mm id column were
transferred to the preparative 50 mm id column using the
method transfer equations from the literature
[3,4]
.
A calibration mixture of ve different compounds of high to low
polarity was prepared to dene six different elution zones for
purication of a test sample. Using a linear gradient slope from
2 to 98 %B on the analytical ow path for 16 minutes, the elution
points of the standard compounds were determined, taking into
consideration the dwell volume and the column dead volume
[2]
.
The elution points of two adjacent peaks from the calibration
mixture gave the start- and endpoints of the focused gradients.
The gradient slope of each focused gradient was 2 %B /min.
Results
5 L of the purication sample were analyzed on the 4.6 mm id
column using the generic gradient prole. The target compounds
of the purication sample fell within elution window #3. Therefore,
focused gradient #3 was applied for the purication process.
With 40 L (18.7 mg) injection volume the separation of the two
compounds was still obtained with a resolution of 1.11. The nal
injection volume for the purication step on the 50 x 243 mm column
was calculated using the scale-up equation to be 4500 L or 2.11g of
mixture. Applying the scale-up, the recovery of the collected fractions
from the purication sample was 85% and 89%.
Conclusion
Successful scale-up from an analytical 4.6 x 250 mm column
to a self-packed 50 x 243 mm Agilent Load & Lock column on a
combined analytical/preparative Agilent SD-1 Purication System
has been demonstrated. A focused gradient facilitated increase of
column load and reduced runtime and solvent consumption.
References
[1] J.W.Dolan, LCGC, 2006, Vol. 24, No.5, 458-466
[2] J.W.Dolan, L.R. Snyder, J. of Chromatography A, 799 (1998) 21-34
[3] D. Guillarme, D. Nguyen, S. Rudaz, J.L. Veuthey, Eur. J. Pharm. Biopharm., 2007, 66, 475-482
[4] D. Guillarme, D. Nguyen, S. Rudaz, J.L. Veuthey, Eur. J. Pharm. Biopharm., 2008, 68, 430-440
[5] L.R. Snyder, J.J. Kirkland, J.W. Dolan, Introduction to Modern Liquid Chromatography,
pp 403-498, 3rd Edition, Wiley, 2010
[6] P. Penduff, Scale-up with the Agilent SD-1 Purication System Analytical and Preparative Runs
on a Single System, Agilent publication number 5991-2012EN, 2013
1 2 3 4 5 6
5
.
3
2
2
1
8
.
1
5
2
1
5
.
1
7
4
9
.
6
7
2
1
2
.
9
6
1
9
.
3
7
9
1
0
.
5
5
7
EP(2) = 34%
EP(1) = 11%
EP(5) = 89%
EP(4) = 68%
EP(3) = 56%
Focused Gradient 3
32-58%
Norm
%B.
20000
100%
15000
75%
10000
50%
5000
25%
0
0 2.5 7.5 5 10 12.5 15 17.5 20 min 22.5
Chromatogram standard mix
Chromatogram target compounds
Programmed gradient curve
Dwell + column void volume
corrected gradient
Elution windows
Time
(min)
0.00
0.01
13.01
13.10
17.00
Flow
(mL/min)
1.5
1.5
1.5
1.5
1.5
% B

2
32
58
98
98
ES257451_LCGC0613_441_FP.pgs 05.29.2013 02:12 ADV
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CATALYST FOR SUCCESS
Publishing & Sales
485F US Highway One South, Suite 100, Iselin, NJ 08830
tel. (732) 596-0276 fax (732) 647-1235
Science Group Publisher Michael J. Tessalone
MTessalone@advanstar.com
Associate Publisher Edward Fantuzzi
EFantuzzi@advanstar.com
East Coast Sales Manager Stephanie Shaffer
SShaffer@advanstar.com
Editorial
Editorial Director Laura Bush
LBush@advanstar.com
Managing Editor Megan Evans
MEvans@advanstar.com
Group Technical Editor Stephen A. Brown
SBrown@advanstar.com
Associate Editor Cindy Delonas
CDelonas@advanstar.com
Art Director Dan Ward
DWard@media.advanstar.com
Vice President Sales Russell Pratt
RPratt@advanstar.com
Marketing Manager Anne Young
AYoung@advanstar.com
Classified/Recruitment Sales Representative Tod McCloskey
TMcCloskey@advanstar.com
Direct List Rental Sales Tamara Phillips
TPhillips@advanstar.com
Permissions Maureen Cannon
MCannon@advanstar.com
Reprint Services 877-652-5295 ext. 121/ bkolb@wrightsmedia.com
Outside US, UK, direct dial: 281-419-5725. Ext. 121
Production Manager Jesse Singer
JSinger@media.advanstar.com
Assistant Audience Development Manager Gail Mantay
GMantay@advanstar.com
Chief Executive Officer Joe Loggia
Chief Executive Officer Fashion Group, Executive Vice-President Tom Florio
Executive Vice-President, Chief Administrative Officer &
Chief Financial Officer Tom Ehardt
Executive Vice-President Georgiann DeCenzo
Executive Vice-President Chris DeMoulin
Executive Vice-President Ron Wall
Executive Vice-President, Business Systems Rebecca Evangelou
Sr Vice-President Tracy Harris
Vice-President, Media Operations Francis Heid
Vice-President, Legal Michael Bernstein
Vice-President, Electronic Information Technology J Vaughn
ES262198_LCGC0613_442.pgs 05.31.2013 23:54 ADV

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ES258512_LCGC0613_443_FP.pgs 05.30.2013 00:58 ADV
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Kevin D. Altria GlaxoSmithKline, Ware, United Kingdom
Jared L. Anderson The University of Toledo, Toledo, Ohio
Daniel W. Armstrong University of Texas, Arlington, Texas
Michael P. Balogh Waters Corp., Milford, Massachusetts
Brian A. Bidlingmeyer Agilent Technologies, Wilmington, Delaware
Dennis D. Blevins Agilent Technologies, Wilmington, Delaware
Peter Carr Department of Chemistry, University of Minnesota,
Minneapolis, Minnesota
Jean-Pierre Chervet Antec Leyden, Zoeterwoude, The Netherlands
John W. Dolan LC Resources, Walnut Creek, California
Michael W. Dong Genentech, San Francisco, California
Roy Eksteen Sigma-Aldrich/Supelco, Bellefonte, Pennsylvania
Anthony F. Fell School of Pharmacy, University of Bradford, Bradford,
United Kingdom
Francesco Gasparrini Dipartimento di Studi di Chimica e Tecnologia delle
Sostanze Biologicamente Attive, Universit La Sapienza, Rome, Italy
Joseph L. Glajch Momenta Pharmaceuticals, Cambridge, Massachusetts
Davy Guillarme University of Geneva, University of Lausanne, Geneva,
Switzerland
Richard Hartwick PharmAssist Analytical Laboratory, Inc., South New
Berlin, New York
Milton T.W. Hearn Center for Bioprocess Technology, Monash University,
Clayton, Victoria, Australia
Emily Hilder University of Tasmania, Hobart, Tasmania, Australia
John V. Hinshaw BPL Global, Ltd., Hillsboro, Oregon
Kiyokatsu Jinno School of Materials Science, Toyohashi University of
Technology, Toyohashi, Japan
Ira S. Krull Northeastern University, Boston, Massachusetts
Ronald E. Majors Agilent Technologies, Wilmington, Delaware
R.D. McDowall McDowall Consulting, Bromley, United Kingdom
Michael D. McGinley Phenomenex, Inc., Torrance, California
Victoria A. McGuffin Department of Chemistry, Michigan State University,
East Lansing, Michigan
Mary Ellen McNally E.I. du Pont de Nemours & Co., Wilmington,
Delaware
Imre Molnr Molnar Research Institute, Berlin, Germany
Glenn I. Ouchi Brego Research, San Jose, California
Colin Poole Department of Chemistry, Wayne State University, Detroit,
Michigan
Fred E. Regnier Department of Chemistry, Purdue University, West
Lafayette, Indiana
Pat Sandra Research Institute for Chromatography, Kortrijk, Belgium
Peter Schoenmakers Department of Chemical Engineering, University of
Amsterdam, Amsterdam, The Netherlands
Kevin Schug University of Texas, Arlington, Texas
Dwight Stoll Gustavus Adolphus College, St. Peter, Minnesota
Michael E. Swartz Ariad Pharmaceuticals, Cambridge, Massachusetts
Thomas Wheat Waters Corporation, Milford, Massachusetts
CONSULTING EDITORS: Jason Anspach, Phenomenex, Inc.; Stuart Cram,
Thermo Fisher Scientific; David Henderson, Trinity College; Tom Jupille, LC
Resources; Sam Margolis, The National Institute of Standards and Technology;
Joy R. Miksic, Bioanalytical Solutions LLC; Frank Yang, Micro-Tech Scientific.
Editorial Advisory Board
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2013 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA-ALDRICH and
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countries. Ascentis is a registered trademark of Sigma-Aldrich Co. LLC. Solutions
within is a trademark of Sigma-Aldrich Co. LLC. Fused-Core is a registered trademark
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ES261997_LCGC0613_444.pgs 05.31.2013 22:37 ADV
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pretreatment, key in high throughput quantitative applications.
Dont accept compromise, choose compact. www.bruker.com
ES257403_LCGC0613_445_FP.pgs 05.29.2013 02:11 ADV
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PEAKS of Interest
Phenomenex CEO
Fasha Mahjoor Awarded
an Ellis Island Medal of Honor
Fasha Mahjoor, CEO of Phenomenex
(Torrance, California), has been
awarded an Ellis Island Medal of
Honor. The awards are given annually
by the National Ethnic Coalition of
Organizations (NECO) in recognition
of leaders who are philanthropic, who
preserve and celebrate the values of
their ancestry group, and who pro-
mote tolerance and acceptance.
More than 10,000 nominations are
received each year, and this year 98
were presented at an awards ceremony
held in the Great Hall on Ellis Island in
New York Harbor. The awards are rec-
ognized by the United States House of
Representatives and the United States
Senate, and are read into the congres-
sional record. Past awardees have
included United States presidents and
Nobel Prize winners.
Mahjoor was selected by NECO for
his dedication to humanitarian efforts
globally. The American Red Cross pub-
licly recognized Mahjoor and his com-
panies for their support of Superstorm
Sandy disaster relief efforts. He recently
abseiled over 1000 feet of the Shard sky-
scraper (London, UK) to raise funds for
The Outward Bound Trust educational
charity, and sponsored the making of
over 200,000 meals for starving children
in developing countries at Phenomenex
companies in the United States and Italy.
Malvern Breaks into
Grinding and Dispersing Market
Malvern Instruments (Malvern, UK)
has entered a three year co-marketing
agreement with Netzsch Grinding
and Dispersing (Selb, Germany). The
two companies serve shared markets
including food, ceramics, pharmaceu-
ticals, nano-technology applications,
and surface coatings through to metal
and mineral mining.
Particle characterization is becom-
ing increasingly important for fine
grinding to the submicron- or nano-
meter range because product proper-
ties often depend directly on particle
Chromatography Market Profile
Supercritical Fluid Chromatography
Supercritical fluid chromatogra-
phy (SFC) makes use of a super-
critical fluid, typically carbon
dioxide, instead of an organic
or aqueous solvent, to carry the
sample through the chromatog-
raphy column. The advantages
of SFC when using carbon
dioxide are that there are no
organic solvents to dispose
of. Separations can also be per-
formed more quickly than with
high-performance liquid chromatography (HPLC), since the diffusion of solutes
in supercritical fluids is about an order of magnitude greater than in liquids.
The nontoxic nature of carbon dioxide as a solvent is a very attractive feature
of SFC when working with products intended for human consumption. SFC is
also a particularly attractive separation method for chiral compounds because
of its efficient use of the chiral stationary phase (CSP) media, and the fact that
SFC provides a compatible environment for CSP media. These factors make SFC
particularly attractive to the pharmaceutical, agriculture, and food industries.
In 2013, SDi conducted a global survey of knowledgeable liquid chromatog-
raphy users. This extensive 60-question survey provides a great deal of insight
into users opinions on various topics relating to their liquid chromatography
usage. For SFC in particular, nearly two-thirds of the SFC users chose the
technology because of its chiral separation ability. Improved resolution and
decrease in solvent usage were also two frequently mentioned benefits of SFC
among the survey respondents. In contrast, the top three limitations or draw-
backs of SFC as mentioned by the participants were high initial system costs,
difficulty in transferring methods, and unproven methods.
The foregoing data were extracted and adapted from SDis recently published
Tactical Sales and Marketing (TSM) report titled HPLC: End Users Influencing
Development for Advanced HPLC Systems, Columns and Chemistry. For more
information, contact Glenn Cudiamat, VP of Research Services, Strategic Directions
International, Inc., 6242 Westchester Parkway, Suite 100, Los Angeles, CA 90045,
(310) 641-4982, fax: (310) 641-8851, e-mail: cudiamat@strategic-directions.com
Reasons for choosing SFC.
size, according to Dimitrios Makrakis,
Netzsch Grinding & Dispersing Busi-
ness Unit managing director. He
added: The correct and reproducible
analysis of particle size is a prereq-
uisite for process optimization and
quality control. As Malvern is a world-
leading manufacturer in particle and
materials characterization, we have
the ideal partner to deliver the best
overall solution for both grinding
technology and particle characteriza-
tion and clients of both companies
will benefit from this partnership.
Under the terms of the agreement,
Malvern instrumentation will be
installed in Netzsch demonstration
and test laboratories in China, Brazil,
Germany, Korea, India, and Russia.
Customer materials in Netzsch facilities
will be routinely tested using Malvern
instrumentation, with Malvern staff
available for consultation about ana-
lyzing data. The companies will work
together to organize seminars and
other initiatives to target key markets.
70
62%
35%
Chiral separation Other reasons Improved resolution Decrease in solvent
usage
35%
23%
R
e
s
p
o
n
s
e

(
%
)

60
50
40
30
20
10
0
ES261995_LCGC0613_446.pgs 05.31.2013 22:37 ADV
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ES257412_LCGC0613_447_FP.pgs 05.29.2013 02:11 ADV
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COLUMN WATCH
Advances in gas
chromatography
(GC), especially gas
chromatographymass
spectrometry (GCMS)
detector technologies,
are pushing the levels of
detection to parts-per-
billion and parts-per-trillion
levels. However, detectors
can only measure the
analytes that reach them.
Because a typical column
contributes a majority of
the surface area, column
inertness has received a
great deal of attention in the
GC community. One must
think of the GC flow path
as a chain of components
linked together to separate
the analytes and deliver
them to the detector.
This installment looks at
contributions of the entire
GC flow path, identifying
the remaining links that
contribute to surface
activity. Improvements in
GC flow path inertness are
highlighted.
The Inert Flow Path Story for
GC and GCMS: Eliminating
the Weakest Links
G
as chromatographymass spec-
trometry (GCMS) and GC
MS-MS systems have increased in
sensitivity dramatically during the last two
decades, especially over the last five years.
In the early days of benchtop GCMS,
single-digit nanogram on-column amounts
were barely detectable in full-scan mode
today sensitivities are in the picogram
to femtogram range in full-scan mode, and
even more so in single-ion mode (SIM)
and multiple reaction monitoring (MRM)
mode with GCMS-MS. This increase
in sensitivity has enabled analysts to reach
lower and lower detection limits. So, what
is the challenge? The MS detector can only
measure analytes that reach it. The lower
the amount of analyte injected, the greater
the chance that this analyte can be lost to
any chromatographically reactive parts of
the GC flow path. The problem for labo-
ratories chartered to detect and measure
trace components is the risk of not detect-
ing trace analytes if the GC flow path is
chromatographically reactive (active). The
purpose of this installment of Column
Watch is to take a look at the entire GC
flow path and identify those system ele-
ments that may contribute to analyte loss
(or extremely bad tailing) and nondetect-
ability. Furthermore, we discuss actions
that have been taken to eliminate (or sub-
stantially decrease) the contributions of the
weakest links in the GC flow path.
What Is the GC Flow Path?
The GC flow path is essentially every
component that the injected sample poten-
tially touches. To keep the scope of this
column installment reasonable, the bound-
ary begins at the sample inlet and ends at
the mass spectrometer source (Figure 1).
While other detection methods (such as
electron-capture detection [ECD], pulsed
flame photometric detection [PFPD], or
sulfur chemiluminescence detection [SCD])
are frequently used in trace analysis, the
focus on MS over other detection systems is
deliberate because it is the most widely used
GC detection method in trace analysis. The
point-of-injection is when the sample leaves
the syringe or sample loop and enters the
carrier gas flow stream. After the separated
components leave the gas chromatograph
itself and enter the source of the MS system,
ionization takes place and the path of the
ions through the MS system is no longer
considered to be part of the GC flow path.
What Is
Chromatographic Reactivity?
In any given chromatographic system at
the molecular or atomic level, there is a
finite number of active sites (for example,
molecules or atoms) on the surfaces of the
chromatographic flow path materials that
may interact with analytes of interest. This
number of active (or reactive) sites is gener-
ally fixed on a new or freshly maintained
system. Here is an example to illustrate
why this is important for trace analysis:
If a system has 10 reactive sites and 100
reactive sample molecules are injected,
there is the potential to lose 10% of the
sample molecules, probably not significant
in routine trace analysis. If only 10 sample
molecules are injected on a system that
has 10 reactive sites, 100% of them could
be lost to the reactive sites in the system.
The end result even though reactive
analytes of interest are injected, they will
never be detected if they are lost on reactive
sites in the GC flow path. The impact of
non-detects can be profound. Our food,
drinking water, environment, and com-
mercial goods are less safe and are of poorer
quality, criminals go free, and warfare
agents go undetected.
Mitch Hastings
is a guest co-author this
month.
Ronald E. Majors
is the editor of Column
Watch
ES262140_LCGC0613_448.pgs 05.31.2013 23:33 ADV
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ES257409_LCGC0613_449_FP.pgs 05.29.2013 02:11 ADV
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Some examples of active sites in a typical
flow stream could be
silanols on the inner surface of a fused
silica capillary column tubing, reten-
tion gaps or guard columns, and inlet
liners
metallic atoms or molecules (such as
metal oxides) on the surface of stain-
less steel weldments or other fabricated
components (such as gold seals or con-
necting tubing)
silanols or other molecular sites on the
glass wool often used in inlet liners
ferrules and unions used to connect
the column or other devices into the
flow stream.
What Parts of the GC
Flow Path Contribute to
Chromatographic Reactivity?
There are a number of sources of activ-
ity that a given sample will encounter in
the sample flow path. Fortunately, a basic
understanding of chemical reaction kinet-
ics enables us to understand the potential
relative contribution of a given GC flow
path component to the loss of a reactive
analyte.
Temperature: The higher the tempera-
ture at the time of analyte contact, the
more likely the system component will be
reactive.
Nature of Reactants: Of the com-
pounds that are typically analyzed by
GC, on one end of the spectrum are
hydrocarbons: they are very nonreactive
at normal GC temperatures (which is one
reason why they are frequently used as
internal standards for trace analysis meth-
ods). On the reactive end, there are polar
analytes like phenols, amines, and ther-
mally labile components (molecules that
have complex structures with inherently
strained bonds).
Catalysts: In the GC flow path there
are really only three relevant general mate-
rial types that are potentially catalytic:
Metals >> Glass >>> Organic coatings
Within these three subsets, there are
varying degrees of activity. Certain met-
als may be more catalytic than others;
sometimes special metal components are
used in GC. Surface silanols on glass or
fused-silica columns and glass wool vary in
acidity (for example, isolated, vicinal, and
hydrogen bonded). The organic coatings
for wall-coated open tubular GC columns
may have a fairly low degree of activity. A
familiar culprit is the deactivation coating
used on glass wool. When glass wool is
deactivated outside of the injection liner
and then a plug is inserted, often fibers
are broken, which exposes active sites that
can cause reactions with certain analytes.
We will discuss this phenomenon in more
detail later.
Physical State: One side of the physical
state equation has been addressed in the
above example of trace analysis. The other
side of the equation is the contribution
of the physical state by the GC system.
One way to understand these physical
contributions is to consider the surface
area of each flow path component. The
greater the surface area of catalytic mate-
rial that a volatilized analyte is exposed
to, the more it is a potential contributor
to system reactivity. All of the exposed
surfaces within the flow stream may not
contribute equally to analytesurface
interactions, but for a first approximation,
exposed surface area is a good place to
start by comparing potential contributors
to analyte loss or peak tailing.
GC Flow Path:
A Chain of Components
When all the above elements are considered,
it is relatively easy to understand which GC
flow path components pose the most risk to
analyte loss. Figure 1 provides an overview
of some components that might be found
in a typical GC flow path. Table I estimates
the contributions of various flow path ele-
ments to potentially exposed surface area
as well as the typical operating temperature
that may enhance any chemical reactions or
interactions that can occur.
We will discuss some of these
contributions in the next section.
Table I: Estimated contribution of various GC flow path components reactivity risk
on a typical GC system
Typical GC
Flow Path Component
Surface
Area (cm
2
)
Operating
Temperature
Catalyst Reactivity Risk
Inlet top seal 0.4 Medium Metal Medium
Septum* 0.008 High Organic* Low
Inlet housing 17 High Metal Medium
Liner without
glass wool
10 High Glass Medium
Liner wool 36 High Glass High
Inlet bottom seal 0.4 High Metal Medium
Ferrule 0.0061 High Metal Medium
Typical guard column
(5 m 0.25 mm)
39 Medium
Glass/
organic
Medium
Typical GC column
(30 m 0.25 mm)
235.5 Medium Organic Medium
GCMS source 3.4 Medium Metal High
*Although the septum initially has a low surface area, septum particles can be cored
with each injection, accumulating in the inlet. Additionally, septa are not 100% organ-
ic, but sometimes contain metal moieties.
Sample
Septum
GC liner
GC column
Glass wool
Inert fow path
Splitsplitless inlet
Inlet seal
Inside GC column
Polyimide outer coating
Fused silica
surface
Stationary
phase
Deactivation
Ferrule
Ferrule
Data
Ion
source
MS system
(or other detector)
Union
Union
Retention
gap
Figure 1: Gas flow path in a typical GCMS system.
ES262144_LCGC0613_450.pgs 05.31.2013 23:34 ADV
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ES257413_LCGC0613_451_FP.pgs 05.29.2013 02:11 ADV
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GC Capillary Columns
As depicted in Table I, the GC column
itself is, by far, the biggest overall contribu-
tor to exposed surface area. More than a
decade ago, even after chemically bonded
and cross-linked GC capillary columns
were introduced and being used, manu-
facturers started to address user problems
associated with GC capillary columns
employed with the increasing detection
sensitivity, particularly with ECD and MS.
Users were often baffled by the baseline
drift during temperature programming
because of stationary phase bleed, loss of
analytes, and tailing of polar compounds,
particularly at lower levels on state-of-the-
art columns of the day. The first problem
that was addressed was column bleed or
baseline drift. Throughout the 1990s and
into the 2000s, new columns with better
bleed specifications were introduced with
manufacturers out-specing each other
such as factors of four lower bleed and
higher temperature limits. For examples,
see the articles on GC columns introduced
at Pittcon by Majors during that time
period (14). Columns began to be speci-
fied with two upper temperature limits (for
example, 225 C and 250 C). The first
number was the upper temperature limit
for isothermal operation and the second
number was the upper temperature limit
for programmed runs in which the column
was not expected to perform for hours on
end because it was normally cooled down
to get ready for the next temperature-
programmed run. New phase chemistries
were developed that had the same or fairly
similar selectivities to the popular phases at
that time, such as 100% dimethylpolysilox-
ane and 5% phenyldimethylpolysiloxane.
Arylene phases were introduced that had
lower densities since the phenyl groups
were more embedded within the polymer
chain than their pendant phenyl counter-
parts, and thus, bleed mass was reduced for
equivalent film thickness columns.
Indeed, most GC column manufactur-
ers developed a second-generation family
of columns that performed better than
the columns that preceded them. Table
II lists some typical column families that
were introduced with improved specifica-
tions, most of them with an MS designa-
tion because of the growing popularity
of this detection mode. Nowadays, ultra-
inert columns are the name-of-the-game
with not only improved bleed specs, but
better analyte recovery and lower tailing
for polar compounds being observed.
The variety of stationary-phase types
offered with extremely inert character-
istics has since expanded from apolar
phases to more polar phases.
Enter Inert Flow Path Components
However, with regulatory agencies driv-
ing lower limits of detection for increas-
ingly active and more complex samples,
other parts of the GC flow system started
to contribute adversely to the analytical
results. Having to repeat or verify suspect
trace analysis wasted the analysts time and
hindered productivity. Fortunately for the
trace analysis community and the stake-
holders served, leading instrument and
consumables companies began to develop
commercially available inert upgrades to
the flow path components that have, for
all practical purposes, made loss of analyte
at trace levels negligible. This has enabled
more accurate results for trace analysis.
The first area to be addressed was the
injector port and, particularly, the inlet
liner itself. Consisting of borosilicate glass
often deactivated with silane reagents and
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SELECTIVE GC
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ANALYSIS
ES262143_LCGC0613_452.pgs 05.31.2013 23:34 ADV
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other surface treatments to prevent ana-
lyte interaction, it was found that some
of the lack of inertness was attributed
to the hydrolysis of the deactivated liner
surface, particularly when moisture was
present in the sample and carrier gases
were not treated with moisture traps to
remove residual water. Because the liner is
at a high temperature, any surface activity
effects were accentuated. Oxygen pres-
ent in the carrier gas had to be scrubbed
via oxygen traps. Contamination of the
liner with sample matrix or bits of septum
material from coring because of needle
design were other problems that had to be
addressed. To combat the first problem,
manufactures came up with faster meth-
ods to change liners (some automated) so
that users didnt have to shut down their
gas chromatographs to change hot inlet
components. To address the second prob-
lem, new septa and injector needles were
designed that minimized the contribution
to flow path activity of cored septum frag-
ments. The exposed surface area of the GC
septum itself, which is considered part of
the flow system, is very small (Table I) and
over the years polymer formulations have
been developed that cause little activity
problems, even in splitless backflash situa-
tions. Nevertheless, the inlet liner itself still
needed to be addressed and most recently
the use of new organic chemical vapor
deactivation processes for glass materials
beyond the typical siloxane chemistry has
allowed the introduction of new inert boro-
silicate liners for improved performance
with trace polar analytes.
26,000
20,000
M
S

S
i
g
n
a
l
1
2
3
4
5
10,000
20.20 20.40 20.60 20.80
Time (min)
21.00 21.20 21.40 21.60
Figure 2: Analysis of drugs of abuse at the 500 ppb level: inert flow path (green trace)
vs. a standard flow path in a GCMS (5). Instrument: Agilent 7890 GC system with
5975 C MSD detector; column: 30 m 0.25 mm, 0.25-m d
f
J&W HP-5ms UI (Ultra-
Inert); gas purifier: Gas Clean GCMS, 1/8-in. kit; temperature program: 100 C (4-min
hold); 100280 C at 10 C/min, 280300 C at 6 C/min (4.67 min hold); carrier: helium
at 52.7 cm/s (2 mL/min), set at 100 C with EPC-Constant flow; inlet: pulsed splitless,
1 L, 35-psi pulse until 0.73 min, 0.75-min purge at 50 mL/min, gas saver 20 mL/min at
2 min; inlet liner: Ultra Inert with wool (inert flow path), standard single taper liner
with wool (standard flow path); seal: UI Gold seal (inert flow path), standard Gold
seal (standard flow path); detector: MSD scan mode 40450 m/z; source temperature:
230 C; quadrupole temperature: 150 C; transfer line temperature: 310 C. Peaks: 1 =
dronabinol, 2 = oxycodone, 3 = temazepam, 4 = flunitrazepam, 5 = diacetyl morphine.
Table II: Typical families of GC columns with improved inertness
Family Name of GC Columns Company
J&W Ultra-Inert (UI) Agilent Technologies
Factor Four Agilent Technologies (formerly Varian)
Advanced Inertness VICI Valco
Optima Plus Macherey-Nagel
Rxi Restek
SLB Supelco/Sigma Aldrich
Trace GOLD Thermo Fisher Scientific
Zebron ZB Phenomenex
BPX SGE Scientific
Condence means knowing your
inertness is superior. Quantify active
analytes with condence using Agilents
industry leading GC/MS instruments,
Agilent J&W Ultra Inert GC columns,
and Inert Flow Path supplies.
ATTAIN
SUPERIOR
INERTNESS
Learn more and get your free
inert ow path poster at:
www.agilent.com/chem/inert
ES262146_LCGC0613_453.pgs 05.31.2013 23:34 ADV
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However, one problem area remained
that seemed to be a continuing saga for
gas chromatographers: the preferred use of
glass wool packed into the inlet liner. Glass
wool serves two purposes: to catch impu-
rities, especially nonvolatiles, that could
enter the GC column and cause further
problems with the column or detector; and
to serve as a mixing volume and a thermal
mass aid and ensure vaporization of the
sample and solvent in the injection port.
It is worth noting that Table I illustrates
the fairly high surface area that an inlet
liner with glass wool has relative to the
other components in the flow path. This
is because of the very small fiber diameter
of the wool. If the glass wool was properly
deactivated and correctly placed in the
inlet liner, then its contribution to surface
activity would be fairly minimal. However,
when glass wool was deactivated externally
and the delicate fibers were then wadded
up and carefully placed in the liner, not
surprisingly, fragments of the underly-
ing high activity surface glass wool were
inadvertently created that contributed to
undesirable effects, particularly for trace
polar compounds. In fact, many pesticide
chemists refused to use glass-wool-packed
liners because their trace pesticides would
certainly be lost and their analytical results
would be compromised.
The solution to this problem was to
develop high purity, high surface area glass
wool, carefully place it in the glass liner
and then deactivate it in situ. Again, new
deactivation procedures using organic coat-
ings were used that gave even better results
than traditional silylation. In this manner,
high-surface-area glass wool fragments
were not created and the new deactivated
glass-wool liners provided a high level
of inertness. These dramatic advances
in organic coatings on high-surface-area
glass wool have enabled their use without
compromising inertness, enabling analysts
to enjoy the benefits of glass wool and all
the associated reduced operational expenses
coming from a longer-lived GC column.
With respect to other elements of the
flow path, the application of more inert
coatings over metal surfaces has enabled
better trace analyte fidelity for inlet
housings, inlet bottoms, and top seals.
Bottom seals consist of stainless steel or
gold-plated stainless steel. Even gold seals
have been shown to provide active sites
and also must be further deactivated to
ensure an optimum surface for trace polar
analytes. In many cases, in the absence of
sample backflash, many of the surfaces in
the injection port arent entirely exposed to
sample vapor and their surface areas dont
contribute to major activity problems.
On the detection side, proprietary metal
deactivation procedures and special metal
alloys are also used to cut down on surface
activity; even detectors with a high metal
content, such as the thermal conductivity
detector, now contribute less to analyte
adsorption than earlier models. For flame
ionization detection (FID), if the capillary
column is correctly positioned there is
almost no exposure to hot metal surfaces
that could cause potential problems. Nev-
ertheless, deactivated FID housings are
also available.
Although not discussed here, Figure 1
also shows some additional parts such as
retention gaps, ferrules, and unions that
can also contribute to the overall system
performance. Indeed, in modern gas chro-
matographs, these components also require
the same deactivation treatments that will
render them harmless. As with all prod-
ucts, inertness claims, proof of inertness
performance, and actual inertness perfor-
mance may vary among manufacturers.
Putting It All Together
Figure 2 shows the comparison of a trace
injection of drugs of abuse into a standard
GC system versus a GC system with
optimized components for ultrainert per-
formance: an ultrainert capillary column,
an ultrainert glass liner with deactivated
glass wool, and all metal parts deactivated
using proprietary procedures. Not only do
the drug peaks in the ultrainert system give
much stronger signals (lower detection lim-
its) and better peak shape, but for temaze-
pam a peak that was lost on the standard
system, gives a nontailing peak on the high
performance GC system. More definitive
information can be found in reference 5.
Conclusions
With respect to trace analysis, the GC and
GCMS instruments of today outperform
those produced only a few years ago. As
the manufacturers of GC instruments and
consumables continue to improve GC flow
path parts exposed to the precious samples,
users performing trace analysis in these
ultrainert systems will have the same degree
of confidence in their analytical results
that general users have had for years in GC
laboratories around the globe. Even simple
procedures such as touchless packaging
can bring major advantages to trace ana-
lysts in the prevention of accidentally con-
taminating frequently replaced flow path
components, such as septa and inlet liners.
Undoubtedly, as regulatory bodies or other
user demands require lower limits of detec-
tion, challenges to further increase system
inertness will continue to be confronted.
References
(1) R.E. Majors, LCGC North Am. 21(4), 332
349 (2003).
358 (2007).
290 (2010).
381 (2013).
(5) K. Lynam, Agilent Application Note 5991-
1859EN, Feb. 2013.
Ronald E. Majors
Column Watch
Editor Ronald E. Majors
is a Senior Scientist
in the Columns and
Supplies Division at
Agilent Technologies
(Wilmington, Delaware),
and is a member of
LCGCs editorial advisory board. Direct
correspondence about this column to
lcgcedit@lcgcmag.com.
The editor of Column Watch:
For more information on this topic,
please visit
www.chromatographyonline.com/majors
Mitch Hastings is
a manager with the
chemistry and supplies
division of Agilent
Technologies located in
Folsom, California. He
learned the majority
of his chromatography
from GC Column pio-
neer Dr. Walter Jennings and from experts
within Agilent and the former J&W Scien-
tific. Outside of work, he enjoys cycling
and watching his son play sports.
This months guest Coauthor:
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LC TROUBLESHOOTING
Gradient methods can
offer unique solutions to
separation problems, but
transferring a gradient
method from the literature,
between laboratories,
or even within the same
laboratory can be a
challenging process.
Gradient Elution, Part IV:
Dwell-Volume Problems
John W. Dolan
LC Troubleshooting Editor
T
his is the fourth installment in
a series of LC Troubleshoot-
ing columns about gradient
elution in liquid chromatography
(LC). Before we started this series,
we considered some techniques for
using a gradient scouting run to speed
the initial investigations in method
development or to quickly obtain a
separation under generic conditions
(1). This series of column install-
ments started with a discussion about
how we could transfer our intuitive
understanding of isocratic separations
to gradients (2), and followed this
with a way to compare isocratic and
gradient methods under equivalent
conditions (3). Last month (4), we
considered some unexpected results,
or surprises, that might occur if we
inadvertently make changes in one
gradient variable without making
compensating changes in another.
This month, well begin looking at
some very practical problems related
to gradient operation with a discus-
sion of how the gradient dwell volume
can impact the results.
What Is Dwell Volume?
There are two general designs of LC
gradient systems, as illustrated in Fig-
ure 1. High-pressure-mixing systems
(Figure 1a) generally comprise two
pumps, with mobile-phase blending
taking place after the pumps (in the
high-pressure region). Such systems
usually are limited to two solvents,
although switching valves may be
included that allow you to switch
from one solvent to another. The
other design uses low-pressure mixing
(Figure 1b), in which two to four sol-
vents are blended before they reach a
single pump (mixing on the low-pres-
sure side of the pump). The design of
both system types results in a measur-
able volume between the point the
solvents are mixed and the inlet to the
column this is the dwell volume,
sometimes called the gradient delay
volume. From a practical standpoint,
the dwell volume is made up of two
parts. The first is the physical volume
of the various components, including
the mixer, any connecting tubing, and
usually the injection loop volume.
The second is the wash-out volume,
which adds to the physical volume
the hydraulic characteristics of the
various components, especially the
mixer, to increase the effective dwell
volume of the system. Well just refer
to the combined dwell volume here,
although well touch on the wash-out
volume brief ly later.
The Consequences of
Dwell-Volume Differences
One of the biggest complaints about
gradient methods is that they are
hard to transfer, whether it is trying
to reproduce a published method,
transferring a method between labo-
ratories, or even moving a method
from one instrument to another in
the same laboratory. Often the prob-
lem can be traced to differences in
dwell volume between the various
LC systems. This is illustrated in the
simulated chromatograms of Figure 2.
In each case, the same reversed-phase
gradient method is run. This com-
prises a 1040% B gradient (where
the B-solvent is the organic solvent)
in 15 min, using a 100 mm 4.6 mm
column run at 1 mL/min. Well con-
sider the run of Figure 2a as the
ES262110_LCGC0613_456.pgs 05.31.2013 23:32 ADV
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.

Mass spectrometry
transformed.
June 9. Be the rst to know.
Sign up now for an advanced announcement: thermoscientic.com/mstransformed
ES257423_LCGC0613_457_FP.pgs 05.29.2013 02:12 ADV
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reference case, where the LC system
has a dwell volume of 1 mL. The
peaks are all separated to baseline
with a separation time of approxi-
mately 10 min. When the method
is transferred to another LC system
with a dwell volume of 3 mL, the
chromatogram of Figure 2b results.
You can see that all the peaks are
shifted to longer retention times and
the separation of the first two peaks
suffers. This occurs even though the
same gradient conditions are set in
the controller.
The practical difference between
the two methods is highlighted in
the gradient overlay above each chro-
matogram. Although the program
is set for 1040% B in 15 min, the
actual gradient that is delivered is
not the same in both cases. In the
first case (Figure 2a), the 1-mL dwell
volume results in a 1-min delay from
the time the gradient program starts
until the gradient reaches the head
of the column. In other words, the
injection takes place 1 min before the
gradient arrives at the column. This
means that the sample experiences
an isocratic hold for 1 min under the
starting conditions. In an analogous
manner, the 3-mL dwell volume of
the second system (Figure 2b) adds a
3-min isocratic hold at the beginning
of the gradient.
The result of the differences in
dwell volume is a shifting of retention
times in the chromatogram. Recall
from an earlier LC Troubleshoot-
ing discussion (4) our oversimpli-
fied description of gradient elution:
A compound sits at the top of the
column until a strong enough solvent
comes along to wash it off, then it
travels through the column at the
same rate as all other sample compo-
nents. If this is true, we would expect
the two chromatograms to be offset
by the differences in dwell time, t
D
.
(The dwell time is just the dwell vol-
ume, V
D
, divided by the f low rate, F:
t
D
= V
D
/F.) In the current case, the
1-mL dwell volume system has a dwell
time of t
D
= 1 mL/1 mL/min = 1 min,
whereas the 3-mL system has t
D
= 3
mL/1 mL/min = 3 min. The differ-
ence in dwell times is 2 min, so we
would expect a 2-min offset between
chromatograms if our assumptions are
true. This is what is seen for the later
peaks in the two runs, as highlighted
by the arrows comparing retention
Figure 1: Schematics of (a) high-pressure-mixing and (b) low-pressure-mixing LC
systems, highlighting differences in dwell volume.
(a)
Controller
Dwell
volume
Mixer
Column
Injector
Pumps
A
B
(b)
Controller
Column
Injector
Dwell
volume
Pumps
Mixer
Proportioning
valve
A
B
0 2 4 6 8 10 12 14 16
0
(b)
(a)
100 mm x 4.6 mm column
1040% B in 15 min
1 mL/min
3-mL dwell
1-mL dwell
2 4 6 8 10 12 14 16
Figure 2: The practical effect of differences in dwell volume for a 1040% B gradient
run over 15 min at 1 mL/min on a 100 mm 4.6 mm column. Simulated chromato-
grams for systems with (a) 1-mL and (b) 3-mL dwell volumes; an overlay of the effec-
tive gradient is shown with each chromatogram.
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ES257411_LCGC0613_459_FP.pgs 05.29.2013 02:11 ADV
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times between Figures 2a and 2b. However, it is a bit naive
to assume that the sample components are totally unaf-
fected by the initial conditions. This is highlighted by the
shift in peak spacing (selectivity) for the first two peaks,
in addition to the approximately 2-min offset. So the first
two peaks of Figure 2a experience a 1-min isocratic hold at
10% B and then approximately 2 min of the gradient. In
contrast, the first two peaks of Figure 2b are subjected to
a 3-min isocratic hold before the gradient starts. This dif-
ference accounts for the change in selectivity that we see
for the first two peaks in each run. So the primary inf lu-
ence of dwell-volume differences is an offset in retention
time, with a secondary inf luence of potential changes in
selectivity. It is easy to see how such differences can make
it hard to transfer gradient methods between systems with
different dwell volumes.
How to Measure Dwell Volume
It is important to know the dwell volume of each LC
system so that we can avoid problems by compensating
for differences in dwell volume when gradient methods
are transferred. This measurement is illustrated in Figure
3. The setup is quite simple: Remove the column and
replace it with a piece of capillary tubing. A good choice
is to use approximately 1 m of 0.125-mm (0.005-in.) i.d.
tubing, which provides enough back pressure to ensure
that the pump check valves will work properly. Replace
the A-solvent with HPLC-grade water and the B-solvent
with HPLC-grade water spiked with 0.1% acetone. Use
a UV detector set at 265 nm. Choose conditions that are
typical of what you run in the laboratory. For example,
with conventional LC systems that run 1030 min gra-
dients at 12 mL/min, a 20-min gradient at 2 mL/min
A
b
s
o
r
b
a
n
c
e
Baseline
50%
Time
t
50
t
D
t
D
1/2 t
D
Figure 3: Illustration of how to measure system dwell volume. See
text for details.
(a)
100
Mixer washout
Dwell offset
S
t
e
p

p
r
o
g
r
a
m
Time
C
o
n
v
e
n
t
i
o
n
a
l

L
C
U
H
P
L
C
0
%
B
(b)
Time
Figure 4: Contributions of dwell volume and wash-out volume to
effective dwell volume: (a) comparison of dwell volume measured
on a UHPLC and conventional LC system, adapted from reference
5; (b) comparison of same high-pressure-mixing system with two
different mixers. See text for details.
Abstract Submission Deadline
June 28, 2013 for Oral Consideration
August 23, 2013 for Poster Consideration
Symposium Co-Chairs
Franois de lEscaille, Analis
Oscar Salas-Solano, Seattle Genetics, Inc.
CE in the
Biotechnology &
Pharmaceutical Industries
OCTOBER 6-10, 2013
Crystal City Marriott at
Reagan National Airport
Arlington, Virginia
15th Symposium on the Practical Applications for the
Analysis of Proteins, Nucleotides and Small Molecules
EARLY BIRD
REGISTRATION
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EST. 1983
ES262142_LCGC0613_460.pgs 05.31.2013 23:34 ADV
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JUNE 2013 LCGC NORTH AMERICA VOLUME 31 NUMBER 6 461 www.chromatographyonline.com
is a good choice. For a system dedi-
cated to liquid chromatographymass
spectrometry (LCMS) or ultrahigh-
pressure liquid chromatography
(UHPLC), a 5-min gradient at
0.5 mL/min may be more typical. In
all cases, use a gradient of 0100%
B. Most of us use the autosampler
in a mode that leaves the injection
loop in the f low stream during the
gradient, so leave the injector in the
inject position for this test if this is
your practice. When the gradient is
run, you should see a baseline trace
that looks similar to that of Figure 3,
rising from 0% B to 100% B over the
selected gradient time.
The dwell volume can be deter-
mined in one of two ways illustrated
in Figure 3. You can print the chro-
matogram, then draw a best-fit line
through the rising baseline, as shown
by the dashed line. In a similar
manner, extend the initial baseline
until it intersects the diagonal. This
intersection should correspond to the
dwell time, t
D
. Alternatively, perform
the measurement on the computer
monitor. Determine the difference
in signal (offset) between the initial
(0% B) and final (100% B) baselines;
divide this by two to find the signal
corresponding to 50% B and locate
this point on the curve. Find the cor-
responding retention time, t
R
; this
should occur halfway through the
gradient, so subtract half this value
(t
R
/2) and the remaining retention
time will be equal to t
D
. For example,
if a 0100% B gradient in run in
20 min at 2 mL/min, the midpoint
should be reached at 10 min into the
gradient. If the midpoint of the gradi-
ent is measured as 11.2 min, then t
D
= 11.2 20/2 = 1.2 min. Convert this
to the dwell volume, V
D
= 1.2 min
2 mL/min = 2.4 mL.
As mentioned in the introduc-
tion, the dwell volume as well as the
wash-out characteristics of a given
LC system can inf luence the resulting
chromatogram. These differences are
illustrated in Figure 4a, where mea-
surements similar to those of Figure
3 are compared for a conventional
LC system and a UHPLC system. In
this case, rather than a sloping gradi-
ent, a vertical step from 0 to 100% B
was used. You can see that the offset
between the UHPLC and LC curves
is different at the beginning of the
step and the end of the step. The
authors (5) argue that the beginning
difference corresponds to the dwell
volume and the additional offset at
the end represents the mixer wash-out
characteristics. Thus, the wash-in and
wash-out curvature is different in this
case. Which measurement represents
the proper dwell volume you should
use when transferring a method? The
choice is somewhat arbitrary, and I
think that the midpoint technique
used in Figure 3 is probably as good
as any, unless complicated conversion
procedures are used. The illustration
of Figure 4b shows that dwell differ-
ences also can occur when a single
system is reconfigured. Each trace
represents two consecutive gradient
steps in a stair-step gradient program.
The same high-pressure-mixing LC
system was used in each case. The
upper trace represents the performance
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ES262112_LCGC0613_461.pgs 05.31.2013 23:32 ADV
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of the conventional LC system using
the factory-installed mixer, generat-
ing a dwell volume measured as 2.2
mL. We modified the system for
use with LCMS by removing the
original mixer and replacing it with
an after-market mixer that reduced
the dwell volume to 0.5 mL, with the
resulting trace at the bottom of Fig-
ure 4b (the difference in line widths
is an artifact of scaling to overlay the
two traces). As with the two examples
of Figure 4a, we see that both traces
show a more rapid wash-in than
wash-out and that the smaller-dwell-
volume system responds much more
quickly to changes in mobile-phase
composition.
Compensating for
Dwell-Volume Differences
Weve seen how differences in dwell
volume can result in differences in
the appearance of gradient chromato-
grams and how to measure the dwell
volume. Next, lets consider how to
compensate for differences in dwell
volume, using the example of Figure
2. Switching from a larger-dwell to a
smaller-dwell system is very simple.
Just add a gradient delay (isocratic
hold) corresponding to the desired
additional offset. For the transfer
of the method of Figure 2b to the
system of Figure 2a, it would mean
adding 2 min of isocratic hold to the
beginning of the program. Now the
program of 10/10/50% B at 0/2/17
min should give the same chromato-
gram as in Figure 2b with the system
of Figure 2a. Of course, you could
add 2 mL of volume to the system
by adding a mixing coil or other
modification, but this is much less
convenient than adding a hold. (And
it can be shown experimentally that
there are slightly different results
when adding an isocratic hold or a
mixing coil, but these are minor in
most cases.)
Transferring a gradient method
from a low-dwell system to a larger-
dwell system is not as simple. There
are several options. First, if you are
developing a new method and know
that it will be used on a larger-dwell
system, use the same technique men-
tioned above by adding an isocratic
hold to the gradient program that
corresponds to the additional dwell
volume likely to be encountered.
Then when the method is set up on
a new system, the isocratic hold time
(volume) can be adjusted so that the
combination of the isocratic hold and
the dwell volume remain unchanged
between the two systems. A second
technique to compensate for dwell-
volume differences may or may not
be available on your LC system.
Many of the newer LC systems allow
you to program the autosampler to
inject after the gradient starts. For
the present example of Figure 2b, you
would program the system to inject
2 min after the gradient program is
started. This would mean that the
injection would take place at the
same point in the gradient as it did
in Figure 2a. A third technique to
overcome dwell-volume differences
is to over-engineer the initial separa-
tion so that a dwell-volume change
is unlikely to cause problems. In the
present case, the method of Figure
2a might be developed so that the
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ES262145_LCGC0613_462.pgs 05.31.2013 23:34 ADV
black
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first two peaks had enough excess
resolution that some loss of resolu-
tion when the method is transferred
will not make any difference in the
analytical results. Remember that the
early peaks in the chromatogram are
the ones that will be most affected
by dwell differences; later peaks usu-
ally are offset in time, but resolution
changes are less likely. Some addi-
tional, more complex techniques are
described in the literature (6), such as
changing the starting %B.
Summary
We have seen that differences in sys-
tem dwell volume can account for
much of the difficulty encountered
when transferring a gradient LC
method from one system to another.
A technique to measure the dwell vol-
ume of an LC system was presented.
This procedure should be performed
at least once on every gradient LC
system so that you know its dwell vol-
ume. Several options were presented
to compensate for dwell volume dif-
ferences when transferring gradient
methods. Because the dwell volume
can be so important in gradient meth-
ods and their transfer, it should be
obvious that you should list the dwell
volume of the LC system as part of
the method description. This will
give the next user knowledge to aid in
adjusting the method to get the same
results on a different LC system.
Next month, well continue our
series on gradients with a look at
other common gradient elution prob-
lems.
References
(1) J.W. Dolan, LCGC North Am. 31(1),
3035 (2013).
204209 (2013).
300305 (2013).
382389 (2013).
(5) M. Woodman and L. Sanford, Poster
P1-G-327-MO, presented at HPLC
2011, Budapest, Hungary, 2011.
(6) L.R. Snyder, J.J. Kirkland, and J.W.
Dolan, Introduction to Modern Liquid
Chromatography, 3rd. ed. (Wiley, Hobo-
ken, New Jersey, 2010).
John W. Dolan
LC Troubleshooting
Editor John Dolan
has been writing LC
Troubleshooting
for LCGC for more
than 25 years. One of
the industrys most
respected profession-
als, John is currently the Vice President of
and a principal instructor for LC Resources,
Walnut Creek, California. He is also a mem-
ber of LCGCs editorial advisory board. Direct
correspondence about this column via e-mail
to John.Dolan@LCResources.com
please visit
www.chromatographyonline.com/dolan
ES262141_LCGC0613_463.pgs 05.31.2013 23:33 ADV
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MS THE PRACTICAL ART
The higher-order
structure (secondary,
tertiary, and quaternary
structure) of a protein
therapeutic is important
for proper biological
function. This article
looks at how hydrogen-
deuterium exchange mass
spectrometry (HDX-MS),
an MS-based labeling
approach, has begun
to serve as a means
for performing routine
biophysical analysis.
Joomi Ahn and St. John
Skilton are the guest
authors of this months
installment. Kate Yu is the
editor of MSThe Practical Art
Editor
T
he higher-order structure
(HOS) of a protein (second-
ary, tertiary, and quaternary
structure) is important for proper
biological function (1). Analytically
measuring the HOS properties of a
biotherapeutic and understanding
the relationships between structure
and function are important, as they
directly inf luence a drugs efficacy
and safety. The significance of
protein HOS was emphasized in a
biosimilar draft guidance that the
U.S. Food and Drug Administration
(FDA) issued in February 2012 (2).
Current analytical and structural
methods provide a comprehensive
understanding of the chemical, phys-
ical, and biological characteristics
of biologics, from primary structure
to HOS (3). Yet detecting changes
in protein dynamics and conforma-
tion can often prove challenging
compared to detecting changes in a
primary structure (3,4).
Tools That Yield
Information About Protein
Higher Order Structure
More than one biophysical analyti-
cal technique is typically used to
broadly characterize protein HOS
(Figure 1). Multidimensional nuclear
magnetic resonance (NMR) and
X-ray crystallography can determine
protein structure with high reso-
lution. Other structural analysis
techniques are more global in infor-
mation content, but are amenable
to high-throughput screening (57).
These include circular dichroism
(CD), differential scanning calorim-
etry (DSC), Fourier transform infra-
red (FT-IR) spectroscopy, intrinsic
f luorescence, light-scattering spec-
troscopy, and UV spectroscopy. Size
exclusion chromatography (SEC) and
analytical ultracentrifugation (AUC)
can be used to profile global struc-
tural distributions of a protein. Each
of the biophysical tools is invaluable
for biotherapeutic HOS analysis
because it provides information that
can be both complementary and
confirmatory. Assembling a biophys-
ical, structural analysis package for
a therapeutic protein is challenging.
One must use structural screening
tools that are sensitive enough to
detect critical structural attributes
as well as more discriminating tech-
niques that enable specific localiza-
tion of the sites of structural change.
Limitations on the throughput
and applicability of protein size such
as those presented by NMR and
X-ray crystallography have prompted
a quest for complementary analytical
tools that can detect local conforma-
tion changes with high sensitivity
and experimental throughput.
Hydrogendeuterium exchange
mass spectrometry (HDX-MS), an
MS-based labeling approach, has
begun to serve as a means for per-
forming routine biophysical analysis.
HDX is a chemical phenomenon in
which labile hydrogens in a protein
exchange with hydrogen atoms in
a surrounding solvent or gas (8).
HydrogenDeuterium
Exchange Mass Spectrometry:
An Emerging Biophysical Tool
for Probing Protein Behavior
and Higher-Order Structure
ES261830_LCGC0613_464.pgs 05.31.2013 18:46 ADV
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ES257410_LCGC0613_465_FP.pgs 05.29.2013 02:11 ADV
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Certain hydrogen atoms, such as
the amide hydrogen (NH) on the
protein backbone, exchange with
deuterium atoms, and the mass spec-
trometer detects where in the mol-
ecule the exchange takes place: at
the protein, peptide, or single amino
acid level (9). HDX-MS analysis has
been widely used to investigate the
f lexibility and dynamics of protein
molecules (1014). HDX is capable
of monitoring domain interaction,
localized protein breathing, and
folding or unfolding in the solution
phase. Information about protein
conformation from an HDX-MS
analysis can quantitatively compare
a control with an analyte by mea-
suring the relative amount of deu-
teration uptake (mass increase) as a
function of time.
HDX-MS recently gained currency
in the biopharmaceutical industry,
specifically in the discovery research
area, for defining the underlying
biology of a cellular process. The
technique is also well received by
process development organizations
for its ability to characterize the
HOS of protein drugs. The tech-
niques increasing popularity and
versatility were recently highlighted
in an article in Chemical &Engineer-
ing News (15). The value of HDX-
MS was also recently espoused in
several industry presentations at the
2nd International Symposium on
Higher Order Structure of Protein
Therapeutics, held in Baltimore,
Maryland, in February 2013 (16).
Speakers from leading biopharma-
ceutical companies (innovators)
demonstrated the utility of HDX-
MS for studying protein conforma-
tion to compare bioprocess changes
(before versus after), assess lot-to-lot
differences, discern the effects of
post-translational modifications, and
establish innovator versus biosimilar
comparability.
Comparability and biosimilar-
ity studies by HDX-MS can reveal
whether protein drugs are consistent
at the HOS level. Such detailed
information is difficult to obtain
because of the considerable time and
effort that they require. Indeed, con-
sistency at the HOS level is one of
X-ray,
NMR
E
a
s
e

o
f

u
s
e
I
n
f
o
r
m
a
t
i
o
n

r
i
c
h
MS-based tools:
HDX MS, ion mobility,
covalent labeling
Chromatography and others:
SEC, AUC, EM
Traditional tools: DSC, CD, IR, light scattering,
FLR, UV, and so forth
1 2 3 4
Low order to higher-order structure of protein
Protein in H
2
O, pH 7, 25 C
Deuterium labeling
in time-points
Exchange reaction Quenched
Injected into HDX manager
On-line pepsin digestion
Fast trapping and
UHPLC at 0 C
ESI Q-TOF
MS
E
Peptide identifcation
Peptide
map
Uptake
comparision
Deuterated uptake
determination
Automated software
Refrigerated
module
Automated
preparation
H
2
O D
2
O
Figure 1: Higher-order protein structure and analytical biophysical tools. Various
analytical biophysical tools including DSC, CD, NMR, and AUC are used to characterize
higher-order protein structures. HDX-MS is an emerging MS-based method for
studying protein dynamics and flexibility.
Figure 2: An streamlined HDX workflow. Peptide HDX analysis consists of several
steps. The labeled protein is quenched to pH 2.50 and directly injected into a
refrigerated module, called the HDX Manager system (Waters). The peptide separation
temperature was set at 0 C, and the digestion temperature can be independently set.
Fast desalting and UHPLC separation were performed, and peptides were acquired on
a electrospray ionization (ESI) Q-TOF instrument in MS
E
mode (Waters). Undeuterated
peptides (purple arrows) were processed to identify the peptide sequence using MS-MS
fragments. Deuterated peptides (red) were used to determine the deuterium uptakes.
ES261842_LCGC0613_466.pgs 05.31.2013 18:47 ADV
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ES257426_LCGC0613_467_FP.pgs 05.29.2013 02:12 ADV
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the most important proof-points that
biotechnology organizations must
demonstrate to regulatory agencies.
These studies can be used as orthog-
onal techniques that facilitate the
approval of process improvements
and establish that a biotherapeutic
organization has properly controlled
the stability of a biotherapeutic
product.
The study of protein interactions
in drug discovery, particularly anti-
bodyantigen binding studies, is
another area in which HDX-MS has
been widely applied. Determining
the interacting regions (such as epit-
opes and paratopes) along with their
partner molecules, constitutes criti-
cally important intellectual property
for both innovator and biosimilar
biotechnology companies. HDX-MS
can localize interacting regions by
determining which among them are
protected from deuterium exchange
upon the partner molecules bind-
ing (17). This information can be
well correlated with other, common
epitope mapping approaches like
NMR, X-ray, phage display, peptide
libraries, and mutagenesis. HDX-
MS provides the proper resolution
to distinguish the contact areas (~5
to 15 residues in length). More-
over, it does so within a relatively
fast turnaround time of a few days,
particularly when using automation
and modern HDX informatics tools
to meet the demands of higher-
throughput screening. In discovery
research, organizations that have
adopted HDX-MS, can use MS to
help fill the therapeutic pipelines, by
rapidly performing epitope mapping
to establish intellectual property for
multiple paratope and epitope inter-
actions. The importance of this has
been underlined in recent freedom
to operate court cases that have
re-examined existing IP with newer
technology, allowing or barring
entrants into a lucrative market (24).
Studies of insulin, an extremely
well-characterized protein, have
recently underscored the importance
of characterizing HOS to ensure
a biosimilar candidate can match
an innovator products efficacy. In
one case, the primary structure of
a biosimilar insulin was fully char-
acterized. Yet the submission failed
when clinical data indicated differ-
ent pharmacokinetics, a difference
that HOS studies may well have
predicted (18).
A D
DM I
Q
Q
S
V D E M I R E A D D D G G Q V N Y E E F V QM I
E I R E A F F D K D G G N Y I S A A E L R H V M N L G E K L T D E E T R V E E
Q
Q A E E A F S L F D K D G G D T T T K E L G T V M R S L G
40 35 30 25 20 15 10 5
45 50 55 60 65 70 75 80
85
125 130 135 140
90 95 100 105 110 115 120
I F K I L
L N N P T E E V D D G G D F P E F L T MM M A R K K D D T T I N A A E
T E E
Figure 3: High-sequence coverage of calmodulin by on-line pepsin digestion (96.5%).
Each blue bar represents an identified peptic peptide. The calmodulin sequence along
with residue number (every fifth residue) is shown in the map. Several overlapping
peptides generated by pepsin digestion assist in narrowing down the location where
the conformation of the protein has changed.
Figure 4: Automated data processing is possible for faster data turn-around
time. DynamX software is shown as an example of peptide HDX data analysis.
The automated informatics package efficiently calculated the deuterium uptakes
of identified peptides (left panel) from extracted spectra (right, bottom panel)
and displays the deuterium uptakes in the uptake plot (right, top panel). In this
calmodulin study, the different deuterium uptakes were observed between apo (red)
and holo (blue) forms.
ES261851_LCGC0613_468.pgs 05.31.2013 18:48 ADV
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The Need for Improved
Instrumentation for HDX
Analysis
The hyphenated technique of liquid
chromatographymass spectrom-
etry (LCMS) has long been used
to determine protein identification,
post-translational modification,
disulfide bond mapping, and various
other applications. Improved analyt-
ical capabilities enable commercial
LCMS systems to address HDX-
MS studies of greater molecular
complexity with increasing sensitiv-
ity to changes in protein structure,
dynamics, and interactions (1921).
These capabilities take the form of
sub-2-m chromatography (ultra-
high-pressure liquid chromatography
or UHPLC) and ion-mobility gas
phase separation.
A streamlined HDX workf low,
from automated sample preparation
to data processing, was previously
well established (9). Figure 2 illus-
trates a typical workf low of HDX
experiments. Proteins, prepared in
H
2
O as a control, are incubated
Apo Calmodulin
(calcium free)
Holo Calmodulin
(calcium bound)
Control
10 s
10 min
Relative % deuterium uptake
240 min
0 10 20 30 40 50 60 70 80 90% Undetermined
ca
+2
ca
+2
ca
+2
ca
+2
Figure 5: Effective HDX data visualization of a 3D structure of a protein. Calmodulin
apo and holo forms were used as an example of a HDX heat map on 3D structures.
Color differences between apo (1CFD) and holo (1PRW) forms can be used to compare
differences in relative % deuterium as well differences that appear over time.
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ES261848_LCGC0613_469.pgs 05.31.2013 18:48 ADV
black
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with D
2
O to produce a labeling
time-course. The labeling reaction
is quenched and the labeled protein
is injected into an HDX Manager
system, a device designed specif i-
cally for this task as part of the LC
instruments sample system and
offers unique functions (Waters,
nanoAcquity UPLC system with
HDX Technology). During the
HDX experiments, the entire pro-
cess of online digestion and analyti-
cal separation of peptides is tightly
controlled by temperature and pH.
Sub-2-m UHPLC separations
occur within a cooled environment
(0 C). If the optimal quenching
conditions (pH 2.5 and 0 C) are
not properly controlled, the deu-
terium tends to reverse exchange
with hydrogen. Therefore, precise
temperature control in a cold path-
way and faster separation time are
important factors in HDX
experiments.
Typically, pepsin, an acidic aspar-
tic protease, cleaves a protein into
short peptides on an on-line diges-
tion format using a pepsin column.
Figure 3 shows the calmodulin
sequence coverage map as an exam-
ple of a pepsin on-line digestion.
This calmodulin study achieved
high coverage (96.5%) and multiple
overlapping peptides, assisting in
localizing where conformation is
changed.
Using Automated Software to
Cope with the Data Burden
Historically, curating HDX-MS data
manually was inefficient because it
required expert interpretation and
hundreds to thousands of spectral
calculations (for peptide analysis).
Despite the requisite expertise, how-
ever, interpretation is a repetitive
process and, like all such processes,
it lends itself to automation. Soft-
ware developers knew that if they
were going to make the HDX-MS
technique attractive to industry,
they must develop an informat-
ics tool that systematically selects
spectra by applying predetermined
criteria and then measuring the mass
change of the deuterated forms (22).
Such software has made HDX-MS a
highly practicable technique even for
biologists and biochemists.
Yet the mere availability of tools
for biologists and biochemists to
process HDX data isnt enough.
Data displays must be intuitive, and
they must directly enable recogni-
tion of regions where structural
differences exist. Consistent with
that requirement, recent software
versions often show conformation as
it relates to differences between two
states of the protein sequence, rather
than as a spectral readout from the
instrument. Figure 4 illustrates vari-
ous data displays in DynamX HDX
informatics tools (Waters). After
processing information from raw
spectra, the identified peptides were
extracted at a particular retention
time. The software automatically
calculates the amount of deutera-
tion and displays results via several
visualization tools: deuterium uptake
curves, sequence coverage heat maps,
and difference (butterf ly) plots.
Recent software development
has enabled the mapping of HDX-
MS results onto three-dimensional
(3D), NMR, or crystal structures
of proteins as colored heat maps.
In Figure 5, HDX-MS data for
calmodulin, conformationally sensi-
tive to calcium ion binding (apo:
calcium free; holo: calcium bound),
are represented with HDX data on
3D structures. Such intuitive plot-
ting schemes offer a rapid, visual
comparison of different states, such
as bound and free forms of the pro-
teins of interest. Additionally, a dif-
ference plot provides a rapid visual
overview of the average, relative,
fractional exchange difference for all
peptides in each labeling time-point,
highlighting at a glance where the
active regions of the protein can be
localized. Thus, the entire polypep-
tide backbone of each form can be
compared in terms of both spatial
and temporal information.
Advanced Work
and Future Directions
As more complex proteins are ana-
lyzed, it becomes increasingly use-
ful to include ion mobility separa-
tions to minimize interfering ions.
Ion mobility spectrometry (IMS)
provides an orthogonal gas-phase
separation, based on the charge,
50
5
0
0
1
0
0
0
1
5
0
0
m
/
z
100
150
Drift time (bins)
Off
710 711 712 713 714 716 717 718 715
On
On
Ion Mobility
Peptide 1
Peptide 2
Z = 1 Z = 2
Figure 6: Isolating interfering ion clusters by charge state in an ion mobility
separation, illustrated with two deuterated peptides from BSA, peptide 1
(ASIQKFGERALKA, z = 2, top, right panel) and peptide 2 (AVEGPKL, z = 1, middle,
right panel). The bottom panel shows the spectrum produced when ion mobility
was turned off, where two peptides were overlapped.
HDX-MS can be
used to compare
bioprocess changes,
assess lot-to-lot
differences, and
establish innovator
versus biosimilar
comparability.
ES261996_LCGC0613_470.pgs 05.31.2013 22:37 ADV
black
yellow magenta cyan
shape, and mass of molecules, that
is capable of detangling ions that in
MS detection share the same mass-
to-charge ratio (20). Figure 6 illus-
trates the ion mobility separation
of two peptides of similar mass that
were eluted at the same retention
time. The two species overlapped
without IMS separation enabled
(bottom panel). By enabling the IMS
separation, the two labeled peptides
were successfully isolated from each
other, making it possible to improve
the robustness of peptide detection
and measures of deuterium uptake
for each peptide.
Electron transfer dissociation
(ETD) is another MS technique
with the potential to increase the
resolution for assigning regions of
structural difference. Recent studies
demonstrate that HDX-MS applica-
tions analyze deuteration to the level
of single amino-acid residues (23).
Future efforts to use this approach
will provide the increased spatial
resolution combined with the classi-
cal, bottom-up HDX-MS workf low.
Summary
The ability of novice and experienced
researchers to conduct HOS studies
with HDX-MS technology has bene-
fitted greatly from combined advances
in separations, automation, MS, and
informatics. The wider scientific dis-
semination of what is achievable for
protein structure analysis has yielded
a quantum leap in the demand for
and development of HDX-MS.
Like a genie, once released from its
bottle, this powerful set of capa-
bilities will not easily be contained.
Indeed, it will only continue to grow
in application and, in doing so, fur-
ther inform our understanding of pro-
tein biotherapeutics.
References
(1) G. Scapin, Curr. Pharm. Des. 12, 2087
2097 (2006).
(2) FDA, CDER, CBER, Biosimi lar draf t
guidance, 2012.
(3) A. Beck et al., Anal . Chem. 85, 715736
(2013).
(4) A. Beck et al., Anal . Chem. 84, 4637
4646 (2012).
(5) A.W. Vermeer et al., Biophysical Journal,
79, 21502154 (2000).
(6) H. Liu et al., Immunol . Lett. 106, 144
153, (2006).
(7) J.F. Neault et al., J. Biomol . Struct. Dyn.
25, 387394 (2008).
(8) A. Hvidt and K. Linderstrom-Lang, Bio-
chim. Biophys. Acta 14, 574575 (1954).
(9) J.R. Engen et al., i n Encycl opedia of
Analytical Chemistry, R. A. Meyers, Ed.
(Wiley, 2011), pp. 217.
(10) T.E. Wales and J.R. Engen, J. Mol . Biol .
357, 15921604 (2006).
(11) C.K. Woodward, Curr. Opin. Struct .
Biol . 4, 112116 (1994).
(12) Y. Bai et al ., Science 269, 192197
(1995).
(13) J. Fang et al., J. Mol . Biol . 398, 4053
(2010).
(14) C.M. Hebling et al., Anal . Chem. 82,
54155419 (2010).
(15) A. M. Rouhi, C&EN 90(17), 1617
(2012).
(16) CASSS Second International Symposium on
Higher Order Structure of Protein Therapeu-
tics, Baltimore, Maryland, February 2013.
(17) Q. Zhang et al., Anal .Chem. 83, 7129
7136 (2011).
(18) L. Hei nemann and M. Hompesch, J.
Diabetes Sci . Technol . 5(3), 741754
(2011).
(19) T. E. Wales et al ., Anal . Chem. 80,
68156820 (2008).
(20) R.E. Iacob et al., Rapid Commun. Mass
Spectrom. 22, 28982904 (2008).
(21) R.E. Iacob and J.R. Engen, J. Am. Soc.
Mass Spectr. 23, 10031010 (2012).
(22) D. Houde et al., J. Pharm. Sci. 100,
20712086 (2011).
(23) K. D. Rand et al ., Anal . Chem. 81,
55775584 (2009).
(24) C.G. Sandercock and U. Storz, Nat .
Biotechnol . 30(7), 615618 (2012).
For more on mass spectrometry, see the
full MSThe Practical Art column at
www.chromatographyonline.com/MSPA.
Also, see our ongoing supplement series,
Current Trends in Mass Spectrometry,
under Publications, then Supplements.
St. John Skilton
is a life sciences
specialist at Waters,
concentrating on
the biologics and
omics fields. He
has a background in
GC, LC, MS, analyt-
ics, and supporting
laboratories in adopting new analytical
technologies. He helped coordinate the
biologics element of the introduction of
the first commercialized HDX platform to
study protein interactions, working closely
with Dr. Ahn to translate theory into an
understandable reality.
Joomi Ahn is a
principal scientist in
pharmaceutical life
sciences at Waters
Corporation in Mil-
ford, Massachusetts.
She joined Waters in
1999 and has broad
experience applying
LCMS technologies in biopharmaceuti-
cal characterization applications. She
received her PhD in chemistry and chemi-
cal biology from Northeastern University
in Boston, Massachusetts. Her research
interests include the understanding of
proteins and protein conformation with
HDX-MS technology.
Kate Yu
MS The Practi-
cal Art Editor Kate
Yu joined Waters
in Milford, Mas-
sachusetts, in 1998.
She has a wealth of
experience in apply-
ing LCMS technolo-
gies to various application fields such as
metabolite identification, metabolomics,
quantitative bioanalysis, natural products,
and environmental applications. Direct
correspondence about this column to
lcgcedit@lcgcmag.com
The study of
protein interactions
in drug discovery,
particularly
antibodyantigen
binding studies,
is another area
in which HDX-MS
has been widely
applied.
ES261994_LCGC0613_471.pgs 05.31.2013 22:37 ADV
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PERSPECTIVES IN MODERN HPLC
I
c
o
n

i
m
a
g
e
:

J
o
e

Z
u
g
c
i
c
,

J
o
e

Z
u
g
c
i
c

P
h
o
t
o
g
r
a
p
h
y
T
his is the second installment
of a new column in LCGC
North America titled Perspec-
tives in Modern HPLC, which will
be published every quarter and fea-
tures fresh perspectives, innovative
approaches, best practices, mega-
trends, and emerging opportunities in
this ever-evolving field of separation
science. The first installment in the
April 2013 issue was devoted to new
high performance liquid chromatog-
raphy (HPLC) products introduced at
Pittcon 2013 (1).
HPLC is a dominant analytical
technique with mature technologies
that have been widely practiced for five
decades. Innovations such as ultra-
high-pressure liquid chromatography
(UHPLC), liquid chromatographymass
spectrometry (LCMS), two-dimen-
sional liquid chromatography (2D-LC),
chiral separations, coreshell columns,
and novel stationary phases have helped
drive HPLC to higher performance
in diverse applications, yielding faster
speed, higher resolution, greater sen-
sitivity, and increased precision. The
practice of HPLC is no longer limited
to specialists or chromatographers, but
is now widely performed by students,
chemists, biologists, production workers,
and other novices in academia, research,
and quality control laboratories. More
than $4 billion of HPLC equipment,
columns, and accessories were sold
worldwide in 2012 (2).
There is no shortage of information
on HPLC (39). Hundreds of books,
thousands of articles, and millions of
web citations are available. My goals
are to reexamine the big picture of
HPLC and its applications from a
users perspective. I will strive to find
approaches to make it more productive
or relevant. I am excited to be a new
columnist for LCGC North America
and promise to dig deeper and com-
ment on ideas to make HPLC more
exciting and less arduous for all practi-
tioners. A listing of my tentative topics
for 2013 and beyond can be found in
the addendum.
In this installment, the essence of
modern HPLC is discussed first,
by examining the reasons that make
HPLC so ubiquitous; second, by
reviewing the fundamental chromato-
graphic principles on how they can
guide the way we conduct HPLC sep-
arations; and finally, by commenting
on some less-obvious opportunities
with far-reaching impacts or imme-
diate job prospects for separation
scientists.
Why Is HPLC So Ubiquitous?
Advantages and Perceived
Limitations of HPLC
Why is HPLC so ubiquitous as prac-
ticed by thousands of practitioners
around the world? Table I lists the
advantages and perceived limitations
of HPLC (3). The reader, without a
doubt, has seen similar lists elsewhere.
Here, I would like to discuss a few
highlighted advantages (in bold) with
an example on a stability study fol-
lowed by a description of its perceived
limitations.
The Essence of Modern
HPLC: Advantages,
Limitations, Fundamentals,
and Opportunities
This article reexamines the
fundamental concepts of
high performance liquid
chromatography (HPLC) to
bring fresh insights to how
we perform HPLC today.
It reviews the prominent
advantages that render
HPLC indispensable and
comments on its many per-
ceived limitations. Several
opportunities with far-
reaching impacts in life
science for the separation
scientist are described.
Michael W. Dong
Perspectives in Modern HPLC Editor
ES262040_LCGC0613_472.pgs 05.31.2013 22:47 ADV
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# thermoscientic.com/Veo
UV at 220 nm
UV at 254 nm
Charged Aerosol
Detection
MS TIC
(+ ion scan 150-500 AMU)
Ketoprofen
Verapamil
Propranolol
Void + Cl
-
Comparison of Charged Aerosol
Detection to UV and MS

2
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ES257425_LCGC0613_473_FP.pgs 05.29.2013 02:12 ADV
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Advantages of HPLC
The dominance of HPLC as a premier
analytical technique is no accident.
The most prominent advantage is its
applicability to diverse analytes types,
from small organic molecules and ions
to large biomolecules and polymers.
The successful coupling of HPLC to
MS gave it an invincible edge as the
perfect analytical tool combining
excellent separation capability with the
unsurpassed sensitivity and specificity
of MS. HPLCMS is rapidly becoming
the standard platform technology for
bioanalytical testing (drugs in biologi-
cal fluids), trace analysis for residues
in food, forensic and environmental
samples, and life science research (36).
Finally, the excellent precision and
robustness of HPLC with UV detec-
tion makes it an indispensable tool for
quality control (QC). This last point is
illustrated by a case study on stability
evaluation of a pharmaceutical product
shown in Figure 1 and Table II.
Figure 1 shows chromatograms of a
retention marker solution and a three-
month stability sample of a drug tablet
formulation. The retention marker
solution contains the active pharma-
ceutical ingredient (API) spiked with
its expected impurities and degradants.
This type of testing is conducted rou-
tinely by pharmaceutical laboratories to
establish shelf lives and storage condi-
tions for API and drug products (5,6).
The HPLC conditions use a multiseg-
ment gradient with ammonium for-
mate buffer and acetonitrile. Peak des-
ignations shown in the chromatograms
are API (SRR, absolute configuration
for the drug molecule with three chiral
centers); SRS and RRR (process impu-
rities-diastereomers); M235, M416, and
M399 (degradants designated by their
MS parent ions); ketone (an oxidative
degradant); and BHA (butyl hydroxy-
anisole, an antioxidant additive). The
bottom chromatogram shows the
extract of a tablet formulation kept in
a stability chamber at 50 C/75%RH
for three months, indicating increased
levels of degradants for M416, SRS,
RRR, ketone, and M399 (data cap-
tured in the stability table in Table II).
The chromatograms and the operating
conditions are fairly unremarkable by
todays standard though they serve
to illustrate some of the less obvious
strengths of HPLC in QC applications,
such as
t the ability to quantitate all compo-
nents (API and all related substances,
including isomers);
t very precise retention times and peak
areas using UV detection (<0.1%
relative standard deviation [RSD] is
routinely achievable for UHPLC ver-
sus 0.20.3% RSD for HPLC);
t highly reproducible assays (robust-
ness) by different laboratories (with
instruments from different vendors
and columns from different batches);
t high-sensitivity assays for trace
impurities ~0.01% in this assay
(limit of quantitation of 0.05% or
0.10% is required by regulations);
t and all components readily identified
by MS for assays with volatile mobile
phases.
Table II is a stability report sum-
marizing data from the three-month
time point of this accelerated stability
study of the oral tablet formulation
under various storage and packaging
conditions, indicating increased levels
of degradants at 40 C/75%RH and
50 C/75%RH, particularly for hydro-
lytic degradant M399. The remarkable
aspect lies in the exceptional quality of
the stability data generated by HPLC,
Table I: Advantages and limitations of HPLC
Advantages Perceived Limitations
Amenable to diverse analyte or sample
types
Lack of an ideal universal detector
Precise and highly reproducible
quantitative analysis
Less separation efficiency than capillary
gas chromatography
LCMS Relatively difficult for novices
Flexible, customizable, automated operation Still arduous for regulatory testing
High separation power with sensitive
detection
175
(a)
150
125
100
A
b
s
o
r
b
a
n
c
e

(
m
A
U
)
A
b
s
o
r
b
a
n
c
e

(
m
A
U
)
75
50
25
-25
2 4 6
Time (min)
Time (min)
8 10 12
2 4
M416
M235
M416
SRS
RRR
M399
API (SRR)
BHA
Ketone
SRS RRR
API (SRR)
M399
BHA
Ketone
5
.
3
0
9
1
.
1
9
6
5
.
3
0
2
5
.
5
0
9
6
.
9
4
1
6
.
6
4
2
6
.
7
2
9
8
.
1
5
3
9
.
7
9
6
1
1
.
1
8
0
1
3
.
3
7
4
5
.
8
1
8
6
.
0
5
2
6
.
5
3
0
0
.
0
0
8
6
.
9
3
8
8
.
1
7
7
8
.
8
6
8
9
.
7
9
9
1
3
.
1
5
2
1
3
.
3
8
0
6 8 10 12
8
(b)
6
4
2
-2
0
0
Figure 1: UHPLC chromatograms of (a) a retention marker solution and (b) a three-
month stability sample (extract of a tablet kept in a stability chamber at 50 C/75%RH).
This is an example of a stability-indicating assay used extensively in the pharmaceutical
industry to establish shelf life. Column: 100 mm 3.0 mm, 2-m d
p
ACE Excel 2 C18;
mobile-phase A: 20 mM ammonium formate (pH 3.7); mobile-phase B: 0.05% formic
acid in acetonitrile; flow: 0.8 mL/min; temperature: 40 C; pressure: 450 bar; gradient:
515% B in 2 min, 1540% B in 10 min, 4090% B in 1 min; detection: UV absorbance at
280 nm; sample: tablet extract in 20% acetonitrile in 0.1 N HCl; injection volume: 3 L.
ES262042_LCGC0613_474.pgs 05.31.2013 22:48 ADV
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ES257452_LCGC0613_475_FP.pgs 05.29.2013 02:12 ADV
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with its ability to track changes of
drug impurities over time. Data are
highly reproducible with a high degree
of confidence and can be repeated by
different labs. This high level of data
reliability and reproducibility is taken
for granted in HPLC applications for
quality control to such an extent that it
becomes mundane a feat less achiev-
able by capillary electrophoresis (CE),
MS, or supercritical fluid chromatogra-
phy (SFC).
Perceived Limitations of HPLC
Limitations of HPLC are rarely dis-
cussed and are, therefore, more inter-
esting. Perceived limitations is the
terminology used here since most have
been mitigated by recent advances and
are no longer real practical issues.
Lack of a Universal Detector
The lack of a universal detector for
HPLC is often mentioned, although
the UVvis detector comes close to
one for chromophoric compounds.
Refractive index detection fits the bill,
but suffers from low sensitivity and
incompatibility with gradient elution.
Evaporative light scattering detection
(ELSD) was a contender, but was sur-
passed by charged aerosol detection
(CAD). CAD uses a nebulizer with
corona discharge detection and has
better sensitivity (low ng) and ease-of-
use than ELSD (3,10).
Mass spectrometry is becoming a
universal detection method for ionic
or ionizable compounds with incred-
ible speed, sensitivity, and selectivity.
The developments of triple-quadrupole
MS-MS, high-resolution MS (for
example, time-of-flight [TOF] and
orbital trap), and hybrid MS (Q-TOF
or ion traporbital trap) (11) in combi-
nation with UHPLC and 2D-LC have
transformed our abilities to develop
and perform bioanalytical assays, mul-
tiresidue analysis of complex samples,
and life science research (9).
Less Separation Efficiency
Than Capillary Gas Chromatography
Conventional HPLC has a practi-
cal peak capacity (P
c
) of ~200 using
columns with ~20,000 plates under
gradient conditions not particularly
effective for very complex samples (3).
The advent of UHPLC has extended P
c
to 4001000 range in a time span of
~60 min (9,1216). 2D-LC can further
increase P
c
for comprehensive analysis
of very complex samples in proteomics
and metabonomics (9,16).
Relatively More Difficult for Novices
The bewildering number of HPLC
modules, columns, mobile phases,
and operating parameters renders
HPLC difficult for the novice. Sur-
prisingly, with a single-point control
of the HPLC system by the data
system, it becomes relatively easy to
teach a new person to run an exist-
ing HPLC method. For example,
just place the sample vial into the
autosampler tray and the assay can be
started with few mouse clicks with
formal-looking reports automatically
printed afterwards. Nevertheless,
substantial experience and scientific
judgment are needed to develop a
new method, interpret a strange
result, or to troubleshoot a problem.
The good thing is that chromato-
graphic principles are well docu-
mented and can easily be explained
by more experienced colleagues in
your laboratory. HPLC is complex,
predictable, and not particularly
complicated to a typical scientist
with a strong chemistry background.
Still Arduous,
Particularly for Regulated Testing
HPLC is versatile, quantitative, sensi-
tive, and extremely precise. It can also
be time-consuming and arduous, par-
ticularly for regulated analysis under
good manufacturing practices (GMP).
For instance, these are the steps in a
typical operation: weighing reference
Table II: Results of a three-month accelerated stability study in a new drug product formulation
Peak ID
M235
(area%)
M416
(area%)
SRS
(area%)
API
(area%)
RRR
(area%)
Ketone
(area%)
M399
(area%)
t
R
(min) 1.20 5.30 6.53 6.73 6.94 8.15 9.80
Temp/RH% RRT 0.18 0.79 0.97 1.00 1.03 1.21 1.46
5 C Coated-yellow tablet, 100 mg 0.01 99.9
25 C/60
Coated-yellow tablet, 100 mg 0.01 0.01 99.9 0.01 0.01
Coated-yellow tablet, 100 mg, 1
dessicant
0.01 0.01 99.8 0.03 0.01
Coated-yellow tablet, 100 mg,
open dish
0.01 0.01 99.8 0.03 0.01
30 C/65 Coated-yellow tablet, 100 mg 0.01 0.01 99.9 0.02 0.02
40 C/75
Coated-yellow tablet, 100 mg 0.02 0.02 99.8 0.04 0.06
Coated-yellow tablet, 100 mg, 1
dessicant
0.03 0.02 99.7 0.05 0.06
Coated-yellow tablet, 100 mg,
open dish
0.02 0.03 99.7 0.01 0.03 0.10
50 C/75 Coated-yellow tablet, 100 mg 0.01 0.02 0.09 99.3 0.06 0.04 0.35
RRT = relative retention time
ES262041_LCGC0613_476.pgs 05.31.2013 22:48 ADV
black
yellow magenta cyan
standards; preparing samples and
mobile phases; setting up the column
and all modules; performing system
suitability testing; injecting standards
to calibrate the system followed by
samples analysis; performing peak
integration; reporting; reviewing; and
sign-offs. Fortunately, most steps are
automated by precision instruments
for routine testing and are therefore
highly reproducible. Compare it with
spectroscopic analysis such as the
identification of raw materials using
a hand-held Raman spectrometer
point the laser to the sample, press a
button and a pass or fail result with
GMP documentation is available in
seconds. One piece of advice: Dont
use HPLC unless you have to quanti-
tate analytes with high accuracy and
precision.
HPLC Fundamentals and
Insights on Performing
HPLC Separations
Lets briefly review the fundamen-
tal principles to look for some fresh
insights on how we perform HPLC
analysis. First, the goal of most HPLC
analysis is to quantitate analytes in a
sample (mixture) by physically separat-
ing its components. It is useful to cat-
egorize samples as simple or complex
since the analytical approach is quite
different. In chromatography, three
factors control the separation or resolu-
tion (R
s
) of several components in the
sample:
Retention or having k values (reten-
tion factor) greater than one
Selectivity () or differential migra-
tion of analytes in the column
Separation power or column effi-
ciency (N) the ability of the col-
umn to separate many components
in the chromatogram.
These factors are defined and
explained in every HPLC textbook
(36). For isocratic analysis in which
the mobile phase composition remain
constant, the relationship of R
s
to k, ,
and N are described by the resolution
equation shown below (3,4).
R
s

=
k
k + 1
1
N
4
[1]
Retention Selectivity Efficiency
Conventional wisdom leads us to the
following rules of thumb for isocratic
analysis.
First, k or retention factor is the ratio
of retention times of the analyte to that
of an unretained component. Keep k
from 1 to 20 by adjusting mobile phase
strength (% organic in reversed-phase
LC). For potency or performance test-
ing of the main component (for exam-
ple, assays of drug substances or prod-
ucts, dissolution, or content uniformity
testing), adjust the k to be ~1 and use a
short column (length = 50 mm) for fast
analysis (<2 min). For multicomponent
analysis of a simple mixture, increase k
by lowering the mobile phase strength
until all components are retained and
separated from each other. If there
are four components and four distinct
peaks are observed, then this can be
a preliminary method condition. If
there is a pair of coeluting or partially
resolved peaks with R
s
< 1.01.5, then
the selectivity () of the two peaks or
critical pair (, which is the ratio of
the two k values) should be adjusted.
Second, a selectivity value of 1.0 of
the critical pair means coelution (that
means interference of an analyte with
another component), which precludes
accurate quantitation because the
method is not specific to that analyte.
In HPLC, it is often easier to change
the mobile phase (organic solvent
type, buffer type or strength, and pH)
because they can be continuously var-
ied. The next step is to change column
type or column temperature. In HPLC,
adjusting or fine-tuning is the main,
and most time-consuming part of the
method development process.
Finally, N or plate count is a mea-
sure of the separation power of the
column and is proportional to column
length and inversely proportional to
particle size (d
p
) (35). N can be rea-
sonably low (for example, 5000 plates)
for simple mixtures using a short
column. Longer columns with higher
N are preferred for more complex mix-
tures or for closely eluting analytes (for
example, isomers). The practical maxi-
mum for N in conventional HPLC
is ~20,000 plates for routine testing;
equivalent to the N of a 150-mm-long
column packed with 3-m particles.
In isocratic analysis, the analyte
peak or band is continuously broad-
ened with higher retention times by
the inherent chromatographic process
(35). Values of k exceeding 20 are
typically not feasible because the peaks
would be too broad for quantitation
and the gain in resolution becomes
negligible. In reversed-phase LC, -log k
is proportional to the solvent strength
of the mobile phase or % organic sol-
vent (%B). These relationships are well-
behaved and very predictable (3,4).
Gradient analysis with increasing
mobile phase strength is typically pre-
ferred for complex samples or mixtures
with diverse polarities or for assays in
which all components must be reported
(such as impurity testing of pharmaceu-
ticals). Gradient analysis yields higher
peak capacity (P
c
, defined as the num-
ber of peaks that can be resolved in
chromatogram with an R
s
value of 1.0;
for example, P
c
is ~200 for gradient vs.
~50100 for isocratic) (2) and sharper
peaks because peak widths are similar
for all peaks irrespective of retention
times. P
c
is useful to measure perfor-
mance under gradient conditions since
one can only measure N isocratically.
Gradient methods are more difficult to
develop because retention and selectiv-
ity (and P
c
) are affected by many fac-
tors, including initial and ending sol-
vent strengths (%B) and gradient time
(t
G
), in addition to the typical mobile
phase factors. Secondary factors are
flow rate (F) and column temperature
(35). Gradient analysis is less suscep-
tible to extracolumn band broadening
because sample band dispersion before
the column and large injection volumes
are inconsequential (for injection of
samples in lower-strength diluents)
an important fact for UHPLC using
columns with smaller internal diam-
eters (9,13).
The advent of UHPLC (systems with
low dispersion and pressure limits of
15,000 to 19,000 psi) together with
the use of smaller internal diameter
columns packed with sub-2-m par-
ticles, accentuated the need for better
understanding of chromatographic
fundamentals such as R
s
, k, N, ,
particle size (d
p
), column internal
diameter (d
c
), column void volume
ES262043_LCGC0613_477.pgs 05.31.2013 22:48 ADV
black
(V
m
), peak volume, peak width, instru-
ment bandwidth or system dispersion,
flow-cell volume, and dwell volumes
(3,9,13). Because UHPLC is becoming
the modern standard HPLC platform,
better understanding of these concepts
will be helpful for efficient operation
and method development and transfer
(9,16).
In summary, the biggest strength
of HPLC is its versatility for reliable
quantitation of analytes in complex
mixtures through physical separations
of the analyte peaks from coeluted
components. To effect separations,
one must have retention (k), selectiv-
ity (), and adequate plate counts (N).
Retention is related to the partition
coefficient of the analyte molecule
between mobile and stationary phases.
This partitioning process is repeated
millions of times down the column
to allow separation of analytes with
minute differential migration ().
Selectivity () can be tweaked by
changing column or mobile phase
parameters. The unlimited number
of combinations of columns, mobile
phases, and controlling factors makes
HPLC complex but gives endless pos-
sibilities for the quantitation of all or
specific components in many sample
types. HPLC works reliably in prac-
tice because of the gentle, predictable
nature of the liquid phase chromato-
graphic processes and the availability
of precise instrumentation with effi-
cient and reproducible columns. Very
complex samples with thousands of
analytes can be separated by brute
force with UHPLC and 2D-LC cou-
pled with UV, MS, or MS-MS (16).
The complexity (versatility) of HPLC
is its greatest strength and also its key
weakness (laborious).
Opportunities for
Separation Scientists
Today, I believe that biology and life
sciences are the research areas that
offer opportunities for separation
scientists to make the greatest impact.
Biology is the Wild West of the 21st
century with a lot of fertile ground
for scientific discovery. Unfortunately,
most biologists are not experts in the
versatile tools for discovery, HPLC or
LCMS (8), and separation scientists
(mostly analytical chemists) dont
usually have the intimate knowledge
of the great biological problems (such
as cell signaling and curing cancer or
Alzheimers disease). It would be ideal
if scientists could straddle both ana-
lytical chemistry and biology to tackle
pressing problems, such as the iden-
tification of disease biomarkers used
for clinical diagnostics in personalized
medicine (1719). Many instrumenta-
tion and pharmaceutical companies
are already investing heavily in this
area (20), though new approaches
are needed such as automated proce-
dures to isolate the key analytes in
these complex matrices (19). Here,
2D-UHPLC coupled with hybrid
high-resolution MS can be a power-
ful generic tool but only for those
scientists with a good understanding
of the problem and the analytical
technologies.
My next comments are some
immediate job opportunities for
separation scientists in our recover-
ing economies. In 2012, six of the 15
top selling drugs were monoclonal
antibodies (mAb) (21). With hundreds
of on-going mAb research projects as
therapeutics, many job opportunities
are available in the characterization
and quality control of mAbs (22,23).
However, analysts experienced in
assessing the critical quality attri-
butes of biological drugs are rare and
graduate students are not trained in
this area because it is not the funding
source of their professors. So, there
appears to be a disconnect between
graduate training and job opportuni-
ties that goes beyond summer intern-
ships in the pharmaceutical industry.
Perhaps a closer collaboration or part-
nership between academia and indus-
try is the right solution.
Summary and Conclusions
In this installment, my first real
column for Perspectives in Modern
HPLC, I have addressed the essence
of modern HPLC by reviewing its
advantages, limitations, and funda-
mental principles. HPLC is the domi-
nant analytical technique because
of its versatility, reproducibility, and
wide applicability in research and
quality control. HPLC is a complex
technique because of its myriad com-
binations of modules, columns or
mobile phases, and operating param-
eters. A deeper understanding of the
principles is becoming more impor-
tant for the effective use of UHPLC,
the new standard platform of HPLC.
Passionate separation scientists with
expertise in LCMS plus an in-depth
understanding of the great problems
in biology are in an excellent position
to develop new approaches to make
real impacts in life science for a better
tomorrow.
Acknowledgments
The author is grateful to Drs. Sam
Yang, Mohammad Al-Sayah, and
C.J. Venkatramani, and Midco Tsang
and Bob Garcia, Jr., of Genentech;
Drs. Davy Guillarme of University
of Geneva, and Ron Majors of Agi-
lent Technologies; and Professors
Kevin Schug of University of Texas at
Arlington and Milton Lee of Brigham
Young University for providing useful
inputs and comments. The opinions
expressed in this column are solely
those of the author and bear no ref lec-
tions on those of LCGC North Amer-
ica or other organizations.
Addendum
Agenda for 2013 and beyond for Per-
spectives in Modern HPLC:
2013
t New HPLC product introductions at
Pittcon 2013
t Essence of modern HPLC
t A three-pronged template approach
for rapid HPLC method development
t Myths in UHPLC
Some potential Future Topics:
t Seven common faux pas in modern
HPLC
t High-resolution UHPLC
t Analytical platform technologies
t Key equations in pharmaceutical
analysis
t HPLC in drug discovery, develop-
ment, and quality control
t Trends in modern food and environ-
mental testing by HPLC
ES262039_LCGC0613_478.pgs 05.31.2013 22:48 ADV
black
t LCMS in clinical diagnostics
t Best practice of HPLC for character-
ization of biopharmaceuticals
Your ideas and inputs are solicited
on areas deserving further investiga-
tion or discussions. Please send your
comments and suggestions to: dong.
michael_w@gene.com
References
(1) M.W. Dong, LCGC North Am. 31(4) 316
325 (2013).
(2) Market Analysis and Perspectives Report
for Analytical and Life Science Instruments
Industry (Strategic Directions Inc., Los
Angeles, California, 2012).
(3) M.W. Dong, Modern HPLC for Practicing Sci-
entists (Wiley, Hoboken, New Jersey, 2006).
(4) L.R. Snyder, J.J. Kirkland, and J. W.
Dolan, Introduction to Modern Liquid
Chromatography, 3rd ed. (John Wiley &
Sons, Hoboken, New Jersey, 2009).
(5) Handbook of Pharmaceutical Analysis by
HPLC, S. Ahuja and M.W. Dong, Eds.
(Elsevier/Academic Press, 2005).
(6) HPLC for Pharmaceutical Scientists, Y.V.
Kazakevich and R. LoBrutto, Eds. (Wiley,
Hoboken, New Jersey, 2007).
(7) C.F. Poole, Essence of Chromatography
(Elsevier Science, Amsterdam, The Neth-
erlands, 2002).
(8) Chromatography: A Science of Discov-
ery, R.L. Wixom and C.L. Gehrke, Eds.
(Wiley, Hoboken, New Jersey, 2010).
(9) UHPLC in Life Sciences, D. Guillarme, J-L
Veuthey, and R.M. Smith, Eds. (Royal
Society of Chemistry, Cambridge, United
Kingdom, 2012).
(10) M. Swartz, M. Emmanuel, A. Awad, and
D. Hartley, Advances in HPLC Systems
Technology supplement to LCGC North
Am. 27(4), 4048 (2009).
(11) Mass Spectrometry for Drug Discovery and
Drug Development, W.A. Korfmacher, Ed.
(Wiley, Hoboken, New Jersey, 2013).
(12) J.E. MacNair, K.C. Lewis, and J.W. Jor-
genson, Anal . Chem. 69, 983989 (1997).
(13) M.W. Dong, LCGC North Am. 25(7),
656666 (2007).
(14) N. Wu and A.M. Clausen, J. Sep. Sci. 30,
11671182 (2007).
(15) D. Guillarme and M.W. Dong, Amer.
Pharm. Rev., (2013) submitted.
(16) M.W. Dong, D. Guillarme, S. Fekete, R.
Rangelova, J. Richards, D. Prudhomme,
and N.P. Chet wyn, J. Chromatogr. A.
submitted.
(17) L. Sannes, Commercializing Biomarkers
in Therapeutic and Diagnostic Applica-
tions Overview, Insight Pharma Report,
May 2011.
(18) A. Tessitore, A. Gaggiano, G. Cicciarelli,
D. Verzella, D. Capece, M. Fischietti, F.
Zazzeroni, and E. Alesse, Int. J. Proteomics
2013, 115 (2013).
(19) F.E. Regnier, LCGC North Am. 30(8),
622623 (2012).
(20) M. Hollmer, 2012s Top 10 Diagnos-
tics Companies, Fierce Medical Devices,
November 27, 2012, www.f iercemedi-
caldevices.com/special-reports/top-10-di-
agnostics-companies.
(21) J.D. Carroll, The 15 best-selling drugs
of 2012, Fierce Pharma, October 9, 2012,
http://www.f iercepharma.com/speci al-
reports/15-best-selling-drugs-2012.
(22) T. Zhang, J. Zhang, D. Hewitt, B.Tran, X.
Gao, Z.J. Qiu, M. Tejada, H. Gazzano-
Santoro, and Y-H. Kao, Anal. Chem. 84,
71127123 (2012).
(23) S. Fekete, M.W. Dong, T. Zhang, and
D. Guillarme, J. Pharm. Biomed. Anal .
submitted.
please visit
Michael W. Dong
is a senior scientist in Small
Molecule Drug Discovery
at Genentech in South
San Francisco, California.
He is responsible for new
technologies, automation,
and supporting late-stage
research projects in small molecule analytical
chemistry and QC of small molecule pharma-
ceutical sciences. He holds a PhD in analytical
chemistry from the City University of New
York and a certificate in Biotechnology from
U.C. Santa Cruz. He has conducted numer-
ous courses on HPLC/UHPLC, pharmaceutical
analysis, HPLC method development, drug
development process, and drug quality fun-
damentals. He is the author of Modern HPLC
for Practicing Scientists and a co-editor of
Handbook of Pharmaceutical Analysis by
HPLC. He is a member of the editorial advi-
sory board of LCGC.
designscientific.com
(800) 572 - 6653
Faster
& More Accurate
Mobile PHase
Preparation
-plus Full-
GMP
documentation
ES262038_LCGC0613_479.pgs 05.31.2013 22:47 ADV
black
yellow magenta cyan
Laura Bush
is the editorial director of LCGC.
Direct correspondence to
lbush@advanstar.com
A Tribute to Ron Majors
From making major advances in column technology to educating several
generations of chromatographers, Ron Majors has played an invaluable
role in the field of separation science.
I
n any scientific field, it is impor-
tant to honor our heroes. In the
separation science community, we
do that in various ways. Yet we some-
times overlook the vital contribu-
tions of leading scientists at analytical
instrumentation companies, whose
achievements are often different from
those of scientists in academia. It is
that sentiment that drove the idea for
this special tribute to longtime LCGC
columnist and editorial advisory board
member Ronald E. Majors.
Majors has spent almost his entire
career in analytical instrumentation,
with 16 years at Varian Associates, 3
at EM Science (now EMD Millipore),
and 23 at what was originally Hewlett-
Packard (HP) and which later became
Agilent Technologies. In that work, he
has made critical scientific advances,
and also educated generations of chro-
matographers around the globe, by
working directly with customers and
giving scientific talks.
But for many, of course, it is for his
work beyond his day job that Majors
is best known, and so greatly admired:
his monthly columns here in LCGC,
columns that he has been writing for
more than 30 years.
At the HPLC 2013 conference in
Amsterdam this June, the editors of
LCGC and Peter Schoenmakers, the
conference chair, will present Majors
with a special award of appreciation
for all of his contributions to the sepa-
ration science community.
In thi s tribute, col leagues and
friends from around the world discuss
what Ron Majors has meant to the
f ield of separation science, and why
the scientific community and the
LCGC community in particular
appreciates him so much.
Early Achievements
Majors began making an impression
on those around him from the very
beginning of his scientific career.
Stuart Cram, vice president of
Strategic Consulting International
in Danville, California, remembers
meeting Majors when they were both
in graduate school. Cram took a trip
from the University of Illinois, where
he was studying, to Purdue University
in Indiana, where Majors was com-
pleting his doctorate under Professor
L.B. Buck Rogers.
During that visit, I could see that
Ron was bound to set a high stan-
dard in chromatography, because he
radiated an exceptional personality
and excel lence in chromatography
research, said Cram. He was clearly
dedicated to making a significant con-
tribution to the field of chromatogra-
phy, starting in the early 1960s.
After Majors completed his doctor-
ate, with a thesis on the synthesis and
characterization of very selective adsor-
bents (molecular imprinted phases) for
liquid chromatography (LC) and sam-
ple preparation, he did an initial stint
in industry, in the separations labora-
tory at Celanese, in Summit, New Jer-
sey. He then made his definitive move
into analytical instrumentation, start-
ing with Varian Associates in Walnut
Creek, California, in 1971.
In Varians research and develop-
ment division, Majors and Cram met
up again and became colleagues, sealing
ES263022_LCGC0613_480.pgs 06.03.2013 13:35 ADV
black
yellow magenta cyan
a friendship that continues today.
Cram has fond memories of that
time, when Majors worked on high
performance liquid chromatography
(HPLC) columns and chemistry and
Cram specialized in gas chromatogra-
phy (GC) instrumentation and auto-
mation. I learned more about HPLC
in those days through Ron than any-
thing I could have learned from sym-
posia and published papers, he said.
Ron distinguished himself in HPLC
column technology, was exception-
ally focused and innovative, and was
totally supportive of the manufactur-
ing department as they began making
smal l-particle HPLC columns, he
continued.
Pat Sandra, professor emeritus at
Ghent University in Belgium, remem-
bers the team of Majors and Cram
well. Once upon a time, in the 1970s,
there were two very important sepa-
ration scientists at Varian in Walnut
Creek: Stuart Cram for GC and Ron
Majors for LC, said Sandra. My boss
at that time, Professor Maurits Ver-
zele, was a consultant for Varian, and
I was often invited to join the meet-
ings. I was impressed by the technical
achievements they made.
Groundbreaking Advances
in LC Column Technology
Majors was indeed making impressive
achievements. When we asked other
separation scientists what they consider
Ron Majorss greatest contribution to the
field, his groundbreaking work in HPLC
columns was cited repeatedly.
Ron was one of the first people
to study and optimize silica particles
and bonding chemistry, said Dick
Henry, an analytical consultant who
has known Majors since they shared a
laboratory at Purdue, when Majors was
a graduate student and Henry was a
post-doc.
Sjoerd van der Wal, a former Varian col-
league who is now retired from DSM and
still a part-time professor at the University
of Amsterdam, summarized Majorss con-
tribution to column science succinctly.
He packed the first real HPLC col-
umn, he said.
Majors described t hat column
packing in a 1972 paper in Analyti-
cal Chemistry (1). It was through that
paper that Roy Eksteen, currently a
market segment manager at Sigma-
Aldrich/Supelco, f irst met Majors.
The manuscript, as Eksteen remembers
well, introduced the concept of using a
balanced density slurry solvent to pre-
vent settling of the particles during col-
umn packing. The solvent consisted of
Figure 1: Bill Hancock (left) presenting
the 1998 CASSS award to Ron Majors.
ES263025_LCGC0613_481.pgs 06.03.2013 13:35 ADV
black
yellow magenta cyan
tetrabromoethane (60.6 wt %) and tet-
rachloroethylene (39.4 wt %), a com-
position that was carefully adjusted
by observing the direction the silica
particles tended to migrate in the vial
when left undisturbed for a period of
30 minutes and adding either tetra-
bromomethane or tetrachloroethylene
to resuspend them.
The packing of that 150 mm
2.1 mm stainless steel column also
advanced another i mportant new
approach: performing column packing
at a pressure of 5000 psi (340 bar, 34
MPa). This is not too different from
what became standard practice in the
industry several years later, and is a
procedure that has not been changed
much since then, notes Eksteen.
The particles Majors packed into
that column were also very small
510 m in diameter and this was
another important advance in funda-
mental concepts of HPLC.
The standard particles at that time
were 3744 m, so the move to 10 m
was a game changer, said John Dolan
of LC Resources, himself a prominent
expert in HPLC and the longtime
author of the LC Troubleshooting
column in LCGC.
The columns Ron packed proved
t he poi nt t hat smal ler part icles
resulted in more-eff icient and faster
separations, points out Eksteen.That
was very much at the start of the pio-
neering days of HPLC.
Lloyd Snyder, now retired from LC
Resources, concurs. This 1972 publi-
cation on small-particle columns was
a major contribution to the f ield of
separation science, he said.
Ron was one of the early trend set-
ters in HPLC, a role that he continued
to play throughout the next 40 years
of his illustrious career, concluded
Eksteen.
Educating Others
Majorss contribution to separation
science is not limited to his ground-
breaking advances in column develop-
ment, however. In fact, Ron Majors
is perhaps more famous for his work
expl ai ni ng separation science to
others.
Ron has a sustained passion for
HPLC column technology, and an out-
standing record of educating others,
through hundreds of seminars, short
courses, published papers, and columns
in LCGC, said Cram. Thanks to his
exceptional contributions sharing his
knowledge and technical expertise, peo-
ple using HPLC in every country in the
world know the name of Ron Majors.
Indeed, an important aspect of
Majorss work at Agilent is providing
technical advice to staff and customers
worldwide about columns and sample
preparation. To do this, he travels
extensively around the world, working
with customers, giving seminars at cus-
tomer sites and in Agilent-sponsored
events, and presenting technical papers
at national and international meetings.
I suspect that no one has given
more talks on LC, solid-phase extrac-
tion, and sample preparation or
logged more miles around the world
than Ron, said Henry.
Maria Matyska-Pesek, an adjunct
professor at San Jose State University,
noted the popularity of those pre-
sentations. Rons lectures on HPLC
columns, techniques, and tips have
always generated large audiences, she
said.
Of course, the reason his talks are
popular is because they are so good.
Ron has a remarkable ability to teach
the complicated matters of running
HPLC columns and systems in a clear
way on a level that novices can under-
stand, said Gerard Rozing, a retired
Agilent research fellow. The practical
Figure 2: Ron Majors in the laboratory at
Agilent, in the early 2000s.
Figure 3: Majors presenting the Best Poster
Award to Kanta Horie of the Kyoto Insti-
tute of Technology of Kyoto, Japan, at the
HPLC 2006 conference in San Francisco.
Figure 4: Majors presenting the Best
Poster Award to Wolfgang Weider of the
University of Innsbruck, Austria, at the
HPLC 2007 conference in Ghent, Belgium.
The columns Ron packed [in 1972]
proved that smaller particles
resulted in more-efficient and
faster separations.
ES263021_LCGC0613_482.pgs 06.03.2013 13:34 ADV
black
yellow magenta cyan
detail that he includes is convincing
because of his broad competence.
Of course, the podium is just one
forum Majors uses for teaching. He is
renowned worldwide for his work edu-
cating separation scientists through
his regular columns in LCGC.
Majors started writing monthly for
LCGC in 1983, in the second issue,
when the magazine was called just LC.
For the first seven years, he wrote just
about columns mainly about LC
columns but also about gas chroma-
tography (GC) columns. After joining
HP (Agilent) in 1990 and starting to
work in sample preparation, Majors
launched a second column, Sample
Preparation Perspectives, in January
1991. He continues both columns to
this day, 30 years later.
As a columnist in LCGC, hes
become known worldwide as the go-to
guy for information on column tech-
nology and sample prep for HPLC,
says fellow LCGC columnist Dolan.
Ron has enormous knowledge
about LC, and about LC columns in
particular, combined with an unri-
valed ability to bring this across to
regular LC users, said Peter Schoen-
makers, a chemistry professor at the
University of Amsterdam. Ron has
raised the competence level of several
generations of LC users.
Hi s communication ski l l s are
impressive, agrees Sandra. In the
long term, I think he will receive most
credit for his LCGC column contribu-
tions on LC and sample preparation.
Majorss work educating LCGC s
readers goes beyond his monthly col-
umn. He has served as guest editor for
many LCGC special issues, on topics
such as LC column technology, sam-
ple preparation, and more recently, ion
chromatography. He has also authored
or coauthored many wall charts, and
designed numerous surveys and ana-
lyzed the results for readers.
In all his writing for LCGC, Majors
has shown an incredible ability not
just to explain the fundamentals of
chromatography columns and sample
preparation, but also to keep readers
abreast of the latest developments in
technologies and methods. He always
has his finger on the pulse of what is
new, and then explains it to readers
in practical terms, so that they know
which concepts are just exciting theo-
ries or early-stage developments and
which ones are proven approaches
they should consider applying in their
own laboratories.
At a recent meeting of the editorial
advisory board of LCGC, time after
time, after someone proposed an idea
for coverage, one of us would say Ron
just covered that, or Ron just invited
that person to write a guest column,
or Ron just brought that expert in
as a contributor to our recent special
issue on that topic. He always knows
what is going on and who can explain
it best on the odd occasion when
that person is not himself !
Majors also has applied his ability
to encapsulate new chromatography
developments to his annual Pittcon
review articles i n LCGC, which
started with his second installment
of his Column Watch column, in
May 1983. In those Pittcon reviews,
Majors not only manages to identify
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ES263024_LCGC0613_483.pgs 06.03.2013 13:35 ADV
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all the new chromatography columns
and sample preparation instruments
introduced at the conference; he also
explains the signif icance of these
developments and analyzes the trends.
This regular Pittcon article became
the standard reference describing the lat-
est developments in LC columns, said
Henry.
Awards
Given al l his accompl ishments, it
i s not surpri si ng that Majors has
received important awards over the
years. He was the recipient of the
1994 Merit Award in Chromatog-
raphy from the Chicago Discussion
Group, the 1998 Award for Distin-
gui shed Contributions i n Separa-
tion Science sponsored by CASSS
(formerly known as the California
Separations Science Societ y), and,
in 2000, the Salute to Excellence
Award from the North Jersey Chro-
matography Discussion Group. He
also received the 2006 Palmer Award
from the Minnesota Chromatography
Forum and, most recently, the Chro-
matographic Societys 2007 Martin
Gold Medal from the United King-
dom, named for the Nobel Prize winner
A.J.P. Martin.
Other Service
In addition to fostering the develop-
ment of chromatography and chro-
matographers worldwide through his
scientific developments, teaching, and
writing, Ron Majors has served the
scientific community in other ways.
One of those has been his involve-
ment with the HPLC conferences,
where he was the poster committee
chair from 1998 to 2008. Ron was
the mastermind behind the (first HP
then) Agilent poster awards at the
HPLC meetings, and I became his
right-hand man, says Schoenmakers.
We worked closely together for quite
a few years. Ron was incredibly com-
mitted to the task, and he did a fan-
tastic job. Majors was also the chair
of HPLC 1986, in San Francisco, Cal-
ifornia, an event that still ranks as the
largest chromatography-only sympo-
sium ever held in terms of attendance.
Majors also has been highly involved
in planning the annual spring sympo-
sium of the Chromatography Forum of
the Delaware Valley and continues to
serve on the Executive Committee. He
was the program chair in 20072008
and president in 20082009. He also has
been a member of the Associate Board of
Directors of CASSS since 2011.
He also has served on the editorial
advisory board of LCGC since its very
first issue in March 1983. To this day,
he continues to provide invaluable and
constant support that goes far beyond
conducting peer reviews, by regularly
helping the editors with numerous
and wide-ranging questions. And his
answers are always clear, concise, and
timely, no matter what part of the
world he sends them from.
Pat Sandra put it succinctly. Rons
self less and endless commitment to
the chromatographic community is
unique and highly appreciated.
A Good Friend and Colleague
So many scientists in the field of sepa-
ration science have warm recollections
of Ron Majors, from hearing him talk,
working with him in industry, collabo-
rating on articles for LCGC, and more.
One of my fondest memories of
working with Ron was traveling with
him to interesting places in the world
to present HPLC and GC technology
seminars, said Cram. He was truly a
scholar and highly respected by cus-
tomers wherever we went. His com-
munications style exemplif ied excel-
lence in HPLC technology, and his
presentations will never be forgotten.
Van der Wal recalls the kindness
Majors showed him when van der
Wal first came to Varian in 1982 for
an interview, arriving on a f light that
had been delayed several hours. His
host, thinking van der Wal was not
going to arrive that evening, had left
the meeting area. As I had no other
contacts in California that I could
reach at that time, I called Ron in
the middle of the night, says van der
Wal. Although I hardly knew him, he
helped me out and made sure my visit
was arranged well. Van der Wal did
swear, however, that he would avoid
such post-midnight calls in the future.
I have kept that promise to this day!
he declares.
Mat yska-Pesek reminisces about
col laborating with Majors on the
HPLC poster committee, and par-
ticularly about how he facilitated the
work. He was very well organized
and made the process fair and seam-
less, she said. Sometimes during the
poster selection process we encoun-
tered opposite opinions about certain
posters from two well known indi-
viduals. Ron was always able to come
to the perfect solution, which was not
easy in such situations.
Dolan shares a long history with
Majors of writing columns for LCGC,
and points out that Ron is the longest-
active person in the LCGC publication,
having started writing his column in
the second issue. (Dolan started a few
Figure 5: Majors speaking at 2011 Desty
Memorial Lecture at the Royal Institu-
tion of Great Britain.
Ron has enormous knowledge
about LC combined with an
unrivaled ability to bring this
across to regular LC users.
ES263023_LCGC0613_484.pgs 06.03.2013 13:35 ADV
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months later, in October 1983). I dont
believe there is anyone in the LCGC
or Advanstar organization who is still
around from those days, he says. We
joke that between us we break in all the
new LCGC editors. Hes been around
for all of them, and I have been around
for all but one.
Dick Henry shared a funny story,
from an event many years ago where
Majors was the keynote speaker. It
was early in the days of converting
to PowerPoint with laptop comput-
ers and data projectors, and Ron was
ahead of the game trying to use his
laptop to project slides, said Henry.
A few slides into Rons talk, we all
were treated to the Blue Screen of
Death which meant that his planned
keynote talk was over. Henry was
also on the program and recalls giving
his talk while Majors quickly left and
brought back a briefcase full of talks
in older slide and transparency for-
mat. He recovered quite well and was
able to present the keynote talk that
we all expected, says Henry. Lots
of things like that seem to happen to
Ron, but he always lands on his feet.
A Passion for Life
Ron Majorss energy and commitment
for his work is equally on display in his
passion for life. He and his wife are avid
birders and travel around the world to
participate in their hobby, including to
remote regions. Majorss life list the
tally that birding aficionados keep of all
the species they have ever spotted con-
tains an incredible 3500 entries. He uses
those birding trips to observe other forms
of wildlife, too. During recent treks to
Uganda and mountainous regions of
northern India he has seen, and taken
incredible photographs of, silverback
gorillas and tigers, among others.
Knowing his busy work schedule and
his vigorous wildlife trips, it is hard to
remember and in fact, most people
dont realize that Majors is over 70.
Few can imagine keeping up with him.
And many dont know that Majors
f inds the time and energy to keep
up with another signif icant hobby:
stamp collecting. He is an expert in
Canadian postal history and a mem-
ber of the American Philatelic Society.
He currently serves on the Board of
Directors of the British North Ameri-
can Philatelic Society and is president-
elect for 2014.
He also manages to f ind time to
help out in his local community. He
served as president of a local civic
safety group, Pennsbury Townwatch
in Pennsbury Township, Pennsylva-
nia, from 2001 to 2004 and as the
organizations publicity chair from
2004 to 2009.
Still Humble and Hardworking
Despite all of his accomplishments and
his worldwide fame, Majors remains
as humble, helpful, and hardworking
as ever.
Mary Ellen McNally, a technical
fellow at DuPont Crop Protection in
Newark, Delaware, remembers hearing
about Majors long before meeting him,
because he was one of the first PhD
students of L.B. Rogers, who was her
post-doctoral research advisor. Ron
was somewhat of an enigma before I
met him, like a rock star of chroma-
tography, she said. But over the years
I have gotten to know him, and he is
just a super nice man. In recent years,
McNally has enjoyed working closely
with Majors on the spring symposium
program for the Chromatography
Forum of the Delaware Valley.
Ron is one of the most accessible
scientists I have ever met, echoes van
der Wal.
Dennis Blevins, a colleague at Agi-
lent, agrees. Ron is always willing to
talk with all colleagues and continues to
support the separation sciences, helping
educate the next generation, he said.
Ron has always been a friend.
I am sendi ng Ron my warm-
est congratulations on his sustained
quest for excellence, HPLC technol-
ogy, and passion for supporting and
educating the HPLC worldwide com-
munity, said Cram. His outstand-
ing career, through his attributes and
contributions, has been accomplished
through his passion for helping others
throughout the world, and has made
him a cornerstone of HPLC technolo-
gies for more than four decades.
References
(1) R.E. Majors, Anal . Chem. 44, 1722
1726 (1972).
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ES263020_LCGC0613_485.pgs 06.03.2013 13:34 ADV

black
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3 m
0 m
Stefan Bruns, Alexandra
Hltzel, and Ulrich Tallarek
Department of Chemistry, Philipps-
Universitt Marburg, Marburg, Germany
Direct correspondence to:
tallarek@staff.uni-marburg.de
Morphological Comparison of Silica-Based
Monolithic and Particulate Beds by Confocal
Laser Scanning Microscopy
Confocal laser scanning microscopy enables the nondestructive, three-
dimensional (3D) imaging of silica-based columns. The key morphological
features responsible for the kinetic column performance can then be
identified directly from the physically reconstructed bed structure,
independent of size and form of the underlying structural element. We
demonstrate this by comparing the bed morphology of a silica monolith
with that of a sub-2-m packing in capillary column format (20 m i.d.).
Our analysis reveals the main structural problem of the silica monolith
and shows the direction along which its optimization should progress
to reach the kinetic efficiency of a sub-2-m packing. The morphological
monitoring enabled by this approach offers constructive guidance to any
optimization efforts in the preparation of silica-based monolithic and
particulate columns.
P
orous silica in the form of par-
ticulate or monolithic beds is the
most popular stationary phase
support for small molecule separations
(1). Monolithic columns have overcome
the limitations of particulate beds with
respect to permeability and mass trans-
fer resistance, but often show disap-
pointing separation efficiencies (2);
presently, even the improved second
generation of silica monoliths (3,4) can-
not compete with well-packed particu-
late beds (57). A similar situation in
which a theoretical concept has not yet
brought the expected outcome in prac-
tice is the size reduction of the struc-
tural elements of a bed. Smaller particle
and domain sizes do not automatically
guarantee better columns, only higher
back pressure (8). Shrinking the struc-
tural elements yields more efficient col-
umns only when the homogeneity of
the bed can be conserved in the process
(911). The bed morphology, specifi-
cally the distribution of the interstitial
void space where eddy dispersion takes
place, largely determines the kinetic
column performance (1214). The het-
erogeneity of a monolithic or particu-
late bed contributes to eddy dispersion
on different length scales (1517): the
size of the individual channels (f low-
through pores) between two neigh-
boring solid elements (transchannel
contribution) (18,19), a size equivalent
to 12 particle diameters or domains
(short-range interchannel contribution)
(18,19), and the column radius (trans-
column contribution) (4,7,2023).
With particulate beds, the size of
each eddy dispersion contribution can
be determined from chromatographic
data and linked to the respective mor-
phological features (14,23,24). The
method cannot be readily transferred
to monolithic beds because the theory
of eddy dispersion was first developed
for discrete spheres as the underlying
structural elements (15). This does
not mean that the origins of eddy
dispersion are fundamentally differ-
ent in monolithic beds, only that the
comparison of particulate and mono-
lithic beds by their column efficiency
ES262012_LCGC0613_486.pgs 05.31.2013 22:37 ADV
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ES257414_LCGC0613_487_FP.pgs 05.29.2013 02:12 ADV
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and hydraulic permeability cannot
get at the morphological roots of the
obser ved chromatographic output
(2528). The same applies to com-
parative studies of silica-based mono-
lithic columns with different domain
sizes, for example, in efforts to reduce
the macropore size (2931). For par-
ticulate beds, the particle size is used
as a normalization parameter when
comparing the chromatographically
determined efficiencies of individual
columns. This normalization works
because the size of the particles and
that of the interstitial f low-through
pores are in a f ixed relationship, so
that when the size of the particles is
reduced, the size of the f low-through
pores also decreases. But the corre-
sponding elements of a monolithic
bed, skeleton thickness and macropore
size, can be altered independently (2);
therefore, the domain size, although
often treated as the analog of the par-
ticle size, is not a suitable normaliza-
tion parameter for monolithic beds.
Yet, the morphology of particulate
and monolithic beds can be analyzed
through three-dimensional (3D) imag-
ing methods: With local resolution,
image analysis delivers an accurate
characterization of the interstitial void
space from individual pores up to the
column cross-section. In our group,
we have used confocal laser scanning
microscopy (CLSM) for silica-based
(hard) materials (4,7,3235) and serial
block-face scanning electron micros-
copy for polymer-based (soft) materials
(36). Using CLSM, we have found the
morphological cause behind the vary-
ing quality in a series of silica mono-
liths (34), investigated the inf luence
of the capillary internal diameter on
the packing morphology (7), and com-
pared coreshell and fully porous par-
ticles with respect to the wall effects in
packed capillaries (35). Excepting one
study (4), our CLSM investigations so
far have been conducted with capillary
columns because this format enables
direct access to the bed morphology;
that is, it is not necessary to extrude
the bed from its conduit and cut the
extruded bed into smaller samples.
Also, a smaller column internal diam-
eter allows reconstruction of longer bed
segments and the amount of CLSM
data involved is still manageable.
In this article, we demonstrate how
the morphologies of chromatographic
beds can be directly and quantitatively
compared by image analysis, indepen-
dent of size and form of the underlying
structural element of the bed. As exam-
ples we selected two capillary columns
of identical internal diameter (20 m):
a silica monolith and a packing of sub-
2-m particles.
Experimental
The investigated fused-silica capil-
lary columns (20 cm 20 m), a tet-
ramethoxysilane (TMOS) monolith
prepared as described in the literature
(30), and a packing of C18-modified,
1.7 m Acquity BEH particles (Waters
Corporation) prepared as shown in
the literature (7) were kindly provided
by Takeshi Hara and Dr. Bernd M.
Smarsly (monolith) of Justus-Liebig-
Universitt, Giessen, Germany, and
(a) (c)
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
(b)
Diameter of inscribed sphere (m)
Sub-2-m packing
Sub-2-m packing
Silica monolith
Silica monolith
Mode
Mean
Standard deviation
1.91 m 0.75 m
0.74 m
0.31 m
1.75 m
0.68 m
P
r
o
b
a
b
i
l
i
t
y

d
e
n
s
i
t
y

(
%
)
3 m
0 m
1.5 m
0 m
Figure 1: Physical reconstruction of the bed morphology of (a) a silica monolith and
(b) a packing of sub-2-m particles by confocal laser scanning microscopy. Segments
(65 m long) of each capillary column (20 m i.d.) were reconstructed from image
stacks of consecutive optical slices acquired parallel to the column axis.
Figure 2: Void space characterization by inscribed spheres. Colors indicate the diam-
eters of the largest spheres that can be ftted into the interstitial void space of (a)
the reconstructed monolithic and (b) particulate bed without intersecting the solid
phase (gray). (c) The probability density distribution of the inscribed-sphere diam-
eters (shown with the corresponding statistical parameters) can be interpreted as a
lower limit of the pore size distribution.
ES262010_LCGC0613_488.pgs 05.31.2013 22:37 ADV
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James P. Grinias, Laura E. Blue, and Dr.
James W. Jorgenson (packing) of the
University of North Carolina at Chapel
Hill. All elements of the experimental
approach have been described in detail
elsewhere (7,3234), so only the salient
points are repeated here. For image con-
trast, the particulate bed was stained
with Bodipy 493/503 (Life Technolo-
gies), a dye that physisorbs to the C18
chains, whereas the bare-silica skeleton
of the monolith was chemically linked
to the dye V450 (32). A 65-m-long
segment from the medium section of
each column was reconstructed from a
set of consecutive optical slices (image
stack, resolution: 30 30 120 nm,
Figure 1) recorded on a TCS SP5 con-
focal microscopy system equipped with
a HCX PL APO 63/1.3 GLYC CORR
CS (21) glycerol immersion lens (Leica
Microsystems). The refractive index
mismatch between lens and sample
was minimized through f lushing and
embedding the investigated column
with (as well as immersing the lens in) a
70:19:11 (v/v/v) liquid mixture of glyc-
erolDMSOwater, whose refractive
index mimics the optical dispersion of
fused silica. The remaining small mis-
match was eliminated by a judiciously
chosen cover slip (type 0, 110 nm
thickness). Contrast, signal-to-noise
ratio, and resolution in the acquired
images were improved by image resto-
ration. The gray-scale images were then
converted to binary data (in which each
pixel belongs to either solid or void
space) through segmentation. Images
of the monolithic column were bina-
rized by high-pass filtering; images of
the particulate column were binarized
by detecting the center of a particle and
fitting a sphere with the appropriate
diameter around it.
Results and Discussion
Figure 1 schematically shows how the
bed morphologies of the two capillary
columns were physically reconstructed
by CLSM. Images were acquired from
parallel planes along the column axis.
The resulting image stack represents a
set of consecutive optical slices through
a column, from wall to wall. The recon-
structed segments cover 65 m of each
column. With the reconstructed mor-
phologies available, our analysis of the
two bed structures progressed from the
pore scale to the column scale.
Pore-Scale Properties
The interstitial void space in a particu-
late or monolithic bed is a network of
f low-through pores whose size deter-
mines the amount of transchannel dis-
persion in the column. Unfortunately,
the concept of linking an eddy disper-
sion term to the scale of individual
f low-through pores is easier to under-
stand than it is to obtain an accurate
pore size distribution (PSD). The stan-
dard experimental method, mercury
intrusion porosimetry (MIP), assumes
a network of cylindrical pores; in real-
ity, the f low-through pores in a col-
umn do not have a constant diameter,
but often widen behind a constricted
entrance (the pore neck). That MIP
measures the diameter of the pore neck
rather than the size of the actual pore
is known as the ink-bottle effect (37).
Considering this drawback, an MIP-
determined PSD should be interpreted
with caution. With the reconstructed
morphology of a packing (Figure 1), an
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ES262011_LCGC0613_489.pgs 05.31.2013 22:38 ADV
black
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accurate image of the interstitial void
space is available, but that still poses
the question: How do you define a
pore within an open pore network or
how do you arrive at the PSD from the
reconstructed morphology? One possi-
bility is the inscription of spheres into
the void space (38,39). Figure 2 shows
the results of this approach applied to
the reconstructed beds. Each void voxel
was assigned the diameter of the larg-
est sphere that could be inscribed at
this location without intersecting with
a solid voxel. Figures 2a and b show a
monolithic and particulate bed, respec-
tively, with the void space colored
according to the diameters of the locally
inscribed spheres. Figure 2c quantifies
the results with the probability density
distribution of the inscribed-sphere
diameters for each voxel location in the
void space. As expected, the inscribed-
sphere diameters in the monolithic bed
are, on average, more than twice the
size of those in the particulate bed. The
sub-2-m packing shows a rather nar-
row and symmetrical distribution of
inscribed-sphere diameters, whereas the
monolith has a very broad, negatively
skewed distribution. The latter ref lects
that f luctuations in skeleton thickness
lead to an irregular solidvoid interface
whose description requires a larger num-
ber of smaller spheres. Although the
inscribed-spheres approach has visual
appeal and targets pores as opposed
to pore necks like MIP, the results
are inaccurate. Inscribing spheres into
the void space probes the adaptation
of the pore space to a fixed, idealized
geometry more than the dimensions
of the f low-through pores themselves,
which makes the resulting PSD only a
lower limit of the range in which the
true PSD lies. An accurate, though less
immediately comprehensible, method
to describe the interstitial void space
distribution in a porous medium was
introduced by Courtois and colleagues
(40) for chromatographic beds. Here,
the particle-to-particle (or skeleton-to-
skeleton) distances in the bed are mea-
sured by chords that are extended in
two opposite directions from random
positions in the interstitial void space
(Figure 3a). Chords are generated and
their lengths collected until the result-
ing histogram, known as a chord length
(a)
(b)
Sub-2-m packing
Silica monolith
0.5
0.4
0.3
0.2
0.1
0.0
0
Chord length (m)
Silica monolith
Sub-2-m packing
Mode 1.97 m
3.60 m
0.91 m
1.78 m
1.94 2.22 k
2 4 6 8 10 12
P
r
o
b
a
b
i
l
i
t
y

d
e
n
s
i
t
y

(
%
)
C
a
p
i
l
l
a
r
y

w
a
l
l
Solid
void
P1
P2
P3
Figure 3: Quantitative void space characterization by chord length distributions.
(a) From random points P1 to Pn in the interstitial void space of the reconstructed
monolithic bed, the linear skeleton-to-skeleton distance (for the particulate bed,
the particle-to-particle distance) is determined in 16 equiangular directions (green
lines); chords that reach beyond the image boundary are rejected (red dashed lines).
(b) k-gamma ft to a distribution of 10
6
chords and the corresponding statistical pa-
rameters that measure the pore-scale properties (average pore size, transchannel,
and short-range interchannel heterogeneity) of the reconstructed monolithic and
particulate bed.
ES262014_LCGC0613_490.pgs 05.31.2013 22:38 ADV
black
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distribution (CLD), remains constant.
The CLDs of the reconstructed beds
(Figure 3b) were established from 10
6

chords each and are statistically over-
determined. The one-dimensional (1D)
chords meticulously scan the complex
geometry of the solidvoid border in a
porous medium, so that the CLD fully
captures the morphology of the recon-
structed interstitial void space. This
accuracy is one essential advantage of
the CLD method over the inscribed-
spheres approach (and MIP). The other
important advantage is that chords may
occasionally reach into the next f low-
through pore; such longer chords then
contain information about the pore
vicinity.
The interpretation of the received
CLDs in terms of eddy dispersion con-
tributions is helped by the circumstance
that the CLDs follow a k-gamma func-
tion. The k-gamma function (41) was
delineated as a descriptor of the void
space distribution in computer-gener-
ated, coagulated colloids of monosized
spheres (42). With the first physical
reconstructions completed, we discov-
ered that the k-gamma function was
also a good fit for the CLDs of the
interstitial void space of experimen-
tal particulate and monolithic beds
(4,3335,43). The k-gamma function is
defined by the mean and the standard
deviation of the CLD:
f (l
chord
)=
exp -k
( )
k
k
(k)
l
k-1
chord
k
l
chord
[1]
where l
chord
is the chord length, is the
gamma-function, denotes the statisti-
cal mean of the distribution, and k =
(/) relates the mean to the stan-
dard deviation of the distribution.
Alternatively, or the mode of the
CLD (which is reasonably close to the
mode of the inscribed-spheres deter-
mined PSD, Figure 2c) can be used
to characterize the size of the f low-
through pores. In the monolithic bed,
the f low-though pores ( = 3.60 m,
mode = 1.97 m) are about twice the
size than those in the particulate bed
( = 1.78 m, mode = 0.91 m). The
size of the transchannel contribution to
eddy dispersion scales with the -value
of the CLD (34). From the compari-
son of the received -values, we can
predict that transchannel dispersion in
the monolith is larger than in the sub-
2-m packing; in fact, it is comparable
to the transchannel dispersion in a ~4
m packing.
The value of k is dominated by the
longer chords that make up the tail of
the CLD and contain information on
the local pore environment. Larger k-values
represent a narrower distribution relative
(a)
(b)
0.9
0.8
0.7
0.6
Silica monolith
Silica monolith
bulk
= 0.70

(
r
)

(
r
)

-

b
u
l
k
IPD = -0.08
bulk
= 0.36
IPD = 0.28
Sub-2-m packing
Sub-2-m packing
Distance from column wall, r (m)
Distance from column wall, r (m)
Bulk 0.5
0.4
0.3
0.2
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2 4 6 8 10
-0.1
-0.2
0 2 4 6 8 10
Figure 4: (a) Characterization of transcolumn heterogeneity by radial porosity pro-
fles. The bulk porosity (
bulk
) of the reconstructed capillary segments was estimated
from the interstitial porosity in the core region (r = 610 m), in which the station-
ary phase material is randomly distributed. (b) Subtraction of
bulk
from the porosity
profles eliminated the large difference in average interstitial porosity between the
monolithic and the particulate bed. The scalar resulting from integration over the
area covered by these curves is the integral porosity deviation (IPD, equation 2). The
IPD quantifes the local porosity variation over the column cross-section relative to
the bulk porosity.
ES262016_LCGC0613_491.pgs 05.31.2013 22:38 ADV
black
yellow magenta cyan
to ; that is, a higher bed homogene-
ity on the length scale of 12 particle
diameters or domains (4,3335,43).
The monolith (k = 2.22) is more homo-
geneous on the short-range interchan-
nel scale than the sub-2-m packing
(k = 1.94), which would translate to a
smaller contribution to eddy dispersion
in the monolithic column. This find-
ing agrees with our previous investi-
gations: Among the capillary columns
we have reconstructed by CLSM so
far the particulate beds have fallen
into the narrow range of k = 1.92.0
(33,35), whereas the silica monoliths
have shown a wider range of k = 1.92.5
(34,43). The maximum observed value
(k = 2.9) was obtained for an analytical
TMOS monolith column of the second
generation (4). Although our sample
range is too small at present to predict
the k-value range of monolithic beds,
it is fairly reasonable to expect a nar-
row k-value range for well-packed par-
ticulate beds. These columns seem to
possess a similar amount of order in the
core region because the constraints of
a dense packing near the random-close
packing limit (44) leave little possibil-
ity for structural variation on the short-
range interchannel length scale (18,35).
Column-Scale Properties
Radial heterogeneities in the interstitial
void space distribution on the scale of the
column diameter are a serious threat to
the separation efficiency of any column,
but are particularly critical in capillary
chromatography. Porosity biases in a col-
umn translate directly into permeability
and velocity biases (21,22). The extent to
which such velocity extremes are experi-
enced by an analyte and, thus, become
apparent in the chromatographic output
depends on the ratio of column internal
diameter to column length (23). Ana-
lytical columns are too short for their
internal diameter to complete equilibra-
tion of the analyte over the column cross-
section; thus, when the analyte arrives
at the column outlet, it has not felt all
transcolumn velocity biases existing in
the bed. Consequently, the efficiency
of analytical columns suffers less from
existing radial heterogeneities than that
of capillary columns, whose kinetic per-
formance discloses every heterogeneity in
the packing (7,16,19,22,45).
We f irst calculated the external
porosity,
ext
, of the reconstructed cap-
illary segments from the amount of void
voxels divided by the total amount of
voxels. The results of
ext
= 0.69 for the
monolith and of
ext
= 0.42 for the sub-
2-m packing are average values, such
as could also be obtained by inverse size
exclusion chromatography or Donnan
exclusion (46), but say nothing about
the void space heterogeneity. Figure 4a
shows the radial porosity profiles (r),
the local porosity as a function of
the distance r from the column wall
(r = 0), obtained for both beds. The
radial porosity profile of the monolith
is relatively f lat compared with that of
the sub-2-m packing, which shows the
typical damped oscillations profile of
particulate beds (7,22,35). Because of
the huge difference in external poros-
ity,
ext
, the porosity profiles of the
two beds cannot be directly compared.
Instead, the local porosity variation
with respect to the average porosity in
the bulk (core) region of each bed is
compared. The bulk region of each bed
(r = 610 m) is indicated in Figure 4a.
In the monolith, bulk (
bulk
= 0.70) and
external porosity (
ext
= 0.69) are very
close in value. In the sub-2-m pack-
ing, the bulk porosity is
bulk
= 0.36,
while the local porosity at the column
wall is = 1. To quantify the local
porosity deviation with respect to the
bulk porosity, we subtracted
bulk
from
the porosity profiles and integrated the
remaining deviation over the column
radius (Figure 4b). The resulting scalar
is the integral porosity deviation (IPD)
(7,35):
( (r)
bulk
)dr
0.5d
0
IPD =
c

[2]
where r is the distance from the col-
umn wall and d
c
is the column internal
diameter. The small IPD value of the
monolithic bed (IPD = 0.08) ref lects
the f lat porosity profile and the nega-
tive sign indicates a lower porosity
at the column wall than in the bulk.
The ~1-m-thick low-porosity region
originates from the wetting layer of
monolithic material at the capillary
wall and is followed first by a region
of higher porosity and then by another
low-porosity region (r = 2.24.6 m).
This often-observed morphological
feature of capillary silica monoliths
(34) is formed during the drying stage,
when the bed shrinks and locally dis-
connects from the material layer at the
wall. The high-porosity region ref lects
where the bed is stretched thin, and the
low-porosity region ref lects where the
material has accumulated. Whereas the
porosity profile of the monolith ref lects
its preparation history, the profile of
the sub-2-m packing and its IPD
value (IPD = 0.28) ref lect the funda-
mental limitations on the packing of
hard spheres against a locally f lat, hard
column wall. For the first 35 m from
the capillary wall, the bed has a much
higher porosity and also higher order
than in the randomly packed bulk (7).
The absolute IPD value of the mono-
lith is 3.5 times lower than that of the
sub-2-m packing. The high radial
homogeneity of the monolithic bed
promises low transcolumn dispersion
and constitutes a substantial structural
advantage of the monolithic over the
particulate format. Unfortunately, col-
umns prepared in the past have often
disappointed in this regard (2), which
has led to reservations against silica
monoliths in general.
Conclusions
We compared the bed morphologies of
two 20 m i.d. capillary columns with
different silica-based support struc-
tures following their physical recon-
struction by CLSM. According to our
analysis, the monolith is more homo-
geneous than the sub-2-m packing
on the short-range interchannel and
on the transcolumn scale, but loses
significantly on the transchannel scale,
with f low-through pores twice the size
of those in the sub-2-m packing. If
the macropore size could be reduced
while maintaining the present level of
short-range interchannel and transcol-
umn homogeneity, the monolith would
become superior to the sub-2-m pack-
ing. The physical reconstruction of the
bed morphology by CLSM allows users
to monitor the morphological conse-
quences of slurry packing and mono-
lith preparation on scales of all lengths
relevant to eddy dispersion: A CLD of
the reconstructed interstitial void space
delivers the parameters and k, which
indicate the average pore size and the
ES262015_LCGC0613_492.pgs 05.31.2013 22:38 ADV
black
heterogeneity in the direct vicinity of
a pore, respectively, while radial poros-
ity profiles track changes in transcol-
umn heterogeneity. Decreasing while
conserving k and the IPD value should
be the goal of monolith preparation.
Reduction of the transcolumn hetero-
geneity as indicated by a drop of the
IPD value would have the strongest
impact on the kinetic performance
of slurry-packed particulate columns.
Beyond all questions, image analysis
provides pivotal insight into the mor-
phological foundations of column per-
formance. Besides guiding academic
and industrial researchers in the prepa-
ration of better high performance liq-
uid chromatography (HPLC) columns,
the CLSM-based physical reconstruc-
tion of chromatographic beds delivers
realistic models for f low and trans-
port simulations to derive accurate
mass transfer relationships for HPLC
(19,47,48).
Acknowledgment
This work was supported by the
Deutsche Forschungsgemei nschaf t
DFG (Bonn, Germany) under grants
TA 268/5 and TA 268/6. The authors
thank Takeshi Hara and Dr. Bernd M.
Smarsly of Justus-Liebig-Universitt,
Giessen, Germany, and James P. Gri-
nias, Laura E. Blue, and Dr. James W.
Jorgenson of the University of North
Carolina at Chapel Hill for generously
providing the studied capillary columns.
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de
ES262013_LCGC0613_493.pgs 05.31.2013 22:38 ADV
black
HPLC Analysis of Very Polar Compounds in
Bioanalysis
Several high performance liquid chromatography (HPLC) approaches can be used in the analysis of polar
small molecules that are classified as pharmaceuticals, metabolites, or biomarkers. Many of these polar
compounds can be challenging for traditional reversed-phase separation methods. One approach for
analyzing these types of samples is the use of columns having polar retention capabilities such as ion-
exchange, hydrophilic interaction liquid chromatography (HILIC), polar-embedded, or polar-endcapped
columns. Another approach is aqueous normal phase (ANP) chromatography. This mode has the advantage
of having both reversed-phase and normal-phase retention. In a recent web seminar, Joseph Pesek, a
professor of Chemistry at San Jos State University (San Jos, California), explained the mechanisms,
advantages, and disadvantages of these approaches to the analysis of polar compounds. Below, he
answers questions raised during the web seminar. A recording of the web seminar is available for free at:
www.chromatographyonline.com/HPLCPolarCompounds
Is silica really a HILIC stationary phase?
Several studies suggest that it is very differ-
ent from bonded phases.
Pesek: It is if you believe that the mecha-
nism for HILIC retention is the presence
of an adsorbed water layer on the surface
that serves as a medium of analyte parti-
tion. It may be different from bonded
phases but for most of these, the presence
of a layer of water at or near the surface is
used to describe retention.
What is your experience with the robustness
of most HILIC columns?
Pesek: Most HILIC columns are less
robust than traditional reversed-phase col-
umns. This results in part from the fact
that the surface can be easily contaminated
and is hard to clean. In other cases, the
bonded group is not very robust and is sus-
ceptible to being cleaved from the surface
in mobile phases used for HILIC retention.
Are HILIC or similar columns compatible
with ultrahigh-pressure liquid chromatogra-
phy (UHPLC)?
Pesek: All of the columns discussed in the
seminar, including aqueous normal phase
(ANP) and HILIC columns, are compat-
ible with UHPLC.
Can I use silica hydride columns for mixed
analysis of polar and nonpolar compounds or
amphiphilic compounds? If so, is there a pos-
sibility that the retention times of polar and
nonpolar compounds might be the same (that
is, their respective affinities are equal), thus
yielding the same retention times?
Pesek: This is relatively unlikely because
that would require that both mechanisms
be operating at equal efficiency for those
particular compounds. In most cases one
mechanism would be more predominant
than the other and retention would be dif-
ferent. However, even in the unlikely event
that both were equal, that would only
occur at one mobile-phase composition.
Therefore, you could switch to a different
isocratic composition or a different gradi-
ent and that would shift the relative contri-
butions of the two mechanisms, allowing
separation.
Why are there different bonded phases using
silica hydride if it is possible to retain both
polar and nonpolar analytes on any silica
hydride column?
Pesek: The degree of retention in reversed-
phase and aqueous normal phase modes
on silica hydride columns depends on the
degree and type of modification. For col-
umns with no or minimal modification the
ANP mode is stronger than the reversed-
phase mode. As the degree of modification
becomes greater that is, in columns
modified with larger groups or that have
Column Types
and Their Properties
If hydrophilic interaction liquid chromatog-
raphy (HILIC) works now, why did it not
work 30 years ago?
Pesek: HILIC was not well understood 30
years ago. However, some of the shortcom-
ings of HILIC were noted at that time and
for that reason it was not pursued vigorously.
How is HILIC different from working with
plain silica columns?
Pesek: Silica is just one column material
used in HILIC. There are many others,
each having somewhat different proper-
ties. Therefore, as in reversed-phase chro-
matography, in HILIC it is important to
select the right type of column to match
the properties of the analyte.
What is the difference between polar-end-
capped and HILIC columns?
Pesek: Polar-endcapped columns are
similar to traditional reversed-phase
columns but instead of using a nonpo-
lar endcapping reagent like a trimethyl
group, a more polar group is used to cover
some of the remaining silanols. HILIC
columns generally are significantly more
polar since they are based on bare silica
or have bonded polar groups that cover a
significant fraction of all of the available
bonding sites.
ES262111_LCGC0613_494.pgs 05.31.2013 23:33 ADV
black
yellow magenta cyan
a greater degree of surface coverage the
reversed-phase properties increase.
What type of columns can be used to separate
both polar and nonpolar compounds?
Pesek: The most versatile columns for
this type of separation are silica hydride
columns because the amount of reten-
tion in both modes can be adjusted by
the type of modification and the mobile-
phase composition. Polar-endcapped and
polar-embedded columns have this same
capability because they are also classified
as mixed-mode stationary phases. The
degree of polar retention depends on the
endcapping group or the embedded group.
What type of gradient do you suggest for
ANP analysis of hydrophilic and hydropho-
bic molecules in the same run? I have a large
hydrophobic active and a prodrug, as well as
five small polar metabolites.
Pesek: The usefulness of ANP separations
is that you have two options in selecting
the gradient. It would be best to try both
a reversed-phase gradient (from 80% to
20% aqueous) and then an ANP gradient
(from 20% to 80% aqueous) as an initial
screening process. From the preliminary
results you can determine which mode is
most likely to produce the best separation.
Once the direction has been selected, then
the gradient can be modified to reach the
desired retention and separation of the
components in your mixture.
How much efficiency do ANP columns
have? Does efficiency have detrimental
effects for biological matrices for liquid
chromatographymass spectrometry (LC
MS) analysis?
Pesek: Normal-phase retention gener-
ally has lower efficiency than reversed-
phased methods. It is highly dependent
on the compound being retained. Effi-
ciency doesnt have detrimental effects
for biological matrices for LCMS anal-
ysis, but sometimes lower ionization of
the compounds is observed in MS detec-
tor when compounds are being analyzed
in biological matrices. The use of inter-
nal standards (usually deuterated com-
pounds) will help to assess how much
lower the peaks are.
Are there other bonded phases for ANP
chromatography?
Pesek: Yes. As stated in the seminar, polar-
embedded, polar-endcapped, and fluori-
nated phases have some ANP capabilities.
Also, graphitized carbon phases have dem-
onstrated some ANP behavior.
Can you tell us about carbon columns?
Pesek: Carbon columns, or porous
graphitized carbon columns, also possess
dual retention capabilities. Their struc-
ture is porous particles composed of flat
sheets of hexagonal carbon. Selectivity
in the reversed-phase mode is somewhat
different than with traditional C18 col-
umns. The absorptive nature of the car-
bon surface results in the polar retention
observed.
What manufacturers make low-carbon silica
hybride columns?
Pesek: The current suppliers of low-car-
bon silica hydride carbons are Microsolv
Technology and VWR in the United
States and Hichrom Ltd. in Europe and
the UK.
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ES263008_LCGC0613_495.pgs 06.03.2013 13:09 ADV
black
yellow magenta cyan
How stable or durable are silica hydride
columns?
Pesek: Silica hydride columns are just as
durable, and in some cases more durable,
than traditional chemically modified
stationary phases based on organosilane
chemistry. This is due to the direct sili-
con-carbon at the surface. For columns
that are used primarily for polar reten-
tion in the ANP mode, these materials
are more durable than typical HILIC col-
umns because the hydride surface is not
as easily contaminated as ordinary silica
and the surface is less susceptible to attack
by aggressive mobile phases because of its
more hydrophobic nature.
Can you compare silica hydride and ionic
liquids? Are there any important advan-
tages of one over the other for retaining
polar analytes?
Pesek: It is assumed you are referring to
ionic liquid stationary phases. All of these
materials contain a charged group that is
part of the bonded material. The charged
site is usually within the bonded chain, but
not always. It is most similar to the polar-
embedded phases discussed in the presen-
tation. They have been shown to retain
both polar and nonpolar compounds. To
date, the range of compounds analyzed
with ionic liquid stationary phases is not
as broad as with silica hydride phases so
the same versatility has not been demon-
strated. A good review of ionic liquids in
chromatography can be found in the book
by Mun and Sim (1).
General
Why are polar compounds retained more
strongly when they are ionized and not
when they are neutral, as in reversed-phase
chromatography?
Pesek: This occurs because the highest
polarity is generally found in ionized com-
pounds, as a result of the fixed charge on
the compound. Therefore, in both aque-
ous normal phase chromatography and
HILIC, maximum retention is attained
for compounds with the highest polarity,
in other words, those that are ionized.
Have you had any issues with ion suppres-
sion when using ion-pairing agents? If so,
how did you remedy the issue?
Pesek: The issue of ion suppression for
the analysis of polar compounds when
using ion-pairing reagents in reversed-
phase chromatography is a prime rea-
son to not use this approach. In general,
ANP chromatography with silica hydride
stationary phases offers the most versatile
approach for the analysis of polar com-
pounds. It has been demonstrated that
ANP chromatography can analyze posi-
tively or negatively charged compounds
having molecular weights below 100 to
larger molecules, such as peptides. Polar
uncharged molecules can also be ana-
lyzed by this approach. In every case the
mobile phases do not contain more than
10 mM of an additive, such as acetic or
formic acids, or ammonium acetate or
formate, all of which are MS-compatible.
Peak shape is usually the main problem I
encounter with HILIC, mixed-mode, or
ion chromatography, even when Im match-
ing the strength of the sample solvent with
the mobile phase. Do you have any tips on
improving peak shape?
Pesek: It is generally better to have the
sample solvent strength stronger that the
mobile-phase solvent, especially at the
beginning of a gradient. In addition, silica
hydride phases are less susceptible to peak
distortions than HILIC. With the proper
gradient, most compounds will give good
peak shape. Certain compounds like poly-
protic acids and phosphate-containing
species have special considerations because
they can be affected by metal ions in the
(HPLC) system. Sometimes the addition
of 0.5% formic acid or ammonia to the
sample (depending on the compound) can
improve peak shape.
Given that ANP media do not appear to
form a water layer, does the ionic strength of
the mobile phase have a significant impact on
the retention characteristics of ANP chroma-
tography, as it does in HILIC?
Pesek: The ionic strength of the mobile
phase has a smaller effect in ANP chro-
matography than in HILIC because it
appears that the mechanism is some type
of competitive adsorption on the surface.
Ionic strength would be expected to have
some impact on this process.
Up to what percent of acid can you use to
acidify silica hydride columns?
Pesek: Generally speaking there is not
much need to go above 0.5% acetic or for-
mic acids for most ANP applications. If it
is trifluoroacetic acid for biological appli-
cations, the usual limit is about 0.1% when
using UV detection. Trifluoroacetic acid is
not recommended when using MS detec-
tion because of ion suppression.
Can you inject aqueous samples using
ANP columns?
Pesek: Yes, it is possible to inject aqueous
samples for many applications. Each spe-
cific analysis would have to be tested to
determine if it is possible in that case.
Are all of these techniques (HILIC, ANP,
and ion-exchange chromatography, and
reversed-phase chromatography with polar-
embedded or polar-endcapped columns) com-
patible with mass spectrometry?
Pesek: Yes, to some degree. It all depends
on the additive and the concentration
of the additive in the mobile phase. In
ANP chromatography, most applications
reported to date use mobile phases that are
MS-compatible.
Could you please provide some guidance on
how to prepare samples for HILIC chromatog-
raphy? For example, what diluent should I use?
Pesek: Mobile phase in approximately the
same ratio as the mobile phase.
Is the use of mobile phases with high amounts
of methanol on phenyl columns considered
ANP chromatography?
Pesek: In ANP separations, methanol is
usually too strong of a solvent for retention
of hydrophilic compounds. It does not
matter what the column is. Only a few very
polar compounds will be retained using
methanol. However, if retention increases
as the amount of methanol is increased in
the mobile phase, then it would be consid-
ered a normal-phase mechanism.
With HILIC, are typical validation param-
eters such as accuracy and precision com-
parable to those when using reversed-phase
chromatography?
Pesek: No. In most cases the average levels
of accuracy and precision in a HILIC anal-
ysis are lower than typical values obtained
by reversed-phase separations. The accu-
racy and precision of a HILIC analysis is
strongly influenced by the equilibration
time if a gradient method is used. It is gen-
erally found that comparable analyses by
ANP chromatography have a higher level
of accuracy and precision.
ES263010_LCGC0613_496.pgs 06.03.2013 13:09 ADV
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How long is the post-run equilibration time
for a typical HILIC column? How does it
compare to the post-run equilibration time
of the other columns you mentioned in the
presentation?
Pesek: Post-run equilibration times for
HILIC columns can be anywhere from
5 to 45 min. For ANP silica-hydride col-
umns, the equilibration never takes more
than 5 min.
Are there any special precautions one should
take regarding water quality when carrying
out ANP experiments?
Pesek: As demonstrated, in ANP and HILIC
the mobile phase must be free of metal ion
contamination. The presence of metal ions,
particularly of copper and iron, will have an
adverse effect on peak shape for certain acids
and phosphate-containing compounds.

What is peak 6 in the right hand chromato-
gram of the five bases on the slide Compat-
ibility of hydride surface for chromatographi-
cally challenging compounds?
Pesek: Actually, there was a mistake on
this figure. Peaks 15 on the left side of
the slide are correct. However, peak 1 on
the right figure is a solvent peak and peaks
26 should actually be peaks 1-5 to cor-
respond to the peaks on the left side. You
will see that if this is done, the retention
times will be very close for each of the five
compounds on the two columns.
Specific Analytes
or Types of Analysis
We are trying to analyze putrescine by LC
MS. Can you recommend a protocol or a col-
umn that can do the job?
Pesek: Being a diamine compound, putres-
cine is very amenable to analysis by ANP
chromatography using a low carbon-bonded
silica hydridebased column (Diamond
Hydride, MicroSolv Technology). A gradi-
ent using water with 0.1% formic acid and
acetonitrile with 0.1% formic acid going
from 90% organic to 40% organic would be
a good start. It can be modified to shorten
analysis time or improve peak shape.
What would be the column of choice to
analyze catecholamines by LCMS? In the
past I had problems with ion suppression
because the catecholamines are not retained
on reversed-phase columns.
Pesek: This is another good applica-
tion for the silica hydride columns
in the ANP mode. You can use 0.1%
acetic or formic acid as the additive
in the mobile phase and will not get
any ion suppression i n your mass
spectrometer.
What is the best way to perform quantitative
analysis of glucuronides?
Pesek: Glucuronides are best retained
by ANP or HILIC modes. For gradi-
ent analyses, ANP mode with a silica
hydride phase wi l l general ly have
faster re-equi l ibration. These are
analyzed with MS detection in the
negative ion mode with the organic
component of the mobile phase 95:5
acetonitrilewater and 10 mM ammo-
nium formate and the aqueous compo-
nent is water with 10 mM ammonium
formate; a starting gradient would
be from 95% organic solvent to 30%
organic solvent.
Paraquat is a very polar compound,
strongly binds with soil matrix, and is dif-
ficult to detach from soil. How can it be
analyzed?
Pesek: You might want to consider
extraction with 0.5 M K
2
SO
4
as pro-
posed by Vance and colleagues (2). If
you can get a reasonable level of extrac-
tion, with good MS or MSMS detec-
tion you should be able to determine
paraquat at low levels using ANP chro-
matography or HILIC. ANP mode has
the advantage when using a gradient of
faster equilibration.
In your opinion, what would be the best
approach to separate sugar phosphates,
particularly C5-sugar phosphates?
Pesek: Sugar phosphates can be sepa-
rated by ANP or HILIC modes. Care
must be exercised to be sure the system
is free of metal ion contamination. For
complex samples requiring gradient
methods, ANP chromatography will
generally have shorter equilibration times
between runs.
Do you recommend a hydride material or
HILIC material for separating cytidine
nucleosides?
Pesek: There have been several reports
in the literature for the separation of
cytidine nucleosides using hydride mate-
rials in MS compatible mobile phases.
One strategy might be similar to what is
found in a recent article in the Journal of
Separation Science (3).
What is your favorite method for analyzing
sugar phosphates, such as those from central
carbon metabolism (G6P, F6P, R5P, X5P,
Ru5P)?
Pesek: Although it is possible to ana-
lyze these compounds by HILIC, the
silica hydride material, particularly the
low carbon-bonded silica hydride, is a
very versatile material for separating
these types of compounds. Care must be
exercised to make sure that the HPLC
system and the solvents are free of metal
ion contamination.
Does ANP chromatography have impor-
tant applications in large-scale metabolo-
mics studies? Or is it more applicable to
targeted approaches?
Pesek: It can be applicable to either
type of approach. A group in New York
has done a large-scale study of bacte-
rial metabolites and another group in
Australia has done a large-scale study
on plant metabolites. There are many
more reports in the literature for tar-
geted studies based on a specific metab-
olite or small group of metabolites.
What percentage of pesticides can be
detected in wines using present HPLC
methods?
Pesek: This is like many generic analyti-
cal problems in that there is no definitive
answer. Obviously, part of the success in
indentifying pesticides will depend on
the separation method used reversed-
phase mode, normal-phase mode, or a
combination of both. Second, it will
depend on the type of detection used.
Some methods are more sensitive than
others and then there is the matter of
how many of the pesticides the detector
responds to.
References
(1) J. Mun and H. Sim, Eds., Handbook of Ionic
Liquids: Properties, Applications and Hazards
(Nova Science Publishers, Hauppauge, New
York, 2012).
(2) E.D. Vance, P.C. Brooks, and D.S. Jen-
kinson, Soil Biol. Biochem. 19(6), 703707
(1987).
(3) M.T. Matyska, J.J. Pesek, J. Duley, M.
Zamzami, and S.M. Fischer, J. Sep. Sci. 33,
930938 (2010).
ES263009_LCGC0613_497.pgs 06.03.2013 13:09 ADV
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PRODUCT RESOURCES
Polymer chromatography system
The Waters Acquity Advanced Poly-
mer Chromatography (APC) system
is designed to provide improved
polymer peak resolution, particularly
for low-molecular-weight polymers
and oligomers. According to the
company, the system has been
optimized for low dispersion, with
the low noise and drift performance
required for accurate integration,
even at low polymer concentrations.
Waters Corporation,
Milford, MA.
www.waters.com
HPLC column
The Hamilton PRP-C18
HPLC column is designed
to provide high-efficiency
reversed-phase separations
over an extended column
life in nearly any mobile
phase or pH. According to
the company, the rigid sta-
tionary phase can be regenerated in high acidic or basic concentrations
(1 M) and is suited for applications in the pharmaceutical, environmen-
tal, food, forensics, and life sciences industries.
Hamilton Company,
Reno, NV.
www.hamiltoncompany.com
Biocompatible tubing and column hardware
Upchurch Scientific PEEK-lined
stainless steel tubing and isola-
tion technologies column hard-
ware from IDEX are designed to
withstand pressures as high as
15,000 psi and column packing
pressures as high as 20,000 psi.
According to the company, tub-
ing dimensional tolerances are
515 m, depending upon the
inner diameter. Column hardware
will initially be available in two sizes when it is formally introduced in the
summer of 2013. IDEX Health & Science/Upchurch Scientific,
Oak Harbor, WA. www.idex-hs.com
HPLC columns
SunShell HPLC columns from Nacalai
are manufactured using coreshell
particle and bonding technologies.
According to the company, the col-
umns provide increased efficiency
and high pH stability from pH 1 to 10,
and stationary phases are available in
C8, C18, C28, Penta Fluoro Phenyl,
Phenyhexyl, and 2-ethylpyridine types.
Nacalai USA,
San Diego, CA.
www.nacalaiusa.com
Water purification system software
Millitrack Compliance software
from EMD Millipore is designed to
enable regulatory compliance for
laboratory water purification system
users. According to the company,
the software was developed for
the pharmaceutical, biotech, and
contract laboratories that follow GxP
regulations (GLP, GCP, or cGmP U.S. FDA regulations) and which are
required to conform to FDA Title 21 CFR Part 11 guidelines, or similar
requirements set by other global regulatory organizations.
EMD Millipore,
Billerica, MA.
www.millipore.com/MillitrackCompliance
Online applications library
To assist customers in finding prod-
ucts, application notes, and other
resources related to market areas of
interest such as food, life sciences,
energy and fuels, mined materials
and metals, and environment and
agriculture, LECO has redesigned its
website. According to the company,
the site is home to hundreds of application notes, journal articles, and
other information.
LECO Corporation,
St. Joseph, MN.
www.leco.com/registration
Vials
L-MARK absolute vials from
LEAP Pal Parts and Consum-
ables are manufactured with a
process that reportedly mini-
mizes the free hydroxyl sites
on the glass surface. According
to the company, the vials allow
measurements to be made
with minimal effects of sample
absorption and pH shifts.
LEAP PAL Parts & Consumables,
Raleigh, NC.
www.palparts.com
GCMS-MS system
PerkinElmers AxION iQT GC
MS-MS system is designed to
perform complex sample analy-
ses for laboratories that need
fast results and more specific,
selective data. According to the
company, the system identifies
compounds and determines
their exact quantity at a rate of
500 compounds per second,
every second.
PerkinElmer,
Waltham, MA.
www.perkinelmer.com
ES262119_LCGC0613_498.pgs 05.31.2013 23:33 ADV
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yellow magenta cyan
powered by
SPE MS GC
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All our chromatography
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ES257424_LCGC0613_499_FP.pgs 05.29.2013 02:12 ADV
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Refractive index detector
The Optilab UT-rEX
refractive index detector
from Wyatt Technology
is designed for use with
UHPLC systems using
columns packed with small
beads. According to the
company, the detector has
no range or gain settings,
and the full range of instru-
ment detection is always
present.
Wyatt Technology Corp.,
Santa Barbara, CA.
www.wyatt.com
Combustion ion chromatography system
Metrohms 881 Compact IC pro fully automated
combustion ion chromatography system is
designed to simultaneously determine trace halo-
gens (F, Cl, Br, and I) and sulfur compounds (as sul-
fate) in any nonaqueous sample mix. According to
the company, after an upstream thermal oxidative
digestion, liberated gases are trapped in an oxidiz-
ing aqueous absorbent that gets injected directly
into the sample loop of the system.
Metrohm,
Riverview, FL.
www.metrohmusa.com
HPLC columns
Shodex HPLC columns
are available in separa-
tion modes that include
normal and reversed
phase, HILIC, size exclu-
sion, multimode, ion
chromatography, ligand
exchange, and other
special purpose columns.
Applications repor tedly
are available from the
companys database.
Showa Denko America, Inc., New York, NY.
www.shodex.net
S ample preparation automation system
The AutoMate-Q40 system
from Teledyne Tekmar is
designed to automate the
QuEChERS sample prepara-
tion workflow. According to
the company, the system is
configured out of the box
to conduct two QuEChERS
sample preparation meth-
ods: AOAC2007.01 and EN
15662.2008.
Teledyne Tekmar,
Mason, OH.
www.teledynetekmar.com/AutoMateQ40
Gas chromatograph
The Tracera gas chromatography
system from Shimadzu Scientific is
designed for high-sensitivity analyses
of organic compounds, permanent
gases, and light hydrocarbons.
According to the company, the sys-
tems barrier discharge ionization
detector creates ionization from a
helium-based, dielectric barrier dis-
charge plasma.
Shimadzu Scientific Instruments,
Columbia, MD.
www.ssi.shimadzu.com
Capillary tubing
Polymicros flexible fused-
silica capillary tubing is
designed with an outer
diameter of 1/32 in.
According to the company,
the tubing mates with
existing 1/32-in. fittings
and is available in a range
of internal diameters from
50 m to 500 m.
Polymicro Technologies,
Phoenix, AZ.
www.polymicro.com
GC system
Agilents 7890B GC system is
designed for reliable, fast perfor-
mance with reduced downtime.
The system reportedly includes
features such as eco-friendly
operation with powergas
management and sleepwake
modes, faster method develop-
ment with integrated GC calcula-
tors and wizards, and end-to-end
inert flow path deactivation. The
system reportedly runs on the
companys OpenLAB CDS software.
Agilent Technologies, Santa Clara, CA.
www.agilent.com/chem/resolve
LC system
The Thermo Scientific Dionex
UltiMate 3000 Biocompatible
Rapid Separation LC system from
Thermo Fisher is designed to
operate at pressures as high as
1000 bar (15,000 psi) and flow
rates up to 8 mL/min. According
to the company, the wide flow/
pressure footprint supports bio-
molecule characterizations that
include development, identifica-
tion, and quality control.
Thermo Fisher Scientific,
San Jose, CA.
www.thermoscientific.com/BioRS
ES262109_LCGC0613_500.pgs 05.31.2013 23:32 ADV
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SPE cartridges
Supel Tox SPE cartridges
from Supelco are designed
to provide sample cleanup
before chromatographic
mycotoxin analysis. The
cartridges reportedly use
an interference removal
strategy that eliminates
wash steps before elution
of alfatoxin, zearalenone,
deoxynivalenol, and tricothecenes (type A and B).
Supelco/Sigma-Aldrich,
Bellefonte, PA.
www.sigma-aldrich.com/supeltox
Sample cleanup workstation
The Freestyle workstation from
Pickering Laboratories is designed
for automated sample cleanup
work flow. The instrument is based
on a suspended rack design, with
an XY robot arm for liquid handling.
The workstation reportedly is able
to handle multiple flask shapes
with volumes ranging from 1 mL to
1 L, and the instruments software
enables users to program multiple sample parameters and to pre-
pare graphical reports and audit logs. SPE, GPC, and evaporation
and solvent exchange modules are available.
Pickering Laboratories, Inc.,
Mountain View, CA. www.pickeringlabs.com
GCMS-MS pesticide analyzer
The Thermo Scientific TSQ 8000
pesticide analyzer is designed
to facilitate multiresidue pes-
ticide analysis. According to
the company, the GCMS-MS
system includes preloaded data
processing methods, TraceGOLD
column and GC consumables
configured for high performance
pesticide analysis, a walkthrough
guide for building customized methods, and a 600+ pesticide com-
pound database with retention times and preoptimized SRMs.
Thermo Fisher Scientific, San Jose, CA.
www.thermoscientific.com/pestanalyzer
Chiral chromatography products website
Regis Technologies website
describes the companys
chiral chromatography prod-
ucts and separation services,
cGMP organic systhesis ser-
vices, and analytical method
development. According
to the company, the chiral
chromatography section also
includes 400 chiral separation
applications of small molecules.
Regis Technologies, Inc.,
Morton Grove, IL.
www.registech.com
Register Free at http://www.chromatographyonline.com/screening
EVENT OVERVIEW:
Residue contamination in foods has generated a global food safety concern. Achieving
accurate and repeatable screening and confrmatory methods is crucial for success-
ful food safety analysis. Thus, unlocking chromatography secrets in LC/MS and GC/MS
method development produces desirable results. In this web seminar we will focus on:

Current trends and needs in screening and
confrmatory methods for foods

Tips and tricks for successfully boosting
chromatography results and column lifetimes

Guidance in choosing an appropriate sample
preparation technique to ft the need

Key Learning Objectives:

Evaluate the current needs in
screening and confrmatory
methods in foods

How to improve chromatographic
results and column lifetime

How to determine the appropriate
sample preparation technique

Who Should Attend:

Scientists and laboratory
managers who work with
HPLC/UHPLC and GC

Anyone interested in developing
or improving screening and
confrmatory methods in foods
PRESENTER:
Dr. Jef Layne
Manager, Product
Management and
Technical
Phenomenex
MODERATOR:
Laura Bush
Editorial Director
LCGC
Sponsored by Presented by
For questions, contact Kristen Farrell at kfarrell@advanstar.com
Tips and Tricks on Screening and Conrmatory
Methods for Residues and Contaminants in Foods
LIVE WEBCAST: Tuesday, June 18, 2013 at 11:00 am EDT

ES262108_LCGC0613_501.pgs 05.31.2013 23:32 ADV
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LC system
The Agilent 1290 Infinity Quaternary LC qua-
ternary UHPLC system is designed to provide
a maximum pressure of 1200 bar and a
maximum flow rate of 10 mL/min. Accord-
ing to the company, the system features the
companys 1290 Infinity diode-array detector
and mixers that incorporate the companys
microfluidic technology.
Agilent Technologies,
Santa Clara, CA.
www.agilent.com
HPLC system
Hitachis Primaide HPLC system is
designed for long-term stable opera-
tion, reliability, and durability. The
system provides front access to all
modules for maintenance, and leak
sensors are built into each module.
According to the company, PC control
is available with an optional UI pad. A
mercury lamp reportedly is installed
in the detector to enable calibration
of UV wavelengths using its 254-nm
emission line.
Hitachi High Technologies
America, Dallas, TX.
www.hitachi-hta.com
Pesticide analysis system
The PesticideScreener system from
Bruker is designed for multiresidue
analysis of pesticides in food and
feed. According to the company,
the system includes UHPLC and
QTOF MS hardware, applications
software, and LCMS methods.
The system also includes a database that provides retention times
for the matched UHPLC method; adduct information; isomer infor-
mation; fragment ion information; isotopic confirmation; and quali-
fier ions for one-shot confirmation in broadband MS-MS mode.
Bruker Daltonics, Billerica, MA.
www.bruker.com
Plate sampling system
MicroLiters plate sampling
system of microplates, glass
inserts, Lmats, and caps with
chromatography-based septa are
designed to provide secure ves-
sels to maintain volatile samples.
According to the company, the
systems polypropylene Lplates
have a variety of closures that
will stabilize the sample during
the analytical sample run and, if necessary, reseal after the sample has
been injected. MicroLiter Analytical Supplies, Inc., Suwanee, GA.
www.microliter.com/
Register Free at www.chromatographyonline.com/avcs
EVENT OVERVIEW:
Just one more turn that has been the chromatographers answer to the age old question of
How tight should I seal my autosampler chromatography vial closure? Unfortunately, for a lot
of chromatographers, this answer has resulted in a myriad of chromatographic problems. Truth
be told, just one more turn is more than likely the absolute
worst thing that one can do to seal an autosampler vial.

The Advance Vial Closure System (AVCS) allows bench
chemists and sam ple prep technicians to remove the sub-
jectiveness out of achieving the optimal compression
when sealing a vial. The beneft of using a vial and closure
designed as a complete system, allows the closure, septum,
and vial to work together to prevent compression from
exceeding the optimal range.

This web seminar will review the impact an innovative vial &
closure design, like AVCS, can have on improving data qual-
ity by providing data that supports how an AVCS-designed
vial & closure eliminates under and over compression of vial
seals while improving robotic autosampler pick-up of vials,
ensuring more centered vial closure positioning and greater
optical recognition of vials placed in autosamplers.

The impact of analyst subjectivity
on vial sealing

Optimal vial/closure sealing and
how it can be reliably achieved

The Advance Vial Closure System
design

Who Should Attend:

Any chromatographer or mass
spectroscopist concerned
improving data quality &
performance

QA/QC looking for ways to
improve data quality

Laboratory managers focusing
on sample throughput and data
integrity
ON-DEMAND WEBCAST
PRESENTER:
Dave Edwards
Sample Handling
Product Manager
Thermo Fisher Scientifc
Detlev Lennartz
Product Manager
Vials and Closures
Chromatography
Consumables and
Specialty Products
MODERATOR:
Laura Bush
Editorial Director
LCGC
Sponsored by
Presented by
Solving One of Chromatographys Biggest Dilemmas
Proper Sealing of Chromatography Autosampler Vials
Using the Advanced Vial Closure System (AVCS)
ES262107_LCGC0613_502.pgs 05.31.2013 23:32 ADV
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Solution preparation system
The Soluprep II solution
preparation system from
Design Scientific, an affili-
ate of Resolution Systems,
is designed to speed up
routine sample preparation
and increase accuracy by
eliminating volumetric mea-
surements. According to the
company, the system automatically prepares samples for LC, GC, ICP,
spectroscopy, viscosity, NMR, and other analyses.
Resolution Systems,
Holland, MI.
www.designscientific.com/soluprep.html
Pulsed discharge helium ionization
detector
VICIs model D-3-1-HP
pulsed discharge helium ion-
ization detector is designed
for plug-and-play installation
on Agilent GC systems.
According to the company,
the detector is optimized
for trace-level analysis in
the helium photoionization
mode and uses the elec-
tronics and power supply of
the host GC system.
Valco Instruments Co., Inc. Houston, TX.
www.vici.com
Coreshell columns
Kinetex 5-m coreshell media from
Phenomenex are designed to provide
90% higher average efficiencies com-
pared to the same size fully porous col-
umns. According to the company, little
to no method development is required
to achieve improved performance on
standard HPLC systems.
Phenomenex, Inc.,
Torrance, CA.
www.phenomenex.com/
Mass spectrometer
The Xevo G2-S QTof benchtop mass spec-
trometer from Waters is a quadrupole
time-of-flight instrument designed with the
companys StepWave ion optics technology.
According to the company, the mass spec-
trometer is up to 20 more sensitive than
earlier generation instruments and is UPLC
compatible.
Waters Corporation,
Milford, MA.
www.waters.com
Register Free at www.chromatographyonline.com/triple
EVENT OVERVIEW:
In recent years, the analysis of residual levels of pesticides has developed into a comprehen-
sive science. Pesticide residues that contaminate foods such as herbal products and teas have
become a worldwide problem with direct implications on human health and international trade.
Routine screening methods to analyze hundreds of potential contaminants are therefore neces-
sary. Multi-residue methods for herbal products and teas
are faced with the additional challenge of being used on
products with worldwide origin and complex matrices of
dried materials.

This webinar explains an application protocol for a highly
sensitive, matrix selective multi-residue pesticide analysis
of herbal products. The protocol uses accelerated solvent
extraction and gel permeation chromatography (GPC) for
sample preparation along with a triple quadrupole GC
MS/MS system for detection and quantifcation. A routine
screening method of more than 200 pesticide compounds
was applied to a wide variety of diferent sample types
including black tea, sage leaves, fennel, thyme, and cham-
omile. This highly automated and easy-to-use, robust
method allows for the analysis of the most challenging
sample types, with minimal system downtime.


Learn about the process of
accelerated solvent extraction
and cleanup for difcult matrices

Gain an understanding of
how a triple quadrupole mass
spectrometer works

Learn how selectivity means
lower detection limits

Who Should Attend:

Food chemists analyzing
pesticides in difcult matrices

Those interested in automation
of extractions to increase sample
throughput

Analysts interested in the
selective power of triple
quadrupole mass spectroscopy
Live Webcast: Tuesday, June 25, 2013 at 8:00 am PDT/ 11:00 am EDT/ 16:00 BST / 17:00 CEDT
PRESENTERS:
David Steiniger
Senior Applications
Scientist

Aaron Kettle
Product Manager,
ASE systems
Marketing
MODERATOR:
Laura Bush
Editorial Director
LCGC
Sponsored by
Presented by
Multi-Residue Pesticide Analysis in Herbal Products Using
Accelerated Solvent Extraction with Triple Quadrupole GCMS/MS
ES262116_LCGC0613_503.pgs 05.31.2013 23:33 ADV
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Application Note 37920312
Keywords
Eclipse 4660 Purge-and-Trap
Sample Concentrator
Hydraulic Fracturing
Methane
Natural Gas
Underground Sources of Drinking
Water (USDW)
Presented at the 2012 Pittsburgh
Conference on Analytical
Chemistry and Applied
Spectroscopy, Orlando, Florida,
March 1215, 2012
Purge-and-Trap GC Analysis of Methane in Water
Samples Associated with Hydraulic Fracturing
Introduction
Shale gas reservoirs, such as the Marcellus shale reserve in Pennsylvania
and Barnett shale reserve in Texas are a growing source of natural gas in
the United States. The U.S. Energy Information Administration (EIA)
estimates that the total natural gas resource base of the United States to be
2,553 trillion cubic feet (Tcf).(1) Shale gas production represents greater
than 20% of the current U.S. supply an increase from 1% in 2000.
Hydraulic fracturing or fracking involves pumping water, sand, and
chemicals at extremely high pressure into deep underground wells to
crack open hydrocarbon-rich shale formation and extract natural gas.
Chemicals used for hydraulic fracturing include potentially toxic
substances such as diesel fuel and disinfectants, which can contaminate
underground sources of drinking water (USDW).
In some cases, methane has been detected in household drinking water
from wells. A study conducted in the Marcellus and Utica shale
formations found methane concentrations were 17-times higher on
average (19.2 mg CH4 L-1) in wells from active drilling and extraction
areas then in non-active areas (1.1 mg L-1 on average).(2) The average
methane concentration found in shallow groundwater in areas of active
drilling and hydraulic fracturing fell within the defined action level (10-
28 mg L-1) for hazard mitigation recommended by the U.S. Department of
Interior.(2)
Methane is not a regulated contaminant under U.S. National Drinking
Water Regulations. In fact, a provision in the Safe Drinking Water Act
passed in 2005 exempts hydraulic fracturing from regulation under the
USEPAa Underground Injection Control Program (except in cases where
diesel fuel is employed for fracking). Consequently, there are no USEPA-
approved testing methods for measuring methane in drinking water and
groundwater.
The application note describes the use of a purge-and-trap gas
chromatography system to analyze methane, ethane, ethene, and propane
hydrocarbons (C1-C3) in drinking water samples.
Experimental
Instrumentation used for this study was an OI Analytical Eclipse 4660
Purge-and-Trap sample concentrator (Figure 1) with a proprietary trap
specifically designed to trap methane. The P&T was interfaced to an
Agilent 7890 GC/FID with a split/splitless injector and a SUPEL-QPLOT
column (30-meter x 0.32-mm I.D.).
A primary, saturated analytical standard was prepared by chilling 1 liter of
Literature
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Chromatography
catalog
Supelcos 2013 catalog reportedly
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Supelco/Sigma-Aldrich,
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sigma-aldrich.com/reserve
Water analysis
application note
An application note from OI
Analytical describes the use of a
purge-and-trap gas chromatogra-
phy system to analyze methane,
ethane, ethene, and propane
hydrocarbons (C
1
C
3
) in drinking
water samples.
OI Analytical,
College Station, TX.
www.oico.com
Application note
An application note from Tosoh
Bioscience describes the analysis of
large metalloproteins and monoclo-
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TSKgel protein C4-300 reversed-
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silica-based butyl (C4) column was
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phy column optimized for protein
analysis. Tosoh Bioscience, King
of Prussia, PA. www.separations.
us.tosohbioscience.com
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Restek Corporation, Bellefonte,
PA. www.restek.com/posters
ES262113_LCGC0613_504.pgs 05.31.2013 23:32 ADV
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THE ESSENTIALS
Excerpts from LCGCs professional development
platform, CHROMacademy.com
More Online:
R
etention times in capillary gas chro-
matography (GC) can change from
run to run or even within the run,
and the underlying causes generally rest
with carrier flow stability, poor tempera-
ture control, or the quality of the station-
ary phase within the column. Some minor
retention-time variability from batch to
batch are perhaps most common and can
be mitigated through the use of retention
factor (k) in calculations or for qualitative
peak identification. Slight differences in car-
rier gas flow rate may arise from incorrect
instrument parameters that would typically
include the nature of the carrier gas or the
column dimensions which will fool
the system into applying an incorrect pres-
sure to generate what it thinks is the correct
carrier flow or linear velocity. Remember,
GC electronic pressure controls calculate
flow from the applied pressure, the nature
of the carrier gas, and the column dimen-
sions. One should also take care to select the
correct operating mode in gradient temper-
ature-programmed GC, because retention-
time differences can be pronounced when
operating in either constant-pressure or
constant-flow modes. Differences in split
flow or column oven temperature pro-
file may also give rise to retention changes
between batches of samples. Changes in k
values may be due to deterioration of the
stationary-phase quality (loss of bonded
phase, especially at the column inlet) via
thermal or chemical decomposition, which
will cause the analyte retention time to vary
relative to the elution time of an unretained
component. In this case one should con-
sider removing up to 5% of the total col-
umn length from the inlet end to eliminate
badly degraded stationary phases. The act of
trimming the column will in itself require a
change in the column length setting in the
instrument (most instruments will calculate
column length based on the elution time of
an unretained peak and the nominal inter-
nal diameter); although the absolute reten-
tion time will change, the retention factor
value should remain constant.
Problems of retention-time variability
between subsequent injections are perhaps
more serious. One of the more insidious
causes relates to the equilibration time
between gradient temperature program
runs. Although the GC oven may be at
the correct temperature for the next injec-
tion, the column has a small but significant
thermal mass and enough time should be
allowed for the column and carrier to reach
and stabilize at the initial gradient tempera-
ture before the next injection otherwise
small retention time variability will occur
from run to run. Usually, 30 s to 1 min is
sufficient for equilibration after the oven
temperature has reached its set point. Injec-
tion-to-injection retention-time instability
can also be caused by faults with the GC
oven temperature controller or the electronic
pressure control module, and these prob-
lems will require attention from your service
provider. Small retention time shifts may
also be caused by irreproducible split flows
(suspect a blockage in the split vent line) or
from small leaks created during the injection
phase caused by a split or cored septum a
further clue here may be a shift in baseline
position before and after the injection phase.
The high efficiency of GC separations is
crucial for successful analysis, especially with
complex samples or when fast analysis is
required. Factors causing reduced efficiency
include compromised stationary phase,
leaks, and dead volumes within the system
as well as poor selection of analysis condi-
tions. Wall-coated capillary GC columns
deteriorate with time because of contamina-
tion and phase bleed and, as such, a gradual
reduction in efficiency is expected. This
process may be accelerated when operating
at high temperatures for prolonged periods,
when using aggressive solvents (acetonitrile
or tetrahydrofuran), or when dirty samples
are injected repeatedly. Trimming the col-
umn inlet is recommended to restore peak,
shape, and efficiency as discussed above.
Dead (unswept) volumes in the system may
be created through poor installation of the
column into the inlet and more especially
the detector (that is, because of incorrect
column insertion depths or distances). Fol-
low vendors column installation instruc-
tions carefully. If the split (purge) is not
initiated at an optimum time post injection
during splitless injection, peaks within the
chromatogram will appear broad. The use
of constant-flow mode during temperature
programming is recommended to avoid loss
of efficiency in later eluted peaks because the
linear velocity of the carrier slows apprecia-
bly with the increase in oven temperature.
Peak tailing in capillary GC is most com-
monly associated with secondary retention
of the analyte by active sites (silanol spe-
cies predominantly) within the GC system.
If analytes are polar, ensure the system is
deactivated by purchasing presilylated lin-
ers (with deactivated packing if required)
and use the most modern, inert, station-
ary phases. Regularly clean and deactivate
the GC inlet and ensure that the column
inlet is regularly trimmed to remove active
sites. Peak splitting problems can arise
from improperly cut GC column ends,
improperly installed columns (incorrect
insertion distances into the inlet or detec-
tor), or a blocked detector jet. Ensure that
column cuts are at 90 to the column wall
and clean with no jagged edges use a
magnifying glass or microscope to assess the
quality of the column cut. Peak shoulders
can occur when the stationary-phase polar-
ity does not match the polarity of either the
analyte or the sample solvent, especially dur-
ing splitless injection.
Happy troubleshooting and for
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ES262102_LCGC0613_506.pgs 05.31.2013 23:31 ADV
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