Journal Separation Science

44
Volume 1 / Issue 3 March 2009 www.sepscience.com Coupling capillary columns in gas chromatography Analytical trends in isoflavone studies Minimizing downtime in QA pharmaceutical laboratories

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.

for research news, technical articles, product updates, jobs and applications visit. . .

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chrom doctor Guest author Sean McCrossen discusses strategies for minimizing downtime in pharmaceutical laboratories

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

Separation Science is published by Eclipse Business Media Ltd, 70 Hospital street, Nantwich,

Cheshire, CW5 5RP, UK. Copyright 2009 Eclipse Business Media Ltd. All rights reserved. No part

of this publication may be reproduced or transmitted in any form or by any means, electronic or

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4 www.sepscience.com

scienti� c advisory

councilPeter Myers

– Chief Scienti� c O� cer

[email protected]

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

Publishing Director

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Stephanie Painter

Associate Publisher

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[email protected]

Kevin McGeehan

Associate Publisher

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Karen High� eld

Financial Controller

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Technical Editor

David Hills

Scienti� c Director

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[email protected]

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Chief Scienti� c O� cer

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[email protected]

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Graphic Designer

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David Hills

– Scienti� c Director

[email protected]

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

[email protected]

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:

For all delegate enquiriesemail Jackie Tan.

26–28 AugustBiopolis Science Park, Singapore

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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Product information

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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.

Shimadzu_Seperation_0309_01 13.02.2009 15:43 Uhr Seite 1

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

[email protected]

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.

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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.

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Eclipse. The ultimate system for the separation of macromolecules and nanoparticles in solution.

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© 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

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technical articles on chromatography?

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March 2009

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Coupling capillary columns in gas

chromatography

Analytical trends in iso�avone

studies

Minimizing downtime in QA

pharmaceutical laboratories

separation driving analytical chemistry forwardsscience

Volume 1 / Issue 1

Febraury 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

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

sepa on scienc

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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

separation driving analytical chemistry forwardsscience

Volume 1 / Issue 1

Febraury 2009

www.sepscienceasia.comwww.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

Volume 1 / Issue 1

separation driving 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