Volume 1 / Issue 5
May 2009www.sepscience.com
A liquid chromatographer’s introduction to mass spectrometry
Analysing synthetic polymers with solvent enhanced light scattering
Minimizing decomposition of components during GC analysis
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contentsVolume 1 / Issue 5
May 2009www.sepscience.com
A liquid chromatographer’s introduction to mass spectrometry
Analysing synthetic polymers with solvent enhanced light scattering
Minimizing decomposition of components during GC analysis
separationdriving analytical chemistry forwardsscience
A liquid chromatographer’s introduction to mass spectrometry
Michal Holčapek
18
features
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 5May 2009
30
research round-up
Packing procedures for high e� ciency, short ion-exchange columns
Quantifying low levels of polymorphic impurity in clopidogrel bisulphate by vibrational spectroscopy and chemometrics
Carbon nanotubes as the sorbent for integrating μ-solid phase extraction within the needle of a syringe
Determination of dissociation constants between polyelectrolytes and proteins by a� nity capillary electrophoresis
Shell and small particles; Evaluation of new column technology
Separation of catechins and methylxanthines in tea samples by capillary electrochromatography
Direct analysis of valsartan or candesartan in human plasma and urines by on-line solid phase extraction coupled to electrospray tandem mass spectrometry
Rr
Cd chrom doctor Guest author Jaap de Zeeuw discusses how to minimize decomposition of components during GC analysis.
for research news, technical articles, product updates, jobs and applications visit. . .
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technology update An overview of recent technology advances in separation science and instrumentation.
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24 Analysing synthetic polymers with solvent enhanced light scattering
Jean-Luc Brousseau and Wei Sen Wong
Separation Science is published by Eclipse Business Media Ltd, 70 Hospital street, Nantwich,
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scienti� c advisory
councilPeter Myers
– Chief Scienti� c O� cer
David Barrow
University of Cardi� , UK
Zongwei Cai
Hong Kong Baptist University
Yi Chen
Chinese Academy of Sciences,
Beijing, China
Gert Desmet
Vrije Universiteit Brussel, Belgium
C. Bor Fuh
National Chi Nan University, Taiwan
Y.S. Fung
Hong Kong University
Xindu Geng
Northwest University, Xi’an, China
Luigi Mondello
University of Messina, Italy
Paul Haddad
University of Tasmania, Australia
Hian Kee Lee
National University of Singapore,
Singapore
Melissa Hanna-Brown
P� zer, UK
Tuulia Hyötyläinen
University of Helsinki, Finland
Gongke Li
Sun Yat-Sen University, Guangzhou,
China
Yong-Chien Ling
National Tsing Hua University,
Taiwan
Klara Valko,
GSK, UK
Jean-Luc Veuthey
University of Geneva, Switzerland
Claudio Villani
Universita’ degli Studi di Roma “La
Sapienza”, Italy
Cheing- Tong Yan
Center of Environmental Safety and
Hygene, Taiwan
Edward Browne
GSK, Singapore
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Paul HaddadDevelopment of Portable Separation Methods for the Identification of Terrorist Explosives by Analysis of Inorganic Residues
Philip MarriottHeadspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples
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RrResearchround-up
Packing procedures for high efficiency, short ion-exchange columnsAustraliaAn optimized packing procedure for the production of high efficiency, short, particle-packed
ion-exchange columns was reported by Professor Paul Haddad from the Australian Centre
for Research on Separation Science at the University of Tasmania in Australia, in the Journal
of Chromatography A [1208 (1-2), 95-100 (2008)]. Professor Haddad and his colleagues are
involved in counter-terrorism studies and the development of methods for the identification
of inorganic improvised explosives, also known as ‘fertiliser bombs.’ Analytical methods
are required for preblast identification in situations such as airport screening, and also for
postblast identification of explosives using analysis of residues left after the explosion. “We
have undertaken extensive studies on postblast analysis and our current focus is on preblast
analysis. In this application we are developing a two-pronged screening procedure. First, a
rapid analysis (20 s) will confirm the presence of a range of indicator ions known to be present
in improvised explosives. This will be followed by a slightly longer (3 min) confirmatory test,
which will identify the particular explosive present. This confirmatory test will be conducted
using ion chromatography (IC) and we, therefore, needed a short column that would provide
the required resolution in the desired time frame. This led us to study the packing of short
(e.g., 30 mm) columns,” Haddad explained.
According to him, the study revealed that normal slurry-packing procedures were not
applicable to very short columns because of inhomegeneities in the packed bed, leading
to variable (and usually poor) efficiencies. “However, we found that by joining a number of
short column segments together and then packing this assembly as a whole, we could use
the middle segments as high-efficiency short columns. In fact, these columns showed similar
efficiency behaviour to that exhibited by longer columns. Using this approach we were
able to make short columns that provided the desired separation for the counter-terrorism
project,” he added.
The team is now in the process of incorporating these short columns into portable
instrumentation in order to apply them in routine screening operations, such as airports,
which involves the simplification of the technique so it can be used reliably by unskilled users.
6 research round-up www.sepscience.com
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Quantifying low levels of polymorphic impurity in clopidogrel bisulphate by vibrational spectroscopy and chemometricsHungaryVibrational spectroscopic methods were developed for quantitative analysis of clopidogrel bisulphate in Form I and
Form II polymorphic mixtures and published in the Journal of Pharmaceutical and Biomedical Analysis [49 (1), 32-41
(2009)]. Results showed that both IR and Raman spectroscopy combined with chemometrics are suitable to quantify
low levels of Form II in Form I, down to 2 and 3%, respectively, with less than 1% limit of detection.
Zoltán Német from the Drug Polymorphism Research Division at the Gedeon Richter PLC in Budapest, Hungary,
explained the aim in performing this research was twofold. “First, detection of the stable form of clopidogrel
bisulphate in the metastable form of the substance, which is the developed product of our company, is a constraint
from quality assurance point of view,” said Német. It needs quantitative solid state method development to meet
this requirement. “Second, there is little knowledge about the relative advantages of different methods suitable for
quantitative determination of polymorphic mixtures of pharmaceutical solids in general. We intended to perform
a comparative study about the possibilities of infrared and Raman spectroscopy combined with chemometrics,” he
added.
The key findings of the study were, on the one hand, the limits of detection and quantitation of the developed
methods, which is considered good compared to results from similar studies, and also to those obtained before
by x-ray powder diffraction for the same polymorphic system. “On the other hand, it was shown that common
multivariate data handling methods give similar results, provided quality of the dataset is high. It was also shown
that general problem of quantitative solid state Raman spectroscopy can be overcome by appropriate sampling
procedure, for which we have developed a special sample holder accessory,” he said.
He believes the idea of the mentioned sampling procedure for the Raman technique can be useful for other research
groups dealing with similar studies. The obtained low limits of detection and quantification may encourage others
to spend time on method development, even if it seems completely hopeless based on univariate data handling.
“As for ourselves, we continue gathering the knowledge about the potential and limitations of quantitative phase
analysis with new polymorphic systems (additional publication in press at JPBA: doi:10.1016/j.jpba.2008.11.033),” he
concluded.
8 research round-up www.sepscience.com
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Carbon nanotubes as the sorbent for integrating μ-solid phase extraction within the needle of a syringeUSAProfessor Somenath Mitra, Chair of Chemistry and
Environmental Science at the New Jersey Institute
of Technology, USA, recently reported on a study
that looked at the implementation of micro-solid-
phase extraction (μ-SPE) in the needle of a syringe for
integrating sampling, analyte enrichment and sample
introduction into a single device. Published in the Journal
of Chromatography A [1216 (12), 2274-2274 (2009)] both
single- and multi-walled carbon nanotubes (CNTs) were
explored as high performance sorbents for μ-SPE in
packed and self assembled formats. The need for such a
sorbent was critical because the needle probe could hold
only a small amount of material (around 300 μg).
“Micro-extraction techniques have been developing
rapidly over the past few years to overcome some of
the limitations of conventional techniques such as
liquid-liquid extraction (LLE) and solid-phase extraction
(SPE). Both LLE and SPE involve multi-step sample
extraction and clean-up procedures that are tedious,
time consuming and result in high levels of dilution. In
addition, these techniques consume substantial amounts
of organic solvents. The development of relatively
simple, fast sampling techniques that require a reduced
amount of solvents is of great importance and will allow
widespread monitoring of trace level contamination,”
said Professor Mitra.
An example of a functionally simple, yet effective
sampling or sample preparation device is solid-phase
micro extraction (SPME), which is an alternative to the
abovementioned methods. SPME relies on passive
equilibrium between the two phases, which leads
to relatively higher detection limits. “The technique
developed here performs the equivalent of SPE. The
sampling is active, i.e., the sample is drawn through the
sorbent in the syringe. The trapping efficiency is relatively
high, leading to lower detection limits. The device itself
performs sample extraction, concentration and sample
introduction into a single procedure,” Mitra explained.
He feels the study showed that since the needle of
a syringe can only hold a small amount of sorbent, it
was important to use a sorbent that has a very high
capacity. The other important issue would be efficient
desorption from the high capacity sorbent. “CNTs were
effective in this application. The main advantage of CNTs
compared conventional carbon sorbents is that they
non-porous, and the solute is held on the surface by
van der Walls type forces,” he explained. This eliminates
the mass transfer resistance related to the diffusion into
the pore structures. The large specific capacity comes
from the nano-scale size of CNTs, while fast desorption is
facilitated by reduced diffusion resistance.
“Derivatization of the nanotube surface can offer not
only a more hydrophilic surface structure, but also a large
number of oxygen-containing polar functional group,
such as, -COOH, -OH, -NO2, and -HSO3 which increases
the ion-exchange and hydrogen bonding capability of
the CNTs. There are numerous unexplored possibilities,”
he added.
In summary, he believes it is possible to implement
sophisticated µ-SPE in the needle of a syringe for easy
sampling, enrichment and injection and that novel
materials offer some unique opportunities. “Our group
has extensive activities in the area of carbon nanotubes,
spanning chromatography to solar cells and we are
excited about these possibilities. Within the separations
area, our work has covered chromatography, microtrap
for air monitoring, and developing an understanding of
these materials as adsorbents. Another application has
been the development of membrane incorporating CNTs.
We plan to continue on the material science as well as
the analytical application of CNTs,” he concluded.
10 research round-up www.sepscience.com
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atlantis_halfpage.indd 1 5/8/09 10:08:47 AM11research round-upseparation science — volume 1 issue 5
Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis
Sweden
A paper in the Journal of Chromatography B [877 (10), 892-896 (2009)] reports on the binding affinity between two
model proteins, human serum albumin (HSA) and ribonuclease A (RNase A), and negatively charged polyelectrolytes,
two different heparin fractions and dextran sulfate, by means of partial filling and affinity capillary electrophoresis.
Main author, Professor Roland Isaksson from the School of Pure and Applied Natural Sciences at the University of
Kalmar in Sweden, explains the main aim of the research investigates the use of polyelectrolytes such as heparin,
dextrane sulphate, etc, in pharmaceutics to formulate peptide and protein based drugs.
“This project included long-term studies of protein polyelectrolyte mixtures, determinations of protein
polyelectrolyte affinities, chemical purities of both proteins and polyelectrolytes as well as sustained release of the
protein from the protein polyelectrolyte complex by use capillary electrophoresis,” Professor Isaksson said.
He believes the key findings of the study are that CE by means of partial filling techniques which mimic the
physiological conditions is a useful complement to other methods to study protein polyelectrolyte interactions. “The
affinity determinations with this technique are relatively simple to perform with only small amounts of (expensive)
proteins,” he said.
“In the future we will combine or use this CE technique as a complement with other methods such as circular
dichroism (CD), FTIR and microcalorimetry, etc. to carefully characterise the protein polyelectyrolyte formulations. Our
technique will also be adopted to follow the release of the drug from its polyelectrolyte complex,” he concluded.
Shell and small particles; Evaluation of new column technologyHungary
The performance of 5 cm long columns packed with shell particles was compared to totally porous sub-2 μm
particles in gradient and isocratic elution separations of hormones (dienogest, fi nasteride, gestodene, levonorgestrel,
estradiol, ethinylestradiol, noretistherone acetate, bicalutamide and tibolone) in a study puiblished in the Journal of
Pharmaceutical and Biomedical Analysis [49 (1), 64-71 (2009)].
“The approach of applying shell type particles in small diameter (2.7 μm) was realized in 2006. We were curious to
know the real (not theoretical) performance of this superfi cial phase under both isocratic and gradient conditions, and
we also wanted to know whether UPLC can be substituted with other techniques,” explained main author, Dr Szabolcs
Fekete from Formulation Development at Gedeon Richter Plc in Budapest, Hungary.
According to Dr Fekete, there are many theoretical assessments about the kinetic effi ciency (plate heights) of both
sub-2 μm totally porous and small shell particles but in this case, his team intended to estimate the time required for
the separation. This is why kinetic plot methods were used to compare the two approaches. “In practice, we mostly
use gradient separations in pharmaceutical applications and in this case the peak capacity is a more suitable measure
for effi ciency,” he added. Peak capacity curves were measured and compared to evaluate the performance of sub-2
μm porous and 2.7 μm shell particles when steep/fast gradient elution (5 – 25 minutes) was applied. Furthermore the
overloading of the column is a critical factor in practical work. “We wanted to evaluate the new column technology
also in this respect. For biological samples, rapid methods are needed for screening purposes or obtaining samples for
high-resolution mass spectrometer,” he said.
The study showed that superfi cial (shell) stationary phase off ers a high separation power with modest operating
pressure. “The performance achieved under both gradient and isocratic condition, is comparable to those obtained
with totally porous sub-2 μm particles. But it is necessary to emphasize that the performance of columns packed
with shell particles is not as high as the theory predicted earlier when high linear velocity (u > 0.3 cm/s) is applied,” he
said. For him, both UPLC and superfi cial phases are adequate tools for screening purposes. Using gradient elution, an
increased injection volume can be applied for sample enrichment in the inlet of the column.
Columns packed with shell particles are worthy of rivaling to any other fast liquid chromatographic techniques
without the requirement and adverse eff ects of ultra-high pressure. Conventional HPLC systems with slight
modifi cations can be applied for fast separations. “In the future we are going to introduce superfi cial phases for
everyday routine applications to achieve fast separations and to save time in method development. We also intend
to apply these columns for environmental analysis. Our researches are focused on micro-pollutants in plastics or in
packing materials and also pharmaceutical residues in drinking and surface water,” he concluded.
12 research round-up www.sepscience.com
14 research round-up www.sepscience.com
Separation of catechins and methylxanthines in tea samples by capillary electrochromatographyItaly
A paper in the the Journal of Separation Science [32 (7), 1002-1010 (2009)] documents the simultaneous separation
of several polyphenols such as (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, theophylline, caff eine in green and
black teas by capillary electrochromatography (CEC). Several experimental parameters such as stationary phase
type, mobile phase composition, buff er and pH, inner diameter of the columns, sample injection, were evaluated
to obtain the complete separation of the analysed compounds by Dr Zeineb Aturki from the Institute of Chemical
Methodologies at the National Council of Research in Rome, Italy.
“The research concerning the analysis of catechins in tea was developed following the aim of our project to analyse
polyphenols in several food matrices with miniaturized techniques including capillary electrochromatography (CEC)
and nano-liquid chromatography (Nano-LC). Our interest in those compounds is due to their nutritional properties
and benefi cial implications for human health. In addition quantifi cation of polyphenols provides useful informations
for food quality control,” explained Dr Aturki.
For him, the key fi ndings of the study include the development of analytical methods using miniaturized
techniques. “They off er several advantages such as high precision, accuracy, sensitivity, short analysis time, low
consumption of samples and reagents, easy coupling to mass spectrometry. For all these purposes, CEC and
Nano-LC can be used as an alternative or complementary technique to high performance liquid chromatography,”
Aturki added.
He believes the results achieved in this research have proved the potential of CEC technique and their future goal is
to determine these compounds by coupling this analytical technique to the mass spectrometer.
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Direct analysis of valsartan or candesartan in human plasma and urines by on-line solid phase extraction coupled to electrospray tandem mass spectrometry
France
As documented in the Journal of Chromatography B
[877 (10), 919-926 (2009)], a direct on-line solid phase
extraction coupled to tandem mass spectrometry was
developed and validated to determine valsartan (5–2000
ng/mL) or candesartan (1–200 ng/mL) in human plasma
and urines.
Lead researcher, Dr Alain Pruvost from CEA, iBiTecS,
Service de Pharmacologie et d’Immunoanalyse in
Gif-sur-Yvette, France, explained this work was initiated
by a well known clinical team ‘Hôpital Européen Georges
Pompidou’ in Paris which investigated what eff ect a
dietary salt intake had on the pharmacokinetics (PK) and
the pharmacodynamic (PD) eff ects of diff erent blockers
of the renin-angiotensin system (RAS) in normotensive
subjects. “As it is known for orally administered drugs
such as verapamil and quinidine, low salt intake can
increase systemic drug availability and consequently
aff ect their PD eff ects. But it was not known whether
such a phenomenon existed with RAS blockers. For that
purpose, we were asked to develop and validate the
most robust, precise, accurate, reliable analytical method
in order to allow revelation of the weakest diff erence in
PK parameters and bioavailability. Indeed, a diff erence in
bioavailability around 15 to 20% may be covered by the
variability of the method if too high. So, we elected a
LC-MS/MS technique to measure drugs in human
plasma,” Dr Pruvost said.
His goals were to develop an analytical method
presenting fi rst, a very short analysis total run time in
order to process a large number of samples in the least
variable conditions and second the least sensitive to
matrix eff ect (large diff erence in samples; many subjects
and many PK time points). “We naturally turned to ‘
on-line’ methods to develop a very fast method,
including sample clean-up. Moreover, we chose not to
use an analytical column which would lower the total
run time resulting in the development of a SPE-MS/
MS method without true analytical chromatographic
separation. To do so and to satisfy the second point
cited above, we were compelled to eliminate the
largest number of endogenous compounds present
in the matrix. For this purpose and because the drugs
were ionisable compounds, we used mixed mode
sorbent packed in a very short column (20 mm),”
Pruvost elaborated. This kind of SPE sorbent allows the
researcher to use hydrophobic and ionic interactions to
retain compounds of interest and allow the use of strong
organic solvent to ‘wash off ’ the sample matrix thus
eliminating a large quantity of unwanted compounds.
Among the key fi ndings, the team cites good effi ciency
of the combination of mixed mode sorbent such as
OASIS MAX (anion exchange) and strong organic solvent
like tetrahydrofuran (THF) for cleaning up samples.
“Indeed, since target compounds are hydrophobic
(log Po/w around 5) and acidic compounds, they are
successfully retained on the stationary phase by ionic
interactions. Moreover, use of THF allows the clean-
up of samples by eliminating lipids, and especially
phospholipids which are known to reduce signals in
electrospray ionization when they are co-eluted with
analytes (matrix eff ect). In these conditions, the method
allows us not to use an analytical chromatographic
column and results in a high total recovery,” he said.
He believes that these kinds of on-line analytical
methods are very useful and successful. “They present
precision, robustness and a very moderate cost per
sample. They also present the advantage of a reduced
manual processing of biological samples and show
satisfactory result dispersion with high throughput. But
they require a very good knowledge of the diff erent
elements of the analytical system when home-build,
and may seem sometimes too complex to use. It is
why, now, to face up to the success of these methods,
some suppliers propose much evolved and ready to use
systems, he concluded.
16 research round-up www.sepscience.com
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18 feature article — MS for chromatographers www.sepscience.com
19feature article — MS for chromatographersseparation science — volume 1 issue 5
mass spectrometer. The sample
introduction system introduces
the solid, liquid or gas sample into
the high vacuum required for ion
analysis and detection, while ion
optics transfer, focus and accelerate
ions once in vacuum. And no mass
spectrometer would be complete
without a noisy vacuum system and
a computer to control the instrument
and provide data handling and
reporting.
Mass spectrometry coupled to chromatographyMass spectrometry alone is best
suited for the analysis of pure
compounds, because the whole
sample is introduced to the ion
source at once. In practice, the
analysis of more complex mixtures is
often required, so we need to couple
the mass spectrometer to some
separation technique.
A liquid chromatographer’s introduction to mass spectrometryMichal Holčapek
Professor of Analytical Chemistry, University of Pardubice, Czech Republic.
Mass spectrometry (MS) is used for the structural elucidation or confirmation of organic, bioorganic and
organometallic compounds, and for quantitative analysis in environmental, pharmaceutical, forensic, food and
other sciences. The first step in MS measurement is the conversion of neutral molecules to charged species (i.e.,
ions), which are then separated according to their mass-to-charge (m/z) ratio in a mass analyser. The relative
abundances of individual m/z values are recorded by a suitable detector to produce what is known as a mass
spectrum. MS can be coupled to both gas-phase and liquid-phase separation techniques, enabling the structural
analysis of complex mixtures after their chromatographic separation without time-consuming off-line isolation.
This article will also include a glossary of relevant MS terminology.
experimental proof of the existence
of electrons and charged particles.
The field of mass spectrometry
has garnered four Nobel Prizes
for chemistry and physics so far.
Nowadays, mass spectrometry
influences and supports research in
many fields of chemistry, biology,
medicine and physics.
Generally speaking, the mass
spectrometer consists of 3 main parts:
Ion source: The role of the ion
source is the conversion (ionization)
of neutral species into charged
particles (ions).
Mass analyser: The mass analyser
then separates the ions according to
their m/z values
Detector: The detector records the
relative abundances of individual
m/z values.
In addition to these basic
parts, several other parts are
essential to the function of the
Actual mass spectra are records of
relative ion abundances (expressed
as per cent) vs mass-to-charge ratio
(x-axis). The most abundant ion in
the spectrum is called the base peak
and is assigned a relative intensity
of 100%. The relative abundances of
other ions in the spectrum are then
normalized to the base peak (Figure 1).
Most of the ions carry just one
charge, so the m/z values correspond
directly to the masses of the
particular ions. In the case of some
ionization techniques (especially
electrospray ionization), ions may be
produced with multiple charges. As
a result, the observed m/z values are
diminished by the factor 1/z.
The current mass spectrometric
nomenclature recommends use of
the Thomson (Th) as a unit for m/z
values, in honor of J. J. Thomson,
who was awarded the Nobel Prize for
physics in 1906 for the discovery and
20 feature article — MS for chromatographers www.sepscience.com
suppression eff ects. Contamination
and suppression eff ects can also be
reduced by using orthogonal ion
source geometry, the standard for
current LC-MS systems.
How can ion suppression infl uence your spectrum?
The analyte may give a diff erent MS
response in a mobile phase without
ionic additives in comparison with
the identical system containing
ionic and/or non-volatile additives,
such as phosphate buff er. The
suppression occurs in the ionizer
when ionic species in the mobile
phase successfully compete with the
analyte for charges at the droplet
surface, thus reducing the ionization
of target analytes. In addition
to ionic additives in the mobile
phase, sample matrix components
can also result in the suppression,
or sometimes enhancement of
response when they co-elute with
target analytes.
What to do with non-volatile compounds?
LC-MS coupling has only one serious
In the past, the target compound
had to be isolated fi rst and then
analysed with a direct insertion
probe, a time-consuming process
that also made the analysis of trace
impurities diffi cult. During the
last several decades, the coupling
of gas chromatography (GC) to
electron ionization (EI) and chemical
ionization (CI) sources has made
MS a routine technique. GC-MS is
used for the analysis of complex
mixtures of gas-phase compounds,
which limits the range of analytes
to relatively volatile, non-polar
molecules .
To prevent extensive fragmentation,
soft ionization techniques are used.
Initially, LC-MS also relied on EI or CI,
but because of the limited sensitivity
and robustness of such devices,
intensive research brought new soft
ionization techniques, which are
ideally suited for LC-MS coupling
because they combine multiple
functions into one step, including:
the interface between the column
and MS system (sample transfer into
gas phase), and sample ionization.
Nowadays, the coupling of HPLC
and MS in analytical laboratories
is very common. MS is compatible
with the whole range of analytical
fl ow rates (from nL/min to 2 mL/min)
and the mobile phase composition
(except for normal phase eluents
without a polar modifi er), thanks to
the innovative ionizers developed
in the last few decades. The
main remaining limitation lies in
the choice of chromatographic
conditions, especially the selection
of buff ers and additives. Non-volatile
additives (e.g., phosphate buff ers,
tetraalkylammonium ion-pairing
agents, etc.) should be replaced
by more volatile analogues (e.g.,
ammonium acetate or formate,
formic or acetic acid, ammonia,
tri- or dialkylammonium acetate,
etc.), which should be used at the
lowest possible concentration
(usually 5-10 mmol/L at maximum)
in order to avoid contamination
of the mass spectrometer or ion
Figure 1
Figure 1: Electron ionization mass spectrum of benzophenone.
Figure 2
© CHROMEDIATo vacuum system
DetectorSource Mass
analyser(sanalyser(s)Inlet
Ions
Datasystem
Figure 2: General schematic of a mass spectrometer.
21feature article — MS for chromatographersseparation science — volume 1 issue 5
limitation concerning the choice of
mobile phase composition, which
is the use of non-volatile inorganic
buff ers and additives, such as
phosphate buff ers, inorganic acids,
non-volatile ion-pairing agents,
cyclodextrins, etc. When the HPLC
method containing such reagents
is converted to LC-MS, non-volatile
additives should be substituted by
more volatile additives.
How can this be achieved without the loss of chromatographic performance?
LC-MS coupling places only one
serious constraint on HPLC mobile
phase composition, ruling out the
use of non-volatile inorganic buff ers
and additives, including phosphate
buff ers, inorganic acids, non-volatile
ion-pairing agents, cyclodextrins, etc.
When an HPLC method containing
such reagents is adapted for LC-MS
coupling, non-volatile additives
should be substituted by more
volatile additives at the lowest
possible concentration, for example:
• Ammonium acetate or formate
(usually up to 5 mmol/l), formic
or acetic acid (up to 0.1%, in some
special cases a little bit more)
• Ammonium hydroxide for basic
pH values (up to 0.1%).
• For analyses employing ion-pairing
reagents, LC-MS is compatible
with di- or trialkylammonium
acetates or formates (for cationic
analytes) at or perfl uorocarboxylic
acids (for anionic analytes), all of
which can be used at
concentrations up to 3mmol/L.
Tandem mass spectrometry (MS/MS)The advantage of so called soft
ionization techniques arises from
the fact that they primarily produce
protonated or deprotonated
molecules and relatively few
fragment ions (thus the term ‘soft’),
which makes the molecular weight
(MW) determination relatively
simple. At the same time, the
absence of fragment ions may be
considered a disadvantage for
structure elucidation because MW
information alone is not suffi cient
to tease out molecular structure.
This drawback may be overcome by
using tandem mass spectrometry
(MS/MS), whereby the fi rst mass
analyser is used for the isolation of
a selected precursor ion (previously
called a ‘parent ion’), which is then
fragmented to give product ions
(originally knows as ‘daughter ions’)
for subsequent MS analysis (Figure 3).
In this manner, we can obtain
information on the sub-structure of
each precursor ion, which should
represent a portion of the molecule.
This process may be repeated for
several precursor ions, typically
using triple quadrupole or ion trap
analysers.
The ion trap analyser
allows repeated isolation and
fragmentation steps, producing
fragments of fragments of fragments,
a technique known as multistage
tandem mass spectrometry (MSn)
(Figure 4). This is valuable for
studying fragmentation paths,
as well as confi rming molecular
structures.
In-source collision induced dissociation (CID) and collision induced dissociation?In-source CID does not enable
precursor ion isolation, so all the
ions present in the ion source at
a given time are fragmented as a
group without prior isolation, which
is feasible with true tandem mass
spectrometry. Another diff erence
is that in-source CID occurs during
the ionization process in the ion
source, while CID in MS/MS occurs
in the ion trap or in the collision
cell, in the case of QqQ (triple
quadrupole) or QqTOF instruments.
The absence of the isolation step for
in-source CID may not be of concern
if chromatographic resolution is
adequate.
Glossary of basic termsAtmospheric pressure chemical
ionization (APCI) = soft ionization
technique typically used for LC-MS
coupling and the analysis of small
organic molecules with low to
medium polarity.
Atmospheric pressure
photoionization (APPI) = soft
Figure 3
© CHROMEDIA
To detectorFromsource
MS1 MS2Collisioncell
Figure 3: Tandem mass spectrometry principle.
22 feature article — MS for chromatographers www.sepscience.com
ionization technique, nearly
identical applications as for APCI,
but it extends the polarity range
slightly towards non-polar or very
labile molecules.
Base peak = peak with the highest
abundance in the spectrum. Its
relative abundance is set to 100%,
relative abundances of other peaks
in the spectrum are related to the
base peak and fall in the range
0-100%.
Chemical ionization (CI) = fi rst soft
ionization technique, used mainly in
GC-MS.
Dalton = a unit of molecular
weight frequently used in mass
spectrometry.
Deprotonated molecule = even-
electron ion [M-H]- with an m/z
ratio one mass unit lower than the
molecular weight. This is typically
the base peak in negative-ion mass
spectra taken with soft ionization
techniques.
Electron ionization (EI) = fi rst
ionization technique generally used
for GC-MS coupling. It is sometimes
referred to as a ‘hard’ ionization
technique because ionized species
can obtain large amounts of internal
energy, which leads to extensive
fragmentation and the complete
absence of the molecular ion
for about 10% of all the volatile
compounds amenable to EI.
Electrospray ionization (ESI) =
the softest ionization technique,
especially useful for polar to ionic
compounds, biopolymers, non-
covalent complexes or any extremely
labile compounds.
Elemental composition = sum
of individual atoms present in
particular ion or molecule.
Fragmentation = process whereby
the ion is cleaved into smaller parts
called fragments.
Exact mass = precise mass of a
particular ion calculated to at least
four decimal places, taking into
account the number of electrons,
used to extract molecular formula
information from highly accurate
and precise mass spectra.
Ion cyclotron resonance (ICR) = very
precise mass analyser requiring
Fourier Transformation of its detector
signal to provide the highest
available resolution and mass
accuracy among mass analysers, but
at the expense of the most rigorous
vacuum requirements and high cost.
Ion trap (IT) = a relatively recent type
of mass analyser capable of iterative
tandem mass spectrometry.
Ionization = the process of
converting a neutral molecule into a
charged species (ion).
Magnetic sector analyser = the
oldest type of mass analyser. Ions
are separated because diff erent m/z
values result in diff erent trajectories
through the magnetic fi eld. Typically
used in series with an electrostatic
analyser to increases resolution.
Mass accuracy = the diff erence
between theoretical and measured
m/z values, reported as ppm.
Mass accuracy better than 5 ppm
is generally considered the
minimum necessary for exact mass
determination, which can allow
determination of the elemental mass
composition.
Mass-to-charge (m/z) = the
quantity graphed on the x-axis of
a mass spectrum. Determines the
interaction of the ion with magnetic
and electrical fi elds, a fact that is
exploited in ion analysers.
Matrix-assisted laser desorption/
ionization (MALDI) = soft desorption
ionization technique not frequently
coupled to separations. Very useful
for biopolymers and synthetic
polymers with high molecular
weights. Sometimes coupled to
HPLC off -line via fraction collection.
Molecular ion = odd-electron ion,
it may be M+. in positive-ion or –. in
negative-ion mode.
Molecular weight (MW) = sum
of masses of the most abundant
isotopes for each atom in
Figure 4
© CHROMEDIA
Ion source
Ionization
Mass spectrum MS/MS spectrum
Analyzer 1
Analyzer 2
Collision induced
dissociation
Fragmentation
Detector
848284
767472
20
40
60
78
Figure 4: Tandem MS allows repeat isolation and fragmentation.
23feature article — MS for chromatographersseparation science — volume 1 issue 5
the molecule. Note that MW
determination using the most
abundant isotopes (used in MS)
diff ers from MW determination
on the basis of averaged isotopic
masses (used in all fi elds of chemistry
except for MS. For example, a
mass spectrometrist should count
bromine as 79 (because 79Br is the
most abundant isotope) rather than
80 (average of 79Br and 81Br isotopes
in the ratio approximately 1:1)
Nominal mass = the integer value of
a particular ion calculated from the
most abundant natural isotopes.
Orbitrap = the newest type of FT
mass analyser introduced in 2005, it
provides high resolution and high
mass accuracy by detecting the
oscillation of ions in an electric fi eld.
Protonated molecule = even-
electron ion with m/z value higher
than the molecular weight by one
mass unit, the [M+H]+ ion is typically
the base peak in positive-ion
mass spectra generated with soft
ionization techniques.
Quadrupole analyser (Q) = low
resolution mass analyser commonly
coupled to chromatography.
Resolution = A measure of the
mass spectrometer’s ability to
distinguish (separate) two adjacent
spectral peaks. There are two basic
defi nitions: (1) the mass of the target
peak divided by the diff erence
between two neighbouring peaks
with the same heights and 10%
valley overlap (R10% valley); (2) the mass
of target peak is divided by the
peak width at the half height of this
peak (RFWHM). The second defi nition
is more widespread and is generally
accepted nowadays, although the
10% valley defi nition is still common
for magnetic sector instruments.
Roughly, RFWHM is approximately half
of R10% valley.
Soft ionization techniques = a group
of ionization techniques with the
common feature that the molecular
ion or deprotonated molecule
usually correspond to the base peak
of mass spectra with the lack or low
abundances of fragment ions.
Tandem mass spectrometry (MS/
MS) = coupling of two or more
analysers (both ion traps and ion
cyclotrons can actually achieve MS/
MS with at single analyser), used
for the isolation of precursor ion, its
subsequent fragmentation, and the
detection of their product ions.
Thomson (Th) = unit for m/z recently
proposed by mass spectrometric
nomenclature.
Time-of-fl ight (TOF) = mass analyser
based on the precise measurement
of the fl ight times of ions accelerated
by an electric fi eld.
Triple quadrupole (QqQ) = tandem
mass analyser consisting of
three quadrupole rods. The fi rst
quadrupole is used for precursor
ion selection, the second one serves
as a collision cell for fragmenting
precursors into product ions, which
are then analysed by the third
quadrupole.
This article was written by Michal
Holčapek, Professor of Analytical
Chemistry, University of Pardubice,
Czech Republic.
Publication of this article was made
possible through collaboration with
Chromedia.
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Recommended Further ReadingClick titles for more information
The Mass Spectrometry Topic Circle
Analysis of PAH’s in foods
LCMS of pesticides
LCMS of lipids
24 feature article — Solvent enhanced light scattering www.sepscience.com
Analysing synthetic polymers with solvent enhanced light scatteringJean-Luc Brousseau and Wei Sen Wong
Viscotek (A Malvern Company)
Solvent enhanced light scattering (SELS) is a gel permeation
chromatography (GPC) technique for the analysis of synthetic polymers.
With SELS two di� erent solvents are used – one to dissolve the polymer,
the other to act as the eluent – allowing users to select the most
appropriate solvent for each function. SELS is particularly valuable for
‘invisible’ polymers, materials with a refractive index close to that of the
solvent used for their analysis.
25feature article — Solvent enhanced light scatteringseparation science — volume 1 issue 5
GPC the polymer or polymer blend
is dissolved in a solvent and then
injected into a fl owing system.
Eluent carries the sample through
a column of porous material,
such as polystyrene gels or silica,
which separates on the basis of
hydrodynamic radius or volume.
Larger molecules fi t into fewer pores
in the packing material and therefore
elute more rapidly than smaller ones.
Detectors at the exit of the column
analyse the resultant size fractions.
temperatures, the melt temperature
of the polymer, for example, and
variables such as stiff ness, strength,
toughness and viscoelasticity, which
determine commercial usefulness
and value.
Gel permeation chromatography
(GPC) is a well-established technique
for the determination of both
molecular weight and molecular
weight distribution, and is widely
used for both macromolecular
research and quality control. With
Synthetic polymers are widely
manufactured to produce a vast
array of items. Containers for food
and pharmaceuticals, furniture, car
parts and clothing are just a few
examples of the many products
routinely made. For all polymers,
molecular weight and molecular
weight distribution are critical
parameters because they determine
the physical and mechanical
properties of the material. Molecular
weight infl uences transition
26 feature article — Solvent enhanced light scattering www.sepscience.com
produce an overall improvement in
the analytical method. The technique
is particularly eff ective for addressing
the following issues:
• Lack of sensitivity with respect to
refractive index i.e. the refractive
index of the polymer and pure
solvent are similar.
• Cost and SHE concerns connected
with the use of a particular eluent.
• The need for high temperature
GPC.
When performing SELS the eluent
no longer needs to solubilize the
polymer; it must, however, support
the polymer solution. In terms of
detection, the polymer is measured
in the eluent and not in the solvent
in which it was initially dissolved,
thus simplifying application of
the technique. Figure 1 indicates
why this is the case, showing how
larger polymer molecules elute
fi rst, followed by smaller polymer
molecules, and then fi nally the
solvent used to solubilize the
polymer.
Column choice is particularly
important for
SELS because
the column must
tolerate diff erent
solvents and rapid
solvent change without
sustaining any damage.
Increasing measurement sensitivityAs ‘like dissolves like’, it is not
uncommon for the refractive index
of a polymer to be close to that of
the solvent used to dissolve it, which
with traditional GPC will also be the
eluent. When this is the case, dn/
dc (the rate of change of refractive
When a GPC system is calibrated
specifi cally with the same polymer
being analysed, then concentration
detection alone is suffi cient, because
the calibration process defi nes
the relationship between size and
molecular weight. Polymers that
elute at a certain time have a known
molecular weight. The simplest GPCs
have only diff erential refractometer
detectors. These determine the
concentration of polymer present
using the diff erence
in refractive index (RI)
between the eluting
fraction and pure
solvent. However, there
are standards for only a
handful of polymers, polystyrene
being the most widely used. Most
polymers are not available as
calibration standards so the GPC
reports relative rather than ‘absolute’
molecular weight, typically a
‘polystyrene equivalent molecular
weight’. With these systems the
relative molecular weight reported is
correct only if the calibration and the
unknown polymers have the same
density.
More sophisticated GPC systems
have triple or tetra detection
that includes UV, light scattering
and viscometry detectors. Such
systems off er an alternative option
for concentration determination
(UV) and direct absolute molecular
weight and viscosity measurement.
Column calibration is not required
for accurate molecular weight
determination.
The SELS conceptWith traditional GPC the solvent
used to solubilize the polymer is the
same solvent used as the eluent.
SELS, in contrast, involves the use of
diff erent solvents for each function.
Consequently the optimum solvent
can be selected for dissolution of the
polymer or elution of the sample, to
Figure 1
Figure 1: Schematic of SELS where a polymer is injected in solution (blue) but the polymer is measured in a di� erent liquid, the eluent (green).
“SELS involves the use of two di� erent solvents: one to solubilize
the polymer and another to act as the eluent”
27feature article — Solvent enhanced light scatteringseparation science — volume 1 issue 5
index (n) with concentration (c)) is
low, making it more diffi cult for a
RI detector to accurately determine
polymer concentration because the
detector signal is low and the signal-
to-noise ratio poor. This has a direct
impact on the quality of measured
data. With triple and tetra detection,
both light scattering and viscometry
detectors may use the concentration
data provided by the RI detector to
determine molecular weight. For a
light scattering detector, scattering
intensity is proportional to the
square of dn/dc, so a low value
will result in a very poor signal; an
increase in dn/dc from 0.01 to 0.03,
for instance, will increase signal
strength ninefold. So, a low dn/dc
compromises the performance of RI
detectors alone, and also advanced
detection systems, particularly those
incorporating a light scattering
detector.
With SELS it is possible to select an
eluent that will give a better contrast
dn/dc than the solvent, even if this
eluent is not a good solvent for the
polymer. It is important to re-iterate
at this point that the polymer will
be detected in the eluent, rather
than the solubilizing solvent,
which is why this approach works.
Figure 2 shows the eff ect of using
SELS in the analysis of polymethyl
trifl uoromethacrylate (PMTFMA).
PMTFMA is soluble in
tetrahydrofuran (THF) but this
system has a low dn/dc value.
PMTFMA is not soluble in acetone
but results show that THF and
acetone can be used successfully
in combination. Solubilizing the
PMTFMA in THF and using acetone
as the eluent triples dn/dc, from
0.03 to 0.09. For the triple detection
system used here this has the
expected benefi cial impact on light
scattering intensity, improving
both the precision and accuracy of
measurement.
SELS has also been used to
measure the molecular weight of
polyhydroxyalkanoate, a synthetic
bioplastic which is soluble in
chloroform. In this case chloroform
was retained as the solubilizing
solvent but THF was used as an
eluent, producing a fourfold increase
in dn/dc. A corresponding increase
in measurement sensitivity was
observed.
Figure 2
Figure 2: Low Angle Light Scattering (LALS) chromatogram for PMTFMA in THF. The black curve is the LALS signal when the solution is injected in a GPC running THF. The green curve is the LALS signal when the same solution is injected in a GPC system running with acetone as the eluent. Columns used were I-Series (Viscotek, a Malvern company), � ow rate 1.0 mL/min.
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28 feature article — Solvent enhanced light scattering www.sepscience.com
instrument, columns and operator,
and is best avoided if alternative
options are available. Olefin rubbers,
for example, are typically analysed
in trichlorobenzene (TCB), a solvent
with recognized SHE issues, at
elevated temperature. Using SELS it
is possible to effect the same analysis
at a much lower temperature (45 oC)
by dissolving the polymer in xylene
and using THF as an eluent.
Conclusion
Solvent enhanced light scattering
is a GPC technique that decouples
the choice of solubilizing solvent
and eluent, by employing a different
solvent for each function. The
introduction of columns that tolerate
multiple solvents and/or a rapid
change in solvent will encourage its
use.
SELS is particularly valuable when
the refractive index of the polymer
and solvent are very similar, a
situation that can make both RI and
light scattering detection difficult.
With SELS, an eluent can be selected
to increase the contrast between
polymer and solvent refractive
index, enhancing signal quality, and
therefore measurement accuracy.
The tailoring of solvent systems is
also valuable for the development
of GPC methods that operate at
lower temperature or which use less
expensive or more benign solvents.
Jean-Luc Brousseau joined Malvern
Instruments in 2007 to work with the
Zetasizer Nano system and is now
a specialist for separation systems.
Jean-Luc received his PhD from
the Université du Québec à Trois-
Rivièeres in 1999 having conducted
research at the University of Miami
on macromolecules and sol-gel.
His post-doctoral work at Tulane
University was on light scattering of
polymers and novel characterization
of polymerization reactions.
Wei Sen Wong has been with
Viscotek, a Malvern company, since
1997 and is manager of analytical
services. He received his PhD in 1978
from Northeastern University under
Professor Barry Karger, and then joined
Shell Development Company where
he headed up the GPC laboratory for
more than 18 years.
Solvent benefitsWith traditional GPC, a solvent that
successfully dissolves the polymer
is used as the eluent, even if this
solvent is costly and/or potentially
hazardous. The amount of solvent
used to dissolve the polymer is just
a few millilitres whereas the eluent
in a GPC system can be several litres
per day. With SELS, the most suitable
solvent can be selected to dissolve
the polymer, and an alternative
solvent, less expensive or giving rise
to fewer SHE concerns, can be used
as the eluent. This is an effective way
of reducing the cost and/or hazard
associated with analysis and solvent
disposal.
For example, hexafluoroisopropanol
(HFIP) is a fluorinated solvent
commonly used to solubilize organic
polymers such as polyamides,
polyacrylonitriles, polyacetals,
polyesters, and polyketones. HFIP
is corrosive and gives rise to safety
concerns. It is also more expensive
than most commonly used GPC
solvents. Experimental work has
shown that with SELS it is possible to
dissolve certain polymers in HFIP, but
use THF or chloroform as the eluent.
The resulting minimization of HFIP
usage reduces potential hazard and
cost.
Reducing GPC running temperatureSome polymers are only sparingly
soluble at room temperature in
the solvents that can be used as an
eluent, in which case it becomes
necessary to operate the GPC
system at significantly elevated
temperature. High-temperature GPC
is not an easy analytical solution
because of higher demands on the
CdThe Chrom
Doctor
Minimizing decomposition of components during GC analysis
Gas chromatography is performed under
conditions where the components to
separated are in the gas phase. We use a
temperature-controlled oven to heat the
column to evaporate components with
higher boiling points. These ovens are
typically used up to 450 °C, which allows
analysis of components with boiling points
of up to 700 °C.
However, not all components are stable
when heated and decomposition can occur.
This can happen when the component
is evaporated during the injection step,
or it can happen when the component is
‘traveling’ through the capillary column.
Decomposition and peak shapeIf a component is thermally labile, or reactive,
we can expect an non-reproducible and
lower response for that component.
In most cases the component decomposes
into a ‘smaller’ product. If the decomposition
happens inside the injector, the response of
the component will be lower, and we will see
sharp decomposition peaks. We can change
injection conditions to minimize this effect.
If the decomposition happens while the
component is traveling through the column
we see a strong ‘leading’ peak (Figure 1).
Sometimes we see in our chromatogram a peak shape that we know is not ‘normal.’ Last month we discussed the overloading phenomena, which directly impacts peak shape. In this instalment we will again look at peak shape, but from a different perspective. If a component is not thermally stable, the peak shape and size may be a good indicator. There are several actions we can take if we observe the phenomena, but we need to recognize it first.
Figure 1
Figure 1: Example of decomposition during chromatographic separation.
The ‘lead’ of the peak is formed by the
decomposition products, as they elute faster.
As these products are formed during the
time the component is inside the column,
these products will not elute as a peak, but
as an elevated baseline.
Components that are known for
thermolability are pesticides (e.g., DDT,
carbamates etc.), and brominated diphenyl
ethers (e.g., flame retardants). Sometimes
unsaturated compounds, such as propadiene
and pentadienes decompose on activated
alumina surfaces.
30 chrom doctor www.sepscience.com
Reduction of component breakdownThe decomposition reaction is strongly
temperature-dependent. Practically, we need
to do the analysis at the lowest possible
thermal stress, meaning creating optimal
conditions for injection port temperature
and elution temperatures while performing
the GC separation.
Injection: Using evaporating injection
systems is always very challenging as the
component is exposed to high temperature
and will decompose. In splitted injection,
the injection takes a fraction of a second,
which usually is not a problem. With splitless
injection, the sample is initially exposed to
high injection port temperature. During
this time, interactions can take place and
components will decompose. Figure 2(a)
shows an example of what can happen
with carbamates when they are introduced
via splitless injection. The carbamates are
broken down into their phenolic esters.
These compounds will elute as sharp peaks
as they are focused on the column.
Figure 2(b) shows the same analysis
using on-column injection. Because of the
absence of thermal stress during injection,
the carbamates are injected onto the column
without decomposition.
If on-column is not an option and splitless
injection is to be used make sure that:
• the lowest possible injection port
temperature is used
• the highest possible flow rate (use
0.32 mm columns) is used
• a pressure pulse is used
• inert liners (siltek or siloxane-deactivated)
are used
• care is taken with glass wool packings as
these may initiate decomposition
This way we can minimize thermal
stress. An alternative injection technique
to consider is ‘programmed temperature
injection’ or PTV. Here the sample is
introduced into a cold liner, and flash
evaporated when the injector is heated.
PTV is not as good as the cold-on-column
method, but better than the splitless
technique.
The capillary separation column: Once
the sample is injected into the column, the
component must pass the whole column
and during this process decomposition
can occur. This decomposition is directly
dependent on temperature, but also on
column activity. If the column is not properly
deactivated, component breakdown will be
much higher.
Figure 2
Figure 2: Impact of injection technique on decomposition of carbamates: (a) = hot splitless, (b) = cold on-column; Peaks: 1 = bendiocarb, 2 = dimethoate, 3 = aminocarb, 4 = dioxacarb, 5 = carbaryl. (Ref: J. of HRC., Vol 13, nov.1990, p. 759.)
Figure 3
Figure 3 : Analysis of BDE according to EPA 1614 using the following EPA protocol: (a) using column as listed in method; (b) equivalent column, but with different deactivation. Both columns under exact similar conditions.
31chrom doctorseparation science — volume 1 issue 5
BDE or ‘flame-retardants’ are brominated
diphenyl ethers designed to be thermally
unstable, so they will act better as flame
retardants. GC analysis will be a challenge,
but it is possible.
Figure 3 shows the analysis of BDE-209
using the EPA 1614 methodology in which
the impact of deactivation on peak response
is shown. Using exactly similar conditions,
the well deactivated column produces less
degradation. Column inertness plays a role.
Even with well deactivated columns,
degradation still occurs as confirmed by
the ‘lead’ on DME-209. The key to setting
methods for thermolabile components is to
reduce the elution temperature.
Figure 4 shows the same column as in
Figure 3, but now the final temperature
does not exceed 295 °C. Consequently,
the decomposition of DBE-209 is greatly
reduced. Figure 5 shows an expansion of the
problem area.
Ways to reduce the elution temperatureThere are several ways to influence the
elution temperature. In the example of
Figure 5, the program did not exceed
295 °C. Typically, this will increase analysis
time as it will take longer to elute heavy
components.
Use higher flow rate, a flow program or a
pressure program: By doubling the optimal
flow rate, the elution temperatures will be
reduced by 20-25 °C. This is usually very
effective with non-MS detection systems. The
higher flow will cause some loss of efficiency,
Figure 5
Figure 5 : Expansion of problem area of BDE-209. Elution temperature has big impact on decomposition process.
Figure 4
Figure 4: Analysis of BDE using lower elution temperatures. Column: 30 m x 0.25 mm Rtx-1614, df = 0.1 μm; Oven: 120 °C (1 min) 295 °C (15 min) @ 15 °C/min; Injection: splitless; Carrier gas: He @ 2.5 mL/min constant flow
32 chrom doctor www.sepscience.com
so it may be a consideration to initiate the
pressure program after the key separations
are obtained.
Use a slower temperature program: By
using a slower temperature-programming
rate, components will elute at a lower
temperature. However, the downside of this
is longer analysis times and peak broadening
(lower response).
Use hydrogen, rather than helium, as the
carrier gas: Because of the higher optimal
flow rate, we can benefit from lower elution
temperatures, while working under optimal
conditions. Here, however, you must
deal with safety issues, which is another
discussion.
Use columns with thinner films: Elution
temperature is directly dependent on the
amount of stationary phase (film thickness).
Use a 0.10 μm film instead of a 0.25 μm one.
Use a 0.32 mm i.d. capillary: A 0.32 mm
capillary with 0.1 μm film will have higher
phase ratio, which results again in a lower
elution temperature. The 0.32 mm column,
however, will be lower in efficiency, so we
may lose some separation efficiency. If the
target components elute with sufficient
resolution from their neighbours, you can
also apply a pressure program. This is very
effective with 0.32 mm columns.
Use shorter columns: The absolute time
components are in the column should be
a short as possible. Shorter columns will,
therefore, give higher response, but will
have lower efficiency, which will impact on
resolution, similar to that discussed using
0.32 mm columns. If we take a 15 m column
instead of a 30 m one, resolution is only
impacted by a factor 1.4. Figure 6 shows
the separation of the BDE. The components
elute below 295 °C and the total time in
the column is now reduced by a factor of
2. To compensate for efficiency loss, one
can choose a smaller diameter column; for
example, a 20 m x 0.15 mm column will
generate the same efficiency as a 30 m x
0.25 mm one.
SummaryFor analysing thermally labile components,
the best injection technique is cold-on-
column. To minimize exposure to the high
temperature environment, we need to
use inert columns with a high phase ratio.
In addition, short columns are preferably
operated with high gas velocity and slow
temperature programming.
This article was written by Jaap de Zeeuw, a
specialist in gas chromatography working for
Restek Corp.
Figure 6
Figure 6: Fast analysis of DBEs using 15 m x 0.25 mm Rtx-1614 column. Shorter time at higher temperature will also result in reduction of decomposition.
33chrom doctorseparation science — volume 1 issue 5
AnApplication
notes
34 application notes www.sepscience.com
UHPLC – Resolution vs E� ciency
Company: Fortis Technologies
Summary: Fortis Technologies has published an application
note on the role of resolution vs e� ciency in UHPLC.
Previously, the increases that can be made from using
smaller particles in UHPLC, have been widely demonstrated,
including improved e� ciency leading to greater resolution,
sensitivity and speed of analysis. However the biggest gains
in resolution come from the use of selectivity. By having a range of phase chemistries
available the analyst can improve resolution which can then also lead to speed
increases. All of these factors are discussed in this current application note.
www.fortis-technologies.com Tel: +44 151 336 2266
Application Note
IntroductionThe current trend towards using high pres-sure in LC is well documented, high efficien-cies, good resolution and fast throughput being the goal that has driven the move towards the use of sub 2um particles.In previous work we have shown that for short fast gradients well packed 3um For-tis columns can provide equivalent, if not
more, peak capacity than other commer-cial sub 2um columns. In this poster we show that for those analysts already work-ing with ultra high pressure LC systems and 2um particle columns that it is im-portant to consider the role of stationary phase selectivity when trying to maximise resolution and not rely on efficiency alone.
Improving ResolutionApproaches to improving resolution involve making changes to one or more of three variables; efficiency, retention and selectiv-ity. The move towards using sub 2um par-ticles has been driven by the theory that
the resulting jump in efficiency will lead to significant improvements in resolution. As can be seen in the Carr Equation (figure 1) that efficiency (N) does play a significant part in improving resolution, however by far the greatest factor is column selectivity.
With new small particle columns being re-leased by column manufacturers it is important that a range of phase chemistries are offered to allow the analyst an opportunity to maxim-ise resolution rather than depending on effi-ciency alone. Also it should not be assumed that all commercial C18 products on the mar-ket have the same selectivity, therefore as well as evaluating new phase chemistries it might be wise to test some alternative C18’s.Figure 2 shows an example of where in-creasing efficiency by moving from 3um Fortis C18 to 2.1um Fortis C18 does not pro-vide the full resolution of two closely related
compounds. However by altering selectivity, using Fortis PhenylTM chemistry, at the same time as decreasing particle size we are able to obtain resolution whilst decreasing col-umn length and as a result analysis time.
Use of an alternative phase chemistry such as Phenyl can significantly reduce analysis time of a set of compounds whilst main-taining resolution of closely eluting peaks. By combining efficiency from small particles with selectivity from stationary phase chemis-tries much better resolving power is available, potentially in a much shorter period of time.
ConclusionThe use of small particles in Ultra High Pressure LC can provide the analyst with increased sensitivity and resolution. We have shown that an important considera-tion when trying to maximise resolution of closely eluting compounds is the role of selectivity. The application of alternative chemistries based on 2.1um particles can provide greater increases in resolution than the application of small particles alone.
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2.1um Fortis Phenyl (100x2.1mm)
Efficiency Selectivity
1. Isonicotinamide 2. Nicotinamide
3.5mins4mins
“it is important that a range of phase chemistries are of-fered to allow the analyst an opportunity to maximise res-olution rather than depending on efficiency alone”
UHPLC - Resolution vs Efficiency
Mobile Phase: 80:20 20mM NH4OAc Flow: 0.2ml/min Temp: 40°C Wavelength: 210nm
N
Efficiency SelectivityRetention
R= k’k’+1
-14N k’
k’+1k’
k’+1-1-
1.00 1.05 1.10 1.15 1.20 1.250.0
0.5
1.0
1.5
2.0
2.5
3.0
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0 5 10 15 20 25
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k
Nk
Res
olut
ion
(R)
1.00 1.05 1.10 1.15 1.20 1.250.0
0.5
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1.5
2.0
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Automated SPE and Fast GC-ECD Analysis of PCBs in Waste Oil
Company: Gerstel
Summary: A fast SPE-GC-ECD method for the analysis of PCBs
in waste oil was developed. A complete pro� le was obtained
following SPE with a 12 minute GC run-time using a modular
accelerated column heater. Full automation of the sample
preparation and analysis (except sample weigh-in) enables
a daily throughput of 100 samples. A wide range of
concentrations can be determined using a dedicated column and electron capture
detection. This work describes the use of automated SPE with the GERSTEL MPS
3 autosampler with SPE option in combination with fast GC-ECD analysis for the
determination PCBs in waste oil.
Ap
pN
ote
6/2
008 Automated SPE and Fast GC-ECD
Analysis of PCBs in Waste Oil
Karine Jacq, Bart Tienpont, Frank DavidResearch Institute for Chromatography, Pres. Kennedypark 26, B-8500 Kortrijk, Belgium
KEYWORDSMACH, Fast GC-ECD, SPE, PCB, waste oil
ABSTRACTA fast SPE-GC-ECD method for the analysis of PCBs in waste oil was developed. A complete profi le was obtained following SPE with a 12 minute GC run-time using a modular accelerated column heater (MACH). Full automation of the sample preparation and analysis (except sample weigh-in) enables a daily throughput of 100 samples. A wide range of concentrations can be determined using a dedicated column and Electron Capture Detection (ECD).
INTRODUCTIONThe offi cial method for the analysis of PCBs in waste oil (DIN EN 61619) is time consuming and labor intensive (dilutions; extraction, column preparation and cleaning; manual solid phase extraction…) and it requires a long GC run (around 40 min).
Speed of analysis in capillary GC can be increased by using fast and ultra-fast temperature programming. In general, peak resolution will be reduced when the temperature gradient is very fast, but for several applications, some loss of resolution can be accepted. Recently, direct resistive heating of the capillary column resulting in very fast heating rates (> 1800 °C/min) has been introduced [1]. The system available via GERSTEL under the name Modular Accelarated Column Heater (MACH, GERSTEL GmbH, Mülheim an der Ruhr, Germany) is mounted onto the door of a standard GC holding up to four modules containing separate capillary columns
35application notes separation science — volume 1 issue 5
ZIC-HILIC Separation of Purines and Pyrimidines
Company: SeQuant
Summary: SeQuant o� ers an application note describing the hydrophilic
interaction liquid chromatography separation of the compounds thymine, uracil,
adenine, guanine and cytosine using a ZIC-HILIC, PEEK 150 x 2.1 mm, 5 μm, 200 Å
column. Chromatographic conditions are outlined, the resulting chromatogram
provided and retention factor and resolution calcualted for the purines and
pyrimidines.
Virus-Like Particle Characterization Using New AF4 Channel Technology
Company: Wyatt Technology
Summary: Virus-Like Particles (VLP) used for vaccination and immune
stimulation, are of growing interest in the pharmaceutical sciences. For quality
assurance there is a tremendous need for techniques that characterize di� erent
VLP fractions (fragments, monomers, dimers, trimers and aggregates). Wyatt has
previously demonstrated that the separation and subsequent quanti� cation of
di� erent VLP species is possible by AF4.
In this application note a stressed VLP sample was analysed by AF4 (equipped with multi-angle light
scattering and UV detection) by using either Wyatt’s standard channel (25 cm) or a smaller channel (18 cm)
with a spacer height of 350 μm.
DAWN®, miniDAWN®, ASTRA®, Optilab® and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation. ©2007 Wyatt Technology Corporation 9/12/07
Light Scattering for the Masses™
irus-Like Particles (VLP) used for vaccination and immune stimulation, are of growing interest in the pharmaceutical sciences. For quality assur-
ance there is a tremendous need for techniques that characterize different VLP fractions (fragments, mono-mers, dimers, trimers and aggregates). We have recently demonstrated that the separation and subsequent quan-tification of different VLP species is possible by AF4. However, some disadvantages, like long equilibration and analysis times, as well as the need for high sample amounts and large eluent volumes, been overcome by using new, shortened channel geometries.
A stressed VLP sample was analyzed by AF4 (equipped with multi-angle light scattering and UV detection) by using either Wyatt’s standard channel (25 cm) or a smaller channel (18 cm) with a spacer height of 350 µm.
Comparative AF4 measurements of VLPs with the standard channel (25 cm) and the new channel (18 cm) revealed almost similar peak heights when 20 µg VLP were injected in the standard channel or when 10 µg VLP were injected in the new channel, respectively (Figure 1). Increased peak heights obtained with the new channel are due to sharper peak resolutions. Thus, analysis is possible with significantly less sample amount (Figure 2). At the same time, analysis time and solvent volume were reduced (Table 1).
The standard channel technology has limitations concerning sample amount and separation time. By con-trast, applying Wyatt’s new channel technology analysis of far lower VLP amounts is possible in clearly shorter time and remarkably lower eluent volumes. Thus, it can be stated that the new channel technology is a clear improvement for VLP characterization as compared to the standard channel.
Virus-Like Particle Characterization Using New AF4 Channel Technology
Submitted June 21, 2007. This note graciously submitted by R. Lang and G. Winter,Ludwig Maximilians University, Department of Pharmacy, Pharma-ceutical Technology and Biopharmaceutics, 81377 Munich
Standard Channel (25 cm)
New Channel (18 cm)
time/run 56 mins 31 minseluent volume/run 159 mL 70 mLinjection amount 20 mg 2.5-10 mg
Table 1. Comparison between standard channel and shorter channel.
Figure 1. Comparison between standard channel and shorter channel.
Figure 2. Different injection amounts compared.
Rapid, High-Resolution, Normal-Phase Isocratic Chiral Separations
Company: Eksigent
Summary: Conventional carbon-centered enantiomericity has become a major
aspect of pharmaceutical drug development over the last twenty years. Although
enantiomeric drug forms have long been known to exist, attention to the relative
bioactivity of the enantiomers was often not addressed. More recently, drug
manufacturers have investigated the pharmacological pro� les of the individual
isomers, and in some cases, found that the bioactivity of the drug substance could
be wholly or substantially attributed to a single enantiomer.
0 1 2 3 4 5-100
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200
300
400
500
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700
800
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orba
nce
(mA
U@
220n
m)
time (minutes)
fenoprofen
application note use of the expressLC™ system for chiral drug analysis
rapid, high resolution, normal phase isocratic chiral separations
introduction Conventional carbon-centered enantiomericity has become a major aspect of pharmaceutical drug development over the last twenty years. Although enantiomeric drug forms have long been known to exist, attention to the relative bioactivity of the enantiomers was often not addressed. More recently, drug manufacturers have investigated the pharmacological profiles of the individual isomers, and in some cases, found that the bioactivity of the drug substance could be wholly or substantially attributed to a single enantiomer.
figure 1. chromatogram of the two enantiomers of fenoprofen figure 2. chromatogram of the two enantiomers of thalidomide
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50
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150
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250
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350
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rban
ce (m
AU
@22
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)
time (minutes)
thalidomide
36 application notes www.sepscience.com
A Cryogen-free Method for Monitoring Trace Greenhouse Gases in Air
Company: Markes International
Summary: In response to the Kyoto Protocol, ‘Clean
Development Mechanism’ regulations are being enacted
in a number of countries to facilitate and control
greenhouse gas emission trading. Trace-level greenhouse
gases of interest include chloro� uorocarbons (CFCs) and
hydrochloro� uorocarbons (HCFCs). Improved methods for
monitoring many such compounds in air have recently been reported using sorbent
tube or canister-based air sampling methods together with thermal desorption (TD)-
GC/MS analysis per US EPA ‘air toxics’ Methods TO-17 and TO-15, respectively. This
note demonstrates detection limits below 100 ppt for all CFCs and HCFCs on the ‘air
toxic’ list using a Markes electrically cooled TD platform with GC/MS running in full
scan mode.
Introduction
In response to the Kyoto Protocol, ‘Clean
Development Mechanism’ (CDM) regulations
are being enacted in a number of countries to
facilitate and control greenhouse gas (GHG)
emission trading. Many of the new regulations
require the monitoring of bulk greenhouse
gases such as carbon dioxide and methane and
some require additional consideration of other
lower level and more analytically challenging
compounds. Examples of this include proposed
amendments to the European Emission Trading
Scheme Directive 2003/87/EC1 and Australia’s
recent government white paper on a low
pollution future2.
Trace-level greenhouse gases of interest
include chlorofluorocarbons (CFCs) and
hydrochlorofluorocarbons (HCFCs). Improved
methods for monitoring many such compounds
in air have recently been reported using
sorbent tube or canister-based air sampling
methods together with thermal desorption (TD)
- GC/MS analysis per US EPA ‘air toxics’
Methods TO-17 and TO-15 respectively (see
Markes TDTS Notes 81 and 86). This work
demonstrates detection limits below 100 ppt for
all CFCs and HCFCs on the ‘air toxic’ list using
a Markes electrically-cooled TD platform with
GC/MS running in full scan mode.
However, not all trace level green house gases
are included on the standard US EPA list of
target ‘air toxics’. Perfluorocarbons for example,
are a class of long lived greenhouse gases, the
most volatile of which, carbon tetrafluoride
(CF4), has a boiling point of -128°C. CF4 is
present in the atmosphere at very low
concentrations, but has more than 5,000 times
the ‘global warming potential’ (GWP) of CO2
and a half life in the atmosphere of many
thousands of years. The extreme volatility of
CF4 makes it very difficult to trap/concentrate
and measure at low levels. Similarly,
hexafluoroethane (C2F6), has a boiling point of
-78ºC and over 10,000 times the GWP of CO2.
Other analytically-challenging greenhouse
gases, which don’t appear on the air toxics list
include CF3Cl, nitrous oxide (N2O) and sulphur
hexafluoride (SF6) – see table 1.
Not all of these ultra-volatile GHGs are readily
available. CF3Cl, for example, is banned in
many countries and cannot be obtained as a
standard. It was therefore decided to evaluate
the applicability of the same cryogen-free TD-
GC/MS technology used for air toxics
monitoring (TDTS 81 and 86) for the most
challenging ultra-volatile GHG species (CF4,
C2F6, SF6 and N2O). If successful, this would
demonstrate that such a monitoring system
could be used for both ultra-volatile GHGs plus
higher boiling CFC & HCFC air toxics and, by
extrapolation, any compound in between.
T D T S Thermal Desorption Technical Support
Note 87: A cryogen-free method for monitoring trace
greenhouse gases in air
Key Words:
Trace green house gases, Kyoto Protocol, CFCs, HCFCs, UNITY 2, CIA 8, air toxics, GHG
ww
w.m
arkes.c
om
Markes International Ltd. T: +44 (0) 1443 230935 F: +44 (0) 1443 231531 E: [email protected]
CompoundBoiling point (°C)
GWP (100 year) 2001 IPCC
Estimatedatmospheric
lifetime (years)
CF4 -128 5700 50000
C2F6 -78 11900 50000
N2O -88 296 114
CF3Cl -81 14000 Info. not available
SF6 -64 23900 3200
Table 1: Greenhouse gases with high GWP, notfound in the regular list of US EPA ‘air toxics’
Edmass: Top-Down Sequence Validation on a Benchtop MALDI-TOF Mass Spectrometer
Company: Bruker Daltonics
Summary: This application note describes a concept called
Edmass, the Top-Down sequence analysis on a benchtop
linear MALDI-TOF (MALDI-TDS) to derive C- and N-terminal
protein sequence information directly in the mass
spectrometer – without initial protein digestion. MALDI-
TDS was applied to the research study 2009 for which the
ABRF-ESRG (Edman Sequencing Research Group of the Association of Biomolecular
Research Facilities) provided two samples and expected N-terminal sequence
assignments from both proteins.
Bruker Daltonics
This study describes the analysis of the 2 samples provided by ABRF-ESRG 2009 using Top-Down Sequencing on a benchtop MALDI-TOF [1]. It highlights how a benchtop MALDI-TOF can efficiently be applied to validate the N- and C-terminal sequences of proteins.
Introduction
The major application of Edman sequencing today is the validation of proper N-terminal sequence expression in recombinant protein production. Here, in fact, the availability of both N- and C-terminal sequences is the most important aspect that could not be addressed by Edman sequencing to date.We describe here a new concept called Edmass™, the Top-Down sequence analysis on a benchtop linear MALDI-TOF (MALDI-TDS) to derive C- and N-terminal protein sequence information directly in the mass spectrometer – without initial protein digestion [2, 3]. The technique is described in greater detail in our Application Note MT-96 [4].MALDI-TDS was applied to the research study 2009 for which the ABRF-ESRG (Edman Sequencing Research Group of the Association of Biomolecular Research Facili-ties) provided 2 samples and expected N-terminal sequence assignments from both proteins.
Experimental
Samples (20 pmol) were prepared using the sDHB matrix (#209813, Bruker) and analyzed on the microflex™ LT benchtop linear MALDI-TOF MS (Bruker) by in-source decay
Application Note # MT-95
Edmass™: Top-Down Sequence Validation on a Benchtop MALDI-TOF Mass Spectrometer
(ISD) as previously described [3, 4]. A method for peptide analysis in linear positive ion mode was optimized by mass range extension and detection gain enhancement. Several hundreds to thousands of shots were accumulated and processed. External calibration was performed using ISD c-fragment ions (average masses) of intact BSA.Linear mode ISD spectra (ISD) were peak picked in flexAnalysis™ 3.0, submitted to BioTools™ 3.2 (both Bruker software packages) and directly analyzed by database searching using a Mascot 2.2 (Matrix Science, UK) inhouse server. A new instrument type “MALDI-ISD” was created on Mascot Server with the following specification: 1+ ions only, a, c, z+2 and y-ions, as this reflects the typical ion types in ISD spectra that we used for MALDI-TDS. All protein sequencing work that was required in this study could be performed through straight MS/MS ion searches, where arbitrary strong ISD fragment ions were specified as “virtual” parent ions in the Mascot search dialog. SwissProt was used for Mascot searches. The N-terminus of sample 1 was identified by searching the NCBI database as it also contains recombinant protein constructs.
Results
Our results on the microflex [5] are summarized in the official ESRG documentation [6,7] as entry “ESRG-015” (Tab 1). Both samples (~ 40 kDa) provided sequence calls from the N-terminus and the C-terminus in the same dataset permitting their identification as ADH1_YEAST and G3P_RABIT. All samples were prepared with the 3 matrices
; ; ;
“If a protein aggregates, but there’s no Wyatt instrument to detect it, does it still aggregate?”
DAWN HELEOS. The most advanced multi-angle light scattering instrument for macromolecular characterization.
Optilab rEX. The refractometer with the greatest sensitivity and range.
ViscoStar. The viscometer with unparalleled signal-to-noise, stable baselines and a 21st-century interface.
Eclipse. The ultimate system for the separation of macromolecules and nanoparticles in solution.
DynaPro Plate Reader. Automated dynamic light sattering for 96 or 384 or 1536 well plate samples.
© 2008 Leo Cullum from cartoonbank.com. All Rights Reserved. DAWN, Optilab, DynaPro and the Wyatt Technology logo are registered trademarks, and ViscoStar and Eclipse are trademarks of Wyatt Technology Corporation.
CORPORATIONCORPORATIONCORPORA
That’s the problem with relying on elution times to characterize macromolecules. You don’t
really know if you’re right—you can only assume. Which is why every major pharmaceuti-
cal and biotechnology company, as well as most federal regulatory agencies are switch-
ing from relative methods to Wyatt Technology’s absolute measurements. Our DAWN®
multi-angle light scattering (MALS) instruments allow you to determine absolute
molecular weights and sizes without relying on so-called standards, or measurements made
in someone else’s lab. Wyatt instruments measure all of the quantities required for deter-
mining absolute molar masses directly. So call 805.681.9009 or visit wyatt.com and request
our free 28-page Ultimate Guide to Light Scattering. You’ll learn
how to end your dependence on reference standards forever,
and start detecting aggregates you never even knew were there.
TuTechnology
update
38 technology update www.sepscience.com
Key
Email the company
Product information
Applications
Additional information Agilent launches 1290 Infinity LC system into the UHPLC market
Manufacturer: Agilent
Manufacturer’s description: Agilent Technologies has introduced the 1290 Infinity Liquid
Chromatography System, designed to deliver greater power, speed and sensitivity for
enhanced performance in the high-end ultra high performance liquid chromatography
(UHPLC) market. Reported features of the system include:
• High separation power and flexibility: The company claims that the 1290 delivers the
industry’s largest analytical power range, enabling users to deploy any particle type, any
column dimensions or any mobile and stationary phases. In addition, it reportedly delivers
the foundations for method transferability from and to any vendor’s UHPLC and HPLC
systems.
• Complementary columns: Agilent has also introduced ZORBAX Rapid Resolution High
Definition (RRHD) columns. The 1.8 μm particle size delivers high resolution and peak
definition for both simple and complex separations.
• MS compatibility: The 1290 Infinity LC is designed to drive even higher levels of performance
from the company’s LC/MS systems. It is claimed that the lowest possible delay volume, low
sample carryover, integrated control and operation with MassHunter MS software, and the
ability to perform fast, ultrahigh resolution LC separations contribute to this performance.
39technology update separation science — volume 1 issue 5
separationdriving analytical chemistry forwardsscience
www.sepscience.com
• Infi nity binary pump: The 1290 Infi nity binary pump module reduces background noise, contributing to the system’s
very high signal-to-noise ratio. Active Damping reduces ‘pump ripples’ and associated UV noise, and the company’s
proprietary Jet Weaver microfl uidic mixing technology, further enhances performance.
• UV Diode Array Detector: This contains a Max-Light Cartridge Cell with optofl uidic waveguides, providing very low
limits of detection and high signal-to-noise ratio. In addition, baseline drift is minimized for more reliable and precise
peak integration, because compromising refractive index and thermal eff ects are nearly eliminated, it is claimed.
• High throughput: The 1290 Infi nity Autosampler and Thermostatted Column Compartment modules contain a
number of usability and high-throughput features, including the ability to confi gure the system to run more than
2,000 samples per eight-hour shift. Alternating Column Regeneration (ACR) reduces cycle time by half compared
to single column confi guration, and throughput can be maximized further using automatic delay volume reduction,
overlapped injections, offl ine data analysis and external needle wash capabilities.
The company claims that the 1200 Series LC portfolio lets customers tailor the exact systems they need, from the
simplest manual isocratic LC through the world’s highest-performance, fastest, most sensitive UHPLC systems.
“Limits of detection for the pharmaceutical impurities were as low as 0.001% relative to the main compound using the
new diode array detector,” said Dr Pat Sandra of the Research Institute for Chromatography in Belgium, another early
access user. “This is more than one order of magnitude lower than required by US FDA.”
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March 2009
www.sepscience.com
Coupling capillary columns in gas
chromatography
Analytical trends in iso�avone
studies
Minimizing downtime in QA
pharmaceutical laboratories
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 1
Febraury 2009
www.sepscienceasia.comwww.sepscienceasia.com
Volume 1 / Issue 1
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 2
February 2009
www.sepscienceasia.com
Exploiting particle size to reduce
acetonitrile consumption
Multiresidue analysis using SBSE
and GC-MS/MS
Chromatographic methods for
Con�rming biological activity
markers in fruit
Asia Paci�c
40 technology update www.sepscience.com
SampleGenie Improves HPLC Fraction Pooling Workflow
Manufacturer: Genevac
Manufacturer’s description: Genevac has
announced a technical study that illustrates
how its SampleGenie technology enables
removal of steps from a centrifugal evaporator/HPLC
fraction pooling protocol to improve workflow.
Traditionally HPLC fraction pooling protocols have involved drying multiple
fractions in a centrifugal evaporator, re-suspending pooled fractions into a single vial and then re-drying before
storage and analysis. Even with modern evaporators such processes typically take 2-3 days to complete. Offering the
ability to automatically pool (without robotics) multiple HPLC fractions into a single small sample vial, SampleGenie
has been designed to simplify the protocol to a single overnight drying step before storage and analysis, claims the
company. Improved speed of evaporation and a reduction in the number of sample transfer steps are highly desirable
in a sample preparation method in order to streamline workflow in busy laboratories.
SampleGenie enables samples in the company’s centrifugal evaporators to be concentrated, dried or fast freeze
dried directly into a single vial, eliminating the need for reformatting of samples after drying. The flasks act like a
funnel and permit multiple large volume samples to be concentrated directly into an HPLC (or GC) autosampler vial.
According to Genevac, the SampleGenie is available to cope with most solvent types.
41technology update separation science — volume 1 issue 5
GE Healthcare Launches Suite of 2-D Electrophoresis Products Manufacturer: GE Healthcare
Manufacturer’s description: GE Healthcare offers a suite of products to improve and simplify
2D electrophoresis workflow from sample preparation through to analysis, and further
enhance quantitation in protein expression studies.
The ready-to-use products offer significant improvement in data quality for 2D
electrophoresis experiments, and also reduce costs and time for protein expression
analysis (at least a threefold cost saving and sixfold time saving with 2-D DIGE), claims the
company. The products are designed to be used individually or as a complete solution to
maximize results from 2D analysis and are also fully compatible with 2D DIGE (Difference Gel
Electrophoresis).
“We have worked to identify and address the challenges that our customers face in 2D
electrophoresis. Most of them relate to unwanted sources of experimental variation,” said
Rita Marouga, Product Manager, GE Healthcare. “These products are designed specifically
to improve consistency and reduce heterogeneity throughout the 2D workflow, thereby
enabling identification of differences and changes in protein expression attributable to
biological variation with high confidence.”
The products include: precast low fluorescent DIGE gels and DIGE buffer kit; repackaged
CyDye DIGE Fluor saturation and minimal dyes that better suit experimental designs; and
reformulated IPG buffer and IPG strips (Immobiline DryStrip gels) that
improve spot resolution. These augment the previously
released 2D Protein Extraction Buffers, IPGbox and
DeCyder 2-D v7 software that contribute
to error reduction and optimization
of the results in the 2-D
experimental workflow.
www.sepscience.com
Low volume SPE assays enhanced by C18 sorbent
Manufacturer: Porvair Sciences
Manufacturer’s description: Porvair Sciences Ltd has announced the
availability of a range of BioVyon C18 silica columns and microplates
for use in low-volume solid phase extraction (SPE) assays.
Packed-bed SPE columns and microplates traditionally perform
relatively ineffi ciently when using the shallow sorbent beds necessary
to get good recovery from smaller sample volumes. By immobilizing
the C18 sorbent within the porous BioVyon polymer the company
claims to have created a novel, high surface area matrix that provides
excellent control of fl ow rate. Further, the immobilized C18 sorbent
cannot form liquid channels and does not require inert frits to
support it thereby minimizing hold-up volume. The combination of
these attributes has enabled Porvair to introduce a this range of SPE
columns and microplates designed to provide higher consistency
and greater recoveries for small sample volume assays.
BioVyon C18 is initially being off ered in 96-well microplates as a
10 mg per well loading suitable for low-volume bioassay preparation
and clean-ups. In the popular 1 mL cartridge format, BioVyon C18 is
available in a choice of 12.5, 25 and 50 mg loadings to suit individual
applications.
42 technology update
Register Now for your 20% Early Bird Discount
Conference Highlights
Singapore
www.sepscience.com
FoodEnviroDay One:
Pat SandraAdvances in Separation Sciences Deriven by the Metabolomics and Pro-teomics Quest for Biomarkers
Y.S. FungMicrofluidic Chip-Capillary Electrophoresis for Biomedical Applications
Eric Chun Yong ChanGC×GC/TOFMS Profiling of Human Bladder Cancer
Manfred RaidaMultidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes
Yi ChenNew Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples
Andrew JennerGC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism
Thomas WalczykElement Separation at the Microscale for High-Precision Isotopic Analysis of Biological Samples
Bioscience
Passion. Power. Productivity.
Passion. Power. Productivity.
Passion. Power. Productivity.
sponsors:
For all delegate enquiries email [email protected]
26–28 AugustBiopolis Science Park, Singapore
Day Two:
Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations
Phil NethercoteThe applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.
Sanjay GargThe Role of Analytical Science and Techniques in Early Phase Drug Discov-ery and Registration for Clinical Studies
Anne GohOnline Solid Phase Extraction-LC-MS in DMPK Applications
Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D
Shawn StanleyTBC
Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines
Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine
Day Three:
Alastair LewisTrace Pollutant Detection in Challenging Environments
Hian-Kee LeeSolvent-Minimized Sample Preparation for Separation Science
Siu Kwan SzeAn Advanced Proteomic Approach to the Discovery of Microbial Enzymes for Biorefining
Gongke LiMolecularly Imprinted Polymers for Trace Analysis of Complicated Samples
Paul HaddadDevelopment of Portable Separation Methods for the Identification of Terrorist Explosives by Analysis of Inorganic Residues
Philip MarriottHeadspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples
Jessie TongMultidimensional Gas Chromatographic Analyses of Flavours and Fragrances
Bahruddin SaadDetermination of Biogenic Amines in Food: Conventional and Nonconventional Approaches
Pharma TCM
44 technology update www.sepscience.com
Zebron ZB-XLB-HT Inferno GC columns for fast melamine analysis
Manufacturer: Phenomenex
Manufacturer’s description: Phenomenex has introduced the Zebron ZB-XLB-HT Inferno
high-temperature GC column designed to enhance routine GC/MS melamine analysis of
milk products. According to the company, the ZB-XLB-HT columns reduce total run time to
less than four minutes. Stable up to 400 ˚C, the high-temperature capability allows bake-
off of matrix contamination, present in milk and other food products, that would otherwise
decrease column lifetime.
Standard fused-silica columns are not engineered to withstand temperatures above 380˚C
and their coating begins to degrade, eventually becoming brittle
and inflexible. Phenomenex states that the Zebron Inferno
non-metal columns incorporate proprietary coating and
bonding technologies, providing stability at high
temperatures, low bleed and low activity.
The company’s Zebron ZB-5ms column is ideal
for routine analysis of milk products using the
FDA-recommended GC/MS method. Howevere, for
customers who need faster results, the Zebron ZB-
XLB HT Inferno GC column has been introduced. If a
laboratory prefers LC analysis, the Luna HILIC column
resolves cyanuric acid and melamine in less than three
minutes, claims the company. Phenomenex also offers complementary
Strata Melamine SPE cartridges.
“Our successful Zebron Inferno columns were the first non-metal columns to provide
stability at very high temperatures,” commented Sky Countryman, product manager for
Phenomenex. “With the addition of these new columns, our offering of products, methods
and expertise is the most comprehensive for melamine analysis.”
Automated dialysis as a sample preparation tool in ion chromatography
Manufacturer: Metrohm
Manufacturer’s description: Ion chromatography (IC) as an analytical technique has seen an impressive surge in
popularity. As for samples in a homogenous ionic form, hardly any preparation steps are required at all. According
to Metrohm, its patented stopped-flow dialysis paves the way for the convenient analysis of demanding samples
carrying high organic loads too.
In complex matrices carrying high organic loads such as waste water, soil eluates or dairy products, extensive
sample preparation is mandatory to prevent destruction of the column. Traditional preparation techniques such as
the Carrez precipitation do not provide a satisfying answer as they cannot be automated and are error-prone.
Metrohm claim that its compact stopped-flow dialysis is an elegant alternative. This fully automated sample
preparation setup is based on the selective diffusion of ions from one liquid (sample/donor solution) to another
(acceptor solution) through a membrane.
Contrary to dynamic dialysis, where two solutions continuously pass through the dialysis module, the acceptor
solution is stopped until its concentration is the same as that in the donor solution, the company states. This patented
stopped-flow procedure takes between 10 and 14 minutes and can be directly coupled to an IC setup. As the dialysis
is performed during the recording of the previous sample’s chromatogram, the overall analysis time is not significantly
prolonged.
45Technology update separation science — volume 1 issue 5
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Volume 1 / Issue 3
March 2009
www.sepscience.com
Coupling capillary columns in gas
chromatography
Analytical trends in iso�avone
studies
Minimizing downtime in QA
pharmaceutical laboratories
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 1
Febraury 2009
www.sepscienceasia.com
液相色谱-质谱联用新方法的建立
微波辅助溶剂萃取与气相色谱-质谱
联用分析太子参中的挥发物
微芯片电泳用于生物医学
分析
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 2
February 2009
www.sepscienceasia.com
Exploiting particle size to reduce
acetonitrile consumption
Multiresidue analysis using SBSE
and GC-MS/MS
Chromatographic methods for
Con�rming biological activity
markers in fruit
Asia Paci�c
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 2
April 2009
www.sepscienceasia.com
固相微萃取-高效液相色谱分析羟烷基喹诺酮
固相萃取和气相色谱与三级四极杆质谱联用测
定熟食品中痕量食品衍生的有害物质
液相色谱填料尺寸对降低
溶剂消耗的影响
中国版
Volume 1 / Issue 5
May 2009
www.sepscience.com
A liquid chromatographer’s
introduction to mass spectrometry
Analysing synthetic polymers with
solvent enhanced light scattering
Minimizing decomposition of
components during GC analysis
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 4
April 2009
www.sepscienceasia.com
A practical review of
multidimensional LC
How to deal with overloading in GC
Asia Pacific
Rapid determination of RRFs
for the quantitation of impurities
in pharmaceuticals