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Putting The Pieces TogetherThe power of 2D LC for
peptide mapping
2 The Power of 2D LC Protein biopharmaceuticals have seen an enormous growth in the last decade,
and as a result, separation scientists are giving increased attention to methods for characterizing biopharmaceuticals. One powerful technique for analyzing proteins is two-dimensional liquid chromatography (2D LC). Gerd Vanhoenacker of the Research Institute for Chromatography (RIC) in Kortrijk, Belgium, has been conducting research into peptide mapping of therapeutic monoclonal antibodies (mAbs) using 2D LC. He recently spoke to The Column about this work.
Cover Story
Features
20 The LCGC Blog: Column Overload in Gas Chromatography with Vacuum Ultraviolet Detection
Kevin A. Schug, University of Texas Arlington Column overload is a very commonly encountered issue in gas chromatography (GC) for
beginners. Changes in peak symmetry, generally observed as peak fronting, can be subtle in the sharp peaks generated by GC, but the result can be significant shifts in retention times, loss of resolution, and error in peak integration. LCGC Blogger Kevin Schug explains more.
15 Extending the Detection Limits for the Analysis of Organotin Contaminants Using Soft Ionization
Laura McGregor, Steve Smith, and David Barden, Markes International This article presents a gas chromatography coupled with time of fl ight mass spectrometry
(GC–TOF-MS) method with soft ionization for trace-level detection and quantitation of organotins in complex matrices.
23 The 10th Balaton Symposium on High-Performance Separation Methods: A Review
Ira Krull, Northeastern University
A review of the 10th Balaton Symposium on High-Performance Separation Methods,
which was held 2–4 September 2015 at the Hotel Azúr, Siófok, Hungary.
Regulars9 News The benefi ts of pattern modulation in GC×GC, a detection method for early-stage ovarian cancer using UHPLC–HRMS, and the latest news in brief are featured in this issue.
12 Incognito Reproducibility of Research — Do We Have a Problem Houston? Incognito talks about reproducibilty in research.
26 CHROMacademy Find out what’s new on the professional learning site for chromatographers.
27 Training Courses and Events
28 Staff
19 January 2016 Volume 12 Issue 1
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The Power of 2D LCProtein biopharmaceuticals have seen an enormous growth in the last decade, and as a result, separation scientists are giving increased attention to methods for characterizing biopharmaceuticals. One powerful technique for analyzing proteins is two-dimensional liquid chromatography (2D LC). Gerd Vanhoenacker of the Research Institute for Chromatography (RIC) in Kortrijk, Belgium, has been conducting research into peptide mapping of therapeutic monoclonal antibodies (mAbs) using 2D LC. He recently spoke to The Column about this work.
Q. In a recent article you mention
that within the current decade you
expect a much larger share of drug
approvals to be of a biological nature,
specifically monoclonal antibodies.1
Why do you think this is the way the
therapeutics market is going?
A: Protein biopharmaceuticals have
emerged as important therapeutics for
the treatment of cancer, cardiovascular
diseases, diabetes, infection, and
inflammatory and autoimmune disorders.
Given their obvious benefits in terms of
safety and efficacy, they are reshaping
the pharmaceutical market. The majority
of these proteins are, and will be,
monoclonal antibodies (mAbs) and many
of these have already received approval in
Europe and the United States.
In a recent supplement from LCGC
Europe, edited by Pat and Koen Sandra,
a brief overview of current and future
trends in drug development and
sales was given.2 Trends for different
classes of pharmaceuticals speak for
themselves: Small-molecule drug sales
are stagnating while recombinant protein
pharmaceuticals sales have increased
by over 25% between 2008 and 2013.
Sales for mAbs have nearly doubled over
the same period of time. Big pharma
organizations are now more and more
focused on biopharmaceutical products.
Of course, they will continue to invest in
the development of key small molecule
formulations, but a major part of
their research is now focused on large
biomolecule drugs in general, and mAbs
in particular.
Q. This shift would obviously
represent an analytical challenge. Do
you feel the pharmaceutical industry
is sufficiently experienced and
prepared for such a shift?
A: The analysis of these big,
2
Q&A: Vanhoenacker 2 The LCGC Blog20Barden et al.15Incognito12News9Balaton Symposium Review23 Staff28CHROMacademy262626 Training & Events27272
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Q&A: Vanhoenacker 2 The LCGC Blog20Barden et al.15Incognito12News9Balaton Symposium Review23 Staff28CHROMacademy262626 Training & Events27272
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heterogeneous biomolecules will often require a shift in analytical approach and technique compared to the analysis of small-molecule pharmaceuticals. This shift in approach is needed at nearly all levels going from sample storage and preparation to analysis and data treatment. Most regulatory guidelines were originally developed for small-molecule drug products and formulations. To enable researchers in academia and industry to set up and validate their methodologies, separate guidelines for biopharmaceutical drug substances and products have been issued such as the International Conference on Harmonization (ICH) Q6B guideline on test procedures and acceptance criteria for biotechnological and biological products, and ICH Topic Q5C, D, and E.
I believe there are opportunities for products and techniques that can automate or accelerate all steps involved in the bioanalytical process. A lot of progress has been made over the last decades, but the present achievements are definitely not the endpoint. Advances towards faster analyses and even more accurate and detailed data generation will be necessary and will be made. Innovations in mass spectrometry (MS), chromatography (particularly
ultrahigh-pressure liquid chromatography [UHPLC] and two-dimensional liquid chromatography [2D LC]), and software are most important. The first two might seem obvious, but the last is equally important. A critical part of biopharmaceutical analysis lies in the data handling and interpretation. Data analysis software is playing a very prominent role in research on various biopharmaceuticals and this is a major difference compared to the analysis of small-molecule pharmaceuticals. It is a field in which various instrument and software developers are investing considerably.
Q. Peptide mapping is obviously a very useful technique, can current one-dimensional (1D) LC methods deal with the large amount of peptides that comprise a large protein drug?A: The power of state-of-the-art 1D LC techniques should not be underestimated. A well-developed (U)HPLC method can be very valuable and robust for peptide mapping. However, when sample complexity increases and the chromatogram becomes populated with a larger number of peptides, as is the case for mAbs, problems with co-elutions will be evident. Method development for one biopharmaceutical product will
Q&A: Vanhoenacker
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Q&A: Vanhoenacker 2 Q&A: Vanhoenacker 2 The LCGC Blog20 The LCGC Blog2020Barden et al.15 Barden et al.1515Incognito12 Incognito12News9 News99Balaton Symposium Review23 Balaton Symposium Review2323 Staff28 Staff2828CHROMacademy262626 CHROMacademy2626 Training & Events272727 Training & Events27
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not necessarily, and will probably not, be
applicable to another product. These are
the limitations to 1D LC that researchers
encounter today.
We need a much larger peak capacity
than the number of peptides in our
digest. If we consider a digest with
100 peptides, a peak capacity of about
10,000 is required to have a good
chance of separating all of them with
chromatography. The introduction of
high-end MS systems has alleviated the
need for this extreme chromatographic
performance but once methods are
transferred from research to routine
use, MS is out of the picture and the
chromatography needs to do the job.
However, for relatively simple digests
1D LC can be suitable. More complex
samples such as digests of very large
biomolecules or mixtures thereof will
seldom not require more than one
analysis to completely profile the peptide
map, even with MS installed. Combining
different separations (that is, selectivities)
is what 2D LC is essentially doing. This
makes it a very valuable means to analyze
such complex samples.
Q. What does two-dimensional
chromatography offer that
one-dimensional chromatography
cannot in the analytical study of large
proteins?
A: The considerably increased separation
power is what it is all about. Achieving
resolution in the second dimension of
compounds that are not separated in
the first dimension is the most obvious
advantage. However, 2D LC can be used
in different modes.
First, there is comprehensive 2D LC
(LC×LC) where the complete effluent from
the first dimension is sampled in discrete
fractions and each of these fractions is
then analyzed in the second dimension.
The main goal here is to increase peak
capacity. In theory, the 2D LC peak
capacity will be the product of each of the
individual peak capacities.
This approach should increase separation
power dramatically. In reality, however,
the total peak capacity needs to be
corrected for what is called undersampling
and incomplete orthogonality. This has
a serious impact and the practical peak
capacity is significantly lower than the
theoretical peak capacity. Nonetheless,
the peak capacity in comprehensive
2D LC will be considerably higher than
what can be achieved with 1D LC. This
high peak capacity gives a comprehensive
view of the sample constituents and
resolves compounds that are not resolved
with only the first- or second-dimension
selectivity. The goal here could be to
create a generic method to screen
mAb digests for differences in amino
acid sequence or post-translational
modifications.
Other 2D LC techniques are based on
heart-cutting approaches where only
one or a number of fractions (peaks) are
transferred to the second dimension.
The advantage of such a method is that
the second dimension analysis time, and
therefore chromatographic performance,
is more or less detached from the first
dimension. In comprehensive LC×LC,
the available time to perform the
second dimension separation is very
limited and, consequently, the full
potential of this second dimension is
unable to be exploited. This is not the
case in heartcutting 2D LC approaches,
so the choice of analytical conditions
(column dimension, flow-rate, analysis
time) can be tailored according to the
analytical needs at hand. Applications
for large-biomolecule separations
include the combination of a first
dimension ion-exchange mechanism
with a second-dimension MS-compatible
reversed-phase separation. The inorganic
salt present in the first dimension is
removed after passing through the
second dimension. In this way, a specific
peak containing one or more peptides or
proteins can be transferred on-line to the
second dimension where an additional
separation can take place before MS
detection. Various ways to do this are
available and the user-friendliness is
superior compared to off-line methods
where the peaks of interest have to be
collected and then re-analyzed on another
system following some manipulation.
Q. Your research tested three
different 2D LC combinations,
namely strong cation exchange ×
reversed-phase LC, reversed-phase LC
× reversed phase LC, and hydrophilic
interaction liquid chromatography
(HILIC) × reversed-phase LC. Did any
of these combinations stand out as
particularly effective for peptide
mapping of large proteins?
A: Both reversed-phase LC × reversed-phase
LC and strong cation exchange ×
reversed-phase LC are very suitable. In
reversed-phase LC × reversed-phase
LC, compatibility of both dimensions is
excellent and effi ciency on both columns
is relatively high. It is the easiest of these
three combinations and results in a robust
method with high peak capacity. One of
the disadvantages is that full orthogonality
Q&A: Vanhoenacker
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is impossible to achieve because of the
similarity in separation mechanisms.
However, there are plenty of tools to
incorporate some orthogonality into this
approach, by modifying factors such as
the stationary phase, mobile phase pH and
organic modifi er type, and temperature.
A simple option is to modify the second
dimension gradient during the analysis. In a
reversed-phase LC × reversed-phase LC set
up this makes sense because compounds
that are more strongly retained on the
fi rst dimension will also require stronger
elution conditions in the second dimension.
Combining all of these features results in
powerful methods for detailed peptide
mapping.
The strong cation exchange ×
reversed-phase LC combination will provide
better orthogonality because the separation
mechanisms are not correlated. Peptide and
protein fi rst dimension retention will be
driven mainly by ionic interactions, while
in the second dimension hydrophobicity
of the compound will be the major factor
for retention and separation. Strong cation
exchange for peptides and proteins is
generally performed using aqueous mobile
phases, which makes the transfer to and
injection onto the second dimension
relatively straightforward. Possible
drawbacks of this approach include the low
chromatographic effi ciency some strong
cation exchange columns can provide.
Also, the high buffer and salt load that is
generally used in strong cation exchange
separation of peptides and proteins will be
introduced onto the second dimension and
potentially the MS detector. The choice of a
good fi rst dimension column can obviously
overcome the fi rst drawback. The potential
interference of inorganic mobile phase
additives can somewhat be minimized by
selecting the relevant window in which
2D LC needs to be performed and by using
an additional diverter valve on the mass
spectrometer.
HILIC × reversed-phase LC is the most
difficult combination of the three. It will
provide good orthogonality but there can
be some compatibility issues. Since HILIC
operates with high acetonitrile amounts,
the transfer of these fractions onto the
reversed-phase second dimension can
cause polar peptides to breakthrough.
However, various modifications can be
made to reduce or even avoid this. It is
definitely an interesting approach but I
would advise this combination only in
the case of specific analytical challenges
such as differentiation of protein and
peptide glycosylation. For general work,
the reversed-phase LC × reversed-phase
LC and strong cation exchange ×
Q&A: Vanhoenacker
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The Column www.chromatographyonline.com
addition to the required packing chemistry
and particle size. This is where column
manufacturers could step in. For the first
dimension column we like to work with a
column internal diameter between 1 mm
and 2.1 mm. This is done to keep the first
dimension flow rate as low as possible
to decrease volume loading onto the
second dimension. The range of available
stationary phases packed in narrow- and
especially in micro-bore format is limited
and frequently the ideal column will not
be found in a catalogue. Custom ordering
can be the solution but this takes time
and can be costly.
Second-dimension columns to be used
in heartcutting 2D LC approaches can
be of any dimension and the choice here
is plentiful. For comprehensive 2D LC
applications this is not the case. The
second dimension column should be able
to provide good quality chromatography
in as short an analysis time as possible
(below 1 min), preferably with reasonably
low fl ow rates and back pressure: Short
columns packed with small particles or with
superfi cially porous particles seem to be the
obvious choice here. However, one has to
be aware that one comprehensive 2D LC
analysis will result in numerous injections
(typically 50–150 modulations/run) of
relatively large volumes (20–80 μL) onto this
reversed-phase LC combinations will be
much easier to work with.
Q. You considered two options
(formic acid and trifluoroacetic acid
[TFA]) when replacing the mobile
phase for MS detection. However, as
noted, both options have potential
drawbacks. Are there any alternatives
that could have been used and, if
so, in what circumstances would you
recommend their use?
A: I would like to go back one step before
answering this. For peptide mapping and
protein analysis with reversed-phase LC,
the use of a water–acetonitrile mobile
phase with trifluoroacetic acid (TFA) is still
frequently used. The use of TFA results in
good retention for polar peptides because
of its ion-pairing properties. However,
it can produce interfering noise on the
baseline when ultraviolet (UV) detection
is used. This is a result of absorbance of
UV light at 214 nm. Formic acid has a
similar though less hindering effect. When
running very fast gradients (generally
around 30 s) as we do in comprehensive
2D LC, the excessive baseline noise and
drift will lead to poor quality 2D LC
plots. It will be difficult to detect small
compounds in such data. This can be
partly overcome by subtracting blank runs
but this is limited and not very convenient.
This is the reason we prefer to use
phosphoric acid in the second dimension
reversed-phase LC. With this additive a
stable baseline is obtained and retention
and selectivity are very close to those
acquired with formic acid. In my opinion
it is an excellent choice for 2D LC when a
UV detector or diode-array detector (DAD)
is used.
When MS is required, the use of
phosphate or other inorganic mobile
phase additives should be avoided. Here
we need to replace phosphoric acid with
a volatile and organic alternative. It is
known that the ion-pairing effect of TFA
can lead to ionization suppression. Formic
acid is an excellent alternative because
it will give nearly identical selectivity
compared to phosphoric acid. So by using
phosphate in UV work and formate in MS
work we can easily compare results and
identify peptides and proteins detected
by UV based on their MS and MS–MS
spectra.
For the best MS sensitivity, when
analyzing peptides and proteins, positive
ionization is preferred in combination
with an acidic mobile phase. This
is also the reason why we use high
pH reversed-phase LC (ammonium
bicarbonate pH 8.2) as the first
dimension in our reversed-phase LC ×
reversed-phase LC setup. Fractions of this
separation are then transferred to acidic
conditions, which is ideal for positive
ionization with electrospray.
Q. The method you developed showed
a particular aptitude for identity,
purity, and comparability assessments
of biopharmaceuticals and
biosimilars.1 Are there any limiting
factors in the use of this method?
A: The main practical limitations of 2D LC
lie in the compatibility of the dimensions
and column availability. For the analysis
of peptides and proteins a combination of
reversed-phase LC × reversed-phase LC or
strong cation exchange × reversed-phase
LC will hardly ever disappoint. While
compatibility in this case is not an issue,
in other combinations the efforts required
to overcome incompatibility between two
dimensions (for example, flow splitting,
mobile phase addition, combinations,
trapping) could make the technique less
accessible for routine use. That is why
we generally start with reversed-phase
LC × reversed-phase LC or strong
cation exchange × reversed-phase LC
combinations.
In some cases it can be problematic to
find a column with suitable dimensions, in
Q&A: Vanhoenacker
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second dimension column. For this reason,
the choice of a stable stationary phase with
a slightly larger particle size, and thus end
frits, could be justifi ed. The small loss in
effi ciency will be largely compensated for
by the improved robustness and column
lifetime. In my opinion the ideal column for
second dimension for biopharmaceuticals
and protein digests is about 2 mm to 3 mm
wide and 30 mm to 50 mm long packed
with 3.5-μm particles. The use of a larger
internal diameter increases loading capacity
from the fi rst dimension but requires
high fl ow rates to keep the analysis time
low enough (typically 3–5 mL/min). Such
high fl ow rates lead to signifi cantly high
solvent consumption (several hundred mL/
analysis), which is a disadvantage from an
environmental as well as an economical
point of view.
Of course, the price of 2D LC
instrumentation and consumables and
dedicated software is also something
to keep in mind. The dedicated data
analysis software for multidimensional
chromatography is powerful and very
useful, but it is not as developed as
software developed for protein and
peptide characterization and identification.
Both software platforms often have to be
used in combination, which can make data
analysis more complicated.
Q. Are you planning to develop this
methodology further and perhaps
attempt to address some of those issues?
A: We have recently published an
application note on strong cation
exchange × reversed-phase LC analyses of
E.coli tryptic digest and intact proteins.3
Strong cation exchange conditions were
optimized compared to the published data
on the mAb digests. With the optimized
conditions we were able to generate a
practical peak capacity of about 2250 in
less than 4 h. This is outstanding and very
useful for complex samples such as large
biomolecule digests.
We have also recently used 2D LC in
host cell protein (HCP) characterization
and in determining the pharmacokinetic
properties of antibody fragments. An
interesting overview of the potential
of 2D LC for the analysis of mAbs has
recently been provided by our group.4
The use of heartcutting techniques
has already proven very useful for
hyphenating methods with inorganic
mobile phases with MS after desalting
on the second dimension separation. A
typical application is identification of
impurities and unknowns detected in the
first-dimension separation. We are now
also investigating the use of heartcutting
for large biomolecules. Several separation
E-mail: [email protected]: www.richrom.com
principles commonly used for protein
characterization (reversed-phase LC,
strong cation exchange, size-exclusion
chromatography [SEC], hydrophobic
interaction chromatography [HIC]) can
be used in the first dimension and
well-defined fractions or peaks can be
transferred on-line and analyzed using
reversed-phase LC–DAD–MS.
Multi-dimensional LC is very powerful
and flexible and new ideas will always
surface as applications are developed. I
am convinced that many applications will
follow in the near future.
References
1. G. Vanhoenacker, I. Vandenheede, F. David,
P. Sandra, and K. Sandra, Analytical and
Bioanalytical Chemistry 407(1), 355–366
(2015).
2. K. Sandra and P. Sandra, Advances in
Biopharmaceutical Analysis 28(s10)
(2015).
3. G. Vanhoenacker, K. Sandra, I. Vandenheede,
F. David, and P. Sandra, Agilent Application
Note 5991-5179EN (2014).
4. K. Sandra and P. Sandra, Bioanalysis 7(22),
2843–2847 (2015).
Gerd Vanhoenacker is
the LC Product Manager
at the Research Institute
for Chromatography
(RIC) in Kortrijk,
Belgium. He studied
Pharmaceutical Sciences
at the Katholieke Universtiteit Leuven and
obtained a Ph.D. degree in Pharmaceutical
Sciences from the Ghent University in
2004.
He is (co-)author of over 40 scientific
papers covering different areas of
separation science. His expertise
includes liquid chromatography (HPLC,
UHPLC), liquid chromatography–mass
spectrometry (LC–MS), supercritical
fluid chromatography (SFC), capillary
electrophoresis (CE), and sample
preparation.
In recent years a significant part of
his activities have included method
development for 2D LC. He has practical
experience with 2D LC for the analysis of
a variety of samples.
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Eastern Analytical Symposium Summary
The 54th Eastern Analytical Symposium and Exhibition was
once again held at Garden State Exhibition Centre in Somerset,
New Jersey, USA, and proved to be another exceptional
meeting of analytical chemists. There was a wide and varied
programme of presentations, short courses, and posters
on offer with a notable plenary lecture by Nobel Laureate
Professor Kurt Wüthrich. The 2015 EAS President Oscar Liu
described the Nobel Laureates talk as “inspiring” and was
pleased with the “enthusiasm and energy of participants
[who] kept the level of engagement high”. However, the EAS
does not just exist to provide a venue for cross-disciplinary
knowledge sharing, career development, and networking,
but also to recognize those individuals who have advanced
their respective fields in an exceptional manner. The following
individuals were all deemed to have enhanced their fields of
study with remarkable work:
2015 American Microchemical Society Benedetti Pichler Award — Apryll Stalcup, Dublin City University, Ireland.
2015 EAS Award for Outstanding Achievements in Mass Spectrometry — Emile A. Schweikert, Texas A&M University, USA.
Outstanding Achievements in the Fields of Analytical Chemistry Award — Professor Chris Enke, Michigan State University, USA.
Outstanding Achievements in Separation Science Award — Professor David S. Hage, University of Nebraska, USA.
Pattern Modulation Offers Alternative to Pulse Modulation in GC×GC New research into fl ow modulation methods in valve-based two-dimensional gas chromatography (GC×GC) has produced
an effective alternative to traditional pulse modulation.1 Described as “pattern modulation”, this new method increases
effl uent to the secondary column with fl ow rates compatible with most chromatographs and spectrometers.
Researcher John Seeley from Oakland University in Rochester, Michigan, USA, said the new approach is easy to
implement with existing instrumentation. “Pattern modulation can be produced with the exact same valve-based hardware
used to conduct conventional pulse modulation separations”, he said, “and requires only simple software commands”.
Standard gas chromatographs require a modulator to produce comprehensive separations. These modulators convert
effl uent peaks emerging from the primary column into a series of sharp pulses injected into the secondary column.
However, pulse generation with valve-based modulation requires a large increase in secondary column fl ow rate or only a
small amount of primary effl uent being transferred to the secondary column.
Today, most GC×GC separations are performed with thermal modulation, but that is an expensive approach. As Seeley
commented, “When cost is not a factor thermal modulation will provide the best performance. But when resources
are tight, valve-based modulation in all of its forms can be an extremely effective tool for generating high-resolution
separations.”
Unlike pulse modulation, where narrow pulses are injected, pattern modulation uses an intricate injection pattern.
This approach allows the majority of the primary effl uent to reach the secondary column. However, the
detector signal generated from this process must be transformed to extract a conventional
pulsed signal. This is key to the analysis.
Initial results using pattern modulation were incredibly positive, but the research
recognizes that there is a point where the complexity of the sample can overwhelm
the signal transformation process. At this point, traditional pulse modulation is
preferred.
Currently, Seeley and his team are trying to establish quantitatively when
pattern modulation is superior to pulse modulation. He said, “We want
to be able to recognize when pattern modulation is the best alternative
for producing valve-based GC×GC separations”. — L.B.
Reference
1. J.V. Seeley and S.K. Seeley, J. Chrom. A 1421, 114–122 (2015).
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Metabolomic Detection of Early-StageOvarian CancerA detection method for early-stage
ovarian cancer has been developed using
ultrahigh-pressure liquid chromatography–
high-resolution mass spectrometry (UHPLC–
HRMS) in combination with tandem
MS–MS.1 Ovarian cancer is the biggest
killer of all gynaecological cancers and the
fi fth-leading cause of death among women
in the United States. Current screening
methods are inadequate, requiring lengthy
procedures that are inaccurate.
A considerable challenge to researchers,
ovarian cancer is “almost always
asymptomatic in the early stages,” noted
researcher John McDonald of the Georgia
Institute of Technology in Atlanta, USA.
Survival rates for late-stage ovarian cancer
is low but if the disease is detected early,
overall survival rates increase dramatically
(>92%). “Being able to accurately detect
the disease at early stages will make a big
difference,” said McDonald.
In developing the new method,
researchers used UHPLC–MS and MS–MS
in combination with a customized support
vector machine to identify 16 diagnostic
markers that produced an accuracy level
of 100% within the test group. “We are
currently initiating studies to determine if our
test can prospectively detect ovarian cancer
at early stages in high risk women, for
example women who are BRCA positive,”
said McDonald, referring to mutations of
the BRCA 1 and BRCA 2 genes, which are
associated with higher risks for breast and
ovarian cancer. “The frequency of ovarian
cancer onset in these women is quite high,
making this a good cohort in which to test
the clinical utility of our test,” he continued.
The study results also provide evidence
for the importance of lipid and fatty acid
metabolism in ovarian cancer. “It’s still not
completely clear what the signifi cance is
of the observed changes in lipid and fatty
acid metabolism in ovarian cancer patients,”
McDonald told The Column. “However, there
is a growing body of evidence indicating
that these changes may play a regulatory
role in fostering a cascade of molecular
changes that promote the development of
ovarian cancer.” It is hoped that targeting
the enzymes involved in lipid and fatty acid
metabolism will be a promising new area for
ovarian cancer therapy in the near future.
The research team are currently planning
to expand this study to include a wider
range of ethnic and racial groups because
of a recognized lack of diversity within
the test group. The learning algorithm
approach used by the team to identify
the biomarkers is designed to identify
which metabolites are optimally predictive
of disease among the group of women
analyzed in the study. “While we strove
to include women from broad geographic
areas in our study, not all ethnic and racial
groups were represented,” he said. “Thus,
at this point, we cannot be assured that
the 16 biomarkers identified in our study
will be 100% accurate in predicting early
ovarian cancer across all women.” This
may prove difficult because ovarian cancer
is rarely identified at the early stages and
therefore serum samples are extremely
rare. — L.B.
Reference1. D.A. Gaul et al., Sci. Rep. 5, 16351; doi: 10.1038/
srep16351 (2015).
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News In BriefLCGC TV Highlights
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A new detection method has been developed for gas chromatography (GC) by researchers from The University of Texas. Vacuum ultraviolet (VUV) detection features full spectral acquisition in a wavelength range of 115–240 nm, where virtually all chemical species absorb. The technique has real world applications in the analysis of fatty acid methyl esters (FAME). The research details the advantages of the detection method and profiles several food oil samples to demonstrate its applicability. doi:10.1016/j.foodchem.2015.08.004
Researchers have investigated the opportunistic foodborne pathogen Cronobacter sp. using gas chromatography–mass spectrometry (GC–MS) to understand its biofilm formation mechanism. Cronobacter sp. are linked to high mortality in neonatal and premature infants as a result of an association with powdered infant formula. Forming a biofilm enables survival in harsh conditions and the resistance of cleaning agents. doi:10.1016/j.foodcont.2015.11.029
Researchers from the University of Seville, Spain, have developed an efficient extraction, clean-up, and analysis method for the determination of environmental contaminants in human placental tissue. Perfluorinated compounds (PFCs) were quantified using UHPLC–MS–MS from 25 randomly selected women. PFCs are a public health concern because of their persistence, bioaccumulation, and toxicity.doi:10.1016/j.talanta.2015.12.020
The LCGC Blog: In Defense of Nitrogen as a Carrier for Capillary GC — There has been much
written about the use of nitrogen as a carrier gas for capillary GC. Formerly, to say it wasn’t any good.
Latterly to say that it’s pretty good and a better alternative to helium than hydrogen from a practicality
standpoint. Read Here>>
Are You Getting the Most Out of Your HPLC Column? — This article provided guidance for
working with the low-dispersion, small-volume columns that were gaining popularity in 2003. These
considerations are still appropriate today with the short, narrow HPLC and UHPLC columns now in
vogue. Read Here>>
What Is “Dead” Volume and Why Should Chromatographers Worry About It? — Dead-volume
effects can cause serious trouble for chromatographers. But if you understand what dead volume is and
how it affects chromatographic results, you can take control of it. Read Here>>
LCGC TV: Bob Kennedy, Part 4: Analytical Chemists in the “Omics” WorldYes, the LC in LC–MS matters in metabolomics and proteomics — it’s not all about the MS. Bob Kennedy discusses how to be an analytical
chemist in a world of biological data.Watch Here>>
LCGC TV: Joe Glajch on Biosimilars, Part 3: Strategies for Demonstrating Similarity of BiologicsHow fully can we characterize a biopharmaceutical? Joe Glajch offers strategies for demonstrating similarity, including statistical approaches, and what
the fi rst US approval of a biosimilar tells us about the FDA’s thinking.Watch Here>>
News The Column www.chromatographyonline.com
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Reproducibility of Research — Do We Have a Problem Houston?Incognito talks about reproducibility in research.
I’ve been trying this week to reproduce some
experiments from a paper by a well-known
research group, and whilst I have results
(fi nally), they would appear to be pointing to
a very different conclusion than that drawn by
the paper’s authors. My aim was to start by
reproducing the results from the paper and
then trying to adapt the methodology to use
a different sampling technique to improve the
sensitivity of the method — a situation, the
like of which, I’m sure, happens globally on a
daily basis. However, as I have found several
times in my career, I was unable to reproduce
the original experiments and therefore unable
to validate my starting position for the new
experiments.
So, one of two things is true: the original
research and data is fl awed, or I am not
capable of replicating that data because of
fl aws in my own experimentation.
I’m unable to tell which is true here — but I
do know that I have wasted a couple of days’
work. The original paper was diffi cult to follow,
with what I thought to be several key variables
and pieces of methodological information
missing. I’m not blaming this — the issues
could well be with my own work — but I’m still
cross, whoever is to blame.
So cross in fact, that I went back to re-read
an excellent recent edition of Nature, regarding
the issues of reproducibility in scientifi c
publications.1 Not the statistical measure of
repeatability, rather the ability of another group
to repeat and substantiate the work of the
originators.
In modern research and development,
it’s all too easy to jump to conclusions and
fi nd patterns in what may otherwise be
considered to be random data, as so often we
have a vested interest in the data — a PhD
thesis, tenure, further funding, advancing a
commercial project, maintaining the reputation
of the department (academic or industrial),
kudos, etc. This makes us sound like a
thoroughly unscrupulous lot, but that’s not
what I’m alleging.
Psychologist Brian Nosek of the non-profi t
Center for Open Science in Charlottesville,
Virginia, USA, which works to increase the
reproducibility of scientifi c research, puts it
much better than I can: “As a researcher,
I’m not trying to produce misleading results, Ph
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but I do have a stake in the outcome.”2 I’m suggesting then that we might be pre-disposed to certain outcomes in our work which leads to actions and decisions that do not give a true re� ection of what the data is telling us, or that we can choose those experiments which lead us to substantiate our theories at the exclusion of other, more rigorous, experiments.
So, what can be done, and what does Nature tell us about what analytical science can do to avoid fooling itself and wasting time?
Well � rst of all, science has always operated on the postulation of a theory or conclusion from experimentation, which has then been repeated and validated or refuted by other groups who will go on to expand upon the original work or postulate an alternative theory. Fine — that’s how things work, but there has been some shocking failures at reproducing academic research on a large scale, which brings into question how much time and resource is wasted producing meaningless data that does not advance science and in fact may even be holding up the good research.
In 2012, researchers at biotechnology � rm Amgen in Thousand Oaks, California, USA, reported that they could replicate only 6 out of 53 landmark studies in oncology and haematology.3 In 2009, workers at the Meta-Research Innovation Center at Stanford University in Palo Alto, California,
described how they had been able to fully reproduce only 2 out of 18 microarray-based gene-expression studies.4
So, what can be done?
Be More Rigorous with Academic PublicationsHere’s the advice to authors on Materials and Methods in a leading academic journal:5 Provide suf� cient detail to allow the work to be reproduced. That’s it. Enough? I really don’t think so. We need checklists to ensure even the most esoteric details are addressed so that reproduction is possible. Checklists would include minor experimental details, experimental design, method performance via statistical analysis, etc. Is there a restriction on space or content for method details? If so — abolish it. What about electronic submission of raw data? In review — blind the author’s names and institutions to avoid bias or deference.
How about replicating papers prior to publication? I can hear the gasps from readers already, but why not? Set aside funding, allow publication of replication studies (more publications for sound science), give replicated publications more research credits, establish replication groups. Where advanced equipment is used, allow replicators to take charge of original equipment to repeat experimentation. Non-replication needn’t
Incognito
1313
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www.gerstel.com
Sample Prep Automation
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Derivatization,
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The Column www.chromatographyonline.com
methods and design prior to any practical
work, perhaps with an “in-principle”
promise of publication. It’s not really any
different to having a methods scrutiny panel
or a good laboratory manager in industry!
t� Adversarial Collaboration — Invite your
rivals to work with you, or on a competing
hypothesis, which will ultimately result in
a joint publication. For all of you thinking,
“They will never agree on enough results
or conclusions to write a joint paper”,
ask yourself if its healthy to have beliefs
so entrenched that pure experimentation
and data analysis cannot be used to prove
or disprove either side’s argument. These
entrenched beliefs are prevalent and deeply
unhealthy.
t� Blinding — There are many ways to “blind”
our data analysis. Mixing up columns in
databases, introducing dummy or biased
data, and mixing up data into different
sets are all possible. Once the data have
been analyzed, the blinds are lifted to
see if the conclusions remain valid. The
obvious example is the use of “placebo”
medications in clinical trials or bioanalytical
work, where the placebo subjects are not
revealed until after the data analysis.
I feel that writing a clever conclusion
to this piece will do nothing other than
detract from the thoughts and theories
contained within. I’d just ask you to
consider if you recognize, in your own
work, any of the problems or biases
presented and if you agree or disagree
with the proposed solutions. Just having
you think about the issue of reproducibility
is a good start; if this piece then allows
you to adopt measures to avoid any of the
problems, then even better.
Oh, and by the way, these issues don’t
just apply to research in an academic
environment!
References
1. Nature 526(7572), 164–286 (2015).
2. Regina Nuzzo, Nature: http://www.nature.com/
news/how-scientists-fool-themselves-and-how-
they-can-stop-1.18517
3. C.G. Begley and L.M. Ellis, Nature 483,
531–533 (2012).
4. J.P.A. Ioannidis et al., Nature Genet. 41,
149–155 (2009).
5. https://www.elsevier.com/journals/journal-
of-chromatography-a/0021-9673/guide-for-
authors
Contact author: IncognitoE-mail: [email protected]
necessarily disbar work from publication, but
some notifi cation of the failure to reproduce
should be made. This approach would
foster and encourage collaboration as well
as introducing rigour and help to underpin
good science. The most I ever learn about
our science is when troubleshooting issues
that arise from method transfers to client
laboratories — would this be any different?
Recognize Cognitive Bias and Build in
Safety Measures
There are many ways in which we can fool
ourselves or have underlying bias in our work.
Even the most ethical of researchers are
susceptible to self-deception; outlined below
are a few of the reasons why:
t� Hypothesis Myopia — A natural inclination
to favour only one hypothesis and look for
evidence to support it, whilst playing down
evidence against it and being reluctant
to adjust or propose more than one
hypothesis.
t� Sharpshooter — Fire off a random series
of shots, then draw a target around the
bullet holes to ensure the highest number
of bullseyes. Getting some encouragement
from your on-going experimental data and
deciding that this must be the correct path
to go down, without realizing that the
data could actually support many different
conclusions from the one you are drawing.
t� Asymmetric Attention (Disconfi rmation
Bias) — Giving the expected results smiling
approval, whilst unexpected results are
blamed on experimental procedure or
error rather than being accepted as a true
challenge to your hypothesis.
t� Just-So Storytelling — Finding rational
explanations to fi t the data after the fact.
The problem is, we can fi nd a story to fi t
just about every type of data — it doesn’t
mean to say the story is true! Also known
as JARKing — “justifying after the results
are known” — because it’s really diffi cult to
go back and start again once we are at the
end of the process.
t� The Ikea Effect — Everyone has a vested
interest in loving the furniture they built
themselves. Is it the same with our
analytical data?
So what strategies might we employ to
overcome these innate biases?
t� Strong Inference Techniques — Develop
opposing or competing hypotheses and
develop experiments to distinguish which is
correct. Not having a favourite child avoids
Hypotheses Myopia and cuts down on the
need for Just-So Storytelling.
t� Open Science — Publication of methods
and raw data for various groups to
scrutinize. Even more radical — publish
and seek approval or revision of research
Incognito
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Extending the Detection Limits for the Analysis of Organotin Contaminants Using Soft Ionization
The inherent sensitivity and selectivity of time-of-fl ight mass spectrometry (TOF-MS) can be augmented by soft electron ionization (EI) to provide ultratrace-level quantitation of organotins in complex environmental extracts. These organotin species are a focus of current concern as environmental contaminants, but analysis using conventional 70 eV ionization energies is made diffi cult by their propensity to undergo extensive fragmentation. The use of soft EI helps to solve this problem by producing simplifi ed spectra with enhanced diagnostic ions.
Laura McGregor, Steve Smith, and David Barden, Markes International, Llantrisant, Wales, UK.
Organotins (stannanes) are anthropogenic
chemicals that are attracting attention from
environmental analysts because of their
high toxicity and ability to interfere with
the endocrine system, along with their
persistence in the environment. Use of
organotins in anti-fouling paints fi rst caused
concern in the 1970s as a result of a decline
in populations of marine molluscs. The use
of such paints has now been restricted or
banned by many countries, but other sources
remain, such as PVC products, disinfectants,
and agricultural pesticides.1
The current annual average of organotins in
water, as stated by the EU Water Framework
Directive, is just 0.2 ng/L, with a maximum
allowable concentration of 1.5 ng/L.2
This means that highly sensitive detection
methods are required, with environmental
analysts constantly seeking improvements to
instrumentation and technological advances
that could lower reporting limits.
Time-of-fl ight mass spectrometry (TOF-MS)
offers signifi cant advantages in such
scenarios, with instruments using direct
extraction (rather than the inherently less Ph
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effi cient orthogonal extraction) providing
a distinct sensitivity advantage. However,
certain analytes remain challenging even with
such technologies because of their propensity
to fragment extensively at conventional
(70 eV) ionization energy.
This problem can now be addressed by the
use of a soft electron ionization (EI) technique
that offers the advantages of soft ionization
for gas chromatography coupled to MS
(GC–MS), without any of its historic
disadvantages — such as hardware
changes and additional reagent gases. The
technique uses an ion source design in the
TOF instrument that allows the ionization
energy to be varied on a sliding scale from
conventional 70 eV to lower energies.3 The
physical properties of most small molecules
means that relatively small differences in
ionization energies between 10 eV and
20 eV can have signifi cant differences in the
fragmentation pattern. Typically, however, an
ionization energy of about 12–14 eV retains
an useful degree of fragmentation while
avoiding a loss in sensitivity relating to the
unavoidable drop-off in ionization effi ciency
as the ionization potential is approached.
This technique has already been applied
to analytes ranging from petrochemical
hydrocarbons4 to emerging environmental
contaminants.5 This article explains how this
soft ionization method can be applied to
organotin compounds. These are particularly
challenging because of the requirement for
high sensitivity to meet the demands of the
low detection limits, in conjunction with their
extensive fragmentation at 70 eV making
speciation diffi cult. Using soft ionization
provides enhanced “diagnostic” ions and
reduced chemical noise, which leads to
lower detection limits, as well as simplifi ed
spectra for more confi dent qualifi cation. A
further boost in sensitivity is demonstrated
by reduced ionization of common
background/carrier gases at low ionization
energies, leading to minimal chemical noise
and improved signal-to-noise ratios for
compounds of interest.
Experimental
Samples: A selection of some of the most
common organotins were analyzed in this
work: dibutyl tin (Bu2SnH2), tributyl tin
(Bu3SnH), monooctyl tin ([C8H17]SnH3),
and tetrabutyl tin (Bu4Sn). Tri-substituted
organotins such as tributyltin are used as
pesticides,6 while mono- and di-substituted
organotins are used as PVC stabilizers,
as catalysts, and in glass coatings.7 The
lower-substituted organotins can also be
formed in the environment by metabolism
and degradation of higher-substituted
analogues.6 Environmental water samples
were spiked with organotins (50 ng/L) and
ethylated with sodium tetraethylborate
prior to extraction to make the tin hydrides
suffi ciently volatile for analysis by GC–MS. A
dilution series (ranging from 0.1–20 ng/L) was
prepared from the stock solution.
GC: Carrier gas: Helium, constant fl ow at
0.9 mL/min; mode: splitless for 2.0 min (then
150 mL/min purge); temperature: 200 °C;
septum purge: on, 3 mL/min; column:
20 m × 0.18 mm, 0.18-μm Rxi-5Silms
(Restek); oven: 50 °C (2.5 min), 20 °C/min to
300 °C (1 min); total run time: 16 min.
Barden et al.
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(a)
Retention time (min)
Retention time (min)
Inte
nsi
ty (
x 1
04 c
ou
nts
)In
ten
sity
(x 1
03 c
ou
nts
)
15
10
5
04 6 8 12
(C8H17)SnEt
Bu4Sn
Bu3SnEt
Bu2SnEt2
1200
900
8.75 8.80 8.85 8.90
600
300
6
2
1
08 10
0
1410
(b)
Figure 1: GC–TOF-MS analysis of the 5 ng/L spiked sample: (a) TIC and (b) EIC (m/z 263+291) highlighting the organotin targets. The inset shows the excellent peak shape for (C8H17)SnEt.
The Column www.chromatographyonline.com
TOF-MS: Instrument: BenchTOF-Select
(Markes International); fi lament voltage: 1.8 V;
ion source: 250 °C; transfer line: 280 °C; mass
range: m/z 40–500; data rate: 4 Hz.
Software: TOF-DS software for BenchTOF
(Markes International) was used for full
instrument control and data analysis.
Results and Discussion
Figure 1 shows the GC–TOF-MS analysis of
the 5 ng/L spiked sample, with an extracted
ion chromatogram (EIC) indicating the elution
order and excellent peak shape and intensity
for the organotin targets, even at this trace
level (5 pg on-column).
Spectral Quality: The spectra obtained for
each of the organotin target compounds
at 70 eV and 14 eV are compared in
Figure 2. The results show that softer
(14 eV) ionization simplifi es the spectra
and increases the intensity of the higher
m/z ions. This is important because these
larger fragments are useful for determining
compound structure.
Furthermore, the preservation of a degree
of fragmentation provides more information
than other soft ionization techniques (such
as chemical ionization) that may produce
spectra containing solely the molecular ion.
Library-searching can therefore be performed
Barden et al.
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100(a) (b)
70 eV 70 eV
14 eV
70 eV 70 eV
14 eV14 eV
14 eV
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
41
41
41
41
56
56
121
121
151
121
121
Bu2SnEt2151
151
151
Forward match 904Reverse match 911
Bu3SnEtForward match 907Reverse match 917
(c) (d)C8H17SnEt3
Forward match 911Reverse match 916
Bu4SnForward match 902Reverse match 923
179
179
179
179
179
149
179179
177
207
207
207
207
235
235
235
235
207
207
263
263
263
235
263
291
291
291
291
291
291
41
56
56
83
83
Figure 2: Spectral comparisons for the organotin targets from the 5 ng/L standard, at 70 eV (top) and 14 eV (bottom), illustrating the simplifi ed spectra and enhanced selectivity achieved by soft ionization. Forward and reverse match factors against the NIST 14 database are indicated. Note that for Bu2SnEt2 and Bu3SnEt m/z 207 is a prominent ion at 70 eV, but because it is a common interference ion from column bleed, it would not be ideal for quantitation.
14 eV
Retention time (min)
Inte
nsi
ty (
x 1
03 c
ou
nts
)
5
4
3
2
1
0
70 eVBu2SnEt2
Bu3SnEt2
C8H17SnEt3
Bu4Sn
7.5 8.0 8.5 9.0
Figure 3: GC–TOF-MS EIC (m/z 263+291) overlays for the organotin targets at 70 eV (blue) and 14 eV (red) for the 5 ng/L spiked sample, showing the improvement in peak intensity achieved at low ionization energy.
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at low eV to add another level of confi dence
in compound identifi cation.
Figure 3 shows the EIC (m/z 263+291)
overlays for 14 eV and 70 eV, illustrating
the improved detection limits at low eV.
Note the reduced baseline at 14 eV, as a
result of reduced ionization of common
background/carrier gases, further improving
signal-to-noise ratios for the peaks of interest.
Linearity: The set of derivatized organotins
was analyzed at six dilution levels by GC–
TOF-MS at ionization energies of 70 eV
and 14 eV. The resulting calibration curves
(Figure 4) display excellent linearity at both
ionization energies, with all R2 values
over 0.997. Note in particular the steeper
calibration curves at 14 eV, which result
in increased analyte response factors and
therefore lower quantitation limits.
None of the organotins at the lowest
dilution level (0.1 ng/L) could be reliably
detected at 70 eV (and so this data point is
Barden et al.
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Inte
nsi
ty (
cou
nts
)In
ten
sity
(co
un
ts)
(a)
1 ng/L
0.1 ng/L(b)
400
60
40
20
0
8.30 8.35 8.40
14 eV
14 eV
70 eV
70 eV
300
200
100
0
Retention time (min)
8.30 8.35 8.40
Retention time (min)
Figure 5: GC–TOF-MS EIC (m/z 291) chromatograms showing overlays of the 70 eV and 14 eV analyses of Bu3SnEt at (a) the 1 ng/L level and (b) the 0.1 ng/L level.
5
4
3
2
1
Bu2SnEt2 Bu3SnEt(a) (b)
(c) (d)
Pe
ak
are
a (
x 1
04 c
ou
nts
)P
ea
k a
rea
(x
10
4 c
ou
nts
)
Pe
ak
are
a (
x 1
04 c
ou
nts
)P
ea
k a
rea
(x
10
4 c
ou
nts
)
14 eV
70 eV
14 eV
70 eV
14 eVR2=0.9991 R2=0.9984
R2=0.9974R2=0.9990
R2=0.9987
R2=0.9995
R2=0.9981
R2=0.9996
Concentration (ng/L)
70 eV
14 eV
70 eV
0
0
1
2
3
4
5
6
7
0 5 10 15 20
Concentration (ng/L)
0 5 10 15 20
Concentration (ng/L)
0 5 10 15 20
Concentration (ng/L)
0 5 10 15 20
3
2
1
0
4
2
0
6
8
10
C8H17SnEt3 Bu4Sn
Figure 4: Calibration curves for the organotin targets at both 70 eV (n = 5) and 14 eV (n = 6).
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not shown in Figure 4), but the enhanced
sensitivity at 14 eV easily enabled detection
at this level (Figure 5).
Conclusion
This article has demonstrated the
performance of this GC–TOF-MS method
for trace-level detection and quantitation of
organotins in complex matrices.
The soft ionization technology used
provided a level of sensitivity and selectivity
not achievable with other methods, and
spectra exhibiting enhanced diagnostic
ions for all organotins at 14 eV. Excellent
linearity was achieved (R2 > 0.997) for
both conventional (70 eV) and soft (14 eV)
ionization. Moreover, the enhancement in
quantitation ions and reduced background
at 14 eV enabled detection limits to be
extended down to just 0.1 pg on-column for
all organotins in this study.
References
1. H.K. Okoro, O.S. Fatoki, F.A. Adekola, B.J.
Ximba, R.G. Snyman, and B. Opeolu, in
Reviews of Environmental Contamination and
Toxicology, (vol. 213, ch. 2, 2011) pp. 27–54.
2. Directive 2000/60/EC of the European
Parliament and of the Council of 23 October
2000 establishing a framework for Community
action in the fi eld of water. See http://
ec.europa.eu/environment/water/water-
framework/index_en.html
3. L. McGregor, N. Bukowski, and D. Barden,
Current Trends in Mass Spectrometry,
supplement to LCGC North America, LCGC
Europe, and Spectroscopy, 12(1), 16–19 (2014).
4. L. McGregor and D. Barden, Analyzing
crude oil: Improving compound speciation,
Hydrocarbon Engineering, July 2014, http://
www.energyglobal.com/downstream/gas-
processing/31072014/Crude-oil-markes-
analysis/
5. L. McGregor, A. Gravell, I. Allan, G. Mills,
D. Barden, N. Bukowski, and S. Smith, The
Analytical Scientist April 2015.
6. S. Dobson and R. Cabridenc, Tributyltin
compounds (Environmental Health Criteria 116),
World Health Organization (1990).
7. S. Dobson and P.D. Howe, Mono- and
disubstituted methyltin, butyltin, and octyltin
compounds (Concise International Chemical
Assessment Document 73), World Health
Organization (2006).
Laura McGregor received an M.Chem.
in chemistry from the University of St
Andrews, UK, followed by an M.Sc.
in forensic science at the University
of Strathclyde, UK. Her Ph.D. in
environmental forensics, also at the
University of Strathclyde, focused on the
chemical fingerprinting of environmental
contamination using advanced techniques
E-mail: [email protected]: www.markes.com
such as GC×GC−TOF-MS. Laura joined
Markes International in 2013 as a sales
support specialist, and is now product
marketing manager for Markes’ TOF-MS
product portfolio.
Steve Smith studied in Bristol, UK,
for both his B.Sc. and Ph.D., which he
obtained in 2008 on innovative work
profiling volatile organic compounds for
disease diagnosis. Following post-doctoral
positions at the University of the West
of England and Bristol University, Steve
joined Markes International as a senior
applications specialist for thermal
desorption and TOF-MS in 2011, where he
now specializes in GC×GC−TOF-MS.
David Barden is a technical copywriter
at Markes International, having joined
the company in 2011. David studied
natural sciences at the University of
Cambridge, UK, and remained there for
his Ph.D. in organic chemistry, which
he received in 2003. A placement at
the European Journals Department of
Wiley-VCH, Weinheim, Germany, was
then followed by seven years in journal
publishing at the Royal Society of
Chemistry, UK.
Barden et al.
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The LCGC Blog: Column Overload in Gas Chromatography with Vacuum Ultraviolet Detection
Column overload is a very commonly encountered issue in gas chromatography (GC) for beginners. Changes in peak symmetry, generally observed as peak fronting, can be subtle in the sharp peaks generated by GC, but the result can be signifi cant shifts in retention times, loss of resolution, and error in peak integration. LCGC Blogger Kevin Schug explains more.
Kevin A. Schug, University of Texas Arlington, Texas, USA.
Split injectors were invented to ensure that
wall-coated open-tubular capillary gas
chromatography (GC) columns are not
overloaded. Because it is not practical to
reduce actual injection volumes much lower
than tenths of microlitres and the capacity
of thin-fi lm stationary phases coated on the
capillary wall surface should not be exceeded,
the ratio of gas fl ows in the injection port
directed through the column versus to waste
(through the split vent) is adjusted to set an
appropriate split ratio. Split ratios are generally
reliable between 10:1 and 400:1 (where the
majority of analyte is split to waste) to ensure
that the target analyte of interest exhibits good
peak symmetry. In fact, column overload is
a very commonly encountered issue in GC
for beginners. Changes in peak symmetry,
generally observed as peak fronting, can
be subtle in the sharp peaks generated by
GC, but the result can be signifi cant shifts in
retention times, loss of resolution, and error in
peak integration. Traditionally, it is preached
that column overloading conditions should be
avoided for those reasons; however, in cases
where the greatest sensitivity is desired to
perform ultratrace analysis, a splitless injection,
where all of the analyte is transferred onto the
Ph
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column (the split vent is closed for a minute or
two during the injection phase of the analysis),
can be performed. One can imagine that the
choice between the use of split or splitless
injection would be driven by monitoring peak
area and peak shape, and then choosing the
conditions that give a nice symmetric peak and
signifi cant signal for the target analyte over the
desired concentration ranges for analysis. In our
experience with the new vacuum ultraviolet
(VUV) absorption detector for GC, these
considerations may not be so critical.
A VUV detector was introduced into the
market in 2014.1 I have written previously
about the concept and the general operational
advantages it provides.2,3 Since then, we
have applied GC–VUV for the investigation of
permanent gases,4 pesticides,5 and fatty acids.6
We, as well as other groups around the world,
are currently looking at several other application
areas. The majority of VUV detectors to date
primarily have been sold in the petroleum and
petrochemical industries.
In short, the principle of VUV spectroscopic
detection is full-range absorption measurements
between 120 nm and 240 nm, where all
chemical entities absorb and have unique
gas-phase absorption signatures. VUV detection
is quite complementary to mass spectrometry
(MS), because it can well discern isomeric
(isobaric) species that are often indistinguishable
based on electron-ionization MS spectra. Further,
standard concepts from Beer’s law apply — the
magnitude of absorption is directly proportional
to the amount of analyte present and its
absorptivity (cross-section), and absorption for
overlapping signals is additive. This latter point
means that unresolved components can be easily
deconvolved if the reference spectra for each
are present in the VUV spectral library. A ton of
effort need not be placed in fully resolving all
peaks of interest. The strength of this capability
is impressive. For example, we are currently
drafting a manuscript on our effort to fully
speciate Arachlor (previously manufactured by
Monsanto) samples, which are complex mixtures
of polychlorinated biphenyl compounds (PCBs).
Each of the 209 PCBs has a unique spectrum —
they can be well differentiated from one another,
even if they chromatographically overlap.
Returning to the question at hand, the
deleterious effects of GC column overload
should be quite well handled for GC–VUV
analyses. Signifi cant peak fronting can
compromise resolution; it can cause overlap of
neighbouring peaks that would be very diffi cult
to deconvolve using any other GC detector. Yet,
we have shown in most of our applications that
two or three (and probably more are possible)
distinct analytes can be completely resolved from
coeluted peaks into their respective contributions
to the overall signal. Assigning the retention time
to the peak apex will cause the retention time to
shift more and more as overloading is increased.
This is not really a major problem, except for
the context of analyzing a number of samples
with a wide range of analyte concentrations.
In such cases, where some peaks might front
(high analyte concentration) and some might be
symmetrical (low analyte concentration), it would
be necessary to understand that there is potential
for the retention time to shift.
At some point, when larger and larger
amounts of analyte are eluted through the
column into the VUV fl ow cell, the absorption
signal might saturate the detector, especially
in regions of the absorption spectrum where
absorptivity of the molecule is very high.
However, because quantitative analysis is typically
performed through averaging the signal across
a wide wavelength range (in contrast to typical
UV quantitation, where quantitation is often
performed at a predefi ned peak maximum),
when one spectral feature goes off scale (that
is, it becomes saturated), the quantitation
performed by the VUV detector can be defi ned
based on absorption across less-absorbing
wavelengths in the spectrum. Because we know
the shape of the absorption spectrum for an
analyte across the full range of 120–240 nm,
and because the ratios of intensities for
electronic transitions across that range remain
constant, an off-scale response in one region
of the absorption spectrum does not matter.
It is a simple matter to model the shape of
the response of that off-scale region based on
the magnitude of response in other regions of
the spectrum. This treatment could effectively
increase the dynamic range of the detector.
These concepts have not been shown
empirically using GC–VUV; these are simply
my thoughts on the subject. However, there
is nothing to prevent methods from being
conceptualized where column overload is used
to achieve lower detection limits in GC–VUV
analysis. Currently, VUV detection is about as
sensitive as MS in a scan mode (50–200 pg
on-column LODs, depending on the analyte
chromophore). In truth, the main limitation might
actually be the volume of the injection port
liner and the thermal expansion of the various
sample solvents used. In other words, you can
only increase injection volume so high (unless
you become quite creative and experienced in
performing methodically slow splitless injections)
before the capacity of the liner will be reached
and vaporized sample solutions will overfl ow
into other parts of the injection port — an
undesirable situation. Of course, there are
large-volume injectors commercially available.
Some more obscure standard methods (I can
think of one for disinfection by-products by GC
with negative chemical ionization MS) require this
type of hardware for ultratrace detection limits.
Further, the dynamic range of the response for
the method can be extremely large, considering
that the strategy above can be used when
detector saturation is reached.
The LCGC Blog
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So, I need to get my students to try this
approach (or perhaps one of you readers with
access to a VUV system could try it): Prepare
a series of solutions ranging from absurdly
dilute analyte concentration up to some
concentrations a few orders of magnitude
higher. Use large volume splitless injection
to see how low you can go and how wide
the dynamic range of response could be. I
purposely use “dynamic range” here, because
nothing says that the response has to be linear
all the way up to high concentrations. As long
as the response changes in a predictable, even
if nonlinear, fashion across the concentration
range, then a calibration curve could be
constructed. The progression of peak shape
and size should go from small and symmetrical
up to very large and fronting. At some point,
the detector will saturate in part of the
wavelength range, but that problem can be
mitigated with some modelling of the response
using intensities in less-responsive regions of
the spectrum. There is some precedent for
this technique in the literature using lower
abundance isotope species in MS,7,8 but it is far
from commonplace. Dare I say, it would be fun
to try it and see what performance would be
like.
References
1. K.A. Schug, I. Sawicki, D.D. Carlton Jr., H. Fan,
H.M. McNair, J.P. Nimmo, P. Kroll, J. Smuts, P.
Walsh, and D. Harrison, Anal. Chem. 86,
8329–8335 (2014).
2. K.A. Schug, The LCGC Blog 11 September 2014.
http://www.chromatographyonline.com/lcgc/
Blog/The-LCGC-Blog-My-New-Obsession-Gas-
Chromatography-/ArticleStandard/Article/detail/853
093?contextCategoryId=50130
3. K.A. Schug and H.M. McNair, LCGC North
Am. 33(1), 24–33 (2015). http://www.
chromatographyonline.com/gc-detectors-thermal-
conductivity-vacuum-ultraviolet-absorption
4. L. Bai, J. Smuts, P. Walsh, H. Fan, Z.L. Hildenbrand,
D. Wong, D. Wetz, and K.A. Schug, J. Chromatogr.
A 1388, 244–250 (2015).
5. H. Fan, J. Smuts, P. Walsh, and K.A. Schug, J.
Chromatogr. A 1389, 120–127 (2015).
6. H. Fan, J. Smuts, L. Bai, P. Walsh, D.W. Armstrong,
and K.A. Schug, Food Chem. 194, 265–271 (2016).
7. H. Liu, L. Lam, L. Yan, B. Chi, and P.K. Dasgupta,
Anal. Chim. Acta 850, 65–70 (2014).
8. H. Liu, L. Lam, and P.K. Dasgupta, Talanta 87,
307–310 (2011).
Kevin A. Schug is an Associate Professor and
Shimadzu Distinguished Professor of Analytical
Chemistry in the Department of Chemistry &
Biochemistry at The University of Texas (UT) at
Arlington, USA.
E-mail: [email protected]: www.chromatographyonline.com
The LCGC Blog
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The 10th Balaton Symposium on High-Performance Separation Methods: A Review
A review of the 10th Balaton Symposium on High-Performance Separation Methods, which was held 2–4
September 2015 at the Hotel Azúr, Siófok, Hungary.
Ira Krull, Northeastern University, Boston, Massachusetts, USA.
The Hungarian Society for Separation
Sciences, headed by Professor Attila
Felinger (Department of Analytical and
Environmental Chemistry, University of
Pécs, Hungary) organized the 10th biennial
Balaton Symposium meeting, which was
held 2–4 September 2015 at the Hotel
Azúr, on the shores of Lake Balaton, Siófok,
Hungary. The Diamond Congress of Hungary
coordinated the symposium, which included
presentations from notable scientists,
plenary lectures, and keynote lectures. In
addition, awards were presented to student
posters that were deemed exceptional.
Technical sessions and poster presentations
were combined with a full social agenda
including meals with unique entertainment
from Zoltán Orosz, a Hungarian accordionist,
and a four-piece band and singer. The
symposium concluded with an evening
buffet-style barbeque by the outdoor hotel
pool, complete with drinks.
The biennial conference was attended
by 285 scientists, mainly from Europe,
but with a few attendees from the USA,
Canada, Asia, and Israel. In addition, there
was a very successful exhibition comprising
14 vendors exhibiting instrumentation,
ultrahigh-pressure liquid chromatography
(UHPLC) columns and accessories, and
supplies related to separation sciences of all
types.
The opening ceremony dedicated
the event to the late Professor Georges
Guiochon, who sadly passed away in
2014. Professor Guiochon was a signifi cant
sponsor and attendee at many past Balaton
Symposia. His wife, Lois Beaver, attended
and participated in the conference.
Following these initial proceedings, the
Halász Medal Award and the Csaba Horváth
Memorial Award were individually awarded
to Professors Janusz Pawliszyn and Peter
Schoenmakers, respectively.
The 2013 Halász Medal Award was
awarded to Professor Nobuo Tanaka, who
could not attend the Balaton Symposium
two years ago. The three awardees Ph
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presented their 30-minute award lectures
before the Thursday sessions began in
earnest. There was further respect and
remembrance for Professor Guiochon
throughout the subsequent sessions, with
many presentations made by his former
students and colleagues.
The emphasis throughout the conference
was on the latest innovative advances in
different areas of separations science. These
included gas chromatography (GC), UHPLC,
high performance liquid chromatography
(HPLC), supercritical fl uid chromatography
(SFC), high performance capillary
electrophoresis (HPCE), and variations
thereof, with numerous applications to
small molecules, biopharmaceuticals,
low-molecular-weight (MW)
pharmaceuticals, and other interesting and
important topics. The talks were generally
followed by meaningful questions for the
speaker, as well as discussions amongst
the attendees, often quite interactive and
thought-provoking. There was also a large
number of poster papers from separation
scientists at many universities and colleges
around the world. This was mainly an
academically oriented conference, but
there were also several presentations from
scientists from industry, instrument and
column vendors, and some government
laboratories.
Two parallel sessions were run
simultaneously over the course of the
conference. The fi rst day included the
aforementioned welcome and awards
ceremony for both the Halász and Horváth
Medals, followed by plenary talks by the
recipients and others to complete each
plenary session. Oral talks were arranged
so that they interrelated with a common,
general topic or theme, which was also
attempted with the poster papers (available
to view on all three days).
Several talks worthy of mention include
one by Gerard Hopfgartner of the University
of Geneva in Geneva, Switzerland, who
spoke on target and non-targeted LC–MS–
MS approaches including differential ion
mobility spectrometry to support -omics
studies. Another combined separations
presentation of interest was that of Dora
Palya of the Wessling International Research
and Education Center in Budapest, Hungary,
entitled “Miniaturized Silica Gel Column
Chromatography Combined with Large
Volume Gas Injection-Gas Chromatography–
Mass Spectrometry as a Chemical
Fingerprinting Method”, a truly hyphenated,
combined chromatography (2D) analytical
approach. A collaborator with Professor
Lindner’s group, Dr. Zs. Gecse of the Institute
of Pharmaceutical Chemistry at the University
of Szeged in Szeged, Hungary, presented
a joint talk on high-performance liquid
chromatographic enantioseparation of cyclic
beta-3-amino acids applying chiral stationary
phases based on cinchona-alkaloids.
This new and novel approach to chiral
recognition described new and improved
chiral stationary phases in HPLC for a wide
variety of natural product analytes. Several
talks included a separation step combined
with on-line MS, such as that by T. Baygildiev
and co-workers of the Department of
Chemistry at the Lomonosov Moscow State
University in Moscow, Russia, which dealt
with simultaneous hydrophilic interaction
liquid chromatography (HILIC) tandem mass
spectrometry methylphosphonic and alkyl
methylphosphonic acids determination
after derivatization with p-bromophenacyl
bromide. This talk dealt with the advantages
of interfacing HILIC-type separations on-line
with MS in terms of improved sensitivity,
lowered detection limits, and improved
identifi cations of trace level impurities,
if present. Finally, several talks centred
around the recent revival of supercritical
fl uid chromatography (SFC) — termed, at
times, as ultraperformance convergence
chromatography (UPC2). This talk was
titled: Chiral Recognition of Dapoxetine
Enantiomers Using Ultraperformance
Convergence Chromatography (UPC2),
and was presented by András Darcsi of
the Department of Pharmacognosy at
Semmelweis University in Budapest,
Hungary, and co-workers. The above is
but a sampling of the total programme
but indicates the breadth and thoroughness
of the meeting, which covered many areas
in analytical chemistry, with specifi c
applications of widespread interest and
importance.
The emphasis throughout was on
UHPLC, HPLC, and high effi ciency and
high throughput separations involving
the same. By and large, the majority of
oral presentations dealt with some form
of LC, involving newer column types
(core–shell, monolithic, charged surface
hybrid, and others), multidimensional
techniques (2D LC, 3D LC), and a few
featuring miniaturized separations (capillary,
nano, micro, and so forth). Many of these
talks dealt with possible applications
for low MW pharmaceuticals and/or
biopharmaceuticals, especially those
from the University of Geneva. One such
example was given by a current graduate
student of Jean-Luc Veuthey and Davy
Guillarme from the University of Geneva in
Switzerland; Aurélie Periat spoke about the
performance of hydrophilic interaction liquid
chromatography–mass spectrometry
(HILIC–MS) versus reversed-phase LC–MS for
the analysis of pharmaceutical compounds.
Balaton Symposium Review
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There was a great deal of emphasis on
HILIC approaches, and how this might be
interfaced with a second dimension, usually
reversed-phase LC. There were a few papers
that discussed preparative or process-scale
chromatography, and several that dealt with
SFC. Very few papers dealt with regulatory
issues or quality-by-design (QbD). There
were also very few orals dealing with
‘omics topics of any type, though, as above,
many dealt with both low and high MW
pharmaceutical analysis.
One disappointing aspect of this
conference was the lack of oral or poster
presentations dealing with any form of
MS. Given that MS has become a premier
separations approach, whether used alone
or hyphenated with an initial separation
step, and is now widely employed in most
industrial and academic laboratories, one
would have expected more MS papers at
this Balaton Symposium. Perhaps next time?
An interesting question came up at this
conference related to the defi nition of
supercritical fl uid chromatography (SFC).
A number of papers discussed the use of
CO2 as a mobile-phase additive, sometimes
in the form of a supercritical fl uid but
not always. In particular, there seems to
be some confusion about whether the
analytical instrumentation actually performs
SFC. The use of UHPLC instrumentation
to perform true SFC is possible only if the
amount of the organic modifi er is about
2–3% or less, with the pressure needed to
maintain a supercritical fl uid as the mobile
phase. However, more and more papers are
appearing in the literature, and at meetings
such as this, that do not really differentiate
between SFC and UHPLC using CO2 as
a modifi er. It is possible that with higher
and higher percentages of an organic
solvent, one is no longer dealing with a
true supercritical fl uid at all but rather, a
CO2-modifi ed UHPLC mobile phase of high
organic content that is performing HPLC.
In summary, this was a very up-to-date
scientifi c conference with quite high
standards, mostly aimed at serving the
European separations community, which
it has done well for 20 years (every other
year). It was a thoroughly enjoyable social,
academic, intellectual, and practical
experience, with a signifi cant amount of
new and important topics presented and
discussed. There were lively discussions
between the speaker and audience after
each oral talk, as well as at the posters,
which were usually presented by graduate
students or postdocs. There was a
successful mix of academics, industrial-, and
government-separations oriented scientists
present, which led to interesting talks and
discussions throughout the meeting.
Having attended innumerable
national, local, and international
separations-oriented meetings over
too many decades (four or five in 2015
alone), I will attest to this event being
one of the best, most stimulating, open,
and technically sophisticated meetings
in my memory. There was an intellectual
atmosphere obvious at all times, and
everyone came away with more knowledge
than they had when they arrived. I am
indebted to the organizers for giving me
the opportunity to attend my first ever
Balaton symposium in 2015. I look forward
to attending the next Balaton Symposium
in 2017. More information about the
specific programme, vendors, and other
registration information can be found at
the website: www.balaton.mett.hu.
Further information is available, via the July
2015 issue of LCGC Europe (volume 28,
issue 7).
Ira S. Krull is a Professor Emeritus with the
Department of Chemistry and Chemical
Biology at Northeastern Univeristy in
Boston, Massachusetts, USA, and is a
member of LCGC’s editorial board.
E-mail: [email protected]
Balaton Symposium Review
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27–29 January 2016 14th International Symposium on Hyphenated Techniques in Chromatography
and Separation Technology (HTC-14)
Ghent, Belgium
Tel: +32 10 454777
E-mail: [email protected]
Website: http://www.ldorganisation.com/v2/content.php?langue=english&cle_
menus=1238916064
20–21 April 2016 XI International Conference ION CHROMATOGRAPHY 2016
Zabrze, Poland
Contact: Rajmund Michalski
E-mail: [email protected]
Website: www.ipis.zabrze.pl
10–13 May 2016 Analytica 2016
Messe München GmbH, Munich, Germany
Tel: +49 89 949 20720
E-mail: [email protected]
Website: http://www.analytica.de
29 May–3 June 201640th International Symposium on Capillary Chromatography and
13th GC×GC Symposium
Palazzo dei Congressi, Riva del Garda, Italy
Tel: +39 334 3612788
E-mail: [email protected]
Website: www.chromaleont.it/iscc
Event News
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