Journal Separation Science

Volume 1 / Issue 4

April 2009www.sepscience.com

Analysing biomarkers in human breath

A review of multidimensional LC

How to deal with overloading in GC

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3contentsseparation science — volume 1 issue 1

contentsVolume 1 / Issue 4

April 2009www.sepscience.com

Analysing biomarkers in human breath

A review of multidimensional LC

How to deal with overloading in GC

Analytical methods in exhaled breath diagnostics

Nicholas C. Strand and Cristina E. Davis

20

features

separationdriving analytical chemistry forwardsscience

Volume 1 / Issue 4April 2009

38

research round-up

Using LC-UV to understand redox conditions

Preparative isolation and puri� cation from natural products by high-speed counter-current chromatography

Determination of serotonin, melatonin and metabolites in gastrointestinal tissue using HPLC-ECD

HPLC determination of 6-mercaptopurine and metabolites in plasma

Partially porous particle columns for use in pharmacokinetic studies with RP-UHPLC-MS/MS

Headspace solid-phase microextraction for determining chloroanisoles and chlorophenols in wine

Improving the performance of simulated moving bed chromatography

Uncovering PAHs in biota samples with GC-MS-MS

HILIC – want to know more?

Rr

Cd chrom doctor Guest author Jaap de Zeeuw discusses the problem of overloading in GC and how to deal with it.

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application notes

technology update An overview of recent technology advances in separation science and instrumentation.

42

46

30 A practical review of multidimensional LC

Tuulia Hyotylainen

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

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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

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Using LC-UV to understand redox conditionsPoland

“Thiols are an interesting family of compounds which fulfil a multitude of functions in living

organisms, and modifications in physiological concentrations of thiols are often a cause

of severe pathological events. An oxidative shift in the thiol/disulfide ratio in intra- and

extracellular compartments is associated with pathological conditions and aging,” said Dr

Edward Bald from the Department of Environmental Chemistry at the University of Lodz,

Poland. Current literature describes variations of thiol/disulfide redox state in plasma, blood

or tissues, but not in urine and Dr Bald and his team undertook a study to measure thiol

redox status, defined as reduced-to-oxidized ratio, of main urinary aminothiols. Published in

Chromatographia [68 (supplement 1), 91-95 (2008)] the study determined both forms of thiols

by liquid chromatography with ultraviolet detection.

The key findings of the research are that the thiol redox state, defined as the ratio of

reduced to oxidized form of two main urinary thiols cysteine and cysteinylglycine, remains

stable in urine ex vivo during the early hours after urine collection. “This means that urine,

kept at room temperature, can be safely analysed for the redox status within four to five

hours. The results of analysis of the first morning urine samples from 45 apparently healthy,

ethnically homogenous, volunteers convince that cysteine redox status in urine is not age

dependent. A significantly positive correlation between cysteinylglycine redox status and

age (P = 0.0013) was observed. Moreover, cysteinylglycine redox status in children was

significantly lower than in adults (P < 0.01),” he explained.

He believes that because in some studies, urinary cysteine and other sulphur amino acids

concentrations mirror the change in plasma, determination of urinary content of these

thiols may be a valuable non-invasive means of oxidative stress monitoring and method of

diagnosis, especially in children.

6 research round-up www.sepscience.com

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Preparative isolation and purifi cation from natural products by high-speed counter-current chromatography

Greece

Alkannin, shikonin (A/S) and their derivatives (acetyl-, isovaleryl-, β,β-dimethylacryl-, β-hydroxyisovaleryl-

ones, etc.) are bioactive compounds with multiple biological properties, such as wound healing, antimicrobial,

anti-infl ammatory and antitumor activities. These bioactive compounds are mainly isolated from the roots

of Boraginaceous species after a multiple-step procedure, followed by column chromatography (CC), or are

synthesized, or are prepared by plant tissue cultures followed by further purifi cation.

“Because of the important biological properties and uses of A/S and their derivatives in pharmaceuticals and

food colourants, and their broad applications, high-purity preparations containing A/S and derivatives are of

great interest. The purity of the above-mentioned compounds is a critical point for determining biological activity

and for subsequent structure–activity relationships,” said Dr Andreana Assimopoulou from the Department of

Chemical Engineering at the Aristotle University of Thessaloniki in Greece, who recently conducted a study to

introduce an effi cient HSCCC method for separation and purifi cation of A/S derivatives from samples containing

A/S derivatives and to compare the purity of fractions separated by HSCCC with those obtained by the

traditionally applied method – CC using as stationary phases silica gel and Sephadex LH-20.

Documented in Biomedical Chromatography [23 (2), 182-198 (2009)], is a reliable HPLC-DAD-MS method for the

identifi cation of HSCCC fraction constituents, and thus for evaluation of the HSCCC separation of monomeric and

oligomeric alkannin fractions from a commercial sample. In addition, described was the separation and isolation

of bioactive A/S derivatives, mainly esters, from a mixture of A/S pigments isolated from A. tinctoria roots.

“An optimization was performed for selection of the two-phase solvent system for separation and purifi cation

of A/S derivatives. When HPLC–vis or DAD alone was used for identifi cation of HSCCC fractions, evaluation of

their purity and separation of A/S derivatives by HSCCC, the results and conclusions were misleading, because

oligomeric compounds that exist in the samples were not taken into account. Therefore, HPLC-DAD-MS was

applied for evaluation of the HSCCC separation,” she explained.

Comparing the purity of monomeric A/S isolated using Sephadex LH-20 (CC), silica gel CC and HSCCC, it was

shown that monomeric alkannin isolated by HSCCC is purer than monomeric shikonin isolated by silica gel CC,

followed by CC with Sephadex LH-20. “HSCCC is an important method for isolating, in preparative scale, A/S and

their derivatives from samples and for purifying commercial A/S samples, because these compounds are used as

bioactive ingredients in pharmaceuticals and cosmetics, as food additives, and as raw materials for synthesis of

other A/S derivatives for pharmaceutical or other purposes,” she said.

In most papers reported on biological activity evaluation, commercial samples of A/S and their derivatives were

used, which contain oligomeric derivatives. Additionally, the study showed that when commercial samples of A/S

derivatives are purifi ed by CC for further biological experiments, monomeric A/S isolated also contains dimers.

Thus, the purity of A/S derivatives examined for each biological activity reported hitherto is in question.

“Additionally, defi ned fractions containing several types of oligomeric A/S compounds (vaforhizins, di-alkannins,

trimeric A/S derivatives and combinations of them) will be further evaluated for biological activity in order to

perform a structure activity relationship between monomeric and oligomeric A/S derivatives,” she concluded.


UK

In a paper in Biomedical Chromatography [23 (2), 175-

181 (2009)], Dr Bhavik Patel from the Department of

Bioengineering at Imperial College London, UK, shows

a simple isocratic chromatographic method for the

detection of serotonin and its precursors and metabolites

in various types of gastrointestinal tissue.

“We conducted this research as we have an avid

interest in understanding the role of signalling molecules

in the gastrointestinal track. We are especially interested

in the relationship between the levels of these signalling

molecules and the function of the tissue,” said Dr

Patel. Serotonin (5-HT) is known to play a key role in

influencing gastrointestinal motility, however through

the application of sensing on the tissue the team learnt

that melatonin was also released from the gut. Numerous

published papers look into the means of measuring

basal levels of serotonin, but only a few measure both

serotonin and melatonin, and none are specific to

gastrointestinal tissue.

“We were able to develop an isocratic HPLC method

that was capable of analysing all the analytes of interest

within 15 minutes, which is a marked improvement

on all other methods that have larger retention times

for these analytes or utilize gradient-based methods.

We used electrochemical detection, rather than the

conventional UV/vis detector, to assure greater selectivity

during recordings,” he said. The team was able to detect

serotonin and melatonin levels in the 100s of nanomolar

range and also showed that storage at -80 °C for a

week was the only means of having stable accurate

responses from tissue. “From a biological perspective,

we demonstrated there were alterations in the level

of serotonin and melatonin in various regions of the

gastrointestinal (GI) tract. Serotonin levels decreased

as you went down the GI tract, whilst melatonin levels

varied,” he added.

He believes this technique is an excellent means of

measuring intracellular levels of signalling molecules

in the GI tract, but it will also be used to measure

basal released levels of serotonin and melatonin.

“This technique will be employed for all subsequent

measurements from gastrointestinal tissue and will be

utilised for studying how these signalling molecules vary

during gastrointestinal disease, and will provide greater

information on the basic physiology of the system and

getting mechanistic information of neurotransmission

during disease,” he concluded.

Determination of serotonin, melatonin and metabolites in gastrointestinal tissue using HPLC-ECD

9research round-upseparation science — volume 1 issue 4


UKAccording to a paper in the Journal of Pharmaceutical

and Biomedical Analysis [49 (2), 401-409 (2009)], an

HPLC method has been developed and validated for the

rapid determination of mercaptopurine and four of its

metabolites; thioguanine, thiouric acid, thioxanthine and

methylmercaptopurine, in plasma and red blood cells.

“Despite the extensive clinical experience with

mercaptopurine/azathioprine, used in the treatment of

several diseases including childhood acute lymphobalstic

leukaeima and inflammatory bowel disease, the

disposition and metabolism of these drugs and their

various metabolites remains only partially understood,”

said Dr Ahmed Hawwa from the Clinical and Practice

Research Group at the School of Pharmacy of Queen’s

University in Belfast, UK.

“Due to the wide inter-individual differences in

mercaptopurine and azathioprine metabolism,

monitoring their metabolites in erythrocytes has been

proposed as a useful clinical tool for assessing treatment

efficacy and toxicity and to ascertain non-adherence to

the prescribed treatment,” said Hawwa.

Numerous HPLC methods have been developed for the

determination of mercaptopurine and its metabolites in

biological fluids. However, these methods were limited

by low recovery, laborious and time-consuming sample

preparation procedures, multiple extraction procedures

and compromised sensitivity. Consequently, this

necessitated the development of a method that would

overcome these limitations. “The present HPLC method

was developed in order to facilitate the pharmacokinetic

modelling of mercaptopurine/azathioprine metabolites

in different age-subsets particularly children and

identify the inter and intra-individual variability in

mercaptopurine metabolism among its competing

metabolic routes as it permits the quantification of the

metabolic end products of these enzymes,” he explained.

The most significant outcomes of the study are that

the developed method was selective and sensitive

enough to analyse the different metabolites in a single

run under isocratic conditions. “The main advantage

of this HPLC methodology, however, is the rapid

simultaneous determination of mercaptopurine and

its four metabolites,” he said. The total run time is only

13 minutes with all peaks of interest being eluted

within seven minutes. Furthermore, the method

allows the measurement of mercaptopurine and its

metabolites using only small amounts of plasma (200

µL) or erythrocytes; such low volume requirements are

particularly applicable to low volume paediatric samples.

“The small volume of blood required together with the

simplicity of the analytical technique makes this a useful

procedure for monitoring mercaptopurine cytotoxic

metabolites concentrations in routine clinical settings as

well as in research studies,” he added.

This newly developed method was recently

employed in the determination of mercaptopurine

and its metabolites in a study investigating population

pharmacokinetics, pharmacogenetics and patient non-

adherence to thiopurine therapy. The aim was threefold;

to assess the possible associations of these metabolites

with the various polymorphic variations in the genes

encoding the main enzymes involved in mercaptopurine

metabolism, to evaluate the kinetic nature of the

branched enzyme system working on mercaptopurine/

azathioprine and facilitate the pharmacokinetic

modelling of mercaptopurine/azathioprine metabolites.

“In addition to its usefulness in pharmacokinetic and

pharmacogenetic assessment of thiopurine therapy, the

developed method would also be used to prospectively

assess adherence to thiopurine medication in patients

receiving such treatment,” he concluded.

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Partially porous particle columns for use in pharmacokinetic studies with RP-UHPLC-MS/MSUK

In late 2007 the Respiratory Drug Metabolism and Pharmacokinetics (DMPK) Department of GlaxoSmithKline (GSK)

replaced its existing ‘conventional’ HPLC systems with UHPLC systems with the capability of operating at system back-

pressures of up to 15000 p.s.i. (~100 Mpa). “As a drug discovery department, our challenge is the rapid generation

of DMPK information from in vitro and in vivo studies on a large number of new chemical entities across many

structurally diverse chemical series,” said Dave Mallett from the GSK Medicines Research Centre, UK.

A paper Mallet authored in the Journal of Pharmaceutical and Biomedical Analysis [49 (1), 100-107 (2009)], presents

data showing the robustness of a partially porous 2.7 μm diameter particle material and the accuracy and precision of

an assay for a typical pharmaceutical in plasma.

“A consequence of the wide chemical diversity and the need for rapid information generation is the requirement

that wherever possible, generic methods of sample analysis are available. These methods had been successfully

implemented for our HPLC systems featuring fast gradient reversed phase chromatography in an approximately 3-4

minutes per sample timescale and had proven to be highly reliable and robust. The UHPLC technology allowed us

to consider reduction of this analysis time and, therefore, a similarly generic UHPLC method was needed with the

fundamental requirement that it be equally robust,” he explained.

According to him, the development of suitable generic gradient reversed phase conditions operated over a <1.5

minutes per sample timescale using common UHPLC technology (<2 µm particulate stationary phases and pressures

>6000 p.s.i.) proved relatively simple. However, the definition of a suitable reliable column proved more difficult.

“In our hands, some <2 µm stationary phase materials operated at very high backpressures (>80% of the specified

maximum operating pressure of both the columns and the UHPLC equipment) and it was felt that continued

operation of systems under these high stress conditions was likely to lead to an unacceptable high occurrence of

system problems; that is, a non-robust methodology. Other UPLC columns that operate at lower but UHPLC-type

pressures were found to perform well for routine analyses at first, but the separations rapidly degraded to unuseable


after 500-600 injections,” he said.

The answer lay in a relatively new stationary phase, a material featuring a non-porous core surrounded by a ‘shell’

of porous stationary phase. This material had been shown to exhibit extremely tight particle size distribution and

consequently excellent separation efficiencies and lower backpressures compared with similar size totally porous

materials. “Applying these columns to our generic methodologies we were indeed able to demonstrate these

excellent separation efficiencies at backpressures of approximately 6000-7000 p.s.i. and furthermore we discovered

that the separation remained excellent over the analysis of many thousands of our typical protein-precipitated plasma

samples. Thus we were able to fully define the highly robust generic UHPLC analytical method required to support our

business needs,” he said.

The methodology has been implemented across the whole department (as well as another DMPK department

within GSK), enabling the successful transference to UHPLC methods of analysis. The consequent reduction from

3-4 minutes per sample to <1.5 minutes per sample has also given the department more analytical capability and

flexibility. “Should one LC-MS system fail, it is possible for an analyst to transfer the methods and samples and run

as an additional batch on another instrument along with a colleague analysing his or her own samples on that

instrument – and both still have their analyses ready for processing by the following morning,” he explained. For

smaller batches of samples, it is even possible to provide DMPK results on the same day they are delivered, extracted

and analysed. Finally, the effectively greater than doubled sample capacity of the systems has reduced and in some

cases removed the need to buy additionally LC-MS systems at considerable financial savings to the company.

The department now has sufficient capacity to meet its needs and so the focus will shift from speed towards

improving sensitivity and developing high efficiency separation methods for lower throughput applications such as

metabolite identification.

13research round-upseparation science — volume 1 issue 4


Headspace solid-phase microextraction for determining chloroanisoles and chlorophenols in winespain

Dr Consuelo Pizarro and colleagues from the Department of Chemistry at the University of La Rioja in Spain have

developed a robustness test for a solid-phase microextraction-based method optimized for the simultaneous

determination of chloroanisoles and acetyl-chlorophenols in wine, using a hybrid experimental design. These

compounds are implicated in the unpleasant taste of corked wine. Documented in the Journal of Chromatography

A [1208 (1-2), 54-61 (2008)], the main aim of this work was testing the robustness of a previously optimized HS-

SPME method (performed by the same research group) and, consequently, ensuring its correct implementation in

the enological industry. “With that objective in mind, some factors, which represent potential sources of variability,

were selected and examined in an interval around the nominal level using experimental design. The experimental

ranges selected were representative of the variations which can be expected when the method is transferred. Once

the eff ects of these small oscillations on the responses considered were evaluated, it was possible to establish if the

analytical procedure was robust with respect to these factors or if they must be more strictly controlled during the

execution of the method,” Dr Pizarro explained.

A 4-factor hybrid design was utilized for robustness testing of the SPME method. This design included 25

experiments with small variations of Vs/Vt ratio, exposition time, extraction temperature and sample incubation

time around the nominal level.

“From the statistical analysis of the results obtained, it was possible to indicate how tightly controlled the

experimental factors should be if the method is to be transferred,” he said, adding that the proposed procedure

can be considered robust for TCA and acetyl-PCP extraction because the changes of the four factors considered

do not signifi cantly aff ect the measured responses for these analytes. “However, for the analysis of the rest of the

compounds it is necessary to keep the Vs/Vt ratio, exposition time, extraction temperature and sample incubation

time under strict control because their infl uence on the extraction yield is critical. These results can be justifi ed

by the fact that the use of SPME as the extraction technique implies the constant maintenance of experimental

conditions, especially when partition equilibrium among the system phases was not reached, as in the present

case,” he said.

Food fl avour represents a key characteristic in quality control and in winemaking particularly, with the quality of

wines being highly dependent on fl avour. “Chloroanisoles (2,4,6-trichloroanisole, 2,3,4,6-tetrachloroanisole, and

pentachloroanisole) are well known for being responsible for ‘cork taint’ in wines. The presence of haloanisoles is

a great enological problem because of their extraordinary low sensory thresholds. As halophenolic compounds

(2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol) may develop into haloanisoles, their

determination is of great interest to wine industries,” he explained.

The proposed HS-SPME method allows the simultaneous determination of chloroanisoles and chlorophenols

in wine samples and the results of the method validation confi rm that it is suitable for the extraction of these

compounds from the wine matrix. “The proposed method showed satisfactory linearity, precision and detection

limits. In the case of anisoles, detection limits were below the odour detection threshold of the compounds,” he

said.

Its application was demonstrated by analysing commercial red wines contaminated with the target compounds.

Pizarro believes that because this method is solvent free, it has a short preparation time and can be easily

automated, and could therefore be a very suitable technique for determining ‘cork taint’ in wines. “Thanks to the

robustness test it is possible to ensure the correct implementation of this method in the enological industry,” he

concluded.

15research round-upseparation science — volume 1 issue 4separation science — volume 1 issue 4

Improving the performance of simulated moving bed chromatography

Germany

Professor Andreas Seidel-Morgenstern from the Max

Planck Institute for Dynamics of Complex Technical

Systems in Magdeburg, Germany, has several years

experience of working with simulated moving bed

(SMB) chromatography, based on exploiting the

elegant principle of simulated moving countercurrent

movement between the stationary and mobile phases.

Having seen the potential for further improvement, a

study was conducted on a novel concept that combines

non-permanent product withdrawal at one or both

outlet ports (leading periodically to a ‘product’ and a

‘non-product’ fraction), with an internal recycle and

re-feeding of the ‘non-product’ fraction in alternation to

the original feed mixture.

“After studying the possibilities to systematically vary

feed concentrations, we examined the concept of partial

product withdrawal and optimized feed-back. The

idea was inspired from reaction engineering concepts

based on recycling not suffi ciently converted reactants,”

Professor Seidel-Morgenstern said about the research,

published in the Journal of Chromatography A 1207 (1-

2), 55-71 (2008)].

Using simulation studies for linear and non-linear

isotherms, it was shown that in terms of process

performance and product recovery, this fractionation

and feed-back approach (FF-SMB) is superior to both

the conventional SMB process as well as to a previously

reported fractionation and discard strategy. “The

key fi nding is the fact that the productivity of SMB

chromatography can be increased signifi cantly, if

the product streams are collected at the outlets only

within certain fractions of the shift times, and if the

not suffi ciently separated fractions are recycled. This

concept allows to process altogether more feed and,

thus, allows to reach higher productivities,” he added.

The results will be tested in the near future for various

applications. “If the theoretical predictions can be

confi rmed, conventional processes might be modifi ed

in order to apply the new FF-SMB concept. For this, just

small modifi cations have to be made in the hardware,”

he concluded.

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Uncovering PAHs in biota samples with GC-MS-MSSpain

A paper in the Journal of Chromatography A [1207 (1-2), 136-145 (2008)] presents the

development of a programmed temperature vaporization-gas chromatography–tandem

mass spectrometry (PTV-GC–MS-MS) method for the determination of PAHs in complex

environmental matrices. “This research was performed to overcome the current problems

in detecting PAHs in complex matrices such as biota samples. The main problem with

the determination of PAHs in those samples is the presence of numerous interfering

substances that cannot be removed by repeated extraction and purifi cation steps,”

explained main author, Dr Soledad Muniategui-Lorenzo from the Department of Analytical

Chemistry at the University of A Coruña, Spain.

As a result of the increasing importance accorded to conservation and environmental

protection, interactions between aquaculture and the environment are subject to

increasingly strict control and regulations. Thus, the European Commission Regulation (EC)

Nº 208/2005 of 4 February 2005 concerning polycyclic aromatic hydrocarbons and more

recently, the Commission Regulation (EC) Nº 1881/2006 of 19 December 2006, have fi xed

a maximum level of 10 µg/kg in wet weight for benzo[a]pyrene in bivalve molluscs. “The

aim of this paper is to develop a method for the qualitative and quantitative determination

of 27 alkylated and no-alkylated PAHs, improving considerably the detection and

quantifi cation limits reaching the legislation requirements and removing any matrix

interferences for complex matrices such as biota samples,” Dr Muniategui-Lorenzo said.

According to her, tandem mass spectrometry (MS-MS) detection provides high reliability

confi dence in the identifi cation of target analytes and low detection limits, so PTV-GC-MS-

MS can detect compounds at low-ppb levels in complex matrices. “The LODs and LOQs of

the method range between 1.9 and 3.6 ng/kg and between 79 and 99 ng/kg, respectively

that are in good agreement with the legislation requirements. The validation of the

method was made with a mussel reference material (SRM 2799) ‘mussel tissue: Organic

contaminants and trace elements’,” she added. In addition, the applicability of the method

to real mussel samples was also studied. “In marine pollution monitoring programs, it is

particularly interesting to analyze ‘source-specifi c marker’ PAHs in order to characterize

spilled oils and relate them to the origin sources,” she said.

She believes the GC-MS-MS method can be applied to other matrices that require low

detection limits. As an example, this method was applied to various types of water samples

(tap, well, superfi cial, and seawater) using SPME. “The proposed SPME-GC-MS-MS method,

extracting only 18 mL of sample, reaches the very restrictive limits fi xed by the 2006/0129

EC proposal for a new water Directive, to be achieved by 2015. [V. Fernandez et al., Journal

of Chromatography A, 1176 48(2007)],” she said.

Moreover, other extraction methods were development to determine PAHs in biota

samples, for example, a soon to be published paper on a new simultaneous matrix solid-

phase dispersion extraction-gel permeation chromatography clean-up method for the

analysis of polycyclic aromatic hydrocarbons in mussel samples. “This method does not

require special instruments or costly equipment, and it is a good alternative technique that

can extract the PAHs and remove lipids, fats and other interfering compounds in one single

step,” she concluded.


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20 feature article — Biomarkers in exhaled breath www.sepscience.com

Analytical methods in exhaled breath diagnosticsNicholas C. Strand and Cristina E. Davis

Mechanical and Aeronautical Engineering, University of California Davis, USA.

21feature article — Biomarkers in exhaled breathseparation science — volume 1 issue 4

The analysis of gaseous chemical

species plays an important

role in environmental studies,

national security and the future of

medical diagnostics. For medical

purposes, the analysis of volatile

and non-volatile compounds in

exhaled human breath and breath

condensate provides a desirable

non-invasive procedure that is

promising for diagnosis of various

Chemicals in exhaled breath appear to be correlated with end-stage

metabolomic processes in the human body, and there is a huge potential

to exploit these putative biomarkers for disease diagnostics and as

markers of exposure to exogenous chemicals. This paper reviews

recent advances in approaches for identifying breath biomarkers,

instrumentation attributes that are important in these studies, and

chemometrics signal-processing methods that may be applied to these

data sets. We will also highlight the technical challenges for advances in

mobile detection platforms in the future.


of these fronts [2, 6-8]. Here we

review recent developments and

trends for each system module

needed for breath analysis systems

with a focus towards miniaturization.

Volatile Organic Compounds (VOCs) as Biomarkers in Exhaled BreathThe field of breath analysis is

predicated on the notion that breath

biomarkers exist that are indicative

of disease and that these biomarkers

can be monitored in large

populations of individuals at any

given time. What this

really means is that

substantial research

must be performed

to identify robust

metabolic biomarkers of

disease that can be measured

in breath, and large-scale trials are

needed to validate these biomarkers

prior to clinical implementation.

There are dozens of VOCs in

human breath that show promise

for diagnosis and management of

diseases, but only modest technical

or clinical research to date [9-11].

Current literature reports >25

exhaled biomarkers other than the

most common breath marker nitric

oxide (NO) in asthmatics and patients

with chronic obstructive pulmonary

disease (COPD) using exhaled breath

condensate (EBC). These markers not

only differentiate between asthma

and COPD, but can be reflective

of disease severity or treatment

response. Some examples include:

increased nitrites in EBC with severe

asthma and COPD [12]; selective

increase of prostaglandin E2 (PGE2)

in EBC with COPD but not asthma

diseases and disorders including

cancer, asthma, respiratory infections

and potentially many others [1, 2].

Although ancient Greek and Chinese

medical texts refer to clinicians

using body and breath odours as

diagnostic indicators, it is only in

recent modern times that we have

begun to apply scientific approaches

to identify specific breath chemical

compounds of interest [3]. We can

point towards several seminal papers

that paved the way for the field that

is often now referred to as simply

“breath analysis”.

Scientific studies

of the array of

compounds in

breath began in

1969 [4]. By 1971,

Pauling et al. [5] used

gas chromatography (GC) to detect

over 200 volatile organic compounds

(VOCs) in human breath. These initial

works set the stage for the modern

breath diagnostics movement.

They identified simple methods for

collecting exhaled breath samples,

and showed that chemical content

could be measured using different

types of chemical analysis measures.

However, despite the availability of

more advanced analytical chemistry

instrumentation in the last three

decades, breath analysis is not yet

commonly used in the medical

field even with growing interest in

the medical community, and there

appear to be several significant

reasons that have limited the growth

of this fascinating field.

One major hindrance to the

clinical use of breath diagnostics is

the mobility and size of the analysis

instrumentation. Mass spectrometric

methods for VOC analysis of

breath are continually improving

resolution and sensitivity, from gas

chromatography-mass spectrometry

(GC-MS) to proton-transfer-reaction

mass spectrometry (PTR-MS)

and selected ion flow tube mass

spectrometry (SIFT-MS). But access to

such equipment is limited because

of its size and expense. In addition,

analysis time can be lengthy.

The benefits of a mobile or

handheld device capable of

detecting and characterizing VOCs in

breath are wide-ranging. In order to

reach this objective, miniaturization

without a reduction in sensitivity

or precision is crucial. We can begin

to categorize advances that must

be made before breath analysis can

be adopted as a clinical measure

into several main areas. As depicted

by Figure 1, a total breath analysis

instrumentation system can be

subdivided into four stages: (1)

sampling, (2) preconcentration and

desorption, (3) chemical analysis

and (4) informatics and biomarker

identification. Each part plays an

important role in the analysis and

resulting output. Ideally, all of

these system components will be

combined into a small hand-held

portable “breathalyser” device

that can be used in clinical point-

of-care settings. It is a tall order to

satisfy, and to date only incremental

advances have been made on each

“The benefits of a mobile or handheld device capable of detecting and

characterizing VOCs in breath are wide-ranging”


[13, 14]; higher concentrations

8-isoprostane and interleukin-6 in

acute COPD exacerbations [15] with

reduction in 8-isoprostane with

antibiotic treatment [16]; leukotriene

B4 (LTB4) increased in EBC with

moderate to severe persistent

asthma [17] and increased during

COPD exacerbation; interleukin-4

and TNF-alpha signifi cantly

elevated in EBC of asthmatics

compared with nonsmoking healthy

subjects [18]; higher macrophage-

derived chemokine (MDC) levels

in EBC of asthmatics on inhaled

corticosteroids than steroid-naïve

asthmatics or controls [19-21];

lower pH in moderate asthmatics

compared with mild asthma and

controls [22]; hydrogen peroxide

(H2O2) increased with COPD

and further increased during

exacerbations, plus was related to

disease severity [23, 24]; ethane

was also found to be elevated in

COPD and asthma, correlated with

FEV1 and airway obstruction, and

was lower in patients treated with

steroids [25]. Together all of these

studies and many others that are

not cited, provide evidence that

biomarkers can be determined that

are clinically signifi cant. In this case,

we report putative biomarkers that

will distinguish between asthma and

COPD; however, biomarkers of other

diseases are also of interest.

Although the scientifi c community

has indentifi ed some putative

breath biomarkers, the long-term

viability of these markers is relatively

unknown. They may be altered by

many potential confounding factors,

such as diet, exercise, stress, age,

gender, unrelated diseases/disorders,

exposure to environmental

chemicals and other potentially

unknown causes. While much

research in this area is still required,

it is feasible for the instrumentation

fi eld to continue advances

toward miniaturization strategies

concurrently with the biomarker

research eff orts.

Instrumentation Platforms Used For Breath AnalysisFor most purposes, on-site

measurement of gas analytes is

not only desirable but necessary.

Such is the case in environmental

applications for providing more

eff ective data concerning pollutant

sources and for avoiding changes

in the data because of time

variation. In security applications,

on-site measurements are required

for instant analysis of potentially

hazardous conditions, such as gas

buildup or terrorist activities. For

breath analysis, there is a need for

real-time monitoring of patients for

rapid treatment and to avoid analytes

being lost during sampling or

transportation to the laboratory [1].

Gas chromatography coupled with

mass spectrometry (GC-MS) is one

of the current methods of choice

for characterizing VOCs in breath

[7]. Conventional single-column gas

chromatography requires the use of

mass spectrometry to characterize

and separate coeluting compounds

towards the end of the column.

This method is especially helpful to

resolve the co-elution of compounds

such as isoprene with pentane [26]

or isopentane with pentane [27].

However, research is still needed to

establish reference biomarker values

because of the inter-individual

variations of human breath [8].

Interpretation of GC-MS data

requires a method for peak

separation and identifi cation. This

stage (informatics) plays a critical

role in breath analysis. While the

prior stages must be accurate,

precise and robust, false positives

or false negatives resulting from

faulty signal processing would

render a handheld “breathalyser”

useless and potentially dangerous

for determining the treatment plan.

Coupled with this stage is the need

for robust biomarker identifi cation.

Elevated concentrations of specifi c

VOCs in breath do appear to

correlate to certain diseases such as

high levels of acetone as a biomarker

for diabetes [28-31], increased

ammonia concentration as a sign of

hepatitis [32] and dimethyl sulfi de

Figure 1

Figure 1 – The major components of a future micro total-analysis diagnostic system for breath.


breath are excreted in concentrations

as low as parts-per-trillion (ppt)

which is often too low for direct

detection [40]. Thus emerges the

need for preconcentration prior

to assay. Preconcentration can

be achieved by several methods:

adsorptive, chemical and cryogenic

trapping. Chemical trapping consists

of breathing through a reagent

which captures target compounds.

The downfalls of this method are a

long collection time, memory effects

of the reaction mechanism and

potential loss of analyte [41,

42]. Cryogenic

trapping

captures

VOCs traveling

through a tube

held at a sub-zero

temperature. However, the low

temperature of the trap generally

freezes water vapor and carbon

dioxide in breath, causing the

collection device to fill with ice. Thus,

the most promising and widely used

method for preconcentration is

adsorptive trapping [40].

Collecting a breath sample directly

onto a preconcentration platform

eliminates an extra analytical step

and simultaneously reduces the

overall system size. Adsorbent

materials for preconcentration

can be divided into three groups:

inorganic, carbon-based and organic

polymers. Inorganic sorbents are

ineffective for breath analysis

because of high water adsorption

but carbon-based sorbents and

organic polymers show promise

for capturing VOCs in breath [43,

44]. Adsorptive materials differ in

their capture efficiency, memory

pointing to cirrhosis [33]. A false

diagnosis for these or any other

disease could elicit unnecessary

treatment or a lack of essential

treatment.

There are several software

packages for deconvolution

of GC-MS data such as AMDIS,

ChromaTOF and AnalyzerPro and a

comparison and review was reported

by Lu et al. [34]. Each package has

advantages and disadvantages

such as an overwhelming display

of false positives by AMDIS, a lack

of detected analytes by

AnalyzerPro and

a mixture

of false

positives and

negatives

from ChromaTOF.

Despite their flaws, the

deconvolution method for a given

application must be carefully

considered as one method may be

the most appropriate. These signal

processing methods are under

continual revision and will someday

help bridge the gap towards a

portable and reliable breathalyser.

Strategies for MiniaturizationThe development of micro total

analysis systems (µTAS) is an active

field, one that has seen considerable

growth in the past few years [35].

Miniaturization is particularly

important in the real-time analysis

of gaseous species. In addition to

increased mobility, miniaturization

of such devices lends itself to

higher sensitivity, decreased power

consumption and faster analysis [1].

A micro total breath analysis system

(μTBAS) as a clinical diagnostic tool

could increase the accuracy while

decreasing the time of diagnosis

to expedite patient treatment. To

achieve this goal, developments for

each of the four major components

of a μTBAS need to be investigated

simultaneously.

A standardized and repeatable

method of sampling is the first major

component to a μTBAS. A healthy

individual exhales approximately

half a litre of breath on average, the

first third of which consists of empty

or “dead space” air. The remaining

“end-expiratory” breath

consists of alveolar air from the

lower airways where gas exchange

occurs. Alveolar air has a higher

concentration of VOCs and is the

optimal portion of expired air for

breath analysis [11, 36]. Tedlar bags

are a popular method of sample

collection [37, 38] but require an

extra step to collect the sample

on a sorbent trap, adding time

and complexity to the system. In

addition, these bags may adsorb

some of the VOCs in breath and can

only store samples for a limited time

while maintaining a decent recovery

rate [39].

In healthy individuals, metabolic

processes produce VOCs such as

acetone, ethanol, methanol and

isoprene. These compounds are

generally emitted in high enough

concentrations for direct detection

[8, 37]. But many compounds in

“In healthy individuals, metabolic processes produce VOCs in high enough

concentrations for direct detection”


effects and breakthrough volume.

No single adsorptive material can

capture all VOCs in breath because of

varying compound size, weight and

boiling point. A table of adsorptive

materials and their properties was

reported by Dettmer et al. [43] and

condensed in Table 1. It is shown

that as a whole, carbon molecular

sieves have a larger specific water

retention volume (Vg H2O) than

organic polymers but can withstand

higher temperatures. These

higher temperature thresholds are

beneficial to ensure full desorption

before adsorbent deterioration can

occur. Sanchez et al. reported that

desorption temperatures up to

300 °C have little effect on the GC

analysis of VOCs [45].

Excess water vapour is problematic

in breath sampling. Groves et al. [46]

report that while water vapour in

exhaled breath does not significantly

absorb VOCs, it needs to be removed

prior to analysis to prevent damage

to the GC column. In addition,

some non-volatile compounds

in breath such as cytokines and

isoprostanes are dissolved in water

vapor making a lower adsorbent

water retention desirable [7, 40].

Carbon molecular sieves (CMS) are

excellent at trapping highly volatile

compounds but adsorb a large

quantity of water vapour [47-49].

One method to partially overcome

this is a multibed sorption trap

consisting of CMS, carbon blacks

and organic polymers, arranged by

increasing sorption strength [38, 45,

48, 50]. The carbon molecular sieve

attracts and separates the water

vapour while various graphitized

carbon blacks are ordered to

maximize collection and desorption

efficiency. Another method to

separate water vapour is to use a

membrane extraction with sorbent

interface (MESI) setup. MESI systems

utilize a hydrophobic membrane to

block moisture from reaching the

sorbent trap, thereby combining

sampling and preconcentration in

one step [6, 51-53]. Recently, Ma et

al. [51] have reported a MESI system

combined with CO2 monitoring for

normalization of measured acetone

levels in breath.

Numerous methods have been

reported for the analysis of VOCs in

breath. Several of these methods,

such as the SAW technique or the

Table 1

Adsorbent Sampling Range Density (g/mL) Spec. Surface

Area (m2/g)

Tmax (°C) Vg H2O @ 20°C

(mL/g)

Carbon molecular sieves

Carboxen 563 C2-C5 0.53 510 >400 778

Carboxen 564 C2-C5 0.6 400 >400 276

Carboxen 569 C2-C5 0.58 485 >400 257

Carboxen 1000 C2-C5 0.44 1200 >400 418

Carboxen 1001 C2-C5 0.61 500 >400 234

Carboxen 1003 C2-C5 0.46 1000 >400 79

Carbosieve SIII C2-C5 0.61 820 >400 378

Carbosphere C2-C5 - 1000 400 779

Graphitized carbon blacks

Carbotrap C5-C12 0.36 100 >400 -

Carbotrap C C12-C20 0.72 10 >400 -

Carbotrap F >C20 0.66 5 >400 -

Carbotrap X C3-C5 0.41 250 >400 -

Carbotrap Y C12-C20 0.42 25 >400 -

Carbograph 5 C3-C5 - 560 >400 -

Porous organic polymers

Chromosorb 106 small molecules - 750 250 173

Tenax TA C7-C26 0.25 35 350 39

Table 1: Commonly used adsorbent materials for pre-concentration of VOCs (adapted from [43])


electrochemical method, have

been previously reviewed [40, 54]

but are generally more selective

and designed to detect individual

compounds. GC-MS is often heralded

as the “gold standard” for gas

analysis, but several improvements

have been suggested. One of the

most promising is two-dimensional

gas chromatography (GCxGC), which

demonstrates a higher peak capacity

and enhanced resolution over single-

column GC and off ers a promising

alternative to GC-MS analysis [50,

55-58]. Seeley et al. [59] report

a dual-secondary column setup

(GCx2GC) for further separation of

coeluting compounds and up to an

85% reduction of analysis time over

traditional GC-MS, and this method

has been generally successful when

applied to breath analysis [37, 50, 59].

Elimination of consumables

in a μTBAS is a major hurdle

aff ecting the portability of analysis

instrumentation. A typical GC-MS

requires electrical power, carrier

gas, fl ame gas and often a cooling

liquid. Libardoni et al. [56] reported

an air-cooled thermal modulator

to eliminate the need for cryogenic

materials and utilized at-column

resistive heating for a 99% reduction

in the power requirement over a

conventional GC oven. More recently,

Libardoni et al. [50] have combined

the GCxGC system with a multibed

sorption trap and fl ame ionization

detector (FID) in a system that

requires no consumables other than

a carrier gas and electrical power.

Future work involves coupling a

miniaturized time-of-fl ight mass

spectrometer to the GCxGC for

faster data acquisition and better

resolution. To further increase system

autonomy, Sanchez and Sacks [60]

demonstrated the use of ambient air

as the carrier gas and developed a

system that requires only electrical

power. While these achievements

have improved system autonomy

and effi ciency, reductions in physical

dimensions are still necessary.

Advancements in the micro-

fabrication of GC components

such as preconcentrators, heaters,

columns and detectors, have paved

the way for smaller systems with

better performance [1]. Agah et

al. [61] and Potkay et al. [62] have

developed low-power micro-GC

columns and Zarejan-Jahromi et al.

[63] has fabricated multicapillary

micro-GC columns for improved

separation effi ciency and sample

capacity. Recently, Zhong et al. [64]

designed a two-dimensional micro-

scale GC capable of separating

31 VOCs in under 7 minutes with

detection limits in the ppt range, and

improvements to increase sensitivity,

resolution and performance

while reducing size are currently

underway. The MicroChemLab

project, which began in 1996,

has developed a 12 x 12 mm GC

chip containing a micromachined

preconcentrator, GC channel and

SAW detector [65]. These micro-

systems have achieved the size

requirement but lack the accuracy

and effi ciency necessary for a reliable

breathalyzer and are undergoing

further investigation.

Future DirectionsContinual advancements in the

aforementioned areas will no doubt

eventually lead to a handheld

breathalyser, potentially within

the next several years. Figure 2

summarizes the current progress in

Figure 2

Figure 2 – Research and advancement in the four major stages of breath analysis required for portable breath analysis systems [references].


June 28 – July 2, 2009

34th International Symposium on High-Performance Liquid Phase Separations and Related TechniquesChairman: Prof. Dr. Christian Huber, Paris-Lodron University Salzburg

Separation Science

ArbeitsKreis

ToPICS:

Advances in Liquid Phase Separation Technology Multidimensional and Hyphenated Techniques Fundamental Aspects of Separations Industrial Aspects of Separations Clinical and Pharmaceutical Analysis Life Sciences Food and Environmental Analysis

DeaDLIneS:

April 30, 2009 Abstract Deadline for Last Minute Posters May 15, 2009 Deadline for Early Registration

Contact: Gesellschaft Deutscher Chemiker e.V. Congress Team E-mail: [email protected]

ww

.hp

lc20

09.c

om

International Congress Center Dresden · Germany


the four stages of a future μTBAS.

Beyond the instrumentation, many

other factors will contribute to

this goal such as standardization

of collection methods [66, 67].

In addition, breath samples from

“healthy” subjects must be defined

and differences resulting from

biological or physiological factors

need to be characterized [8, 36].

Collection efficiency and recovery

during preconcentration and

desorption must be estimated and

overall instrumentation reliability

and reproducibility must be

standardized. While addressing these

challenges will be difficult, doing so

will greatly advance the field and

hasten the day when hand-held

breathalysers for clinical medicine

are a reality.

AcknowledgementsNicholas Strand received fellowship

support from the Science,

Mathematics And Research for

Transformation (SMART) Scholarship

for Service Program established

by the Department of Defense

(DoD). Cristina Davis received grant

support from the Defense Advanced

Research Projects Agency (DARPA),

the California Industry-University

Cooperative Research Program and

the California Citrus Research Board.

The contents of this manuscript

are solely the responsibility of the

authors and do not necessarily

represent the official views of the

funding agencies.

References

1. Ohira SI, Toda K: Analytica Chimica Acta 2008, 619:143-156.2. Lechner M, Rieder J: Current Medicinal

Chemistry 2007, 14:987-995.3. Liddell K: Postgraduate Medical Journal 1976, 52:136-138.4. Jansson BO, Larsson BT: Journal of Laboratory and Clinical Medicine 1969, 74:961-&.5. Pauling L, Robinson AB, Teranish.R, Cary P: Proceedings of the National Academy of Sciences of the United States of America 1971, 68.6. Lord H, Yu YF, Segal A, Pawliszyn J: Analytical Chemistry 2002, 74:5650-5657.7. Mukhopadhyay R: Analytical Chemistry 2004, 76:273A-276A.8. Phillips M, Herrera J, Krishnan S, Zain M, Greenberg J, Cataneo RN: Journal of Chromatography B 1999, 729:75-88.9. Phillips M: Scientific American 1992, 267:74-79.10. Phillips M, Greenberg J: Clinical Chemistry 1992, 38:60-65.11. Zarling EJ, Clapper M: Clinical Chemistry 1987, 33:140-141.12. Corradi M, Montuschi P, Donnelly LE, Pesci A, Kharitonov SA, Barnes PJ: American Journal of Respiratory and Critical Care Medicine 2001, 163:854-858.13. Montuschi P, Kharitonov SA, Ciabattoni G, Barnes PJ: Thorax 2003, 58:585-588.14. Montuschi P, Ragazzoni E, Valente S, Corbo G, Mondino C, Ciappi G, Barnes PJ, Ciabattoni G: Inflammation Research 2003, 52:69-73.15. Carpagnano GE, Resta O, Foschino-Barbaro MP, Spanevello A, Stefano A, Di Gioia G, Serviddio G, Gramiccioni E: European Journal of Pharmacology 2004, 505:169-175.16. Biernacki WA, Kharitonov SA, Barnes PJ: Thorax 2003, 58:294-298.17. Csoma Z, Kharitonov SA, Balint B, Bush A, Wilson NM, Barnes PJ: American Journal of Respiratory and Critical Care Medicine 2002, 166:1345-1349.18. Matsunaga K, Yanagisawa S, Ichikawa T, Ueshima K, Akamatsu K, Hirano T, Nakanishi M, Yamagata T, Minakata Y, Ichinose M: Journal of Allergy and Clinical Immunology 2006, 118:84-90.19. Ko FWS, Lau CYK, Leung TF, Wong GWK, Lam CWK, Hui DSC: Respiratory Medicine 2006, 100:630-638.20. Ko FWS, Lau CYK, Leung TF, Wong GWK, Lam CWK, Lai CKW, Hui DSC: Clinical and Experimental Allergy 2006, 36:44-51.21. Ko J, Yun CY, Lee JS, Kim DH, Yuk JE, Kim IS: Life Sciences 2006, 79:1293-1300.22. Kostikas K, Papatheodorou G, Ganas K, Psathakis K, Panagou P, Loukides S: American Journal of Respiratory and Critical Care Medicine 2002, 165:1364-1370.23.Kostikas K, Papatheodorou G, Psathakis K, Panagou P, Loukides S: European Respiratory Journal 2003, 22:743-747.24. Kostikas K, Papatheodorou G, Psathakis K, Panagou P, Loukides S: Chest 2003, 124:1373-1380.25. Paredi P, Kharitonov SA, Barnes PJ: American Journal of Respiratory and Critical Care Medicine 2000, 162:1450-1454.26. Springfield JR, Levitt MD: Journal of Lipid Research 1994, 35:1497-1504.27. Mitsui T, Naitoh K, Tsuda T, Hirabayashi T, Kondo T: Clinica Chimica Acta 2000, 299:193-198.28. Rooth G, Ostenson S: Lancet 1966, 2:1102-&.29. Tassopou.Cn, Barnett D, Fraser TR: Lancet 1969, 1:1282-&.30. Trotter MD, Sulway MJ, Trotter E: Clinica Chimica Acta 1971, 35:137-&.31. Levey S, Jung R, Medrano V, Balchum OJ: Journal of Laboratory and Clinical Medicine 1964, 63:574-&.32. Manolis A: Clinical Chemistry 1983, 29:5-15.33. Kaji H, Hisamura M, Saito N, Murao M: Clinica Chimica Acta 1978, 85:279-284.34. Lu HM, Dunn WB, Shen HL, Kell DB, Liang YZ:

Trac-Trends in Analytical Chemistry 2008, 27:215-227.35. Dittrich PS, Tachikawa K, Manz A: Analytical Chemistry 2006, 78:3887-3907.36. Mendis S, Sobotka PA, Euler DE: Clinical Chemistry 1994, 40:1485-1488.37. Sanchez JM, Sacks RD: Analytical Chemistry 2006, 78:3046-3054.38. Sanchez JM, Sacks RD: Analytical Chemistry 2003, 75:2231-2236.39. Mochalski P, Wzorek B, Sliwka I, Amann A: Journal of Chromatography B, 2009, 877:189-196.40. Cao WQ, Duan YX: Critical Reviews in Analytical Chemistry 2007, 37:3-13.41. Henderson MJ, Karger BA, Wrenshall GA: Diabetes 1952, 1:188-&.42. Teshima N, Li JZ, Toda K, Dasgupta PK: Analytica Chimica Acta 2005, 535:189-199.43. Dettmer K, Engewald W: Analytical and Bioanalytical Chemistry 2002, 373:490-500.44. Ligor T: Critical Reviews in Analytical Chemistry 2009, 39.45. Sanchez JM, Sacks RD: Analytical Chemistry 2003, 75:978-985.46. Groves WA, Zellers ET: American Industrial Hygiene Association Journal 1996, 57:257-263.47. Gawlowski J, Gierczak T, Jezo A, Niedzielski J: Analyst 1999, 124:1553-1558.48. Buszewski B, Ligor T, Filipiak W, Vasconcelos MT, Pompe M, Veber M: Toxicological and Environmental Chemistry 2008, 90.49. Helmig D, Vierling L: Analytical Chemistry 1995, 67:4380-4386.50. Libardoni M, Stevens PT, Waite JH, Sacks R: Journal of Chromatography B, 2006, 842:13-21.51. Ma W, Liu XY, Pawliszyn J: Analytical and Bioanalytical Chemistry 2006, 385:1398-1408.52. Yu YF, Pawliszyn J: Journal of Chromatography A 2004, 1056:35-41.53. Liu XY, Pawliszyn J: Analytical and Bioanalytical Chemistry 2007, 387:2517-2525.54. Cheng WH, Lee WJ: Journal of Laboratory and Clinical Medicine 1999, 133:218-228.55. Lewis AC, Carslaw N, Marriott PJ, Kinghorn RM, Morrison P, Lee AL, Bartle KD, Pilling MJ: Nature 2000, 405:778-781.56. Libardoni M, Waite JH, Sacks R: Analytical Chemistry 2005, 77:2786-2794.57. Dimandja JMD: Analytical Chemistry 2004, 76:167A-174A.58. Marriott P, Shellie R: Trac-Trends in Analytical Chemistry 2002, 21.59. Seeley JV, Kramp FJ, Sharpe KS, Seeley SK: Journal of Separation Science 2002, 25:53-59.60. Sanchez JM, Sacks RD: Journal of Separation Science 2007, 30:1052-1060.61. Agah M, Lambertus GR, Sacks R, Wise K: Journal of Microelectromechanical Systems 2006, 15:1371-1378.62. Potkay JA, Lambertus GR, Sacks RD, Wise KD: Journal of Microelectromechanical Systems 2007, 16:1071-1079.63. Zarejan-Jahromi MA, Ashraf-Khorassani M, Taylor LT, Agah M: Journal of Microelectromechanical Systems 2009, 18:28-37.64. Zhong Q, Steinecker WH, Zellers ET: Analyst 2009, 134:283-293.65. Lewis PR, Manginell RP, Adkins DR, Kottenstette RJ, Wheeler DR, Sokolowski SS, Trudell DE, Byrnes JE, Okandan M, Bauer JM, et al: MicroChemLab.Sensors Journal 2006, 6:784-795.66. Glaser RA, Arnold JE, Shulman SA: American Industrial Hygiene Association Journal 1990, 51:139-150.67. Phillips M: Analytical Biochemistry 1997, 247:272-278.


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30 feature article — Multidimensional LC www.sepscience.comwww.sepscience.com30 feature article — Multidimensional LC www.sepscience.comwww.sepscience.comwww.sepscience.comwww.sepscience.com

31feature article — Multidimensional LCseparation science — volume 1 issue 4

Multidimensional methodsA method is considered

multidimensional if the separation

mechanisms in different dimensions

are different and if analytes that are

separated in one dimension remain

separated in the other dimensions;

that is, if two peaks are separated

in the first column, they will remain

separated in the second column.

LC-LC: The heart-cut techniques are

applied if only a small portion of the

components (from selected peaks)

is selected from a complex matrix

(Figure 1).

A typical example of heart-

cut LC (LC-LC) is the analysis of

drugs in biological sample (e.g.,

urine), and the first column is used

mainly for selective clean-up and

concentration. LC-LC is perhaps the

A practical review of multidimensional LC

Tuulia Hyotylainen, University of Helsinki, Finland

Many samples contain such a large amount of compounds that a single chromatographic separation does

not provide sufficient separation efficiency. In such cases the separation power can be enhanced using a

multidimensional chromatographic technique. Two-dimensional LC can be divided into two categories; namely

in heart-cut (LC-LC) and comprehensive techniques (LCxLC). A two-dimensional separation is called ‘heart-cut’

(LC-LC) if only a few fractions of the first separation are transferred to the second separation. Comprehensive

two-dimensional liquid chromatography (LC×LC) is a form of liquid chromatography in which the entire sample

is subjected to two different separations, to yield a contour map or colour map that is representative of the entire

sample.

most widely used multidimensional

chromatographic method. The

main applications of LC-LC are

analyte purification and enrichment,

and improvement of separation

efficiency and sensitivity of analysis.

LCxLC: In comprehensive techniques,

the whole sample is separated in all

separation dimensions, this is in two

dimensions as shown in Figure 2.

A two-dimensional

separation can only be called

‘comprehensive’(LCxLC) if:

• Everypartofthesample

is subjected to two different

separations

• Equalpercentages(either100%

or lower) of all sample components

pass through both columns and

eventually reach the detector

• Theseparation(resolution)

obtained in the first dimension is

essentially maintained.

Comprehensive two-dimensional

liquid chromatographic techniques

(LCxLC) are used, when information

is required from all sample

components. An example of this

type is the screening of metabolics in

urine.

Heart-cut LC-LC Instrumentation is based on a

normal LC instrument equipped

with an extra pump(s) and a

switching valve. The two columns

are connected via a multiport

switching valve(s). The effluent from

the first column can be directed,

by switching the valve, to waste, to

32 feature article — Multidimensional LC www.sepscience.com

detector or to the second column.

This is called the transfer.

How it works: The transfer can

be achieved in diff erent ways

depending on the application. The

transfer mode can be altered by

changing the valve confi guration

(see Figure 3). Typically,

a sample is injected into a (short)

fi rst dimension column using a weak

eluent. The analytes of interest are

retained on the column while matrix

components are fl ushed directly to

waste. The analytes of interest are

then eluted to the second dimension

column either by changing the

eluent or with the same eluent.

It is also possible to transfer more

than one fraction by using stopped-

fl ow mode. Usually, only one

detector is used in LC-LC, although

it is possible to use two detectors,

the fi rst one for monitoring the fi rst

column separation and the second

for the second column separation.

During method optimization,

however, this second detector is

also connected to the fi rst column

to enable optimization of the

separation and the fraction to be

transferred. The fraction should be

as narrow as possible to keep the

fraction volume as small as possible.

Solvent strength and peak shape:

An important parameter that

aff ects directly the volume of the

transferred fraction is the diameter

of the fi rst column. The smaller the

column i.d. is, the smaller is the

volume of the fraction. For example,

if the column diameter is decreased

from 4.6 mm to 2.1 mm, the size of

the fraction is decreased from ca. 1

mL to 0.2 mL. However, the sample

capacity also decreases if the i.d.

decreases. A good compromise

of reasonable sample capacity

and small fraction size is to have a

column with an i.d. of 2-3 mm for the

fi rst dimension. The dimensions of

the second column are not as critical,

but it should not have an i.d. smaller

than the column used for the fi rst

dimension.

Comprehensive LC (LCxLC)The instrument used for LCxLC is

very similar to that used for LC-LC;

that is, the interface is based on a

multiport switching valve. The LCxLC

systems are based mainly on two

Figure 1

Figure 1: Heart-cut mode.

Figure 2

1 dimensionseparation

st1 dimensionst1 dimension2 dimension separationnd2 dimension separationnd2 dimension separation

1 dimensionseparation

st1 dimensionst1 dimension 2 dimension separationnd2 dimension separationnd2 dimension separation

Figure 2: Comprehensive mode.


methods:

•Theuseofan8-or10-portvalve

equipped with two sample loops

that allow continuous transfers

from a primary micro-bore LC

column to a second fast column.

The use of a valve that allows

transfer from a conventional

column to two parallel fast

secondary columns, without the

use of storage loops.

Typically, some 15-90 fractions are

taken and analysed in each analysis.

This can be achieved if the second

dimension separation is very fast

(typically 20 s – 2 min). Figure 4

shows both the continuous and the

valve switching concept.

In both setups, the idea is to

take a fraction continuously from

column 1 and analyse each fraction

in 2nd dimension column. LCxLC

analyses are normally performed

so that the 2nd dimension column

is able to perform separations very

fast. It is also possible to perform

LCxLC analysis in so called stop-fl ow

mode (the eluent fl ow in column

1 is stopped while the analysis of a

fraction takes place in column 2, and

then started again). However, this

approach has several disadvantages,

such as very long analysis times

and loss of separation in the 1st

dimension column resulting from

diff usion during the stop fl ow mode.

Combining diff erent LC modes: In

principle, the same parameters that

are described for heart-cut LC have

to be considered in the selection

of LC modes for LCxLC. However,

because the transferred fractions

have typically much smaller volumes

(15-100 μL), the eluent miscibility or

eluent strength are not as critical in

LCxLC. For example, it is possible to

combine NPLC with RPLC, although

careful selection of conditions is

required to avoid band broadening.

Setting the LC parameters: Usually,

the 1st dimension column has a

relatively small i.d. (1-2 mm) and a

low fl ow rate is used (0.05-0.2 mL/

min) to keep the volume of the

fraction small. In the 2nd dimension,

either a monolithic column or a very

short (1-5 cm) column packed with

small particles (1.5-2.5 μm) is used.

With monolithic columns, back

pressure is not a problem and very

high fl ow rates (up to some 10 mL/

min) can be used. However, the

high fl ow rate can cause problems.

The higher the fl ow rate, the more

the fraction is diluted and the

poorer is the sensitivity. In addition,

interfacing with, for example, MS

requires splitting, if the fl ow rate is

very high, decreasing the sensitivity

further. The high fl ow rate also

means that the eluent consumption

is high. In packed columns, the

maximum fl ow rate and thus the

analysis time, is limited by the

high back pressure created by the

column. Conventional packing

materials should be operated under

some 225 bar – novel types of

stationary phases are available that

can withstand very high pressures (>

500 bar). Alternatively, the maximum

pressure of conventional LC pumps

is some 400 bar; however, some

manufacturers have developed

pumps that are capable of

generating ca. 1000 bar pressure.

Selection of modulation period: The

modulation period is determined

by the analysis time of the second

column, and is kept constant during

the whole analysis. To be able to

maintain the separation achieved in

the fi rst column, each peak should

be sampled at least three times. This

means that if the peak width in the

fi rst column is 60 s, the maximum

analysis time in the second column is

then 20 s. However, in practise this is

not always possible.

Figure 3

Figure 3: Animation of the heart-cut technique.

•


Presentation of the data: In LCxLC,

only one detector is used and the

chromatogram obtained actually

consists of several second dimension

chromatograms. This is not a

feasible way to interpret results and,

therefore, data are converted to a

contour or colour plot (Figure 5).

Set-up of the modulation valve: LC×LC systems can be easily

constructed using conventional

commercial high-pressure liquid

chromatographic setups equipped

with extra pump(s) and column(s)

and an automated switching

valve. In practice, there are several

instrumental setups for LC×LC, but

inmostcases8-portor10-port

switching valves have been used.

Best results are obtained when the

loop volume is clearly larger than

the volume of the fraction being

collected. For example, if the fraction

volume is 100 µL, an appropriate

loop size is 125 to 150 µL.

There are two approaches that

should be tested first, namely

symmetrical and asymmetrical

configuration, as shown in Figure 6.

Bothuseeitheran8-portor10-port

valve.

configuration – the flow direction

is the same for both loops. (b)

Asymmetrical configuration in which

each loop is flushed in a different

direction into the second dimension.

L1 = loop 1; L2 = loop 2; LC = from

the first dimension pump; P = from

theseconddimensionpump;SEC=

to the second-dimension column.

The symmetrical configuration

allows flushing of the fractions to

the second dimensions in the same

direction in which they were loaded,

thus making both positions identical.

There are also more complex

solutions to be used. A modification

of the sampling loop interface is the

use of two guard columns instead

of loops. The effluent from the

first column is alternately trapped

and sampled onto the secondary

columns through a guard column

interface. When one guard column

traps the eluate, the other injects

the previously trapped components

onto a secondary column. This cycle

is repeated throughout the analysis.

The guard column is of the same

material as the second-dimension

column. A similar approach, utilizing

18solid-phaseextraction(SPE)

columns, has also been developed.

In this system, the use of several

SPEcolumnsallowedlongertimes

for second-dimension separation

without compromises in sampling

frequency.

It is also possible to use two second-

dimension columns connected in

parallel or even use two modulator

valves with two second-dimension

columns. Also a stopped-flow

approach utilizing two 6-port valves

has been developed.

LCxLC-MSThe same MS systems that are used

in conventional one-dimensional

LC-MS can be used also in LC×LC-MS.

However, there are a few additional

points that must be considered in

the combination of LC×LC with MS:

1. Because the second-dimension

eluent must be compatible with

MS some LC×LC combinations are

not suitable with MS detection;

for example, ion-exchange

chromatography is not the best

option for the second dimension

separation.

2. As the second-dimension

separation in LC×LC is typically

Figure 4

Figure 4: Continuous and valve-switching concepts in LCxLC.


very rapid and the peak widths

can be only a few seconds, the MS

instrument must be capable of rapid

detection. The minimum scanning

rate of MS is about 5 Hz. Commercial

TOF instruments are available with

scanning rates as high as 200 scans/s,

and are thus a good choice for LCxLC.

Commercial single quadrupole

instruments with high scanning rates

have also recently become available

and these can be used in LCxLC.

However, triple quadrupole and ion-

trap type analysers are currently too

slow for most LCxLC applications.

3. The second dimension separation

should be preferably less than ca.

120 s and this can be achieved by

using high flow rates in the second

dimension separation, up to several

mL/min. As the performances of

many MS instruments suffer from

high flow rates and the typically

recommended flow rates are clearly

lower (up to 1 mL/min), it is often

necessary to use flow-splitting.

Several LC×LC-MS combinations

have been developed, utilizing

atmospheric pressure chemical

ionization (APCI) or electrospray

ionization(ESI)sourcesasinterfaces.

Of-line combination via MALDI

has also been applied. TOF and

quadrupole mass spectrometers

have been utilized in on-line

combinations. TOF-MS is a good

choice as the detector for LC×LC,

because it has a very high scanning

rate and offers high resolution,

which allows precise empirical

formula assignments for unknown

compounds. In combination with the

two independent retention times

obtained from the LC×LC separation,

it provides additional assurance for

positive identification in quantitative

work.

Quantitative analysisQuantitation with LC×LC requires

more complex data-handling

procedures than single column LC.

In LC×LC a single compound eluting

from the first column is divided

into several fractions. Typically 1-4

fractions are collected from each

peak eluting from the first dimension

column. In the quantification,

summed peak areas or volumes

can be used in, in a manner similar

to that of the quantitative GC×GC

analysis. Determination of the peak

area for a target compound in LC×LC

requires summing up the areas of

the individual fractions. This can

be done manually, but automatic

procedures are also available. The

volume calculation is performed

with special software and both

methods give good results.

Tuulia Hyötyläinen is professor

of environmental chemistry and

Figure 5

Figure 5: Data treatment, generation and visualization.

1D chromatogramat first column

outlet

Modulation A large series of high-speed second-dimension chromatograms is collected.

Raw 2D chromatogram

at second column outletTransformation

Second-dimension chromatograms side by side. One dimension represents the retention on the first column and the other the retention time on the second column.

VisualizationPeaks displayed by means of:

Contour plot 2D plot In which similar signal intensities are connected by means of ‘contour’ lines.

Colour 2D plotIn which colour (or shading) indicates signal intensitiy.

1

1st dimention 2nd d

imen

tion

3D plot.Signal intensities shown by vertical axis.

2

3

12

12

3

3


environmental analytical chemistry at the University of Helsinki.

Her main research fi eld is the development of multidimensional

chromatographic methods and on-line sample pretreatment techniques.

Her main applications areas are environmental and food samples.

Figure 6

Figure 6: (a) Symmetrical confi guration – the fl ow direction is the same for both loops. (b) Asymmetrical confi guration in which each loop is fl ushed in a diff erent direction into the second dimension. L1 = loop 1; L2 = loop 2; LC = from the fi rst dimension pump; P = from the second dimension pump; SEC = to the second-dimension column. The symmetrical confi guration allows fl ushing of the fractions to the second dimensions in the same direction in which they were loaded, thus making both positions identical.

(a)

(b)

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Doctor

How to understand and deal with overloading in GC

Overloading in gas-liquid vs gas-solid chromatographyIn gas-liquid chromatography (GLC), in which

we use polysiloxane (liquid)-type stationary

phases an overloaded peak will show itself

with a slow-raise and a sharp end. This is also

known as fronting.

In gas-solid chromatography (GSC),

in which the stationary phases used are

adsorbents such as alumina, molecular

sieves and porous polymers, an overloaded

component will show a fast rise but slow tail.

This is also known as tailing (Figure 1).

To deal with these phenomena, it is vital to

know whether we are dealing with GLC or

GSC.

Gas-liquid chromatographyIdeally, a component that is separated in a

capillary should elute with a symmetrical,

Gaussian peak shape. However, this will

only occur if with sufficient loadability. The

maximum amount that can be injected onto

a particular capillary column depends mainly

on:

When operating capillary columns, we must run at optimal conditions of linear gas velocity to maximize

separation power. However, when the amount injected into the column is too high, peaks become non-

symmetrical – this effect is referred to as overloading. An overloaded peak is generally not a problem for

quantification, but when it starts to affect the separation of a neighbouring component, corrective action is

required. In addition, overloading can change the retention time of the component. As the stationary phase

is saturated, the component itself will also act as a stationary phase resulting in ‘strange’ chromatograms.

Overloading mainly occurs in stationary phases for which there are several parameters for manipulation. For

appropriate corrective action, it is important is to understand the type of stationary phase being used; that is, a solid

or a liquid.

Figure 1

b

(s

g l g s

Figure 1: Peak shape of overloaded peak in gas-solid and gas-liquid chromatography.

1. The polarity of the stationary phase:

Polar components will dissolve better in a

polar stationary phase than in a non-polar

stationary phase. Figure 2 shows the elution

of an acidic component, 2-ethylhexanoic

acid from two different columns. On the

non-polar column (a) the peak is strongly

overloaded, while on the polar column (b),

the peak is high and symmetrical. Also on

this phase, the acid peak elutes after the

methyl dodecanoate peak.

38 chrom doctor www.sepscience.com

2. The amount of stationary phase present

within the column: The main parameter here

is film thickness. Thicker films allow more

sample to be while keeping symmetrical

peaks. Also, if we use wider diameter,

longer columns, we will benefit from

higher loadability. However, be aware that

with increased film thickness and column

length, retention times and column bleed

will increase proportionally. Figure 3 shows

the impact of increased film thickness on

loadability. For comparison reasons, the test

mixture on the thicker film was analysed at a

higher temperature for which the retention

factors are similar.

3. The retention (factor) of the component

on that particular column: Components

with a higher retention will often show

quicker overloading phenomena than early

eluting components. Note that we can

influence retention factor with the oven

temperature. If a late-eluting peak shows

sign of overloading, run the analysis at a

higher temperature and/or increase the

temperature program rate.

Gas-solid chromatographyIn gas-solid chromatography there is

generally little flexibility in the amount

of stationary phase in porous layer open

tubular (PLOT) columns – the layers are

already very thick to generate maximum

retention for volatiles. Overloading in gas-

solid chromatography is visualized by a

strong tailing of the component. As the

capacity of adsorbents is usually lower than

liquids, the overloading phenomena is

observed much faster.

When peaks start to tail using a PLOT

column, try to inject less sample. Figure 4(a)

shows hydrocarbon overload on a PLOT

column, in which the polar hydrocarbons,

methyl-acetylene and 1,3 butadiene, tail

significantly. The overloaded hydrocarbons

also elute earlier, which can result in a false

identification. Injecting less sample, as

shown in Figure 4(b), improves the peak

shape considerably.

If injection of less sample is not possible,

because of GC setup restrictions (for

example, when a fixed sample loop is used

with direct injection), or for detecting a trace

analyte, consideration should be given to a

0.53 mm type PLOT column, perhaps even a

longer one.

Another approach is running at a higher

temperature as this will improve peak shape

significantly using PLOT columns. Figure 5

Figure 2

(a) (b)

Time (min) Time (min)

Figure 2: Effect of solubility on loadability. Columns: (a) Rtx-1, (b) Stabilwax-DA; dimensions = both 30 m x 0.53 mm with 0.5 μm film.

Figure 3

Time (min) Time (min)

Figure 3: Effect of liquid stationary phase film thickness on loadability. Column: Rtx-1, 30 m x 0.25 mm i.d.

39chrom doctorseparation science — volume 1 issue 4

shows the separation of C1-C5 hydrocarbons

using Al2O3, but operated at different

temperatures. The peak shapes for both

1,3-butadiene and methyl acetylene improve

at higher temperatures. It should also be

noted that changing temperature will initiate

another effect – the alumina will become

less ‘polar.’ The result being that unsaturated

(polar) hydrocarbons will elute faster relative

to saturated (non-polar) hydrocarbons. This is

why, in the example, both peaks elute closer

to the pentane peaks.

In practiceTo solve an overloading issue that impacts

on quantification a new column with more

capacity or solubility can always be applied.

Initially, it is preferable to overcome the

challenge using the existing column by

reducing the absolute amount of sample

compound injected on to the column. This

can be done via:

• Injectingless,smallersamplevolume,

higher split ratio (Figure 6).

• Dilutingofthesamplepriortoinjection.

By doing this a higher sensitivity setting of

the detector must be used. However, if this

does not solve the problem, a column with

more capacity/solubility is required.

AcknowledgementsSpecialthankstoBillBromps,RestekR&D

Figure 5

1 2 3 4 5 6 7 80

50

100

150

0

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.00

25

50

75

100

125

Figure 5: Impact of temperature on peak shape of 1,3-butadiene and methyl acetylene. Column: 30 m x 0.53 mm Rt-Alumina BOND/Na2SO4. Peaks: 1 = 1,3-butadiene, 2 = methyl acetylene.

Figure 4

Time (min)

(a)

(b)

Figure 4: Impact of absolute sample load on solid stationary phase/PLOT column. Column: 30 m x 0.32 mm Rt-Alumina BOND/KCl: (a) overloaded injecting 50 ng per component, (b) injecting 5 ng/component. Peaks: 1 = pentane; 2 = methylacetyene; 3 = pentane; 4 = 1,3-butadiene.

40 chrom doctor www.sepscience.com

and Tom Vezza, Restek PLOT specialist for supplying

chromatograms.

Jaap de Zeeuw is a specialist in gas chromatography

working for Restek Corp.

Figure 6

20 ng/component on the column

10 ng/component on the column

Split ratio was increased bya factor of 2

Figure 6: Example of practically improving the separation by decreasing the injected amount on the column by a factor 2. Column: 30 m x 0.25 mm Rxi-5Sil MS, fi lm = 0.25 μm.

separation science — volume 1 issue 4 41chrom doctor

Symposium Chair: Brett Paull, Dublin City University, Dublin, IRL

Symposium Co-Chair: Miroslav Macka, Dublin City University, Dublin, IRL

Local Organizing Committee: Damian Connolly, Dublin City University, Dublin, IRLJeremy Glennon, University College Cork, Cork, IRLKieran O’Dwyer, Dublin City University, Dublin, IRL

21 - 24 September 2009Grand Hotel Malahide

Dublin, Ireland

21st international

ion chromatography symposium

Baily lighthouse near Dublin

For More InformationFor program updates, abstract submission, information on exhibiting and/or sponsoring, hotel information and on-line registration, please visit the Symposium Web site frequently at www.casss.org. For additional information, please contact the CASSS offices in the USA or Germany as follows:

AN INTERNATIONAL SEPARATION SCIENCE SOCIETY

CASSS

AnApplication

notes

42 application notes www.sepscience.com

Running fast LC within USP Limits

Company: Agilent Technologies

Summary: Recent revisions in United States Pharmacopeia (USP)

general chapter <621> allow for adjustments to be made in

monographs to enhance the quality of the chromatogram in

meeting system suitability requirements. These adjustments can

be made use of to produce a fast method utilizing the Agilent

1200 Series Rapid Resolution LC (RRLC) system and Agilent

Method Translator software. There are various ways to produce a fast method and one of

them is to start with an established method such as a USP method.

This Application Note shows how to start from a USP method and incorporate the

allowed adjustments to produce a fast method that meets system suitability requirements.

Comprehensive GCxGC(qMS) of PCBs using the rapid scanning GCMS-QP2010 Plus in EI and NCI mode

Company: Shimadzu

Summary: Comprehensive GCxGC is a technique which

o� ers high resolving power in complex separations. In

this technique a cryo modulator is placed in between two

analytical columns. In this application an RTX-1 30 m, 0.25

mm, 0.25 μm column was mounted in the � rst dimension

connected to a BPX50 1 m, 0.15 mm, 0.15 μm in the second.

The cryo modulation is performed by a hot and cold nitrogen

gas jet.

Running fast LC within USP limitsCase study with USP pravastatin sodium chromato-

graphic purity method using the Agilent 1200 Series

Rapid Resolution LC system

Abstract

Recent revisions in United States Pharmacopeia (USP) general chapter <621> allow

for adjustments to be made in monographs to enhance the quality of the chro-

matogram in meeting system suitability requirements. These adjustments can be

made use of to produce a fast method utilizing the Agilent 1200 Series Rapid

Resolution LC (RRLC) system and Agilent Method Translator software. There are vari-

ous ways to produce a fast method and one of them is to start with an established

method such as a USP method.

This Application Note shows how to start from a USP method and incorporate the

allowed adjustments to produce a fast method that meets system suitability require-

ments. The chromatographic purity test for pravastatin as per the USP method sug-

gests a 30-minute gradient with a 3.5 µm particle size column and 1 mL/min flow rate.

These chromatographic conditions can be adjusted to use a 1.8 µm particle and

1.5 mL/min flow rate, which will provide faster run times. Besides particle size and

flow rate, column dimensions and column temperature can also be adjusted to pro-

duce a faster method. The transition to the fast method was quickly and effectively

achieved by the use of Agilent Method Translator. Such fast methods derived from the

USP can be used in high-throughout environments as the new method is closest to

the validated monograph method.

Author

Syed Lateef

Agilent Technologies

Bangalore, India

Application Note

Manufacturing QA/QC

Application Note

Comprehensive GCxGC(qMS) of PCBs using the rapid scanning GCMS-QP2010 Plus in EIand NCI mode

Comprehensive GCxGC is a technique which offers high resolving power in complex separations. In this technique a cryo modulator (ZOEX, USA1) is placed in between 2 analytical columns. In this application a RTX-1 30 m, 0.25 mm, 0.25 µm column was mounted in the first dimension connected to a BPX50 1 m, 0.15 mm, 0.15 µm in the second. The cryo modulation is done by a hot and cold nitrogen gas jet. The hot jet is switched on typically every 4 sec for a duration of 375 msec. This results in freezing and remobilising the analytes in between the columns. Typically 3 to 4 Fractions of a first dimensional peak are so “injected” into the second column and show very narrow peak widths of about 250 msec. Figure 1 shows the loop modulator setup. A part of the second column is used as a loop and it is winded so that 2 cold and 2 hot spots are realized (2 stage modulator).

SCA_280_071 www.shimadzu.eu

Fig. 1: Loop modulator in freezing (left) and remobilising (right) mode

Negative chemical Ionization has become very popular in the recent years for the analysis of electrophilic analytes like PCBs. This ionisation mode show high selectivity for PCBs against matrix and also a higher sensitivity than EI for those compounds. In NCI/GCMS using CH4 as reagent gas 2 reaction channels with PCBs are observed: Resonance electron capture and dissociation in combination with electron capture. Figure 2 shows a modulated chromatogram (NCI GCMS) recorded with a PCB standard (25 pg each). PCB congeners below hexa show mainly a dominant 35 fragment (Cl) while congeners with higher chlorine content show dominant electron capture processes.

Fig. 2: TIC data (34-500 amu) obtained in an GCxGC(qMS) experiment in NCI mode

43application not es separation science — volume 1 issue 4

Simultaneous determination of melamine and cyanuric acid using LC-MS with the Acclaim Mixed-Mode WAX-1 column and mass spectrometric detection

Company: Dionex

Summary: Current methods for quantitative determination

of melamine and cyanuric acid include gas chromatography

mass spectrometry (GC-MS) and liquid chromatography

mass spectrometry (LC-MS). GC-MS requires derivatization

which is labour intensive and the reported LC-MS methods

generally involve a long gradient chromatographic run as

well as column clean up.

This presentation introduces a sensitive, simple, and high-throughput method for

simultaneous determination of melamine and cyanuric acid by LC-MS and uses stable

isotope labeled internal standard (ISTD) for quanti� cation.

HPLC Analysis of lysozyme in di� erent types of wine

Company: Tosoh Bioscience

Summary: Hen’s egg white lysozyme is used in winemaking

to eliminate lactic acid bacteria and to control malolactic

fermentation. As eggs and derivatives are regarded as

potential allergenics the European Commission issued a

directive that wine labels must include information about

egg derived ingredients like lysozyme. As a consequence

the International Organisation for Wine and Vine (OIV) published a standard

method for measurement of lysozyme in wine by HPLC, which is based on a method

developed at the University of Bologna.

This HPLC-FLD method is based on the separation of the components on a polymer

based TSK-GEL Phenyl-5PW reversed phase column with 1000 Å pore size, which

is especially suited for the separation of proteins. Fluorometric detection of the

lysozyme resulted in an increased sensitivity compared to UV based HPLC methods. It

allows the quanti� cation of lysozyme independently of the enzyme activity.

‘A bottle of wine contains more philosophy than allthe books in the world’. It was Louis Pasteur (1822-95) the famous French chemist and microbiologist,who stated this. Wine was one of his early objectsof study and he solved the mystery of fermentationwhen he identified and isolated the specific microorganisms responsible for normal and abnormalfermentations in winemaking. His work remains tobe the basis of today’s winemaking technology.

THE ROLE OF EGG LYSOZYME IN WINEMAKING

Lysozyme (E.C. 3.2.1.17) isolated from egg whiteshas a long tradition as an antimicrobial agent usedin food industry1. It is an enzyme with muramidaseactivity which degrades the cell wall of gram-posi-tive bacteria such as Oenococcus, Pediococcus, andLactobacillus. In the past it was used mainly as pre-servative in cheese making, to prevent spoilage bymicro organisms. Although micro organisms aremostly regarded as food spoilage some are essen-tial for fermentation processes. During winemakingthe primary fermentation process converts thegrape sugar to alcohol by yeast. The so called mal-olactic fermentation occurs shortly after the end ofthe primary fermentation. Lactic acid bacteria con-vert L-malic acid, to, L-lactic acid in such a way act-ing as biological deacidifiers. In red wines, malolac-tic fermentation could hence results in a more bal-anced wine, while for white wines, where the acidicnotes should be preserved, its intervention is oftento be avoided. A tight control of this process is how-ever necessary because the onset of malolactic fer-mentation in the bottle is undesirable as theprocess could proceed to further metabolize otheracids (citric and tartaric acids), thus increasingacetic acid amounts. Furthermore the wine willappear to the consumer to still be fermenting andthe wine may also lose its flavour integrity and takeon an unpleasant lactic aroma.

CONTROL OF MALOLACTIC FERMENTATION

In the past cooling and the anti-microbial agent sul-phur dioxide were used to inhibit the growth of lac-tic acid bacteria. Sulphur dioxide can provoke aller-gic reactions like headache in susceptible individu-als. In Europe wines which contain more than 10ppm of sulphur have to be labelled accordingly. Aslysozyme can lyse the cell wall of wine lactic acidbacteria, it provides a practical means for delayingor preventing the growth of Oenococcus oeni andconsequently the onset of malolactic fermentation2.To control the malolactic fermentation during vinifi-cation and subsequent bottling the addition of up to500 mg lysozyme per liter is permitted since 2001by OIV. Lysozyme cannot replace sulphur dioxidecompletely because it is lacking the anti-oxidativeeffect of sulphur dioxide. It can, however greatlyreduce the amount of sulphur dioxide needed toachieve microbial stability over the life of both redand white wines.

HPLC Analysis of lysozyme in different types of wine

ANALYSIS

Claudio Riponi1), Fabio Chinnici1) and Regina Roemling2),University of Bologna, Food Science Department, Bologna, ItalyTosoh Bioscience GmbH, Stuttgart, Germany

� � � + $ � � � � � ( � � % � � � ) $ * ) � � � � $ � & $ � � � � � ( � � � � � � � � � % � � � � � � � � % � � � � � % � � � � � � � � � � � % � # � � � � � � � % � � � % # � � � � � � � � % � �� # � � � % � % � � � � � $ � � � � $ � � � � � � � # � ' � % � ' � $ � � # � � # � � � # � � � � � $ � ! % � � % � � � � � � � � # � � � � � $ % � � � & # ! � � � � � � � $ $ � � � $ $ & � �� # � � % � ' � � % � � % � ( � � � � � � � � � $ � � & $ % � � � � � & � � � � � � # � � % � � � � � & % � � � � � � � # � ' � � � � � � # � � � � � % $ � � � � � � � ) $ * ) � � � � � $ � � � � � �$ � " & � � � � � % � � � � � % � # � � % � � � � � � # � � � � $ � % � � � � # � � � � � � � � � � � � � � � � � � � � � ! & � � � $ � � � � � � $ % � � � � # � � � � % � � � � # � � � � $ & # � �� % � � � � ) $ * ) � � � � � � ( � � � � � ) � � � � ( � � � � � � $ � � � $ � � � � � � � � � % � � � � � ' � � ! � � � � % � % � � � � � � ' � # $ � % ) � � � � � � � � �

Figure 1

Chromatogram of a model solution (water and tartar-ic acid 2g/l) spiked with pure lysozyme (final concen-tration 150 mg/l). A: UV @ 280nm; B: Fluorescence@ 276ex/345em

Application Note

pplication note WineAppl.qxd:Flyer_Vorlage 26.06.2008 14:24 Uhr Seite 1

Fatty acid methyl esters in B100 biodiesel by gas chromatography

Company: PerkinElmer

Summary: The production and consumption of biofuels

continues to increase as more attention is paid to the

environment and the depletion of fossil-fuel resources.

Biodiesel is a substitute for petroleum-diesel fuel. The quality

criteria for the production of biodiesel are speci� ed in EN

14214.

Within EN 14214, method EN 14103 speci� es the fatty acid methyl ester and

linolenic acid methyl ester content, which is used to pro� le the vegetable or animal oil

feedstock used in biodiesel production.

This application note discusses the analysis according to method EN 14103. In

addition to the methodology speci� ed in EN 14103, a simpler and more accurate

method will be presented.

Reliable analysis of glycerin in biodiesel using a high-temperature mon-metal GC column

Company: Phenomenex

Summary: Arguably the most critical test for biodiesel is the

measure of glycerin content. Glycerin is the major byproduct

of the biodiesel production process, called transesteri� cation,

where oils and fats are reacted with an alcohol to produce

fatty acid methyl esters (FAMEs). High glycerin content can

lead to a number of fuel problems, such as clogged fuel � lters

and fuel pressure drops, and its presence must be minimized. ASTM D 6584 outlines

testing methods measuring total amount

glycerol in a biodiesel.

This article compares the lifetime and stability of the Zebron Inferno column with

the leading fused-silica columns and presents analysis results on the Zebron column

using ASTM Method D 6584.

Figure 1. Linolenic acid.

AP

PL

IC

AT

IO

N

NO

TE

GA

S C

HR

OM

AT

OG

RA

PH

Y

Fatty Acid Methyl Esters in B100 Biodiesel by Gas Chromatography (Modified EN 14103)

Introduction

The production and consumption of biofuels continues to increase as more attention is paid to the environment and the depletion of fossil-fuel resources. Biodiesel, a fuel from natural oils such as soybean oil, rapeseed oil or animal fats, is a substitute for petroleum-diesel fuel. The quality criteria for the production of biodiesel are specified in EN 14214.

Within EN 14214, method EN 14103 specifies the fatty acid methyl ester (FAME) and linolenic acid methyl ester content (Figure 1), which is used to profile the vegetable or animal oil feedstock used in biodiesel production. EN 14103 calls for calibration of all FAME components by relative response to a single compound, methyl heptadecanoate. This requires the measurement of accurate weights for each sample and the addition of an internal standard. The range of FAMEs for which the method is intended lies between C14:0 and C24:1.

This application note will discuss the analysis according to method EN 14103. In addition to the methodology specified in EN 14103, a simpler and more accurate method will be presented. The modified method uses commercially-available calibration and test mixtures for precise peak identification and quantitative accuracy, while streamlining the sample preparation and calculations. Reporting is based on area % of all components after the solvent – as a result, the sample weight does not impact the calculations.

Authors

Timothy Ruppel

Timon Huybrighs

PerkinElmer, Inc.

710 Bridgeport Avenue

Shelton, CT USA

w w w. p e r k i n e l m e r. c o m

www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department by telephone, fax or email: [email protected].

[email protected]

tel.:fax:

email:

[email protected]

Canada(800) 543-3681(310) [email protected]

Ireland01 247 5405+44 [email protected]

Italy051 6327511051 [email protected]

New [email protected]

Denmark4824 80484810 [email protected]

France01 30 09 21 1001 30 09 21 [email protected]

[email protected]

Puerto Rico(800) 541-HPLC(310) [email protected]

United [email protected]

USA(310) 212-0555(310) [email protected]

tel.:fax:

email:

Zebron™ ZB-5HT Inferno™

Agilent® J&W® DB-5ht

Longer Lifetime!

Ret

entio

n Ti

me

(min

)

0 20 40 60 80 hours

Hours at 400 ˚C

8.0

7.5

7.0

Reliable Analysis of Glycerin in Biodiesel Using a High-Temperature Non-metal GC ColumnNgoc Nguyen, Kory Kelly, and Sky CountrymanPhenomenex Inc., Torrance, CA, USA

IntroductionIn the past decade, biodiesel has emerged as a leading alternative fuel source because it is easily derived from common feedstocks and can be used in unmodified diesel engines. The relative ease of biodiesel production can mask the importance of maintaining high quality diesel fuel standards. To support the growth of the biodiesel industry, the United States’ American Society for Testing and Materials (ASTM) and the European Deutsches Institut fur Normung (DIN) recently outlined physical and chemical tests and specified the minimum quality standard for biodiesel fuel used in modern diesel engines.1

Arguably the most critical test for biodiesel is the measure of glycerin content. Glycerin is the major byproduct of the biodiesel production process, called transesterification, where oils and fats are reacted with an alcohol to produce fatty acid methyl esters (FAMEs).1 High glycerin content can lead to a number of fuel problems, such as clogged fuel filters and fuel pressure drops, and its presence must be minimized.

ASTM D 6584 outlines testing methods measuring total amount glycerol in a biodiesel.2 Although GC is the standard analysis technique for this method, it has several inherent challenges. These tests run at very high temperatures and standard fused silica columns are not engineered to withstand temperatures above 380 °C. In fact, at temperatures above 380 °C, the polyimide coating of most fused silica columns starts to degrade, eventually becoming brittle and inflexible.

The alternative, metal columns, present other challenges. While metal columns can withstand higher oven temperatures, they are inflexible, difficult to use, and require special tubing cutters. In addition, they often develop leaks due to the expansion and contraction that occurs during oven heating cycles and are highly active to acids and bases. Thus, using metal columns might compromise the accuracy of the biodiesel analysis.

Phenomenex, Inc., has recently developed unique fused silica columns designed specifically for high-temperature analysis. These columns, called the Zebron™ ZB-1HT and ZB-5HT Inferno™, are specially processed to be thermally stable up to 430 °C. As a result, their stationary phases and a polyimide coating are more rugged and can withstand higher temperatures than other conventional columns. This article compares the lifetime and stability of the Zebron Inferno column with the leading fused-silica columns and presents analysis results on the Zebron column using ASTM Method D 6584.

GC TN-2030

MethodsLifetime ComparisonFor the high temperature lifetime comparison, three columns were compared: Agilent’s (J&W) DB-5ht, Varian’s VF-5ht, and Phenomenex’s Zebron ZB-5HT. The columns were held at 400 °C for 2 hours. After lowering the oven temperature to 120 °C, 1.0 μL of pentadecane was injected and its retention time was measured. This process was repeated 50 times, totaling 100 hours at 400 °C for each column tested.

Figure 1. Lifetime comparison of the DB-5ht and ZB-5HT Inferno. The lifetime and bleed profile comparisons were performed on new, unused DB-5ht and ZB-5HT GC columns. Careful measures were taken to ensure that all conditions were similar for both columns.

Bleed ProfileBleed (pA) was measured using a flame ionization detector (FID) as the GC oven program increased from 120 °C to 400 °C. The GC oven was held at 120 °C for 3 minutes then increased to 320 °C at 30 °C/minute. A null injection was made at 250 °C.

Biodiesel AnalysisCalibration standards, sample preparation, and GC analysis were performed as per ASTM Method D 6584 (Reference ASTM Method). In brief, the samples were derivatized with N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA).

44 application notes www.sepscience.com

TuTechnology

update

46 technology update www.sepscience.com

Key

Email the company

Product information

Applications

Additional information

Phase Optimized Liquid Chromatography (POPLC)

Manufacturer: Bischoff

Manufacturer’s description: The most important tool in HPLC method development is

stationary phase selectivity. The number of commercially available RP phases shows the

importance of the stationary phase. These are approximately 750 diff erent stationary phases

today, and every year new packing materials are introduced. The user now has the task to

select the right column for their application from this large number of HPLC phases. This

can be very diffi cult. Very often it involves a costly and time-consuming optimization of the

mobile phase.

The optimization of a separation becomes much easier once the optimal stationary phase

has been found. Bischoff states that ‘Phase Optimized Liquid Chromatography’ (POPLC) uses

a completely new approach in method development and optimization. After a rough fi rst

choice of mobile phase, only the stationary phase needs to be optimized.

POPLC is based theoretically on the ‘PRISMA Model’, which has been used before to

optimize mobile phases in LC [Szabolcs Nyiredy, K. Dallenbach-Tölke and O. Sticher, Journal of

Planar Chromatography, 1, (1988), 1241 – Figure 4].

First retention times are determined with isocratic chromatographic runs of the analytes

on diff erent stationary phases using the same mobile phase which is chosen by experience

or trial. The stationary phases used for these basic measurements should be of strongly

diff erent selectivity. For example C18, C18 with enhanced polar selectivity and Phenyl, C30

or Cyanopropyl could be used for this purpose. The retention of the analytes on any of these

phases is diff erent, because of diff erent mechanisms of interaction. The individual retention

times are then used for calculations that are performed by optimization software. The

software calculates the combination of column segments.

47technology u pdate separation science — volume 1 issue 4


www.sepscience.com

Clarity Chromatography Station

Manufacturer: DataApex

Manufacturer’s description: Clarity is an advanced chromatography

data station (CDS) with optional software modules for data

acquisition, data processing and instrument control. Its wide range of

data acquisition interfaces (A/D converters, LAN, USB, RS232) allows

connection to virtually any chromatograph, claims the company.

Up to four independent chromatography systems, each of which can

be equipped with up to 12 detectors (signals), can be simultaneously

connected. Clarity can be used with any GC or HPLC. Together with

optional control modules and extensions it provides laboratories with

complete chromatography data handling capabilities. The control modules provide integrated control of selected

instruments, extensions provide functions for specifi c separation techniques, such as PDA or GPC analysis.

Clarity comes with extensive free support from DataApex as well as from the growing community of users in the

Clarity Conference.

48 technology update www.sepscience.com

ISOLUTE SLE+ Supported Liquid Extraction Plates

Manufacturer: Biotage

Manufacturer’s description: ISOLUTE SLE+

Supported Liquid Extraction plates offer an

efficient alternative to traditional liquid-liquid

extraction (LLE) for bioanalytical sample

preparation, extracting up to 400 µL aqueous

sample. ISOLUTE SLE+ plates provide high analyte recoveries, eliminate emulsion formation, and cut sample

preparation time in half, claims the company.

Reported features include:

• Efficientextraction:Supported-liquidextractionmechanismisveryefficient,deliveringhigheranalyterecoveries

and cleaner extracts than the equivalent LLE method.

• Noemulsionformation:Emulsionscannotformbecausethesampleandwaterimmiscibleextractionsolventare

never in direct contact, preventing contamination and maximizing analyte recovery.

• Easytoautomate:ISOLUTESLE+platesprovideaneasy-to-automatealternativetotraditionalliquid-liquid

extraction. No manual ‘off-line’ steps (capping/mixing/centrifuge/de-capping) required. All procedural steps can be

fully automated with no manual intervention necessary.

• Goodflowcharacteristics:TheISOLUTESLE+plateispackedwithanoptimizedgradeofdiatomaceousearth,

providingreproducibleflowcharacteristicsfromwell-to-well.Aqueoussamplesandextractionsolventsloadevenly

across the plate, an important feature for automated sample preparation procedures, where well blockage can lead

to loss of valuable samples.

• Transferablemethods:ThewaterimmiscibleextractionsolventsusedinLLEcanalsobeusedforISOLUTESLE+

procedures. Sample pretreatment conditions are also the same, meaning existing LLE methods are easily

transferable to ISOLUTE SLE+, reducing method development time.

ISOLUTE SLE+ is available in the industry standard 2 mL fixed well ‘SPE’ plate format and is compatible with all

commercially available automated liquid handling systems. Two ISOLUTE SLE+ plate sizes are available. The 200 mg

plate allows a total aqueous sample load of up to 200 µL; the 400 mg plate allows aqueous sample load of

200–400 µL.

49technology update separation science — volume 1 issue 4

Sep-Pak Cartridges

Manufacturer: Waters

Manufacturer’s description: Waters claims

that its Certified Sep-Pak SPE cartridges

are quality tested to the lowest level of

extractables in the industry. Manufactured

using strict performance and cleanliness

specifications and QC-tested for extractables

and leachables, Certified Sep-Pak sample

preparation products reduce interference

and increase sensitivity by eliminating

contaminants introduced by the cartridge

hardware and sorbents.

Ideally suited for low-level GC and LC

analysis, Certified Sep-Pak SPE cartridges

are chromatographically tested for

cleanliness and performance to provide

superior extracts for residue analysis in

environmental, food, chemical and biological

samples, it is claimed.

Certified Sep-Pak cartridges are available in

the following chemistries:

• C18

• Silica

• Florisil™

• AluminaA,B,N

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Asia Paci�c

Swafer Micro-Channel Wafer Technology

Manufacturer: PerkinElmer

Manufacturer’s description: PerkinElmer’sSwafermicro-channelflowtechnologyisaninnovativeanduser-friendly

approachforflow-switchingapplications.Itdelivershardwareandapplicationflexibility,expandingthecapabilitiesof

capillary gas chromatography.

This technology can enhance most analytical laboratories’ productivity, claims the company – in specific

configurations and high-throughput environments, the Swafer offers a fast return on investment. From simple

techniques such as connecting two detectors to one column, and removing unwanted material from a column, to

sophisticated multidimensional separations on complex samples, its capabilities cover a wide range of applications,

including the detection of pesticides in food products and the analysis of complex matrices (e.g., petroleum or natural

products). With tools, such as detector switching or heartcutting capabilities, the Swafer delivers good separations of

complex samples, yielding additional, more reliable data.

The Swafer platform includes 13 user-interchangeable configurations which deliver over 15 possible modes of

operationforunparalleledapplicationflexibility.Theseconfigurationsareavailablethroughthefollowingoptions:

• D-Swafer:basedontheclassicalDeans’Switchprinciple

• S-Swafer:ascalablesplittingdevicedesignedforsample-streamsplittingbetweenarangeofdetectorsorcolumns

Both Swafers can be configured in multiple ways, offering a variety of additional capabilities.

Swafers are inert and easily installed on any existing or new PerkinElmer Clarus 500 or 600 GC with programmable

pneumatics. In addition, Swafers are very easy and quick to configure, exchange and maintain, not requiring service

intervention.

50 technology update

Symbiosis Pharma

Manufacturer: Spark Holland

Manufacturer’s description: Symbiosis Pharma is Spark Holland’s

solution for integrated online SPE-LC-MS automation (XLC-MS). The

systemofferslargeflexibilityinprocessingdifferenttypesofsamples

selecting one of the three fully automated operational modes LC-

MS (direct LC without SPE); XLC-MS (online SPE coupled to LC-MS);

AMD (advanced method development). The combination of a highly

selective online SPE and a leading top line autosampler makes this

dedicated high-throughput Pharma system the best tool for direct

injection of raw biological samples, the company claims. The zero

sample loss injection mode offers the best possible results even

when there is little sample volume available (5 µL injection out of a

8 µL sample).

All Symbiosis online SPE Systems process multiple batches/assays

fast and reliably, with high analytical performance, fully unattended.

They also have all the tools to perform method development using a

systematic approach which generates a working online SPE method

in just a few days.

51technology u pdate separation science — volume 1 issue 4

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