Journal Separation Science
Transcript of Journal Separation Science
Volume 1 / Issue 3
March 2009www.sepscience.com
Coupling capillary columns in gas chromatography
Analytical trends in iso� avone studies
Minimizing downtime in QA pharmaceutical laboratories
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3contentsseparation science — volume 1 issue 1
contentsVolume 1 / Issue 3
March 2009www.sepscience.com
Coupling capillary columns in gas chromatography
Analytical trends in isoflavone studies
Minimizing downtime in QA pharmaceutical laboratories
Critical aspects and recent trends in the analysis of food soy iso� avones
M.A. Rostagno
22
features
separationdriving analytical chemistry forwardsscience
Volume 1 / Issue 3March 2009
30
research round-up
Unravelling parasite pharmacokinetics
Evaluation of solid-phase microextraction methods for atmospheric analysis
The � rst step towards epitope-based vaccines
Finding contaminants in packaged food using LC-MS/MS
LC enantioseparation of β-amino acids
LC-MS/MS method aids pain research
Discovering a deeper shade of red
Solid-state analysis: Raman vs x-ray powder di� raction
Molecularly imprinted polymer analysis of agricultural pesticides
Estimation of adenosine and metabolites in brain tissue using high-performance thin-layer chromatography–densitometry
GC-MS odour pro� ling of buckwheat
Rr
Mr meeting report MSB 2009 chairman, Dr Jonathan Sweedler, answers questions about the recent conference.
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technology update An overview of recent technology advances in separation science and instrumentation.
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24 Development and evaluation of an instant column connector for capillary gas chromatography
Hans-Gerd Janssen and Daniela Peroni
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4 www.sepscience.com
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
contactsDean Graimes
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Scienti� c Director
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– Scienti� c Director
More health concerns
Just as I start to write this editorial another analytical
challenge is hitting the headlines - following on the heels
of the recent melamine and methamidophos scares, it
now seems that issues have been raised again about the
safety of bisphenol A.
Manufacturers of plastic baby bottles in the US are going
to remove bisphenol A (BPA) from their products following
growing concerns about the chemical’s e� ects on human
health. However, according to reports, it would appear that
these same manuafacturers will continue to use BPA in similar
products sold in other countries.
BPA is commonly used in the manufacture of plastics
and is the key monomer in polycarbonate plastics and
epoxy resins. Polycarbonate plastic properties, including its
shatterproof nature and transparency, have made it ideal for the
manufacture of drinking bottles (including baby bottles), sports
equipment, medical and dental equipment, CDs and DVDs, and
various household electronics. In essence, its use is widespread
in our society.
The dangers of BPA to human health through exposure
to these objects is still unclear, but we do know that BPA is
an endocrine disruptor in high enough doses. Endocrine
disruptors are substances that have the potential to interact
with the human hormone systems, and particularly with our
reproductive systems.
BPA is used extensively in food containers, either within
the plastic or as part of internal coatings to stop hazardous
substances leaching into food (e.g., metals from cans). However,
it is apparent that BPA can itself leach into food in small
quantities, particularly if food containers are heated. What has
yet to be clari� ed is whether this happens at levels that can
a� ect health.
Obviously, determination of bisphenol A is well within the
realm of modern analytical methodologies, so expect to see
technical articles and application notes on the subject over the
next few months.
David Hills
Book your place today!Conference Highlights
Singapore
www.sepscience.com
FoodEnviro
Day One:
Milos NovotnyThe Role of Capillary Separations and Mass Spectrometry in the Search for Cancer Biomarkers
C. Bor FuhImmunoassays Using Functional Magnetic Nanopaticles for Biochemical Analysis
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
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Confirmed sponsors:
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Pharma TMC
Day Two:
Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations
Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines
Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine
Tung-Hu TsaiMethods and Strategy of Microdialysis for Pharmacokinetics in Herbal Medicine
Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D
Shawn StanleyTBC
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
Key
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RrResearchround-up
Unravelling parasite pharmacokinetics
Czech RepublicBenzimidazole carbamates are widely used both in human and in veterinary medicine
because of their anthelmintic activity. Although the mode of their pharmacodynamic action
is known, the pharmacokinetics continues to be studied. A very important and interesting
aspect, which is worthy of studies, is an investigation of the resistance of frequently occurring
helminths; for example, Haemonchus contortus. This was the topic of a study led by Dr
Vladimír Kubíček from the Department of Biophysics and Physical Chemistry at Charles
University Prague, Czech Republic.
Published in Chromatographia [68 (9-10), 865-867 (2008)], a validated LC method is
proposed for the analysis of flubendazole and its metabolites in biological samples
of Haemonchus contortus. Two detectors were used — photodiode-array and
spectrofluorimetric. The native fluorescence of reduced flubendazole, the key substance
investigated during biological experiments, was used for its fluorimetric detection with a very
low limit of quantification (0.63 nmol/L).
“Haemonchus contortus is one of the most pathogenic parasites of small ruminants. The
treatment of haemonchosis is complicated because of relatively strong resistance of the
organism to common anthelmintics. Such pharmacokinetic research requires a reliable
method for analyses of biological samples to determine both the parent substance and its
metabolites. As the metabolites are usually present in very low concentrations in biological
samples, their determination is a challenging analytical task in the pharmacokinetic
investigation,” Dr Kubíček explained.
The research showed that flubendazole is benzimidazole exhibiting a significant
anthelmintic activity, but during the therapeutic action it is transformed by helminths to
form two main metabolites; for example, reduced flubendazole and hydrolysed flubendazole.
“According to our previous experiments, observations based on reduced flubendazole
concentration levels in Haemonchus contortus are desirable in the study of the resistance of
the helminth,” he said.
According to him, the usefulness of the photodiode-array detection (coupled in tandem with
fluorescence detection) was confirmed during their research because a verification of peak
purity of the analytes (by evaluation of UV spectra) was required many times.
“Using the proposed chromatographic method we are able to study metabolic processes
leading to lowering of the effectivity of flubendazole action during the treatment of
haemonchosis,” he said. Consequently, procedures can be tested suppressing the metabolic
degradation of flubendazole. Their next challenge is to develop further sensitive analytical
methods for on other benzimidazole anthelmitics.
6 research round-up www.sepscience.com
A paper in the Journal of Chromatography A [1209 (1-2), 44-54 (2008)]
describes how a method for trace analysis of a wide range of aldehydes
(saturated/unsaturated aliphatic, aromatic aldehydes, including hydroxylated
species, and dialdehydes) in an aqueous solution was optimized. Dr Alena
Kubátová from the Chemistry Department at the University of North,
Grand Forks, USA, whose research focus is atmospheric chemistry, explains,
“Aldehydes are signifi cant constituents in air particulate matter (PM) and due
to their reactivity may play an important role in reactions occurring in the
atmosphere. Although, aldehydes are well recognized in air samples (mainly
gas phase) their characterization in PM (solid phase) is limited due to their
trace concentrations.”
Three diff erent solid phase microextraction (SPME) methods coupled with
derivatization using pentafl uorobenzyl hydroxylamine (PFBHA) for analysis
of trace concentrations of wide range of aldehydes (aliphatics, aromatics,
hydroxylated and dialdehydes) were evaluated in the study. “Based on
the optimization of temperature and time, and determination of limits of
detection and linear range, the derivatization with PFBHA followed by liquid
phase SPME at 80 °C for 30 min was found to be optimal for the majority of
aldehydes, with detection limits reaching 0.1 µg/L,” Dr Kubátová added. The
study also revealed that the method commonly used for the determination
of aldehydes in various matrices, SPME with PFBHA on-fi bre derivatization,
was not able to recover hydroxyl aromatic aldehydes and dialdehydes. “These
species were found in wood smoke and diesel exhaust PM extracts and
thus on-fi bre SPME would not be suitable for determination of aldehydes in
extracted PM. Also, the EPA 556 method was not sensitive to allow for the
quantifi cation of aldehydes in PM. Thus, samples had to be re-concentrated,
which resulted in evaporative losses of low molecular weight derivatized
aldehydes,” she explained.
She and her team intend to fi nd the source of discrepancies in dialdehyde
profi les observed between two methods (liquid SPME and EPA 556) in
future research, which will enable a better understanding of PM structure
and processes occurring in the atmosphere. “We will investigate this with
respect to the possibility that a portion of PM can be composed of aldehyde
oligomers, which may be released as monomers during extraction,” she
concluded.
Evaluation of solid-phase microextraction methods for atmospheric analysisUSA
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7research round-upseparation science — volume 1 issue 3
Czech Republic Specifi c allergen immunotherapy is frequently associated
with adverse reactions. Several strategies are being
developed to reduce the allergenicity while maintaining
the therapeutic benefi ts. Peptide immunotherapy is
one such approach. Methods for the simple and rapid
identifi cation of immunogenic epitopes of allergens (i.e.,
allergenic epitopes) are ongoing and could potentially
lead to peptide-based vaccines.
A study published in the Journal of Chromatography
A [1206 (1), 64-71 (2008)], documents how an epitope
extraction technique, based on biofunctionalized
magnetic microspheres self-organized under a magnetic
fi eld in a channel of a simple microfl uidic device
fabricated from polydimethylsiloxane, was applied
to the isolation and identifi cation of prospective
allergenic epitopes. Similarly to chromatographic
column separations, the easily replaceable plug of self-
organized beads in the channel benefi ts especially from
an even larger surface-to-volume ratio and an enhanced
interaction of the surfaces with passing samples.
Dr Barbora Jankovicova and her team from the
Department of Biological and Biochemical Sciences at
the University of Pardubice, Czech Republic, originally
performed the research as part of a national project
for the development of bioaffi nity reactors for on-
chip determination and analysis of epitopes with
immunogenic or allergogenic potential. “The major
goal of this project was to develop and verify the new
methodological strategy for rapid and cost-eff ective
identifi cation of signifi cant immunogenic epitopes
through the combination of chip-based magnetic
enzyme reactors (IMER) with immunoaffi nity capturing
(IMAR) of specifi c peptides. Our strategy was based
on a combination of a simple microfl uidic device
fabricated from polydimethylsiloxane (PDMS) with
biofunctionalized magnetic microspheres integrated into
the microfl uidic channel. We used this method for the
analysis of food allergens (ovalbumin),” Dr Jankovicova
explained.
According to her, the key fi nding was that the chip-
based epitope extraction technique, already validated
in their laboratory for low-molecular weight antigens, is
also applicable to the rapid and low-volume structural
characterization of high-molecular antigens, represented
by the food allergen, ovalbumin. “The peptide fragment
of ovalbumin HIATNAVLFFGR (m/z: 1345.75, position:
371–382) was identifi ed as a relevant allergenic epitope
in this way, which could be potentially used for peptide
immunotherapy of patients allergic to ovalbumin,” she
said.
The study concluded that such a microfl uidic magnetic
force-based epitope extraction technique applied in
the epitope mapping of ovalbumin has the potential
to be a signifi cant step towards developing safe and
cost-eff ective epitope-based vaccines. “This novel
analytical concept off ers many advantages and can be
an appropriate tool for the rapid and simple structural
characterization of antigens and for design of risk-free
immunotherapy in general. We believe that the work
described here is broadly applicable and provides a
fl exible and effi cient analytical approach for screening
of allergens, autoantigens and other immunogenic
biomolecules,” she said.
The fi rst step towards epitope-based vaccines
8 research round-up www.sepscience.com
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10 research round-up www.sepscience.com
SpainA paper in the Journal of Chromatography A [1208 (1-2),
182-188 (2008)] documents the successful separation of
isopropylthioxanthone (ITX), a common photo-initiator
in UV inks used in paper- or plastic-based packaging
materials, using a pentafluorophenylpropyl column
(HS F5).
“My research group is involved in developing new
analytical methods (mainly LC-MS/MS and GC-MS/
MS) to determine contaminants in environmental and
food samples,” explained main author, Dr Encarnación
Moyano from the Department of Analytical Chemistry
at the University of Barcelona, Spain. Having recently
started a new line of research to study compounds that
migrate from packaging to food, the group found that
the analytical methodology for the determination of ITX
at very low levels in food needed improvement.
A gradient elution with acetonitrile and a 25 mM formic
acid–ammonium formate at pH 3.75 are required to
provide an Rs of 1.3 between the two compounds. The
fragmentation pattern of ITX was studied using two mass
analyzers, an ion trap (IT) (multi-stage fragmentation)
and a triple quadrupole mass analyser of hyperbolic rods
[accurate mass (AM) measurement]. Instrumental quality
parameters of three acquisition modes provided by the
triple quadrupole mass analyser were studied and good
run-to-run precision (relative standard deviation, RSD,
lower than 10%) and limits of detection (LODs) down to
0.8 pg injected in the LC–MS/MS system were obtained.
The LC–MS/MS method using H-SRM Q1 acquisition
mode was used to analyse 2- and 4-ITX in a range of
food samples. The use of highly selective selected
reaction monitoring (H-SRM on Q1) resulted in improved
selectivity without sensitivity loss.
“Our key outcome was the development of a fast LC-
MS/MS method (6 mins) using a fluorophenyl column
that allows the individual determination of each ITX
isomer with high sensitivity and selectivity. Until now,
only one described method could chromatographically
separate both ITX isomers, but in more than 20 minutes,”
Dr Moyano said.
He believes the developed method is a useful tool
for quality control in analytical laboratories, because
it enables throughput analysis with a high degree of
sensitivity and selectivity. “This method also provides
enough identification points for confirmation analysis,”
he concluded.
Finding contaminants in packaged food using LC-MS/MS
LC enantioseparation of β-amino acidsHungaryIn a study to develop new analytical methods for the
effi cient separation of β-3-homoamino acids, Dr Péter
from the Department of Inorganic and Analytical
Chemistry at the University of Szeged, Hungary,
discovered that direct HPLC separation is possible when
applying a crown ether-based column. Published in
Chromatographia [68 (supplement 1), 13-18 (2008)], the
research, which entailed developing reversed-phase
high-performance liquid chromatographic methods for
the enantioseparation of ten unusual β-3-homo-amino
acids, also showed that optimization of the analytical
methods are crucial to have reliable results.
The underivatized analytes were separated on a
chiral stationary phase containing (+)-(18-crown-6)-
2,3,11,12-tetracarboxylic acid as chiral selector. The
eff ects of organic and acidic modifi ers and the mobile
phase composition on the separation were investigated.
The structures of the substituents in the β position
substantially infl uenced the retention and resolution.
The elution sequence was determined in some cases:
the S enantiomers eluted before the R enantiomers.
“The results mean that chiral purity of pharmaceuticals
can effi ciently be characterized by direct HPLC
methods,” Dr Péter said, adding “there is no universal
HPLC column for the enantioseparation of chiral
compounds, and method development is always
necessary.”
11research round-upseparation science — volume 1 issue 3
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CanadaA selective and sensitive quantitative method for the determination of capsaicin by LC-ESI/MS/MS was developed by
Dr Francis Beaudry, from the Laboratory of Mass Spectrometry and Medical Chemistry and the University of Montreal,
Canada. Capsaicin is the most abundant pungent molecule present in red peppers and it is widely used for food
flavouring, in pepper spray in self-defence devices and, more recently, in ointments for the relief of neuropathic pain.
Capsaicin is a selective agonist of transient receptor potential channel, vanilloid subfamily member 1.
Published in Biomedical Chromatography [23 (2), 204-211 (2009)] the method consisted of a protein precipitation
extraction followed by analysis using liquid chromatography electrospray quadrupole ion trap mass spectrometry.
Dr Beaudry explained the study was prompted by the treatment of pain following traumatized nervous tissue
leading to hyperalgesia and allodynia in neuropathic animals, which was tested with several drugs with disappointing
outcomes. The vanilloid receptors (TRPV) comprise a family of proteins restricted to a subpopulation of primary
sensory neurons. TRPV1 is a non-selective cation channel and functions as an integrator of painful chemical and
physical stimuli, including noxious heat and low pH. TRPV1 antagonists exhibit analgesic effects on both inflammatory
and neuropathic pain.
Capsaicin and resiniferatoxin are two ligands of the vanillyl group that are use for the treatment of neuropathic pain.
“Our research group is particularly interested in the biomolecular mechanisms involved during the various stages
of neuropathic and chronic pain establishment, development and evolution, as well as the development of new
medicine. Recently, we studied the expression of several neuropeptides associated with neuropathic and chronic
pain mechanisms,” he said. Peptides like substance P, CGRP, VIP, neurotensin and dynorphin A were investigated
as potential biomarkers and efficacy markers using LC-MS/MS methods. Moreover, a recent study has shown that
a selective TRPV1 antagonist inhibited the release of substance P and CGRP in the spinal cord of neuropathic rats.
“However, interestingly, there was no DMPK study performed on capsaicin that demonstrates drug bioavailability. The
first step was to develop a robust quantitative method and verified metabolic stability of the drug in order to select an
adequate route of administration,” he added.
According to Beaudry, a quantitative method can be developed and validated to support a pharmacokinetic
study but more importantly, the metabolic stability of the drug is a concern. “With a half life of less than five minutes
in hepatic microsomes, it suggests that the clearance will be very high and the drug
exposure will be limited. Consequently, the response to the treatment could
be compromised. Despite the obvious problem associated with oral
administration of capsaicin, extensive first-pass metabolism will
significantly impact drug bioavailability,” he said.
Previous studies showed that capsaicin is very pungent,
however, results suggest it is extensively metabolized
in the liver and potentially, metabolites may
interact with TRPV1 or other receptors.
“In fact, we are particularly interested in
vanillylamine following the administration
of capsaicin and potential interaction with
TRPV1 and TRPV4. Our research group
has already demonstrated the analgesic
and anaesthetic effect of analogues
of vanillylamine such as eugenol and
vanillin,” he concluded.
12 research round-up www.sepscience.com
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14 research round-up www.sepscience.com
Discovering a deeper shade of red Portugal According to a paper in the Journal of Chromatography A, [1209 (1-2), 153-161 (2008)], a simple and rapid liquid
chromatographic method with diode-array UV/VIS spectrophotometric detection has been developed for the
authentication of dragon’s blood resins from Dracaena and Daemonorops trees. Dr Maria João Melo from the
Department of Conservation and Restoration at the New University of Lisbon in Caparica, Portugal, whose primary
research fi eld is colour in nature and art, explained: “Anthocyanins are nature’s glamorous palette, and the fl avylium
may be their ancestors”. Dragon’s blood is the generic name for the red resin obtained from species of Dracaena
(Dracaenaceae), Daemonorops (Palmae), Croton (Euphorbiaceae) and Pterocarpus (Fabaceae). “It is a complex resin,
used for centuries for medical and artistic purposes and much is still to be discovered about its use in ancient artistic
production,” Dr Melo added.
According to her research, dragon’s blood was originally produced from a species of Dracaena, namely the
dragon tree Dracaena draco. But because of the over exploitation of Dracaena, species of Daemonorops, Croton and
Pterocarpus were used as substitutes. As a result, both Dracaena cinnabari and Dracaena draco are endangered and
currently cited in the IUCN Red List of Threatened Species.
“In our study we discovered that the fl avylium chromophores, which contribute to the red colour of these resins,
could be used as markers to diff erentiate among species; and we developed a method for the authentication of
dragon’s blood resins from Dracaenaceae and Daemonorops trees,” she explained.
She believes the HPLC-DAD method developed in the study constitutes a breakthrough in the analysis of complex
samples containing dragon’s blood resin, including aged samples. It is anticipated that this could be used to monitor
the trade in endangered species of dragon’s blood, validate the species used in traditional Chinese medicinal
formulations, as well as in the area of cultural heritage to establish which species were used as dyes,” she said.
The HPLC-DAD method was also successfully applied to 37 samples of dragon blood resins from the historical
samples in the Economic Botany Collection at the Royal Botanic Gardens in Kew, UK; having identifi ed anomalies in
how samples in this collection had been labelled.
“One of the driving forces for this research is the fundamental study of how anthocyanins and fl avylium
chromophores are related. Could we prove that fl avylium are the anthocyanin ancestors? Presently, compared with
anthocyanins natural fl avylium are rare. Are there any chemical reasons for this natural selection?” she asked, adding
that she enjoyed the opportunity to collaborate with the team of the Kew Gardens, namely with Frances Cook and
Monique Simmonds, to study their botanical collection and to know that this new method could be used to monitor
the trade in endangered species of dragon’s blood.
15research round-upseparation science — volume 1 issue 3separation science — volume 1 issue 3
Solid-state analysis: Raman vs x-ray powder diff raction
New Zealand A study described in the Journal of Pharmaceutical and Biomedical
Analysis [49 (1), 18-25 (2009)] aimed to develop a reliable
quantifi cation procedure for mixtures of three solid forms of
ranitidine hydrochloride using X-ray powder diff raction (XRPD)
and Raman spectroscopy combined with multivariate analysis.
The eff ect of mixing methods of the calibration samples on
the calibration model quality was also investigated by main
author, Dr Jaakko Aaltonen from the School of Pharmacy at the
University of Otago in Dunedin, New Zealand.
“It is very common for small drug molecules to exhibit
polymorphism (i.e., diff erent solid forms of the same chemical
compound). Our group had a need to quantify diff erent solid
forms of a drug, ranitidine HCl, during a milling process, so we
had to fi nd a way to build a method for reliable calibration and
quantifi cation of the three known solid forms (form I, form II and
the amorphous form). We also wanted to know which one of
the two ‘reference’ methods for solid state analysis, x-ray powder
diff raction or Raman spectroscopy, would be better suited for the
job,” Dr Aaltonen explained.
According to him, when dealing with dry powder samples,
several things can aff ect the measurement, and sample
preparation and sampling is a science on its own. “We tested
diff erent sample preparation and sampling techniques and
their eff ect on sample homogeneity and whether the sample
preparation techniques induce unwanted changes to the
sample,” he said. The team found that the sample preparation
method does have eff ects on the calibration model performance,
and that the multivariate analysis methods we used (principal
component analysis, partial least squares regression) are suitable
for these purposes.
“In terms of application of these techniques, our results build
up the knowledge on analysis and quantifi cation of multiple
solid forms and amorphous content of solid samples. The
phenomena have been known for a long time, but streamlined
methods of analysis are not yet well established, however,
evolving quite rapidly. We are already using these methods as
standard practice, but of course, developing them further all the
time,” he concluded.
Shimadzu_Seperation _0309 17.02.2009 16:11
16 research round-up www.sepscience.com
Molecularly imprinted polymer analysis of agricultural pesticidesFrance Molecularly imprinted polymers applied to the determination of the residual mass of atrazine and metabolites
within an agricultural catchment (Brévilles, France) is documented in Journal of Chromatography A [1206 (2), 95-104
(2008)] by Dr Laurence Amalric from the Department of Metrology at Monitoring and Analyses for BRGM, France. The
company has extensive experience in the assessment of the vulnerability of aquifer resources to contamination by a
range of inorganic or organic pollutants. Vulnerability assessments have been typically based on monitoring activities,
laboratory and field experiments, expert knowledge and/or modelling activities.
“In our field study (located in Brévilles, 70 km west of Paris) the groundwater of the spring still exhibits a recurrent
contamination by atrazine and desethylatrazine, despite the fact that the atrazine application was stopped in the
Brévilles watershed after April 1999,” Dr Amalric said.
One of the hypotheses raised to explain these observations is the existence of a stock of atrazine and
desethylatrazine in the soils of the recharge area that would continuously feed the infiltrating water with these two
substances. The concentrations of each molecule would be particularly low, as the team’s routine methods did not
allow their detection in solid samples from different depths. “The estimation of the residual mass of pesticides in the
soils is hindered by problems of detection limit for these substances in solid matrices. As the matrix of soil samples are
often complex, a partial co-extraction of interfering substances can take place. Therefore, there was a great interest to
introduce selectivity during the sample pretreatment,” he explained.
Several reviews published in recent years report the interest of immunoextraction as a selective sample treatment
method. “This approach has been successfully applied by Valérie Pichon‘s French team from ESCPI (Paris) to the azines
from environmental liquid samples and for the clean-up of soil extracts, as well as molecularly imprinted polymers
(MIP), which are also selective sorbents with molecular recognition sites designated for a particular analyte. The MIP
prepared with terbutylazine developed by ESCPI seemed particularly applicable to our topic,” he said. Molecularly
imprinted polymers were used here in order to eliminate the interferences observed in LC-MS/MS (ion trap) and to
decrease the limit of quantification of triazines in soil samples.
Amalric believes the most significant outcomes include: matrices effects caused by the increase of the solid mass
were suppressed using terbutylazine MIP cartridges. “MIP improved the limit of quantification by a factor 1.5, 3.9 and
25 for atrazine, desethylatrazine and desisopropylatrazine, respectively, compared with the classic pressure liquid
extraction coupled with LC–MS/MS method. It showed one more time the potential of MIP for the selective extraction
of triazines and metabolites from complex samples, and therefore their use as a rapid clean-up method,” he added.
The dedicated extraction procedure and quantification applied to the soil samples proved successful in quantifying
atrazine and its metabolites down to 60 cm below soil surface. “The residual masses of atrazine and its metabolites,
estimated at 1.4, 0.52 and 0.25 kg for atrazine, desethyl-atrazine and desisopropyl-atrazine in the top 60 cm of the
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soil of the watershed, will most likely lead to long-term (decades) inputs to the groundwater even though all use of
atrazine in the watershed stopped seven years before the soils were sampled. A fraction of this mass available for
leaching could generate water infi ltrating with concentrations higher than the drinking water limit,” he said.
For him, this study contributed to demonstrate the potential of MIPs for the rapid clean-up of extracts from complex
samples such as soil extract, fruit juices and waste waters. MIPs present several advantages compared with antibodies
with respect to their ease, cost and time of preparation. In addition, their high capacity indicates a great potential for
the miniaturization of the analytical system.
“Nevertheless, MIPs can present limits in terms of selectivity because of the nature of selective interactions taking
place during the extraction. MIPs are not intrinsically selective. Their selectivity results from the combination of a
polymerization procedure that gives rise to specifi c cavities for the target analytes together with the association of an
extraction procedure involving solvents able to develop interactions that should only take place into the cavities,” he
added.
The synthesis of MIPs still needs improvements for the selective extraction of polar molecules that are not soluble
in conventional solvents. There is also an increasing demand for the development of MIPs for high molecular weight
compounds, such as proteins or micro-organisms. A recent review presenting the current state of-the-art in synthetic
macromolecular receptors synthesized via molecular imprinting methods for the selective recognition of biologically
relevant molecules shows the possibility of the MIPs for SPE applications. “The combination of the high selectivity and
the high capacity of MIPs that can be obtained by in situ polymerization with microfl uidic systems already developed
for sensors will certainly constitute a very promising approach for developing low-cost analytical methods to face very
complex matrices,” he concluded.
17research round-upseparation science — volume 1 issue 3
18 research round-up www.sepscience.com
Estimation of adenosine and metabolites in brain tissue using high-performance thin-layer chromatography–densitometry IndiaAs published in the Journal of Chromatography A [ 1209 (1-2), 230-237 (2008)], a rapid and sensitive high-performance
thin-layer chromatographic (HPTLC) method with densitometry was developed and validated for the concomitant
estimation of purines, including adenosine (Ade) and its major metabolites, inosine (Ino) and hypoxanthine
(Hypoxan) in rat brain tissue preparations. The HPTLC method was chosen to generate better resolution and evade
the tedious and prolonged sample preparation methods necessarily performed with HPLC methods when analysing
biological samples.
“The study was designed to highlight the unexplored aspect of high performance thin-layer chromatography
(HPTLC) in the analysis of purines in biological tissue samples. Taken together, an efficient separation of adenosine,
inosine and hypoxanthine was achieved in this novel method, which was economical in terms of cost/efficiency ratio
for the quantitative determination in intricate brain tissue samples,” said Prof. S.K. Kulkarni from the Pharmacology
Division at Panjab University in Chandigarh, India.“In line with usual HPLC methods, the present HPTLC method
showed good intraday (<8%) and interday (<7%) accuracy and precision in the estimation of purines in the brain
tissue samples. The HPTLC method was highly specific compared with the existing HPLC methods for the estimation
of adenosine, inosine and hypoxanthine,” Prof. Kulkarni said.
According to him, this was evident from the baseline resolution where all these purines were completely separated
without any interference from the biological matrix. The HPTLC method was highly sensitive (pmol) and showed good
recovery (>95%) of the components. “Parameters such as repeatability and stability were also met successfully with
this method. Moreover, the new sample preparation method involving an appropriate mixture of an acid and a base
(described in the present manuscript) resulted in complete extraction of the purines which lead to better resolution
without any chromatographic interference from the complex biological matrix,” he added.
The results for the first time show that this method established for the flexible estimation of Ade, Ino and Hypoxan
by planar chromatography has good linearity, accuracy, precision, sensitivity and selectivity, and is simple, rapid and
economical to produce maximum resolution in brain tissue preparations. “Overall the HPTLC-densitometry method
described in the manuscript was found to have considerable application on par with other chromatographic methods
in the biomedical analysis of tissue samples,” Kulkarni said.
For Kulkarni and his co-authors, this method has simplified the process for the estimation of purines in brain tissue
samples and has paved the way for the successful application of planar chromatography in future biomedical analysis.
“HPTLC-densitometry was shown to be the most economical approach to the biomedical chromatography of purines
in rat brain tissue samples. With appropriate modification, if necessary, this method can also be applied for the
estimation of purines in other biological samples. Thus this planar chromatographic method has revolutionized the
aspect of effective purines’ separation and estimation in biomedical analysis,” he concluded.
In the future, this method will be applied to append their neuropharmacological experiments to study the possible
correlation of purines in several neurological disorders and their modification by drugs.
Slovenia Buckwheat is an important crop in many countries.
From a medical perspective, it contains rutin, which
strengthens capillary walls, reducing haemorrhaging
in people with high blood pressure and increasing
microcirculation in people with chronic venous
insufficiency. Buckwheat also contains d-chiro-inositol,
a component involved with insulin signal transduction
and deficient in Type II diabetes and Polycystic Ovary
Syndrome patients. It is currently being studied for use
in treating Type II diabetes and has shown promising
results. In addition, a buckwheat protein has been found
to bind cholesterol tightly and is being studied for
reducing plasma cholesterol.
Buckwheat has a high nutritional value (e.g., proteins
with favourable amino acid composition, starch with
low glycaemia index, fibres, minerals, antioxidants,
and lipids high in monounsaturated fatty acids), and a
characteristic aroma. Surprisingly little was known about
GC-MS odour profiling of buckwheat
20 research round-up www.sepscience.com
GC-MS odour profiling of buckwheat
the substances responsible for buckwheat aroma.
In Food Chemistry [112 (1), 120-124 (2008)], Damjan
Janeš and colleagues from the University of Ljubljana,
Slovenia used GC-MS to characterize aromas from
Fagopyrum esculentum Moench (buckwheat). Janeš
explained, “Salicylaldehyde (2-hydroxybenzaldehyde)
was identified as the characteristic component
of buckwheat aroma. A selection of other aroma
compounds were also found, including 2,5-dimethyl-
4-hydroxy-3(2H)-furanone, (E,E)-2,4-decadienal,
phenylacetaldehyde, 2-methoxy-4-vinylphenol, (E)-2-
nonenal, decanal and hexanal .
All have odour activity values greater than 50, but in
isolation, none of these compound aromas resembled
buckwheat.” The odour activity value is a measure of
how important one particular substance is to the overall
aroma of a sample. It is calculated as the ratio between
the concentration of an individual substance in a sample
and the threshold concentration of the same substance
(where the threshold refers to the minimal concentration
that can be detected by the human nose).
“Our second finding was that sample preparation by
methanol extraction and distillation gave very different
results, and combining the two was the only way to get
the full picture on buckwheat aroma,” Janeš continued.
A headspace solid-phase microextraction method was
found to be unsuccessful.
“Further development in analytical methodologies
will enable an objective evaluation of aroma in different
buckwheat samples; for example, how milling procedures
influence aroma, how different buckwheat cultivars differ
in aroma, how the aroma is lost during storage etc. It is
the intention of our future research to address some of
these questions, as is the optimization of our sample
preparation procedures,” finished Janeš.
21research round-upseparation science — volume 1 issue 3
Critical aspects and recent trends in the analysis of food soy isoflavones
Enormous worldwide efforts
are being directed towards the
evaluation of isoflavone composition
in foods, and to identify their
relation with possible health effects
of soybeans and products intake.
During the last decade, several
extraction and analysis methods
have been developed generating
huge amounts of scattered
information that is sometimes not
correctly used for quantification of
these compounds in a wide variety
of foods, leading to erroneous
measurements and calculations.
In a recent article published in the
Journal of Chromatography A [issue
1216, 2-29, (2009)] the current state
and recent advances and future
trends in sample preparation and
analysis for the quantification of
isoflavones from soybeans and soy
foods were reviewed.
Quantification of isoflavones in
foods is a complex task because
there are several aspects that can
influence the results obtained in
a given analysis or determination.
Sample storage, extraction solvent
and time, sample-to-solvent ratio,
isoflavone stability during extraction
and more importantly, the extraction
technique used, can dramatically
affect isoflavone profile and recovery
from a given sample.
The sample itself is one of the
critical points. There is a large
variation in isoflavone profile and
concentration in similar products,
meaning it is difficult to obtain
standardized and “universal”
methods for the determination of
these compounds in all types of
samples. There is enough evidence
indicating that sample matrix
(protein content, particle size, etc)
dictates extraction efficiency and,
therefore, extraction methods
developed for a given sample may
not be applicable even to similar
matrices. Consequently, adjustment
of extraction conditions for each
sample is likely to be necessary to
achieve quantitative recoveries. This
implies that sequential extractions
are recommended when using
“general” extraction methods.
Although the need for more than
one extraction considerably reduces
sample output, it has the advantage
that different solvents can be used to
maximize the extraction efficiency of
all chemical forms of the isoflavones
present in the sample.
In general terms, extraction
solvent choice is one of the most
important parameters in the
sample preparation step. There are
several reports suggesting that
high recoveries can be achieved
using common solvents (methanol,
ethanol and acetonitrile with small
amounts of water), given that
extraction conditions are optimized
M.A. Rostagno
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Universitario “Duques de Soria”, Soria, Spain
22 feature — isoflavone analysisticle — CE in biomedical analysis www.sepscience.com
sufficiently for a given sample.
However, the natural tendency
nowadays, because of environmental
and economical concerns, is to
use less expensive and less toxic
solvents, such as ethanol, instead of
methanol and acetonitrile.
Sample stability is also a
critical aspect that should not be
overlooked. Some isoflavones are
relatively unstable and storage of
foods and extracts before actual
analysis can affect isoflavone
profile and concentration, which
is particularly important when
conducting quantification studies.
Although, isoflavone analysis is
still evolving and new techniques,
materials and strategies are
constantly being developed, used
and tested, there are some trends
that can be anticipated. Recent
research points towards minimizing
sample preparation by avoiding
hydrolysis of glucosides, to the
use of modern techniques such
as ultrasound-assisted extraction,
pressurized liquid extraction and
solid phase extraction (especially
when combined), and coupling on-
line with the analysis technique.
Using modern sample preparation
techniques means that extraction
times can be reduced to a minimum
while remaining highly efficient
with the advantage of using less
solvent. By minimizing pre- and
23feature — isofl avone analysisticleseparation science — volume 1 issue 3
post-extraction steps, fast extraction
and analysis techniques can be
fully explored while reducing errors
resulting from sample manipulation.
Furthermore, recent developments
in chromatography suggest that
fast, reliable and highly sensitive
analysis methods, either through
new equipment (such as UPLC) or
materials (monolithic and small-
particle-packed columns) will soon
become the new standard for the
isofl avones analysis in foods. In fact,
there are already some reports using
these technologies in which analysis
time was reduced from the usual
50-60 mins to less than 10 mins.
These new analysis methods
considerably increase sample
output (which indirectly minimizes
degradation of some isofl avones),
while achieving similar, or even
Rapid and convenient extraction directly into your MS
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better, analytical performance than
conventional approaches.
Although in the last decade
there have been advances and
an impressive increase in the
information available regarding
isofl avone determination there is
still a need for further research in
this area that will enable researches
to correctly and accurately evaluate
concentration and distribution of
these compounds in diff erent types
of food.
This article was written by Dr Mauricio
Ariel Rostagno.
Dr Rostagno can be contacted at
24 feature article — capillary GC instrumentation www.sepscience.com
Development and evaluation of an instant column connector for capillary gas chromatographyHans-Gerd Janssen and Daniela Peroni
Polymer-Analysis Group, van ‘t Hoff Institute for Molecular Sciences, Nieuwe Achtergracht 166, 1018 WV Amsterdam,
The Netherland
25feature article — capillary GC instrumentationseparation science — volume 1 issue 3
Coupling capillary columns in gas chromatography (GC) is not an
operation that is only relevant for the users of the most advanced capillary
GC methods. Simple single-column separations can also benefit from
a properly selected and installed precolumn. Precolumns connected
upstream of the main column can increase the life-time of the column,
efficiently refocus the analyte bands to result in better separations and can
be used for more rugged on-column injection in medium-bore columns.
Additionally coupling devices can also be used for other applications such
as connecting two columns when a higher resolving power is needed, to
connect columns of different stationary phase chemistries for optimizing
selectivity or to mend a broken column. Finally, column connectors are
also needed for the more advanced applications of GC such as heart-cut
two-dimensional chromatography, comprehensive GCxGC or column
back-flushing.
There are many more GC
applications that could benefit from
the use of good column connectors.
Many GC users are reluctant to use
column connectors because in
practice they are not always reliable.
Indeed a poor column connector
can destroy much more than a good
connector could deliver! Moreover,
coupling capillary columns has never
been an easy task. Many connectors
available to date have suffered
from clear disadvantages. The very
popular glass press-fit connectors
are easy to install, cheap and can be
26 feature article — capillary GC instrumentation www.sepscience.com
# of connections Tailing factor at 10% of height Peak width at 50% of height (s)
0 1.06 0.21
6 1.07 0.23
11 1.08 0.23
Table 1. Dead volume of 6 and 11 Meltfit connections in a narrow-bore column, (RESTEK RTX-5 column, length 10 m, i.d. 100 μm, film thickness 0.4 µm), p = 3.5 bar.
Table1
• purchaseprice
• costofownership
• leaktightness
• riskofdowntime
• deadvolume
• inertnessoradsorption
• extracolumnbandbroadening
• thermaldegradation
• size
• mechanicalstability
Recently, an instant column
connector has been developed that
combinestheexcellentproperties
of glass connectors with the leak-
tightness and mechanical stability
of metal connectors. In principle,
this Meltfit technology uses heat
and gas-pressure to shrink a glass
tube around the column. According
to the founders of the company
the idea was born in a brain-storm
session where people from the
packed-column years of GC jealously
reflected on the early days when
packed columns were connected
using heat-shrinkable Teflon tubing.
A low melting glass was used to
replace the Teflon and the Meltfit
technology was born. The principle
of the technique is shown in
Figure 1.
Leak-tightnessLeak-tightness was tested both
under elevated pressure conditions
and under vacuum operation. Leak
detection for operation at elevated
pressures was performed visually
used with a wide range of column
diameters. Unfortunately they
are prone to leaking especially in
temperature programmed operation
or at high temperatures.
Metal connectors with ferrules
have a significantly reduced risk
of leakage, but are much more
expensiveandneedtobeclosely
adaptedtothesizeofthecolumn.
They can also induce metal-catalysed
degradation and are difficult
to install. As a result of all these
problems many users have avoided
the use of connectors whenever
possible. Only if their use cannot be
avoided have connectors been used.
The decision to use either, press-fit
or metal is a compromise of many
factors including:
n
C
M
Y
CM
MY
CY
CMY
K
Figure 1. Principle of the Meltfit device. Columns are inserted into a small glass tube made of low-melting proprietary glass (softening temperature 380 ˚C). After heating, a small gas pressure (typically between 1 and 2 bar) is applied. The softened glass then closes tightly around the connector. The insert shows a schematic drawing of a connection and a photograph of a real connector. In our laboratory the Meltfit connectors were subjected to a series of critical tests. The test protocol included tests for leak tightness, inertness (both in terms of adsorption as well as with regard to surface catalysed degradation) and dead-volume.
Figure1
n
C
M
Y
CM
MY
CY
CMY
K
27feature article — capillary GC instrumentationseparation science — volume 1 issue 3
performance. Air ingress is absent
as indicated by the relative levels of
waterversusnitrogenandoxygen
and the low absolute values of the
last two components.
using leak-detection fluid. Tests were
performed up to pressures of 20 bar
using helium as the carrier gas. Even
at this very high pressure no leaks
were seen. Air ingress under vacuum
conditions was evaluated using
mass spectrometry (MS). Even in the
most critical MS, the ToF analyser,
no ingress of air was detected.
Theresultsoftheseexperiments
are shown in Figure 2. In MS it is
standard practice to monitor the
leak tightness by looking at the
signalsofnitrogen,oxygenand
water. After the installation of a new
column these are very high, but they
should decrease rapidly. A very good
indicator for a fully leak-tight system
is the level of water. For a good
connection the water level should be
above that of air and nitrogen.
Figure 2 shows a comparison of a
standard press-fit connection and
the Meltfit technology. At vacuum
conditions press-fit connectors will
always show a small, but not very
relevant, leakage.
The Meltfit connector
demonstrates a much better
n
C
M
Y
CM
MY
CY
CMY
K
Figure 2. Leak tightness of a Meltfit connector versus a standard press-fit coupling. (vacuum operation courtesy Jan Blomberg, Shell, Amsterdam).
Figure2
n
C
M
Y
CM
MY
CY
CMY
K
Figure 3. Results of the Grob and Donike test for multiple numbers of Meltfit connectors. Grob test: RESTEK RTX-5 column, length 10 m, i.d. 100 μm, film thickness 0.4 µm), p = 3.5 bar. Donike test: RESTEK Rxi-5ms column, length 30 m, i.d. 0.32 mm, film thickness 0.25 µm). p = 1.6 bar. Amounts introduced onto the column: 5 ng/compound.
Figure3
28 feature article — capillary GC instrumentation www.sepscience.com
Figure 4
C18:0
C16:0
C18:1 C18:2
8 8.5 9 9.5 10 10.5 11 11.5 12
Time (min.)
Olive oil
No connections
3 Meltfits
0 5 10 15 20 25
Time (min.)
Diesel
3 Meltfits
No connections
C18:0
C16:0
C18:1 C18:2
8 8.5 9 9.5 10 10.5 11 11.5 12
Time (min.)
Olive oil
No connections
3 Meltfits
0 5 10 15 20 25
Time (min.)
Diesel
3 Meltfits
No connections
Figure 4. Fatty acid methyl ester analysis and mineral oil analysis. A comparison of a system with 0 and 3 Meltfit connectors. Conditions FAME analysis: Varian CP-Wax 52 CB column (length 10 m, i.d. 100 µm, film thickness 0.2 µm), Tinj. = 250 °C, p = 3.5 bar, Split ratio = 1:50, Oven: 70° C (0.5 min) to 325 °C (5 min) at 20 °C/min. Conditions Mineral oil analysis: RESTEK Rxi-5ms column (length 30 m, i.d. 0.32 mm, film thickness 0.25 µm), Tinj. = 250°C, p = 1.6 bar, Oven: 45°C (2 min) to 275°C (5 min) at 10°C, Split ratio = 1:50. 100.
Dead volumeA key factor in the performance
of couplings for capillary GC is
the absence of any dead volume.
This is because even the slightest
dead volume will be detrimental
to the separation power of the
capillary column. Dead volumes
are particularly critical for narrow-
bore columns and compounds
that have low retention factors, or
more in general, for narrow peaks.
Table 1 shows the results of dead-
volume tests. The test conditions
applied here were selected to be
as critical as possible: the column
used was a 10 meter 100 μm inner
diameter narrow-bore column; the
test analyte was methane which on
this column was unretained and will
not be refocused by temperature
or stationary phase effects. The
number of connections made was
6 and 11, respectively. Even with 11
connections no significant peak-
broadening was seen. Dead volume
was also assessed by monitoring
the peak shape of the solvent
peak. Even a slight dead volume
would immediately result in severe
tailing of the solvent. In this case no
evidence of any dead-volume was
seen.
Inertness: adsorption and thermal degradationA serious problem with metal
connectors is their poor inertness.
Many compounds adsorb on
the metal and unstable analytes
are easily lost through (metal-
catalysed) degradation reactions.
Similar effects, although usually less
pronounced, can also occur because
of the presence of metal impurities
in the column or stationary phase,
or as a result of surface silanols on
the wall of the fused-silica capillary.
In the past, two very critical tests
for adsorption and thermo-catalytic
degradation in chromatographic
columns have been developed
by Grob and Donike, respectively.
These tests, developed decades ago,
are still used today for assessing
the inertness of chromatographic
systems.
The Grob adsorption test consists
of several different acids and bases.
Only on perfectly neutral surfaces
is a good performance obtained
for the test analytes. On poorly
deactivated surfaces either the
acids, the bases, or both, show
tailing.TheDoniketestmixture,
named after the famous sports
doping investigator, uses unstable
trimethylsilyl-esters of higher fatty
acids to monitor chemical inertness
of the chromatographic system. Even
the slightest activity in the system
results in losses of the unstable
Meltfit Meltfit
29feature article — capillary GC instrumentationseparation science — volume 1 issue 3
Prof. Hans-Gerd Janssen is group
leader Chromatography and Mass
Spectrometry at the Unilever Food
and Health Research Institute in
Vlaardingen, the Netherlands. He
is also a part-time professor in
biomacromolecular separations
at the University of Amsterdam,
Amsterdam,the Netherlands.
Mrs Daniele Peroni is a PhD
student at the Van ‘t Hoff Institute
for Molecular Sciences, University
of Amsterdam, Amsterdam, the
Netherlands. Her research subject
is the development of novel
methodsand systems for single-
and multidimensional capillary
gas chromatography and liquid
chromatography.
Publication of this article was made possible through collaboration with Chromedia.
“We are a worldwide community of experts with a mission. We cooperate to off er you Chromedia, a fast growing database of peer reviewed information with tutorials and solutions for the day-to-day questions in your lab at aff ordable cost.”
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esters. Figure 3 shows the combined
results of the Grob and Donike tests.
ApplicationsAll the fundamental tests performed
to assess the quality of Meltfi t
connections showed an improved
performance. Based on these
fi ndings, systems incorporating
one or several of these connectors
were applied for a large number
of applications from the area of
petrochemical, food, environmental,
fl avour and fragrance, and
pharmaceuticalanalysis.Anexcellent
performance was seen in all GC and
GCxGCapplicationsintheseareas.
Two relevant applications from food
and mineral oil analysis are shown
in Figure 4. The fi gure shows the
comparison of the chromatograms
obtained with 0 and 3 Meltfi t
connectors,themaximumnumber
that will normally be used.
ConclusionsThere are many situations where
capillary GC would benefi t from
coupling an additional column
or a pre-column to the analytical
separation column. As a result
oftheexperimentaldifficulties
associated with the use of column
connectors however, analysts have
been reluctant to use coupled
column systems. This connector
development eliminates the
drawbacks of the current column
connectors available and will allow
many more researchers to benefi t
from the advantages of coupled
columns in GC.
MrMeeting report
MSB 2009
The programme covered all aspects of
capillary-scale separations, hyphenated
detection methods, micro� uidics,
fundamental aspects of micro/nano� uidics
and applications related to systems
biology, pharmaceutical sciences, and
clinical diagnostics. In addition, there were
sponsored sessions on low-� ow liquid
chromatography interfacing, applications of
capillary electrophoresis mass spectrometry,
single cell analysis, and regulatory issues.
Dr Jonathan Sweedler, MSB 2009 chairman,
spoke to Separation Science about the event
highlights.
What were the most important outcomes of
this year’s event?
Jonathan Sweedler: The conference included
an outstanding programme that ranged from
fundamental academic research to more
applied biotechnology and pharmaceutical
presentations. While a number of talks
were presented by regular attendees of
MSB, an equal number were presented by
individuals new to the conference. This year’s
conference had excellent industrial support
and a number of important sessions were
supported by industrial partners. This broad
mix of support helps to ensure the success of
MSB.
The 23rd International Symposium on Microscale Bioseparations took place between
1-5 February 2009 in Boston, USA. Besides o� ering participants a range of plenary and
keynote speakers, MSB 2009 was a platform for industrial and academic practitioners to
exchange knowledge and practical advice.
What were the highlights of the
conference?
Jonathan Sweedler: The meeting schedule
was arranged such that each day had
two tracks, some of which were unique
to this MSB. For example, we had several
sessions on capillary electrophoresis/mass
spectrometry and interfacing capillary
separations using nanoelectrospray
that were well received and showed the
exciting progress made in interfacing mass
spectrometry to capillary scale separations.
On another day, there were several
sessions of interest to biopharma: Heparin
Contamination - Lessons Learned and
Implications, Applications of CE in the
Biotech Industry, and panels on Regulating
Supply Chain in the Flat World – Role of the
Analytical Chemist, and CE in the Regulated
Pharmceutical Industry Over The Last
Decade: The (Un) Ful� lled Promise. These
events provoked lively discussions between
researchers from various sectors.
Perhaps one of the conference highlights
was an entire day session devoted to all
aspects of next generation DNA sequencing
from the inventors and leaders of this � eld.
There are few measurement areas that
have evolved as quickly and as impressively
as sequencing, and this was highlighted
in this dynamic session arranged by
Professor Annelise Barron from Stanford
University. Other sessions included capillary
30 meeting report www.sepscience.com
electrophoresis for systems biology research,
microfabricated devices for integrated
solutions to biochemical measurement
problems, and high throughput
measurement approaches. All of these were
informative and well received.
What role does MSB 2009 play in serving
the needs of this industry?
Jonathan Sweedler: I asked a number of
attendees why they attended MSB 2009.
Some of the reasons participants gave
are obvious, such as keeping up with new
developments in small-volume separations
and understanding the capabilities of a
new measurement platform. I was told by
several participants from CE Pharma and
by instrument vendors that their interests
include meeting students, as they are their
future employees and customers.
What can participants expect from next
year’s conference?
Jonathan Sweedler: This conference normally
alternates between North America and
Europe, with next year’s European meeting
being held in Prague, Czech Republic.
However, before that, there is a special MSB
2009 in Dalian, China in October. MSB Dalian
will feature speakers from around the world
and will give participants the chance to
meet the separation experts from Asia. Thus,
before MSB returns to the USA in early 2010,
there are unique opportunities to listen
to the e� orts of researchers in microscale
bioseparations throughout the world. Details
on these future meetings are found on the
CASSS website.
Professor Jonathan V. Sweedler
holds the James R. Eiszner Family
Chair in Chemistry at the University
of Illinois, is associated with the
Beckman Institute, is the director
of the UIUC Biotechnology
Center, and has appointments
in the Neuroscience Program, the Department of Physiology
and the Bioengineering Program. His research interests are in
bioanalytical chemistry, and focus on new metabolomic and
peptidomic technologies for assaying small volume samples, and
in applying these methods to study novel neurochemistry. He
and his group are developing new sampling methods interfaced
to capillary scale separations, nanoliter volume NMR, single-cell
mass spectrometry, information rich spectroscopic detectors for
capillary-scale separations, and hybrid nano� uidic/micro� uidic
devices for neuronal sampling. Using this suite of technologies,
he is investigating novel neurochemical pathways, and the roles
that peptide hormones, neurotransmitters and neuromodulatory
agents play in behaviour, learning and memory. He has received
numerous awards including the Merck Prize, the Instrumentation
Award from the Analytical Division of the American Chemical
Society, the Gill Prize and the Benedetti-Pichler Award for
Microanalysis, and he is an associate editor for Analytical Chemistry.
*Photograph by Roger Kautz (Barnett Institute, Boston, MA, USA)
31meeting reportseparation science — volume 1 issue 3
*
CdThe Chrom
Doctor
Minimizing Downtime in QA Pharmaceutical Laboratories
Take, for example, the two key and common
tests run in drug substance and drug product
laboratories. Reversed-phase HPLC content
(assay) and related substances (impurity
profile) analyses are both susceptible to
unwelcome chromatographic events such
as rogue injections and ghost peaks. To
make these analyses more secure the analyst
should focus on aspects that are within his
control. Here are some suggestions for good
method practice that can be applied at
different phases of drug development
(Figure 1).
Methods in Development —The Developer’s RoleMethods development is a huge subject
with many good articles abundant. Methods
must be well designed from the outset;
that is, from preclinical development
stage onwards. Impurity methods must
have good specificity and sensitivity; and
should be developed to maximize the
available detector range, particularly when
the main peak is on scale cf. high-low
chromatography. Sound analytical methods
having demonstrable robustness and
ruggedness (reproducibility) stand the test of
time. Robustness testing is a key component
of a method’s development that allows
the analyst to see the effects of intentional
parameter changes. Knowledge from the
robustness exercise can be used to build in
effective system suitability requirements to a
method. During method development some
preliminary method validation is worthwhile.
Assessing a method’s accuracy from recovery
data is particularly useful — recovery is a
discriminating test that alerts the developer
to potential issues, especially for low-level
impurities analysis. During development
the analyst should also consider a method’s
routine use; for example, setting appropriate
equilibration periods for gradient elution
operation — equilibration is often
overlooked and insufficient. Assay methods
tend to be developed based on the acquired
knowledge from the impurity profile method
so are generally more straightforward
to develop. If a method is designed and
developed well then issues downstream
should be minimized.
Methods in ManufacturingMethod transfer: When a method enters
QA laboratories for the first time it presents
Laboratory managers and analysts cannot predict the unexpected; however, there are
certain measures that can be used to reduce the likelihood of analysis downtime. One
of the biggest “time-stealers” is when work has to be repeated, particularly in the GMP
environment; for example, when testing to specification. Repeat analysis is a big overhead.
In GMP laboratories an atypical or out-of-specification result cannot be ignored — it
spawns rigorous experimental investigation, data interpretation challenges and treatment
of results. So, how can repeat analysis be minimized?
32 www.sepscience.comchrom doctor
a good learning opportunity to integrate
method development with the development
laboratory. It is often the initial time when
the method is loaded under a “stress”
condition; that is, by a different user having
a lack of method familiarity working with
different laboratory systems. Testing
in receiving (or different) laboratories
should be performed as early as possible
before methods are finalized, validated
and documented. Successful methods
transfer relies on a strong inter-laboratory
partnership that may lead to method
improvement or sometimes re-development.
System Suitability: Set meaningful system
suitability tests and acceptance criteria.
Rigorous system suitability can save a lot
of headaches if designed and applied well.
System suitability should have a resolution
value as a minimum; for example, between
a critical impurity pair. Concentrations
of analytes in system suitability test
mixtures and the calculation method
of assessing resolution should both be
fixed. A tailing factor offers limited system
suitability information compared with a
resolution value but may be required by
pharmacopoeia.
Instrumentation: Modern autosamplers
deliver excellent precision; however, there
are times when rogue injections occur that
defy understanding! For assay methods,
the use of an internal standard should be
considered. The cost of developing an assay
method with an internal standard may be
favourable compared with the business cost
of repeat testing. Having scrupulously clean
injection systems is important for impurity
analysis; preventive measures should be
employed to ensure carryover is minimized,
particularly when using concentrated
solutions. Some methods requiring very
Figure 1
Review
Improve
Method development
cycle
Post Approval -commercial phase
(QA - lab)
Monitor method performance
QA lab (e.g. manufacturing)
Pre Approval - clinical development phase
R&D lab (e.g. preclinical)
Figure 1. Representation of method developmet processes during development phases.
33chrom doctorseparation science — volume 1 issue 3
short UV detection wavelengths
(<215 nm) may, in some cases, require
dedicated equipment to minimize issues.
Column: The column is the heart of the
separation and must be in good working
order. A column’s history of use (column
record) provides essential data (e.g.,
efficiency) that monitors past performance
and it is worth the effort as it gives early
warning signs of potential trouble.
Restricting a single column for a single
method is also a good practice as the
column is subjected to only the same eluent
and samples each time. Cleaning (column
flushing) procedures should be developed
and shown to be effective in removing all
potentially interfering components that may
reside on the column; for example, after
isocratic operation.
Injection sequences: It is worth investing
time to design good sequences; that is,
the order of test injections and number of
replicates. Impurity profile analyses are prone
to the effects of artifact peaks because of
the sensitivity requirement needed, typically
0.1% relative to the main peak. This target
level means that often impurity peaks are
monitored close to a method’s quantitation
limit where traces of unknown/known eluted
material can interfere. For impurity analyses,
it is worthwhile running two or three blanks;
this gives confidence in the stability of the
whole chromatographic system; and is a
good way to spot artifacts, ghost peaks and
assess baseline repeatability. Where possible,
system suitability injections should be
scrutinized and assessed before committing
samples during attended analysis. System
suitability injections are best run throughout
long sequences as samples bracketed
by these injections can be valid and data
rescued when the unexpected happens;
for example, such as the occurrence of a
power down, column failure etc. In addition,
the stability of the chromatographic
system across the entire analysis set can be
monitored.
GMP: Staff training (sometimes specific
method training) and well-maintained
equipment is implicit and a prerequisite.
Operation of balances requires first-rate
technique as weighing is a key source of
human error. Some materials are awkward
to weigh, especially electrostatic powders,
so a proprietary device for coping with this
problem is advisable.
Methods — Post-ApprovalFollowing drug approval when methods are
in routine operation continuous monitoring
of method performance and critical
assessment of data are valuable activities.
Only when there is a sufficient body of data
available can method issues, bias or trends
be spotted.
In summary, the approaches discussed
maybe useful to the practising analyst in
considering ways to circumvent potential
issues during day-to-day operation of
methods in the QA laboratory and may help
prevent rejection of good batches of material
or product that fail testing due to analytical
errors.
This article was written by Sean McCrossen,
Crawley, West Sussex, UK.
34 chrom doctor www.sepscience.com
5chrom doctorseparation science — volume 1 issue 3 31chrom doctorseparation science — volume 1 issue 3
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
TuTechnology
update
36 technology update www.sepscience.com
Key
Email the company
Product information
Applications
Additional information
7890A GC System
Manufacturer: Agilent
Manufacturer’s description: Agilent’s 7890A GC features advanced separation capabilities,
powerful new productivity enhancements and real-time self-monitoring instrument
intelligence. According to the company, faster oven cool-down and robust backflushing
means more work in less time, at a lower cost per sample. 5th-generation electronic
pneumatics control (EPC) and digital electronics enhanced pressure setpoint and retention
time locking precision (0.001 psi).
Agilent states the capillary flow technology enables leak-free in-oven connections,
enhances productivity and data integrity and offers versatile, robust solutions for complex GC
analyses. GCxGC flow modulation is now available.
Key features include Agilent Lab Advisor Software, which tracks usage of supplies, monitors
chromatographic quality and alerts you to problems before they happen. The system also has
a new Turn-Top design built into each split/splitless (SSL) inlet, allowing users to change liners
in less than 30 seconds — without special tools or training. The complete selection of options
and accessories lets you configure the exact system to meet your lab’s needs, and easily adapt
to changing application and throughput requirements.
In addition the chromatographer-friendly GC software simplifies methods setup and system
operation, and minimizes training time. Built upon proven 6890 inlets, detectors and GC oven,
users can transfer methods to the 7890A GC with complete confidence.
37technology update separation science — volume 1 issue 3
separationdriving analytical chemistry forwardsscience
www.sepscience.com
ACD LC Simulator
Manufacturer: ACD Labs
Manufacturer’s description: The ACD/LC Simulator is LC method
optimization software that performs calculations, using experimental
chromatograms, to optimize concentration gradient, temperature
and resolution during method development. The programme will also
predict analyte pKa values from chemical structure to fi nd the optimal pH for a separation, and predict retention times
for new compounds for existing methods.
ACD/LC Simulator is a powerful chromatographic method development tool that saves development time,
eluent, and money by choosing and refi ning an optimal separation method and a column prior to the fi rst injection,
according to the company.
Its key capabilities allows users to optimize gradient, solvent concentration, temperature, pH, and more, convert
gradient methods to isocratic methods, predict pKa from chemical structure, predict retention times for new
compounds under existing methods, automatically match peaks between LC/UV (DAD, PDA) datasets based on
spectral similarity and visualize the eff ect of changing your method conditions with predicted chromatograms.
ACD/LC Simulator works hand-in-hand with ACD/ChromManager to form ACD/Method Development Suite for LC/MS.
38 technology update www.sepscience.com
separationdriving analytical chemistry forwardsscience
Get your FREE subscription to
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technical articles on chromatography?
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applications of new technology?
information on product developments?
market trends and opinions?Volume 1 / Issue 3
March 2009
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Coupling capillary columns in gas
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pharmaceutical laboratories
separation driving analytical chemistry forwardsscience
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Volume 1 / Issue 2
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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
Focus SPE
Manufacturer: Varian
Manufacturer’s description: Focus is a solid phase extraction solution for extracting developmental pharmaceutical
compounds from blood plasma and other biological matrices for subsequent analysis. The patent-pending Focus
technology delivers good recoveries for polar as well as non-polar analytes using a simple extraction procedures.
Cleaner extracts are also achieved resulting in reduced LC/MS/MS ion suppression and ultimately faster LC run times.
Focus features a unique polar-enhanced sorbent technology that makes it a good choice for any drug at any stage
of drug development. By combining multiple polar and hydrophobic retention mechanisms into a single sorbent it
is possible to specifi cally retain all of the following analyte functionalities. Simple generic methods can be used to
rapidly screen new chemical entities in the discovery phase. Alternatively, methods can be easily optimized to provide
the selectivity and cleanliness required for preclinical and clinical assays.
According to Varian, Focus is the perfect choice for simultaneously extracting both polar and non-polar analytes.
Used in early bioanalysis, it can eff ectively extract broad range of polar and non-polar drugs. Used in DMPK and
clinical studies, it is especially eff ective for quantitatively extracting both parent drug and metabolites at ultra low
levels.
The Power Rinse delivers cleaner extracts, reduced ion suppression, faster LC run times, better quantitation for polar
analytes, basic and neutral analytes and acidic analytes.
39technology update separation science — volume 1 issue 3
30 Amino Acid Analyser
Manufacturer: Biochrom
Manufacturer’s description: The Biochrom 30 Amino Acid Analyser is designed to provide accurate quantitative analysis
of amino acid mixtures. The Biochrom 30 uses modern technology to achieve fast, accurate, reproducible results
with a minimum of operator involvement. The software package controls the operation of Biochrom 30 whilst
continuously monitoring various parameters, thus providing an indication of any fault conditions,
according to the company.
The Biochrom 30 is ergonomic and features load and GoPost-column photometric detection
of amino acids with no additional derivatization procedures, integrated software enables
automatic sample set-up, flexible sample analysis, networking and exporting of
data, a wide variety of sample types can be analysed using the available range of
programmes and columns, and a benchtop design requiring minimum lab
space.
After each sample analysis, the column is regenerated by pumping a
strong base through the column followed by buffer which equilibrates
the column prior to the next analysis. Operation of the Biochrom 30
Amino Acid Analyser is completely automatic, and all functions of the
analyser are controlled by the software. Various analytical standard
programmes are supplied with the instrument, these programmes
contain all the information required to perform the analysis. The instrument package consists of the operating unit,
which includes electronics unit, pumps, detection system, cooled autosampler.
YMC-Pack Preparative HPLC Columns
Manufacturer: YMC
Manufacturer’s description: YMC-Pack preparative columns, packed
with YMC Gels, are available in a broad variety of column sizes to
accommodate virtually any preparative separation, according to
the company. YMC use a high-pressure slurry technique to pack all
preparative columns in optional 1,000 or 2,000 psi pressure rated
hardware. Standard column dimensions range from 50 to 200 mm
inner diameter and 250 to 1000 mm in length.
YMC strongly recommends the use of guard columns in order to
6 technology updatewww.sepscience.com32 technology updatetechnology updatetechnology updatetechnology updatetechnology updatetechnology update
rationdriving analytical chemistry forwardssciencationdriving analytical chemistry forwardsscienc
separationdriving analytical chemistry forwardsscience
6 technology update326326 technology updatetechnology updatetechnology updatetechnology update
technical articles on chromatography and related technologies?
updates on recent research studies?
practical advice on routine analysis?
applications of new technology?
information on commercial productdevelopments?
market trends and opinions?
extend column life. Matching YMC-Pack preparative
guard columns are available for all the standard column
sizes in either 50, 100 or 200 mm in length, depending on
their diameter.
Product features include direct scale up from analytical
and semiprep YMC-Pack columns, standard column
diameters: 50, 70, 100, 150 and 200 mm, standard
column lengths: 250, 300, 500, 1000 mm, and pressure
resistant up to 2,000 psi.
The fl anged stainless steel hardware of the YMC-Pack
preparative columns comprises a unique distributor-frit
assembly that permits an effi cient, uniform distribution
of sample across the entire diameter of the column bed,
which allows maximum sample loading via maximum
utilization of the packed bed. Similarly, eluent fl ow is
directed uniformly across the entire column diameter
providing good peak symmetry.
All YMC-Pack preparative columns are subject to
an individual chromatographic test and have to
meet stringent specifi cations to ensure high column
performance and column-to-column reproducibility.
A Column Test Report is included with each YMC-Pack
preparative column specifying the theoretical plate
number and asymmetry factor. Any of the YMC-Pack
columns is guaranteed to perform consistent with its
Column Test Report.
Separations developed on YMC-Pack analytical or
semi-preparative columns can be directly scaled up to
preparative size YMC columns. For the most convenient
scale up YMC recommends the YMC R&D Scale up Kit,
consisting of a preparative column and a matched
analytical column both packed with material from the
same lot.
41technology update separation science — volume 1 issue 3
Microfl ex MALDI-TOF MS
Manufacturer: Bruker Daltonics
Manufacturer’s description: The microfl ex is designed
as an aff ordable, yet powerful benchtop system that is
convenient for many life-science laboratories. According
to Bruker Daltonics, the unique design of the microScout
ion source and the gridless refl ectron give the microfl ex
superior resolution, as well as high mass accuracy and
outstanding sensitivity.
The modular design off ers several instrument
confi gurations to fulfi l diff erent requirements for
peptide/protein identifi cation and characterization,
biomarker discovery or oligonucleotide analysis.
The system can be upgraded to meet new analytical
challenges. The microfl ex features integrated Compass
software environment for easy operation in multi-
instrument laboratories. All functions from automated
acquisition to in-depth analysis via highly sophisticated
bioinformatics software are provided.
The proprietary AnchorChip technology provides
homogeneous, exactly-positioned samples on the MALDI
target for robust and rapid automated data collection,
as well as a sensitivity boost by up to two orders of
magnitude. Compatibility with laboratory robots (e.g,
map II) is assured by the new microScout MALDI target
plates, featuring 96 position geometry to sample from
industry standard microtiter plates.
The microfl ex identifi es proteins by MALDI-TOF MS
peptide mapping. The data quality directly translates
into high success and reliability of identifi cation.
Thus even faint spots from 2D gels can be identifi ed.
Optional MS/MS (autoPSD) capability enables detailed
structural investigation of proteins, including crosslinks
and modifi cations. Alternatively the MALDI-TOF MS
screening step can be followed by high-content ion trap
LC-MS/MS within the PROTEINEER workfl ow. As part of
the GENOLINK package, the microfl ex is the ideal tool
for routine analysis of oligonucleotides for genotyping
projects and quality control of oligonucleotide synthesis.
As part of the CLINPROT solution, the microfl ex
supports the discovery of biomarker patterns and
the identifi cation of individual biomarkers in clinical
proteomics applications. High system performance
enables detection of biomarkers throughout the typical
mass range for peptides and proteins.
42 technology update www.sepscience.com
Proteomics MDLC
Manufacturer: Dionex
Manufacturer’s description: Using the x2 dual-gradient technology in combination with the
UltiFlow technology for nano and capillary flow generation, the UltiMate 3000 Proteomics
MDLC offers a wide flow rate range and high flow flexibility, which allows the system to
perform an extensive range of applications.
Taking full advantage of the UltiMate 3000 Proteomics MDLC system, fully automated
multidimensional LC (MDLC) is performed easily. This orthogonal separation mechanism,
often using ion-exchange combined with reversed-phase chromatography, can be applied
using on-line or off-line separation strategies, and provides excellent opportunities for
identification of low-abundance proteins with outstanding reproducibility.
On-line MDLC strategy: well-matched for further separation of digested 1D gel bands or 2D
gel spots. In addition, this approach provides good results for the analysis of highly complex proteomic samples, such
as purified biological fluids or PTM complexes.
Fully Automated Off-line MDLC strategy: specifically designed for highly complex proteomic samples such as cell
lysates and tissues. Using the advanced technology of the WPS-3000 well plate autosampler, protein pre-fractionation
can be performed, and then followed by re-injection of each fraction without any manual handling of the sample. This
automated approach to sample preparation allows for the identification of low-abundance proteins or biomarkers.
2D LC Separations for the Analysis of Intact Proteins: for the separation of complex proteomics samples,
a combination of high-resolution columns is available, allowing intact protein separations. The UltiMate 3000
Proteomics MDLC system provides a unique x2 dual-gradient systems that enable ion-exchange and reverse-
phase separation on one system. In addition, it features the UltiMate 3000 well plate sampler for both injection and
fractionation, providing fully automated off-line 2D LC and protein prefractionation. Extended functions include
nanoflow capabilities for the final analytical stage, reduced solvent consumption without compromising application
flexibility, on-line fraction collection on MALDI targets with the Probot Microfraction Collector/Spotter, complete
range of column types for your separation needs and fractionation control and visualization of 2D retention map
using Chromeleon chromatography management software.
It also has a DCMSLink for single-point control of UltiMate 3000 through Xcalibur (Thermo Fisher), Analyst (AB/
Sciex) and HyStar (Bruker Daltonics) for easy ESI coupling. According to Dionex, the easy automation of separation
processes leads to higher sample throughput, more reliable results and improved laboratory productivity. In order to
provide the highest confidence in protein identification, especially when analysing PTM peptides and proteins, the
UltiMate 3000 Proteomics MDLC is also available in a biocompatible set-up.
43technology update separation science — volume 1 issue 3
TLC/MS interface
Manufacturer: Camag
Manufacturer’s description: CAMAG TLC-MS Interface is a versatile instrument to extract compounds from a TLC/
HPTLC plate and feed them into a mass spectrometer for substance identifi cation or structure elucidation. It can be
connected to any brand of LC-coupled mass spectrometer.
According to Camag, surveys have shown that not all samples may be processed by HPLC-MS or HPLC-DAD systems
because of no or low detection of the compounds or impurities in the UV range, a heavy matrix load or a lack of MS
compatible solvents, however necessary for the HPLC separation. alternatively HPTLC is a very fast and convenient
method to separate samples. In the past, unknown substances were scraped off from the TLC/HPTLC plate, eluted
into a tube and transferred into the MS. The universal TLC-MS Interface can now semi-automatically extract zones of
interest and direct them online into any brand of HPLC-MS system.
The interface is quickly and easily connected (by two fi ttings) to any LC-coupled mass spectrometer without
adjustments or mass spectrometer modifi cations. Questioned substances are directly extracted from a TLC/HPTLC
plate and sensitive mass spectrometric signals are obtained within a minute per substance zone. The interface
extracts the complete substance zone with its depth profi le and thus allows detections comparable to HPLC down
to the pg/zone range. The interface has been proven to be one of the most reliable and versatile interfaces for TLC/
HPTLC-MS coupling.
The interface features plug-and-play installation by two HPLC fi ttings at a given HPLC-MS system, semi-automatic
instrumentation involving automatic piston movement for pressure seal of the TLC/HPTLC zone on both glass plates
and aluminium foils, extraction directly from the plate using a suitable solvent delivered by the HPLC pump, and
automatic cleaning of the piston between the extractions and online transfer to the mass spectrometer.
The instrument extracts circular zones of 4 mm diameter from a TLC/HPTLC plate; for example, with methanol or
any other appropriate solvent, using the standard fl ow speed of the HPLC-MS system (e.g. 0.1 mL/min). Additional
extraction head geometries for a reduced or an enlarged layer thickness
or oval geometry will be available. Materials include plates or
aluminium foils up to 20 x 20 cm can be positioned accurately
and analysed zone by zone. The semi-automatic instrument
involves automatic piston movement, automatic cleaning
of the piston, manual positioning and switching.
extraction head geometries for a reduced or an enlarged layer thickness
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Coupling capillary columns in gas
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pharmaceutical laboratories
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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
Volume 1 / Issue 1
separation driving analytical chemistry forwardsscience
Volume 1 / Issue 2
February 2009
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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