journal separation science

Volume 1 / Issue 2

February 2009www.sepscience.com

Chromatography in biomarker

UHPLC-MS/MS analysis of pesticides

Chromatography in biomarker analysis

Micro� uidic chip capillary electrophoresis for biomedical analysis

2 section name www.sepscience.com

Parker Balston is the leading provider of Analytical Gas Systems for the LC/MS instrument market. Generators are specifically designed to meet the stringent gas requirements for all the leading LC/MS instrument manufacturers including Agilent, Thermo Scientific, Waters, Applied Biosystems, Bruker, Varian and Shimadzu.

Utilising Parker’s proprietary hollow fibre membrane technology, there are more than 10,000 systems installed worldwide. Membrane technology offers some unique performance benefits for LC/MS including phthalate free nitrogen, silent operation, no moving parts and no electrical requirements. It is technology you can trust.

www.parker.com/lcmsTel: 00800 27 27 53 74

[email protected]

Click here

to find out m

ore in

formatio

n

on our range of g

as genera

tors

for LC/M

S

Together, we can reduce nitrogen costs for LC/MS. Pure and simple

3contentsseparation science — volume 1 issue 1

contentsVolume 1 / Issue 2

February 2009www.sepscience.com

Chromatography in biomarker

UHPLC-MS/MS analysis of pesticides

Chromatography in biomarker analysis



Y. S. Fung and Z. Nie

20

feature

separationdriving analytical chemistry forwardsscience

Volume 1 / Issue 2February 2009

28

32

research round-up

Advance in hepatorenal tyrosinaemia biomarker analysis

Supercritical � uid extraction of a� atoxin B1 from soil

Single cell analysis in with UV laser-induced � uorescence detection

Methods for authenticating honey

A� nity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems

SPME sampling for the evaluation of volatile organic compounds emitted from building materials

Determination of toxic dyes in water using LC-vis/FLD and con� rmation by MS/MS

Study investigates the stability of mixtures used for patient-controlled analgesia

SPE-HPLC method for accurate and precise quanti� cation of HIV integrase inhibitors

Comparing sample treatment strategies for the analysis of thyreostatic drugs in thyroid samples

Hollow � bre liquid phase microextraction-UHPLC-MS/MS analysis of pesticides in alcoholic beverages

Determination of niacin in plasma using LLE and LC-MS/MS

2D TLC for chromatographic � ngerprinting of furanocoumarins

Rr

Cd

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

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

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

mechanical including by photocopying, recording or information storage and retrieval without

permission from the publisher, Eclipse Business Media Ltd.

Applications for the copyright owner’s permission to reproduce any part of this publication should

be forwarded in writing to Permissions Dept, Separation Science, Eclipse Business Media Ltd, 70

Hospital street, Nantwich, Cheshire, CW5 5RP, UK.

Separation Science does not verify any claims or other information appearing in any of the

advertisements contained in the publication, and cannot take any responsibility for any losses or

other damages incurred by readers in reliance on such content.

chrom doctor Exploiting particle size to reduce solvent consumption in analytical HPLC

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

Regulars

06

06

07

08

09

10

11

12

14

14

15

16

17

18

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

4 www.sepscience.com

scienti� c advisory

councilPeter Myers

– Chief Scienti� c O� cer

[email protected]

David Barrow

University of Cardi� , UK

Zongwei Cai

Hong Kong Baptist University

Yi Chen

Chinese Academy of Sciences,

Beijing, China

Gert Desmet

Vrije Universiteit Brussel, Belgium

C. Bor Fuh

National Chi Nan University, Taiwan

Y.S. Fung

Hong Kong University

Xindu Geng

Northwest University, Xi’an, China

Luigi Mondello

University of Messina, Italy

Paul Haddad

University of Tasmania, Australia

Hian Kee Lee

National University of Singapore,

Singapore

Melissa Hanna-Brown

P� zer, UK

Tuulia Hyötyläinen

University of Helsinki, Finland

Gongke Li

Sun Yat-Sen University, Guangzhou,

China

Yong-Chien Ling

National Tsing Hua University,

Taiwan

Klara Valko,

GSK, UK

Jean-Luc Veuthey

University of Geneva, Switzerland

Claudio Villani

Universita’ degli Studi di Roma “La

Sapienza”, Italy

Cheing- Tong Yan

Center of Environmental Safety and

Hygene, Taiwan

Edward Browne

GSK, Singapore

contactsDean Graimes

Publishing Director

+44 1270 629496

[email protected]

Stephanie Painter

Associate Publisher

+44 1634 855 296

[email protected]

Kevin McGeehan

Associate Publisher

+44 208 398 1750

Karen High� eld

Financial Controller

Bo Zhang

Technical Editor

David Hills

Scienti� c Director

+44 1270 629496

[email protected]

Marita Kritzinger

Assistant Editor

+44 151 494 0971

[email protected]

Professor Peter Myers

Chief Scienti� c O� cer

+44 151 601 2020

[email protected]

Will O’Keefe

Graphic Designer

[email protected]


Keep up to date with the latest analytical methods and chromatographic applications

in the Pharmaceutical , Environmental or Food science Industries by taking a FREE subscription

to the Separation Science Reports.

THE NEW SEPARATION SCIENCE REPORTS ARE NOW AVAILABLE...

October 2008

Enviro ReportOrganochloride analysis using graphitized carbon black

Automated Disposable Pipette Extraction of Pesticides from Fruits and Vegetables

WWEM 2008 Preview

Featured Books

iPod Touch

Recommend your colleague(s) for a free

subscription to Separation Science Reports and enter a

1of 5draw to win

November 2008

Food ReportMSPD-GC-ECD analysis of lindane residues

A novel sample treatment for the LC-MS detection of peanut protein in foods

Evaluating chondroitin sulfate in dietary supplements

Screening for mycotoxins with LC-MS/MS

Please turn the pages using the control panel below


subscription to Separation Science and enter a draw to win

1of 5

iPod Touch

Pharma ReportCharacterization of drug metabolites in epidemiological studies

Interview with Karsten Fjärstedt, GE Healthcare

SPME in high-throughput drug analysis

EDQM Symposium: Pharmaceutical Reference Standards

October 2008

Separation Science Singapore 2009 is a cross-discipline, cross-culture chromatography meeting aimed at scientists, engineers, business and technical experts from the diverse analytical instrumentation industries. Presentations will cover issues of vital importance to application chromatographers working in drug discovery and development, molecular diagnostics, food and agriculture, traditional Chinese medicine, forensics and security, process analytics and energy science industries.

Confirmed speakers include:

Professor Gert Desmet “Current and Future Approaches to Speed

Up HPLC Separations”

Professor Ping Li “HPLC and Hyphenated Techniques for

Analysis of Constituents in Herbal Medicines”

Professor Alastair Lewis “Trace Pollutant Detection in Challenging

Environments”

Milos Novotny, (Indiana University, USA), Philip Marriott, (RMIT, Australia), Edward Browne, (GSK R&D Singapore), Peter Myers, (University of

Liverpool, UK), Hian Kee Lee, (National University of Singapore), Yi Chen, (Chinese Academy of Sciences, Beijing, China), C. Bor Fuh, (National Chi Nan University,

Taiwan), Y.S. Fung, (Hong Kong University), Yizeng Liang, (Research Center for Modernization of TCM, Changsha, China), Tung-Hu Tsai, (National Yang-Ming

University, Taiwan), Yong-Chien Ling, (National Tsing Hua University, Taiwan), Paul Haddad, (University of Tasmania, Australia), Siu Kwan Sze, (Nanyang

Technical University, Singapore), Eric Chan, (National University of Singapore), Thomas Walczyk, (National University of Singapore), Manfred Raida, (Singapore).

Singapore

Professor Gert Desmet Professor Ping Li Professor Alastair Lewis

Submitted contributions are invited on all aspects of separation science pertinent to the theme, and specifically on the following topics:

Call for Papers

www.sepscience.com

Deadline for oral contributions: 15 MayDeadline for poster contributions: 12 June

Instructions for submissions and further information can be found at

proteomics, lipidomics, metabolomics, biomarker research, human/equine doping control, forensics

high-resolution separation methods for petrochemical, hydrocarbon and biodiesel applications

air, soil and water testing, portable field analysis, sample preparation protocols, industrial emissions

contaminants, traceability, ingredients testing, flavours and fragrances, marine toxins

high-throughput separations, chiral separations, ADME/PKPD studies, biopharmaceutical analysis

Pharma

Food

Energy

Enviro

Bioscience

TMC TCM chemistry, fingerprinting, metabolism, quality control

Passion. Power. Productivity.



Confirmed sponsors:

For all programme enquiriesemail David Hills.

For all delegate enquiriesemail Jackie Tan.

26–28 AugustBiopolis Science Park, Singapore

Key

Email the author

Article link

Product information

Comment

Saudi ArabiaHepatorenal tyrosinaemia is an inborn error

of metabolism, in which the body cannot

eff ectively break down the amino acid

tyrosine. Symptoms mainly include liver and

kidney disturbances. Other distinct inborn

errors of tyrosine metabolism include Type-II

and Type-III tyrosinemia. Succinylacetone is

an abnormal metabolite that is elevated in

individuals with hepatorenal tyrosinaemia,

and is the only specifi c marker for the serious

inherited metabolic disease. This disease

is treatable and serious complications will

be prevented if treatment starts before

symptoms appear. Current available

analytical methods for succinylacetone

are based on GC-MS and are lengthy and

tedious.

Dr Osama Y. Al-Dirbashi and research

team (National Laboratory for Newborn

Screening, King Faisal Specialist Hospital

& Research Centre in Riyadh, Saudi Arabia)

has developed an improved method

to determine succinylacetone in dried

blood spots for the diagnosis of type 1

tyrosinaemia using UPLC-MS/MS [Biomedical

Chromatography, 22 (11), 1181-1185 (2008)].

“We found that derivatizing

succinylacetone with dansylhydrazine

signifi cantly improves its ionization and

fragmentation pattern in MS/MS, and

would enable an analysis time of less than

one minute using real chromatographic

separation,” explained

RrResearchround-up

Al-Dirbashi. “ The method was sensitive

enough to detect succinylacetone in

newborn screening blood spots at

physiological and pathological levels with

excellent sensitivity and specifi city,” he

continued.

Looking forwards, Dr Al-Dirbashi

concluded, “This approach may pave the

way to include hepatorenal tyrosinaemia

in newborn screening programmes. It also

provides an excellent example of how

suitable derivatization of biological markers

can improve their determination.”

It is his hope that the approach may be

extended to other biological markers.

Advance in hepatorenal tyrosinaemia biomarker analysis

6 research round-up www.sepscience.com

Visítenos en Expoquimia 2008(20-24 octubre 2008, Barcelona)

• Normal and reversed phase silica basedcolumns

• Normal and reversed phase polymerbased columns

• Size exclusion columns (GPC and GFC)

• Ion chromatography and Ion Exclusioncolumns

• Columns for affinity chromatography

• Columns for quiral chromatography

Shodex®

Capturethethe EssenceEssence

Shodex Europe GmbH Tel.: +49 89 9399 6237

www.shodex.de

Check out our monthly offersCheck out our monthly offers

WE WANT YOUAS A CUSTOMER

www.shodex.de

click on our catalogue

huge application database online

Supercritical � uid extraction of a� atoxin B1

from soilUSAA� atoxin B1 is a fungal secondary metabolite that is acutely toxic and

carcinogenic. A� atoxin in soil has important human health implications

via ground water contamination, availability for uptake by plants, and

aerosolization of dusts. The two fungi that are largely responsible for

production of a� atoxin B1 are frequently isolated from farmland top soils. A

paper on the development of a supercritical � uid extraction (SFE) method

to recover a� atoxin B1 from forti� ed soil was published in the Journal of

Chromatography A [1209 (1-2), 37-43 (2008)].

“This research was performed because previous studies (liquid-liquid

extraction) in soil were limited by methods which were able to account for

only 18% of applied a� atoxin B1. We believed that SFE would prove to be

a much more e� ective extraction technique and, therefore, provide the

foundation for more conclusive research,” said lead author Dr James Starr

from the US Environmental Protection Agency at the National Exposure

Research Laboratory in North Carolina, USA.

The study assessed the e� ects of temperature, pressure, modi� er (identity

and percentage), and extraction type. Using the optimized SFE conditions,

the mean recovery from air dried soil was 72%. “SFE provided a sensitive and

speci� c method for the extraction of a� atoxin B1 from forti� ed soil. Using

the optimized SFE conditions, we were able to a� ect a fourfold increase

in recovery over previously reported literature values and could analyze

soil extracts with no additional puri� cation. The variables associated with

changes in recovery of a� atoxin B1 in these experiments were: co-solvents,

static extraction and temperature,” Dr Starr explained.

He feels this method is useful because it enables them to account for the

majority of applied a� atoxin in laboratory research. “We will combine the SFE

method with hyphenated analytical techniques to better assess the kinetics

and fate of a� atoxin B1 in soil. We need to more clearly understand the

potential for groundwater contamination and uptake by plants,” he added.

There is evidence that the binding of a� atoxin to soil is reversible and under

certain conditions a� atoxin undergoes a chemical transformation in the soil.

The method can be used for assessment of a� atoxin B1 and its transformation

products in adulterated soils or by researchers studying mineral adsorption

as a way to detoxify a� atoxin-contaminated agricultural products. “In doing

so, SFE will help provide a better understanding of the interaction of the

toxicant with soil and potential for human exposure,” he � nished.

7research round-upseparation science — volume 1 issue 2

In order to investigate the individual and inhomogenous

cellular response; e.g., to external stimuli, single cell

analysis is mandatory and may provide new cognitions

in proteomics as well as in other fields of systems biology

in the future. In the Journal of Chromatography A [1206

(1), 83-88 (2008)], Dominik Greif and colleagues from

Experimental Biophysics and Applied Nanoscience

at the Physics Department of Bielefeld University,

Germany, report on novel chip architectures for single

cell analysis based on full body quartz glass microfluidic

chips (QG chips) that extend their previous studies

in polydimethylsiloxane (PDMS) chips, and enhance

the detection sensitivity of native UV laser-induced

fluorescence (UV-LIF) detection.

“We extended our previous studies in PDMS

(polydimethylsiloxane) chips by the usage of quartz

glass based QG chips in order to improve the detection

sensitivity especially for single cell experiments. With

native UV laser-induced fluorescence detection (UV-LIF)

detection, we were able to increase the detection limit

for the amino acid tryptophan (Trp) and to separate

three proteins at rather low concentrations, as well as to

perform single cell electropherograms from Spodoptera

frugiperda (Sf9) cells,” said Greif.

According to him, whole body QG chips were produced

via deep reactive ion etching of silicium dioxide (DRIESO)

process. Label-free UV-LIF detection was performed

using the 4th harmonic of a Nd-YAG Laser at 266 nm

combined with an inverted microscope and adapted

optical components for excitation and emission in the

UV range. It could be shown, that a 10 nM Trp solution

could be detected with an S/N ratio of 11.9, which gives a

theoretical detection limit of 2.5 nM (with S/N=3).

“The three proteins α-chymotrypsinogen ovalbumin

and catalase each at a concentration of 0.1 mg/mL

were injected in a mixture, resulting in three nearly

baseline separated peaks. Single cell analysis of Sf9

insect cells was performed in four steps. The isolation of

an individual cell of interest by optical tweezers. Then

the cell was guided to the intersection position of the

microdevice, where obstacles act as a physical trap,”

he explained. At this position the cell was lysed by an

electrical pulse and the protein content (proteome) was

separated via electrophoresis on chip followed by UV-LIF

detection. In the resulting single cell electropherograms

higher peak intensity is observable in contrast to those

conducted in PDMS chips. Furthermore, fluorescence

spectra (λex=266 nm) clearly demonstrate the very high

UV transparency and nearly not observable background

fluorescence of the QG chips as compared to PDMS chips

and PDMS quartz window (PQW) chips.

“The detection limit for Trp in QG chips created by

a dry etching process was significantly lower than

previously shown in other fused-silica chips made

by wet etching, where only a 2 µM Trp solution was

detectable with UV-LIF detection,” he added. Moreover,

to our best knowledge we thereby reached the lowest

limit of detection reported for Trp with native UV-LIF

detection in microfluidic channels. “As the applicability

of the QG chips for single cell analysis was proven and

the increased detection sensitivity as well as the reached

detection limit should allow even the detection of

low abundant proteins from a single cell we will strive

towards label-free single cell protein fingerprinting on

chip in future,” he concluded.

Single cell analysis with UV laser-induced fluorescence detection Germany

A,


Methods for authenticating honeyCroatiaThe EU Commission encourages the development

of analytical methods for the verification of quality

specification compliance for food matrices. As one

example, assessment of honey botanical origin is of great

concern, because authenticity guarantees its quality and

economic value.

“A wide assortment of honeys is available in Croatia as

the result of production in different regions with specific

climatic conditions and a wide range of floral sources, but

with no determined chemical fingerprints or nutritional

properties,” explained Igor Jerković (Faculty of Chemistry

and Technology,University of Split, Croatia).

“Paliurus spina-christi unifloral honey is a product with

limited production and distinct fragrance characteristics

in comparison with other ubiquitous unifloral honeys

from Croatia (sage, rosemary and others). Little

information about this honey is available and there are

only a few papers on its floral source.”

“As it is known, volatile aroma profile is one of the most

typical features of a food product, for both organoleptic

quality and authenticity. Therefore, we have performed

research on headspace, volatile and semi-volatile

patterns of Paliurus honey as markers of botanical

origin,” (as part of the research project Evaluation

of Unifloral Honeys - Chemical Fingerprinting and

Nutritional Properties, funded by the Ministry of Science,

Education and Sports, Republic of Croatia, Unity through

knowledge fund, UKF No. 25/08).

Published in Food Chemistry [112 (1), 239-245

(2009)], Jerković’s research group analysed samples

of Paliurus honey by means of headspace solid-phase

microextraction (HS-SPME) and ultrasonic solvent

extraction (USE) followed by gas chromatography and

mass spectrometry (GC–MS) to obtain complete patterns

of headspace, volatile and semi-volatile compounds.

Jerković explained, “The results indicate that there is

great variability in the identified volatiles, depending

on the procedure employed. The HS-SPME technique

selectively isolated headspace volatiles, while the USE

method isolated volatiles and semi-volatiles. Different

solvents applied for USE enabled the extracts with

components of different polarity that contributed

to more complete overall analysis to be obtained. In

headspace pattern the most abundant compounds

and possible markers were nonanal, four isomers of

lilac aldehyde, decanal, methyl nonanoate, hexanoic

and 2-ethylhexanoic acids. Although the main

components of USE extracts were higher saturated

aliphatic hydrocarbons, higher aliphatic alcohols and

acids, they cannot be considered reliable biomarkers

because of their probable origin from bee wax or bee

cuticle. Although present in small quantities, the more

reliable markers in the extracts were benzene derivatives

(particularly 4-hydroxy-3,5-dimethylbenzaldehyde,

4-hydroxybenzoic acid and 4-methoxybenzoic acid),

together with lower aliphatic acids (butanoic, hexanoic,

octanoic and nonanoic).”

He described how the techniques are very promising

for further research because they provide different

profiles of headspace, volatile and semi-volatile patterns

on honey flavour, without thermal artefacts.

“Those patterns are important in the search for specific

biomarkers of honey botanical origin, as well as for the

comparison with other ubiquitous honeys. In addition,

those techniques could be applied for the detection of

chemical fingerprints for food traceability. Food quality is

a primary target in today’s world and it involves different

aspects as safety, health, origin and control,” Jerković

concluded.

A,



Dr Jesus Sanz and colleagues from the Institute for

Molecular and Celular Biology at Miguel Hernández

University in Alicante, Spain, present a novel procedure

for affinity partitioning of recombinant proteins fused

to the choline-binding module C-LytA in aqueous two-

phase systems containing poly(ethylene glycol) (PEG)

in the Journal of Chromatography A [1208 (1-2), 189-196

(2008)].

“For several years we have been working with the

choline-binding modules (CBMs) present in several

surface proteins of Streptococcus pneumoniae. These

modules are polypeptidic sequences that recognize

choline in the cell wall, but they are flexible enough

to bind to other tertiary and quaternary amines. We

took advantage of this to construct chimeric proteins

containing these CBMs that can be easily immobilized

and/or purified in a single-step procedure using simple

supports such as DEAE-cellulose. Furthermore, we have

found that many other solid supports derivatized with

choline analogues are also effective in immobilizing

CBM-tagged proteins; for example, 96-well plates, DEAE-

paper, etc.,” Dr Sanz explained.

Sanz and his team took a step further to see whether

they could employ the high affinity for choline displayed

by CBMs to set up an efficient partitioning procedure for

CBM-tagged proteins in ATPSs that could be modulated

by the addition of their natural ligand (choline).

“Poly(ethylene glycol) is one of the polymers most used

for ATPSs. We first found that CBMs specifically interact

with PEG with a certain affinity. Therefore, in ATPSs

containing PEG such as aqueous PEG/phosphate or PEG/

dextran mixtures, CBM-containing proteins are localized

in the upper, PEG-rich phase, almost quantitatively. This

already allows a protein purification higher than 80%

directly from an Escherichia coli extract. Nevertheless,

addition of choline, the natural ligand of the protein,

displaces PEG from the binding sites and shifts the

location of the protein to the lower, PEG-poor phase,” he

said.

According to him, the result is a protein >95% pure

as deduced from SDS-PAGE analysis, and with a yield

higher than 70%. As a proof-of-concept they checked the

procedure with four different hybrid proteins containing

CBMs, with similar results. The whole purification

procedure, starting from an E. coli extract, never lasted

longer than one hour.

“The novelty of our work is that it may transform a

rather empiric phenomenon (protein partitioning in

ATPSs) into a fully predictable technique that is tightly

modulated by the simple addition of choline,” he

said. This allows the easy purification of CBM-tagged

recombinant proteins using only a few centrifugation

steps. In the future they would like to apply this system to

biotransformation processes with enzymes of industrial

interest. “We think that our ATPS CBM-based approach,

given its simplicity, may be useful for several types of

proteomic high-throughput methods,” he concluded.

Affinity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems

Spain

11research round-upseparation science — volume 1 issue 2© 2008 Waters Corporation. Waters, Oasis and The Science of What’s Possible are trademarks of Waters Corporation.

Simplify and improve your sample preparation with Oasis SPE products.

■ Highest SPE Recovery■ Cleanest Extracts■ Lowest Matrix Effects■ Lowest Method Variability

Learn how the Oasis family of products can enhance your system performance.

Visit www.waters.com/oasis

selectscience_oasis_island.indd 1 2/3/09 8:55:17 AM

FranceAccording to the Journal of Chromatography A [1208

(1-2), 10-15 (2008)], a new sampling method was

developed for a simple and fast evaluation of volatile

organic compounds (VOCs) emitted at trace levels from

building materials. The device involves an emission cell

coupled with solid phase microextraction (SPME) for

diffusive sampling.

“For the past 10 years, we have developed SPME

sampling methods for VOC traces in air as alternatives

to traditional techniques. In recent studies, we

applied it successfully to indoor air analysis. For these

environments, the priority to reduce VOC levels consists

in controlling sources, especially building materials,”

said lead author Dr Valérie Desauziers from Laboratoire

Génie de l’Environnement Industriel, Ecole des Mines

d’Alès, France. In Europe, the standardized evaluation

procedure for new products is time-consuming and

not easy to use on site. To generalize audits to the

manufacturers and to make easier measurements on

site, a passive SPME sampling coupled to an emission

cell was performed.

“The developed methodology shows relevant

performance in terms of sensitivity and reproducibility.

As it involves passive sampling, neither pump nor flow

control is required, making the method particularly

convenient for on-site application. Moreover, the

sampling time can be adjusted according to the

analytical objective; for example, a rapid screening of

the major VOCs emitted by new building material can

be performed in five minutes,” Dr Desauziers explained.

For her, this new sampling method provides the

means to rapidly and easily evaluate the VOCs emitted

by building materials (a study is in progress for floor

coverings in collaboration with O. Ramalho from

the CSTB), and to identify VOCs sources in indoor

environments. “Regarding the SPME technique, the

key step is to achieve a reliable quantitative analysis

and to simplify the calibration procedure. In this aim,

modelling approaches for VOCs adsorption on the

SPME fibre are studied in collaboration with Dr P.

Mocho (University of Pau),” she said.

Spain

SPME sampling for the evaluation of volatile organic compounds emitted from building materials

Determination of toxic dyes in water using LC-vis/FLD and confi rmation by MS/MSPolandA liquid chromatography with visible and fl uorescence detection (LC-vis/FLD) method for

screening and a liquid chromatography with mass spectrometry (LC-MS/MS) method for the

confi rmation of malachite green (MG) and its major metabolite, leucomalachite green (LMG)

residues in fi sh farming and river water have been described in the Journal of Chromatography

A [1207 (1-2), 94-100 (2008)]. Dr Kamila Mitrowska and colleagues from the National

Veterinary Research Institute in Pulawy, Poland, conducted the study using water samples

which were preconcentrated on diol solid-phase extraction columns. Chromatographic

separation was achieved using a phenyl-hexyl column with an isocratic mobile phase

consisting of acetonitrile and acetate buff er (0.05 M, pH 4.5) (70:30, v/v).

“The optimized methods provide rapid analysis of MG and LMG with minimal sample

preparation. The LC-vis/FLD method for screening and LC-MS/MS for the confi rmation

allows for quick and sensitive analysis of MG and LMG in water samples. The separation

and determination conditions used enable the detection and identifi cation of not only MG

and LMG, but also their N-demethylated products formed as a result of photo-oxidative

decomposition in water observed in stability tests performed at 20 °C,” Dr Mitrowska

explained.

According to her, these fi ndings prove that N-demethylated products of MG and

LMG, reported as potential carcinogens, may be formed in living fi sh, not only during

enzymatic action but also during photo-oxidative degradation in water. “As there are both

environmental and human health concerns about bioaccumulation of MG and LMG in

terrestrial and aquatic ecosystem, the proposed methods have been successfully applied to

the regulatory control analysis of water samples additionally collected at fi sh farms during

investigation performed as a result of founding MG residues in fi sh,” she concluded.


Melamine contamination is headline news

and food safety is forefront in the minds

of the public. Dionex delivers proven

chromatography and sample prepara-

tion solutions for food safety analysis of

melamine and most other inorganic and

organic contaminants.

Dionex–reliable food safety solutions.

Learn more about food safety solutions at

www.dionex.com/foodsafety

Food Safety First!


Study investigates the stability of mixtures used for patient-controlled analgesia

SpainThe stability of combinations of different drug solutions used in clinical practice to relieve postoperative pain using

the Patient Controlled Analgesia (PCA) technique was tested at the hospital pharmacy of the University Hospital

San Cecilio in Granada (Spain) on non-commercially available mixtures frequently used in hospital treatment. Dr

Luís Fermín Capitán-Vallvey from the Department of Analytical Chemistry, University of Granada, Spain used liquid

chromatography for the separation of the chemicals present and possible degradation products at the concentration,

typically high, used in hospital treatment.

Published in Chromatographia [68 (9-10), 767-772 (2008)], Dexketoprofen (D-KTP), tramadol (TMD), and haloperidol

(HLP), two analgesic agents and an adjuvant, respectively, were analysed simultaneously in 0.9% NaCl and 5% glucose

by use of a new, rapid LC–DAD method. Chromatographic separation was achieved on a C18 column with 50:50 (v/v)

acetonitrile–sodium dihydrogen phosphate (pH 4.10; 0.1 M) as mobile phase at a flow rate of 1 mL min−1. Ultraviolet

diode-array detection was used. The absorbance of the eluate was monitored at 210 nm. The retention times of the

three compounds were 2.0, 4.0, and 8.3 min for TMD, HLP, and D-KTP, respectively; the total run time was 9 minutes.

The method was validated for linearity, accuracy, reproducibility, and limits of quantification and detection.

According to Dr Capitán-Vallvey, the tests revealed good chemical stability and physical compatibility for 30 days

of the widely used analgesic mixture composed of the two analgesic agents (D-KTP and TMD) and an adjuvant (HLP),

prepared in different solvents and under different storage conditions for light, temperature and containers, using a

rapid and validated LC method.

“Liquid chromatography is the most convenient technique for learning about the chemical stability of drugs used

in normal clinical practice. We are currently conducting studies on the stability of other mixtures of drugs used in

oncology and analgesia. Additionally, we are interested in the analysis of residues of different drugs in bodily fluids

and of drugs for veterinary use in different foods,” he concluded.

SPE-HPLC method for accurate and precise quantification of HIV integrase inhibitors

USANaser Rezk and colleagues from the Center for AIDS Research, University of North Carolina

at Chapel Hill, USA have developed an accurate and precise high-performance liquid

chromatography method for the rapid quantification of raltegravir in human blood plasma

after solid phase extraction, according to Analytica Chimica Acta [628 (2), 204-213 (2008)].

Raltegravir is an antiretroviral (ARV) drug produced by Merck & Co, used to treat HIV

infection. Its mode of action is to target integrase, an HIV enzyme that integrates the viral

genetic material into human chromosomes, a critical step in the pathogenesis of HIV. It

received FDA approval in October 2007, the first of a new class of HIV drugs, the integrase

inhibitors, to receive such approval.

“In our clinical pharmacology and analytical chemistry laboratory (CPACL) we always

develop our assays for analysis to assure the accuracy and quality of our clinical

pharmacology studies,” explained Rezk. “It is extremely important to have good separation

for raltegravir from all ARVs, which now total more than 20 individual drugs, particularly as

therapy is always a combination approach.“

“Optimizing separation conditions and developing a robust extraction method were the key

challenges in this work. We were able to successfully separate raltegravir using low ion-paring

reagents. Once optimized the method demonstrated a high extraction efficiency and very

Comparing sample treatment strategies for the analysis of thyreostatic drugs in

thyroid samplesIn an article in Journal of Chromatography A [1207 (1-2), 17-23 (2008)], a method based on ultra-performance liquid

chromatography–electrospray ionization-tandem mass spectrometry (UPLC–MS/MS) for the determination of six

thyreostatic drugs in thyroid tissue was optimized and validated in accordance with Decision 2002/657/EC. Professor

Ramón Companyó from the Department of Analytical Chemistry at the University of Barcelona, Spain, explained the

study emerged from the need to simplify analytical methodologies in order for laboratories to process large volumes

of thyroid and urine samples of animals intended for human consumption for the presence of thyreostatic drugs.

In the study, sample extraction was evaluated in methanol and in ethyl acetate, the latter which gave better results.

Two clean-up strategies were compared: one based on silica cartridges (SPE), and the other, on gel permeation

chromatography (GPC).

“In a previous paper [Analytica Chimica Acta, 617, 184 (2008)] we proposed a method that significantly shortens

the LC methods found in the literature. This arises from two experimental features, namely, the avoidance of a

derivatization step, which simplifies the sample treatment, and a shorter chromatographic separation based on the

application of ultra-performance liquid chromatography (a run time of 13 min in front of the 25-40 min of the former

methods). Moreover, we clarified a controversial issue of previous methods, namely the

significant losses of analytes that take place when the extracts are evaporated to dryness,”

Professor Companyó explained.

In this paper, Companyó and his team proposed the use of gel permeation

chromatography (GPC) as an automatable technique for the clean up of extracts. In

comparison with the manual SPE procedure applied in their previous work, the GPC

approach leads to savings in operator time of approximately 50%. According to him, the

absolute recoveries of all analytes except Tapazol are significantly improved, as well as the

accuracy and precision.

The method, after being validated in accordance with the European Decision 2002/657/

EC, was implemented in the routine analytical work in the laboratory of the Agència de

Salut Pública de Barcelona. “Many other European laboratories are showing an interest in

our work,” he added.

The team is now assessing a GPC column with a smaller diameter that uses a lower

mobile phase flow rate (1 mL/min vs 5 mL/min). “This would allow performing the clean-

up step with a lover solvent consumption, rendering the method cheaper and more

sustainable from an environmental point of view,” he concluded.


high reproducibility,” continued Rezk.

Within the research group three clinical studies are currently running this HPLC method

to analyse raltegravir in human plasma. “We are confident that this extraction/separation

method will enable several clinical studies to evaluate raltegravir in genital tract secretions

and tissues from male and female HIV-infected populations. We believe that the successful

bioanalytical work should be extensively validated in order to assure the method applicability.

Our research group has tested the quality of the data in terms of accuracy and precision of

raltegravir in patients who receive anti-HIV therapy in different combination therapy.”

Improvement in HIV-therapy and prevention are the overriding aim of the research group.

Spain


Hollow fi bre liquid phase microextraction-UHPLC-MS/MS analysis

of pesticides in alcoholic beveragesSpainA paper in the Journal of Chromatography A [1208 (1-2), 16-24 (2008)] presents an alternative

method to determine more than 50 pesticides in alcoholic beverages using hollow fi bre liquid

phase microextraction (HF-LPME) followed by ultra-high pressure liquid chromatography

coupled to tandem mass spectrometry (UHPLC–MS/MS), without any further clean-up step.

Led by the Department of Analytical Chemistry at the University of Almeria, Spain, Dr Roberto

Romero-González initially conducted the research to fi nd an alternative to conventional

techniques of extracting pesticides that are time consuming and require large amounts

of solvents. “New extraction techniques must be developed in order to increase sample

throughput and reduce analysis time,” Dr Romero-González said.

HF-LPME provides several advantages, such as low cost, simplicity and high enrichment

factor, but it is seldom used for the determination of polar pesticides in alcoholic beverages.

“The set up used in this research was very simple and minimized handling, improving the

precision associated with the extraction method. This was based on the use of an acceptor

phase immobilized in the pores of a hollow fi bre, and after the extraction, the fi bre was

desorbed in methanol, which allows the desorption of the pesticides, providing clean

solutions to be injected into the chromatographic system,” Romero- González explained.

Furthermore no matrix eff ect was observed, indicating that the extraction procedure is not

aff ected by the selected matrices (red, white, sparkling wine, beer and non-alcoholic beer).

The optimized method was applied to real samples and carbaryl, triadimenol, spyroxamine,

epoxiconazole, trifl umizol and fenazaquin were detected in some of the analysed samples.

According to him, the combination of HF-LPME with UHPLC increases sample throughput and

the suitability of the developed method for routine analysis. “Bearing in mind the advantages

of this technique, our current work is mainly focused on the development of new extraction

procedures based on HF-LPME for other groups of compounds and the combination with

other separation techniques such as GC-MS/MS,” he concluded.

Phenomenex provides complete solutions including SPE tubes & 96-well plates, protein precipitation plates, syringe filters, vials and more.

• Quality products

• Responsive customer service

• Complete technical support

• Worldwide availability with fast delivery

Request a free sample pack of Phenex™ syringe filters visit www.phenomenex.com/sample

Phenomenex provides complete solutions including SPE Phenomenex provides complete solutions including SPE tubes & 96-well plates, protein precipitation plates, tubes & 96-well plates, protein precipitation plates, syringe filters, vials and more.

• Quality products

• Responsive customer service

• Complete technical support

• Worldwide availability with fast deliveryWorldwide availability with fast delivery

Request a free sample pack of PhenexRequest a free sample pack of Phenex™ syringe filters visit www.phenomenex.com/samplewww.phenomenex.com/sample

PA66

2012

08_L

For more information visit:www.phenomenex.com/info/spePhenomenex products are available worldwide. Email us at [email protected].

Sample Preparation Solutions

© 2

009

Phe

nom

enex

Inc.

All

right

s re

serv

ed.

Determination of niacin in plasma using LLE and LC-MS/MS

USAPublished in Biomedical Chromatography [22 (11), 1272-1278 (2008)] is an article from H. Thomas Karnes and

colleagues (Virginia Commonwealth University Medical Center, Richmond, Virginia, USA) outlining the assessment of

matrix eff ects and determination of niacin in human plasma using liquid-liquid extraction and liquid chromatography-

tandem mass spectrometry.

“We originally performed the work because we wanted to look at the pH dependence of the extractability of ion

suppressing phospholipids from biological samples. Niacin provided an appropriate model analyte,” explained Karnes.

“Our key fi ndings were that acidic extracts provided the lowest ion suppression eff ects from the phospholipids

and that the improvement in sensitivity resulting from the obviation of these ion supression eff ects was more than

enough to compensate for the lower extraction recovery of niacin at acid pH,” he continued.

Karnes concluded, “The implications for future studies is that additional care must be taken to avoid ion suppression

matrix eff ects when using basic extracts as opposed to neutral and acidic extracts.”



2D TLC for chromatographic fi ngerprinting of furanocoumarins

PolandThe numerous taxonomic classifi cations of the genus Heracleum need revision and

morphological identifi cation is diffi cult to perform, as there are minimal characteristic

diff erences between each Heracleum species, varieties and forms. Furanocoumarins are

characteristic compounds from the Apiaceae family, found in the whole genus in large

quantities. However, furanocoumarins are diffi cult to separate, because of their similar

chemical structures and physicochemical properties. In a paper published in the Journal

of Chromatography A [1207 (1-2), 160-168 (2008)]a simple method is proposed for the

discrimination of selected species, varieties and forms of the genus Heracleum.

“The aim of this study was to verify as to what degree graft thin-layer chromatography

(TLC) can be applied for constructing chromatographic fi ngerprints of furanocoumarins

for distinguishing selected varieties and forms of Heracleum species,” said Prof. Monika

Waksmundzka-Hajnos from the Department of Inorganic Chemistry at the Medical University

in Lublin, Poland.

The research proved that two-dimensional chromatography with adsorbent gradient

(silica + octadecylsilica) was suitable for complete resolution of a mixture of 10 structural

analogues. “It was well suited for fi ngerprint construction and the proposed method provides

the possibility to distinguish diff erent species, varieties and forms of the Heracleum genus.

The method is characterized by high specifi city, precision, reproducibility and stability values.

This is the fi rst time that a unidimensional multiple development (UMD) technique along with

graft-TLC was applied for fi ngerprint construction,” Prof. Waksmundzka-Hajnos explained.

She believes that the importance of two-dimensional separations in the analysis of natural

compounds will continue to grow. There is a trend of combining diff erent chromatographic

techniques in orthogonal directions, of which the use of diff erent unidimensional separations

along with two-dimensional techniques is the most popular. “The potential of such methods

is enormous, as they enable the separation of compounds being structural analogues or of

diff erent polarity. The application of these methods will also develop and new procedures will

be introduced for the resolution of complex natural mixtures,” she added.

Because two-dimensional chromatography turned out to be useful in the analysis of

complex mixtures of natural origin, she and her team will focus future research on the

application of special techniques of development in the analysis of very complex samples

(e.g., methanolic plant extracts, herbal preparations, etc.). “In the near future we would

like to work out new chromatographic procedures for constructing and evaluating plant

fi ngerprints,” she concluded.

Ion Chromatographyfor ProfessionalsMetrohm sets the new standard in ion chro-matography. The 850 Professional IC is thefirst ion chromatography system entirelymade of intelligent components, whichmeans:

automatic recognition of all system components including columnsautomatic parametrization of all components permanent monitoring of hardware,fluids, columns, calibration, results and service intervals

Your benefits: more convenience, less risk of errors and complete traceability of youranalyses.

www.professional-ic.com

Eckn

auer

+Sc

ho

ch A

SW

Ins_Metrohm_IC850_A4e

20 feature article — CE in biomedical analysis www.sepscience.com

Microfl uidic chip capillary electrophoresis for biomedical analysis

IntroductionA rapid advance in microfl uidic chip

development for biomedical analysis

has occurred in recent years [1].

Most studies have concentrated on

proof of microfl uidic concept for

intended application and very few on

applications with real world samples

[2]. The major reason for this is that

research are mostly performed by

technology providers and not by

users. The fi rst phase of development

of microfl uidic research at Hong Kong

University, in collaboration with the

Dalian Institute of Chemical Physics,

Chinese Academy of Sciences, has

focused on technology development

and the creation of a supporting

infrastructure.

In the past few years, our research

work has entered a second phase

with a focus on medical application

of microfl uidic technology. To

facilitate integration with intended

applications, collaboration with users

in related biomedical fi elds starts at

the beginning of the projects and a

list of current research in developing

microfl uidic devices for biomedical

application in Hong Kong is given in

Table 1. The works proceed in two

directions to speed up the pace of

implementation with the fi rst based

on using commercially available

instrumentation and the second

based on exploration of microfl uidic

devices for biomedical applications.

For methodology development,

this includes coupling microfl uidic

devices to electrophoretic separation,

fabrication of microfl uidic chips in

plastic material-substrate of choice for

disposable clinical use, and research

exploring detection modes for

bedside monitoring. Laser ablation

is used in the fabrication process

to enable a quick proof of concept

for a given microchip design [3].

Research is currently focused in the

following two areas: fi rst, portable

systems for on-chip mixing to ensure

a complete chemical reaction, and

second, sensitive detection modes

capable of operating under fi eld

conditions. Successful application

of nano-magnetic fl uids for on-chip

mixing and detection based on dual

electrode and immobilized quantum

dots are currently under development

in our laboratory.

Of the various biomedical

applications listed in Table 1, details on

Micro� uidic devices based on commercially available equipment have been developed for biomedical analysis in Hong Kong by coupling micro� uidic chips to capillary electrophoresis (CE) with on-column UV detection. A CO2 laser was used to ablate the desired channel pattern on poly(methyl methacrylate) substrate prior to hot press bonding to fabricate the microchips. Details on two micro� uidic CE devices are given and their applications in Traditional Chinese Medicine analysis and investigation of bilirubin-albumin interaction in human serum albumin are discussed.

Y. S. Fung and Z. Nie,

Department of Chemistry, The University of Hong Kong, Hong Kong SAR, China.

21feature article — CE in biomedical analysisseparation science — volume 1 issue 2


Current research in development and application of microfluidic devices for biomedical analysis in Hong Kong.

A) Development and coupling of microfluidic devices to electropho-retic separation

1) Fabrication of plastic microfluidic chip

2) Application of nano-magnetic fluids for on-chip mixing

3) On-channel detection by dual electrode and immobilized quan-tum dots

B) Areas of biomedical application

Clinical diagnosis and assessment of herbal medicine

Biomedical research

1) Clinical Manage-ment of Unbound Serum Bilirubin in Newborn Babies at Queen Mary Hospi-tal, Hong Kong

1) Characterization of mitochondria for Oocytes

2) Quality Assess-ment of Complex Herbal Preparation

2) 2D protein sepa-ration for on-chip captured single cell

3) Binding of metals with milk and other proteins

C) Collaboration with researchers in related biomedical field

1) School of Traditional Chinese Medicine for quality assessment of herbal medicine

2) Department of Paediatrics & Ado-lescent Medicine for free bilirubin determination in cord bloods for newborns

3) Department of Obstetrics & Gynaecology on protein analysis

4) Department of Anatomy on characterization of mitochondria for Oocytes

Table 1

channels during operation. An eight-

channel high-voltage power supply

with adjustable voltages from 0 to

+3500 V (model MP-3500-FP, Major

Science, Taiwan) was used to control

the sequence of applied high voltage.

A UV/Vis detector from CE Resources

(model CE-P2, Singapore) was used

to detect analytes on-column after

separation at the silica capillary.

Fabrication of the PMMA

CE microchip: The double-T

microchannels with identical

dimensions [Figure 1(a)] were ablated

by a CO2 laser at 10.6 µm wavelength

onto a 30 × 40 × 0.15 mm PMMA chip

(Ensinger Ltd, UK). All channels were

ablated to a depth of 100 µm and a

width of 150 µm at upper channels

[Figure 1(b)]. The 3 mm double-T

injector was designed to introduce

a large sample plug and the 8 mm

channel to connect the four double-T

injectors at equal distance from the

embedded silica capillary. A capillary

with 50 µm i.d. and 13 cm length

(detection window 4 cm from the

other capillary end) was used as a

separation capillary with an effective

In the past few years, our research work has entered

a second phase with a focus on

medical application of microfluidic

technology

the following two areas will be given

in the results section:

1. Quality assessment of complex

herbal preparations

2. Clinical management of unbound

serum bilirubin in newborn babies at

Queen Mary Hospital. Approval from

the Hospital Authority in Hong Kong

for clinical trials of microfluidic devices

for management of unbound serum

bilirubin in newborn babies at Queen

Mary Hospital has been obtained with

an execution plan set for the coming

year.

Experimental Chemicals and reagents: Dried

herbs of Liu Wei Di Huang Wan,

namely, Cortex Moutan (Mu dan

pi), Radix Rehmanniae (Di huang),

Fructus Corni (Shan zhu yu), Poria

(Fu ling), Rhizoma Alismatis (Ze xie)

and Rhizoma Dioscoreae (Shan yao),

were purchased from Tong Ren Tang

Pharmaceutical shop in Hong Kong.

They were ground into powders and

mixed thoroughly according to the

formula of Liu Wei Di Huang Wan in

the following weight ratios: Cortex

Moutan:Radix Rehmanniae:Fructus

Corni:Poria:Rhizoma Alismatis:Rhizoma

Dioscoreae = 3:8:4:3:3:4, respectively

[4].

Bilirubin 4α and human serum

albumin (fraction V) purchased

from Sigma Chemicals (St.

Louis, MO, USA) were used

without further purification. The

running buffer was made up to

10 mmol/L sodium phosphate

and

1 mmol/L EDTA before adjusting

to

pH 7.4. All chemicals used were

analytical reagent grade except

otherwise stated. All buffer solutions

were freshly prepared, filtered through

0.45 µm membranes and degassed

before use.

Instrumentation and facilities:

The CO2 laser engraver (V-series,

Pinnacle, USA) was controlled by

software (CorelDRAW 10) to create

the desired channel pattern on the

PMMA polymer chip. The hot press

bonding machine (up to 500 °C

and 1 MPa pressure) was purchased

from Guangju Machinery Company,

China. A sample workstation with

fine control of the X, Y and Z position

of the microchip was used to enable

microscopic observation of the


tn” = (tn115 – t*) (1)

Hn” = Hn115 / H* (2)

where tn115 and Hn

115 were the

migration time and peak height,

respectively, for peak n at 115% of one

of the ingredients. The range of ±1.96

SD for both the relative migration time

and corrected peak height of 100%

Liu Wei Di Huang Wan was used as

the standard for testing. The symbol

length of 9.8 cm. The capillary was

sandwiched between two PMMA

plates and located at the double-T

microchannel intersection. A thermally

controlled hot plate press was used

for bonding of the microchip under

constant pressure and temperature of

0.6 MPa and 92 °C, respectively for 15

minutes.

Results and DiscussionQuality assessment of Traditional

Chinese Medicine: Under optimized

conditions, 24 diagnostic peaks were

found from the electropherogram of

Liu Wei Di Huang Wan (Figure 1(c)). To

reduce the variability and improve

the repeatability of the fingerprinting

pattern, the migration time and

peak height were normalized using

benzoic acid as the internal standard.

The change in repeatability of peak

height measurement corrected by the

internal standard is shown in Table 2.

After normalization with the internal

standard peak, most of the peaks

showed appreciable reduction in RSD,

except peaks 17 and 22, which gave

a small increase in RSD. As the use of

a smaller RSD could lower the noise

and increase the chance to detect

small but significant differences in RSD

resulting from small changes in the

ingredients of Liu Wei Di Huang Wan,

the corrected peak height was used

for fingerprinting. As a result of its

much smaller RSD, it provided a more

sensitive parameter compared with

the change in the migration time.

To provide a quantitative measure

for fingerprinting the six-component

Liu Wei Di Huang Wan, preparations

were made using five full ingredients

and one with 115% of the ingredients

compared with the original recipe.

The diagnostic pattern obtained

for fingerprinting Liu Wei Di Huang

Wan were shown in Table 3. Three

test criteria used in the table were

based on the change in the migration

time and peak height. The relative

migration time, tn”, and corrected

peak height, Hn”, for peak n from

each sample with 100% Liu Wei Di

Huang Wan plus 15% of the targeted

ingredient were calculated using the

following formulae:

0 5 10 15 20 25 30-0.010

-0.008

-0.006

-0.004

-0.002

0.000

0.002

0.004

0.006

0.00823

8

2422

*

21

20

19

1817

16

151413

1211109

765

432

1

Abso

rban

ce (A

U)

Migration Time (min)

Capillary

SR3 mm

PMMA Chip

BW

DW

8 mm

5 mm

BR

SW a) Layout of the PMMA microfluidic chip

b) Cross section of the microchannel

c) The electropherogram of Liu Wei Di Huang Wan

Figure 1. Schematic diagram showing a) the layout of the PMMA microfluidic chip coupled with the fused silica separation capillary, b) cross section of the micro-channel and c) the electropherogram of Liu Wei Di Huang Wan. (CE conditions: Buffer: 0.01 M borate, 0.05 M SDS with pH adjusted to 9.3, Methanol/H2O: 15% (V/V), Detection: 210 nm; Capillary: 0.05 mm i.d x 13 cm (window 9 cm) * Internal Standard Peak (1.0 x 10 –4 M Benzoic Acid)Injection: 20s with 2100 V applied between SR and SW, while BR and BW were floated; Separation: 3500 V applied to BR, while BW maintained at ground, SR and SW kept at 2100 V. BR, buffer reservoir; SR, sample reservoir ; SW, sample waste reservoir; BW, buffer waste reservoir; DW, Detection window.

Figure1


+ or – indicated the deviation of tn’’

or Hn’’ beyond ±1.96 SD of tn’ or Hn’

(100% Liu Wei Di Huang Wan). The

space was left as blank when tn’’ or Hn’’

was found within ±1.96 SD of tn’ or Hn’,

respectively.

The change to 15% of one of the

ingredients of Liu Wei Di Huang Wan

is shown to produce a noticeable

change in the fingerprinting pattern

that can be used to trace back to a

particular herb, such as a selective

shift in some of the peaks listed in

Table 3. Thus, the change in the

fingerprinting pattern can be used to

indicate change in composition of one

of the ingredients of Liu Wei Di Huang

Wan when the change is greater than

15%. From the pattern change, one

can identify which of the six herbs

has changed its composition under

favourable conditions.

Determination of unbound serum

bilirubin for clinical management

of neonates under critical

conditions: The current practice for

treating jaundice neonates to reduce

neurologic abnormalities, such as

kernicterus (a serious brain injury that

can affect future IQ development),

depends on measuring elevated levels

of total serum bilirubin to provide an

important indicator for intervention

[5]. However, free bilirubin instead of

total serum bilirubin provides a more

reliable indicator to predict the toxicity

of hyperbilirubinemia [6-8]. Moreover,

the equilibrium between bound and

free bilirubin can be disturbed during

measurement.

To determine free bilirubin, current

methods use an enzyme horseradish

peroxidase, either alone [9] or in

combination with a diazo dye [10],

to obtain kinetic data for indirect

estimation of the original level of free

bilirubin at the start of the kinetic

experiment. Although the method is

sensitive, it depends on the validity of

a kinetic model for indirect calculation

of free bilirubin. The results can be

easily influenced by interferents with

a strong effect on reaction rates. In

addition, a sizable volume of blood

(approximately 25 µL) is required to be

taken from neonates.

Frontal analysis (FA) coupled with

capillary electrophoresis (CE) has been

established as an efficient method

for studying non-covalent molecular

interactions. In FA, a relatively large

sample plug is injected into the CE

capillary to give rise to a flat plateau

with measurable height for analyte

concentration under equilibrium

with albumin. It provides a suitable

procedure to investigate bilirubin/

albumin interaction and determine

free bilirubin concentration without

disturbing the equilibrium established

in a bilirubin/albumin mixture [11-13].

However, problems occur during

clinical implementation because

of the need to automate the rather

complicated CE/FA procedure

involving a series of titrations at

microliter volume between albumin

and bilirubin. A CE/FA microchip

device capable of integrating

both titration and electrophoretic

Effects of using internal peak to normalize peak heights from capillary electropherograms of Liu Wei Di Huang Wan

Peak height1 corrected peak height2 Diff3

peak No. Hn(cm) SD RSD(%) Hn’(cm) SD’ RSD’(%) (%)

1 14.43 0.3957 2.74 0.86 0.0215 2.51 0.23

2 3.81 0.0781 2.05 0.23 0.0041 1.80 0.25

3 2.83 0.0608 2.15 0.17 0.0033 1.95 0.20

4 7.68 0.1457 1.90 0.46 0.0075 1.65 0.25

5 4.96 0.1823 3.67 0.30 0.0101 3.41 0.26

6 1.41 0.0525 3.71 0.08 0.0029 3.49 0.22

7 2.84 0.1014 3.57 0.17 0.0056 3.32 0.25

8 7.52 0.1965 2.61 0.45 0.0116 2.60 0.01

9 3.46 0.1106 3.19 0.21 0.0060 2.93 0.26

10 1.70 0.0404 2.37 0.10 0.0021 2.11 0.26

11 2.43 0.0585 2.41 0.14 0.0031 2.15 0.26

12 7.66 0.1814 2.37 0.46 0.0103 2.26 0.11

13 4.48 0.0802 1.79 0.27 0.0041 1.52 0.27

14 1.82 0.1053 5.79 0.11 0.0060 5.53 0.26

15 2.58 0.1266 4.90 0.15 0.0074 4.79 0.11

16 3.70 0.1357 3.67 0.22 0.0076 3.43 0.24

17 15.69 0.2577 1.64 0.93 0.0178 1.91 -0.27

18 57.99 1.6254 2.80 3.45 0.0890 2.58 0.22

19 0.75 0.0251 3.34 0.04 0.0014 3.07 0.27

20 1.64 0.0651 3.96 0.10 0.0036 3.69 0.27

21 1.62 0.0416 2.56 0.10 0.0022 2.30 0.26

22 1.34 0.0360 2.69 0.08 0.0024 2.95 -0.26

23 13.29 0.4540 3.42 0.79 0.0250 3.15 0.27

24 4.75 0.1735 3.65 0.28 0.0096 3.38 0.27

Note: 1) SD(Standard Deviation) & RSD(Relative Standard Deviation) of Original Peak Height, n=3; H = Original Height of Peak n; 2) SD’(Standard Deviation) & RSD’(Relative Standard Deviation) of Corrected Peak Height, n=3; Hn’ (Corrected Height of Peak n) =Hn/H* (Height of Internal Standard Peak); 3) Diff. = RSD-RSD’.

Table 2


Peak No. A b c d e f

tn’’ Hn” tn’’ Hn” tn’’ Hn” tn’’ Hn” tn’’ Hn” tn’’ Hn”1 - - - - - - - + - + -2 - + - + - - + + + -3 - - - - - - + - + -4 - - - - - - - + - + -5 - - - + - - + -6 - + - + - + + + +7 - - + - - -8 - - - - - + -9 - + - + - + + +10 - + - - - - - - -11 - - - - - - - -12 - - - - - - - - -13 - + - - - - + + - -14 - + - + - + + + - + +15 - + - - - + + - + -16 - - - - - - + - - - -17 - - - - - - - - - -18 - - + + - - + + + -19 - - - + - + - + - + + +20 - + - + - + - + - + + +21 + - + + + - + + + + +22 + - + + + + + + + + +23 + - + + + - + + +24 + - + + + + + + + +

Diagnostics pattern in electrophoretic peaks for fingerprinting of Liu Wei Di Huang Wan

Note: Samples: Five ingredients of Liu Wei Di Huang Wan plus A) 115 % Cortex Moutan; B) 115 % Radix Rehmanniae; C) 115 % Fructus Corni; D) 115 % Poriae; E) 115 % Rhizoma Alismatis; F) 115 % Rhizoma Dioscoreae. tn’’(Relative migration time of peak n from sample A to F) = tn115 (Original migration time of peak n from sample A to F) -t* ( Migration time of internal standard peak ) , where tn115 refers to migration time for peak n at 115%. Hn’’(Relative peak height of peak n from sample A to F) = Hn115(Original peak height of peak n from sample A to F) /H*( Peak height of internal standard peak), where Hn115 refers to peak height for peak n at 115%. The symbol + or – refers to deviation of tn’’ or Hn’’ beyond the range of ±1.96SD of tn’ or Hn’ (100% Liu Wei Di Huang Wan). The space is left as blank when tn’’ or Hn’’ was found to be the within the range of ±1.96SD of tn’ or Hn’ respectively.

Table 3

separation is needed to enable

an urgent check on free bilirubin,

especially during an important early

measurement at 24th hour after birth

[8] for decision to take intervention

measures.

The new microfluidic chip for

clinical implementation of the CE/FA

procedure for bilirubin management

of neonates under critical conditions

is shown in Figure 2(a) with results

given for five consecutive injections

of a given bilirubin-albumin mixture

using one double-T channel as

shown in Figure 2(b). A sharp peak

at 4.4 min resulting from albumin-

bound bilirubin and a small plateau

at 5.2 minutes due to free bilirubin

were found with RSDs at 2.3% and

2.5%, respectively, for peak heights

of albumin-bound bilirubin and

free bilirubin. The repeatability was

obviously improved compared with

literature results using a simple

microchip structure with a RSD of

5.11% [14].

The CE buffer was optimized as

follows: 10 mmol/L sodium phosphate

and 1 mmol/L EDTA (added to inhibit

bilirubin oxidation) adjusted to

physiological pH at 7.4 prior to CE run.

The detection wavelength of

440 nm was selected as, at which,

both free and albumin-bound bilirubin

showed maximum absorption. Under

the optimized conditions, the linear

working range was found to vary from

10 to 200 µmol/L with RSD ranging

from 2.1% to 5.0% for n = 3, and

detection limit at 9 µmol/L for S/N =

3, sufficiently sensitive to indicate the

onset of jaundice in infants [15].

Conclusion 1. The CO2 laser ablation method

is shown to provide a satisfactory

means to fabricate PMMA microfluidic

chips for proof of a given microchip

design for intended applications in

biomedical analysis.

2. A PMMA chip with embedded

capillary enables the use of existing

commercially available equipment

to facilitate the implementation

of a microfluidic device for clinical

diagnosis and biomedical research.

3. The applicability of microfluidic chip

capillary electrophoresis for quality

assessment of Traditional Chinese

Medicine has been demonstrated

using peak pattern based on the

change in the relative migration time

and corrected peak height of 24

diagnostic peaks identified from the

electropherograms for fingerprinting

of Liu Wei Di Huang Wan. The use

of benzoic acid as the internal

standard was found to improve the

repeatability of the results and assist

the fingerprinting process.

4. The capability of microfluidic chip

capillary electrophoresis to develop

a device for bedside monitoring of

free bilirubin for its management in

neonates under critical conditions has

been shown under the CE/FA format.

The linear working range for free

bilirubin was found to vary from 10 to

200 µmol with RSD (n = 3) from 2.1%

to 5.0%, and detection limit (S/N = 3)

at 9 µmol/L with sufficient sensitivity

to indicate the onset of jaundice in

infants.


AcknowledgementsWe thank the Hong Kong Research

Grants Council of the Hong Kong

Special Administrative Region, China

and the University of Hong Kong

Research and Conference Grants

Committee for their fi nancial supports.

References1. J. West et al., Anal. Chem., 80, 4403-

4419 (2008).

2. A.W. Martinez et al., Angew. Chem.,

Int. Ed., 46, 1318-1320 (2007).

3. H. Klank, J.P. Kutter and O. Geschke,

Lab Chip, 2, 242-246 (2002).

4. X.S. Meng, Zhong Cheng Yao Fen Xi

(2nd Ed.), People’s Health Press, Beijing,

China, pp. 211-229 (1998).

5. V. Moyer et al., J. Ped. Gastronenter.

Nutrit., 39, 115-128 (2004).

6. C.E. Ahlfors, Anal. Biochem., 279, 130-

135 (2000).

7. S.U. Sarici et al., Pediatrics, 113, 775-

780 (2004).

8. C.Y. Yeung, J. Clin. Med., 110, 448-454

(1997).

9. J. Jacobsen and R.P. Wennberg, Clin.

Chem., 20, 783-789 (1974).

10. R.G. Martinek, J. Am. Med. Technol.,

32, 697-741 (1969).

11. Y.S. Fung, D.X. Sun and C.Y. Yeung,

Electrophoresis, 21, 403-410 (2000).

12. C.Y. Yeung, Y.S. Fung, Y. S., Sun, D. X.,

Sem. Perinatol. 2001, 25, 50-54.

13. Yeung, C. Y., Fung and D.X. Sun, 4th

Asia-Pacifi c Int. Symp. on Micro. Sep. &

Anal. Shanghai, China, pp 131 (2002).

14. X.J. Liu et al., Electrophoresis, 27,

5128-5131 (2006).

15. A.B. Michael and C. Michael, Oski’s

Essential Pediatrics, Lippincott Williams

& Wilkins, Philadelphia 2004.

Comment on this article

Figure 2

D

PMMA multi-channelmicro�uidic chip

13 cm capillary

BW

BR

BR BR

BR

SW

SWSW

SW

10 mm S4

S1

S2

S3

5 mm

5 m

m5

mm

3 mm

8 mm

0 5 10 15 20 25 30 35 40-0.005

0.000

0.005

0.010

0.015

0.020

2

1

Abso

rban

ce A

U

Time (min)

b) CE/FA electropherograms by UV detection

Five continuous injection on the electrophreogram of an albumin mixture by Microchip CE/FA. Running buff er 10 mmol/L sodium phosphate buff er, 1 mmol/L EDTA, pH 7.4; detection 440 nm, sample 120 µmol/L albumin and 396 µmol/L bilirubin

Peak 1: albumin bound bilirubin, Peak 2: free bilirubin Injection, 2000 V was applied between SR and SW, while BR and BW were fl oated

Separation, 3500 V was applied to BR, while BW were maintained at ground , SR and SW at 2100 V

a) Layout of the multi- channel microfl uidic chip

Figure 2. Schematic diagram showing a) the layout of the multi-channel microfl uidic chip coupled with separation capillary and b) its performance in CE/FA using an UV detector

sepa on dsscienc

technical articles on chromatography and related technologies?

updates on recent research studies?

practical advice on routine analysis?

applications of new technology?

information on commercial product developments?

market trends and opinions?

... then get your FREE subscription to


dsencon scaration

driving analylyl ticticti al chemistcal chemistc ral chemistral chemist y ry r fofof rwawaw rdsrdsrscienceenal chemistrry rsepa atioatioaratio

iPod Touch



1of 5

cecece

CdThe Chrom

Doctor

Exploiting particle size to reduce solvent consumption in analytical HPLC

Introduction

As the supply of acetonitrile becomes

more limited, high performance liquid

chromatography (HPLC) operators are

coming under increasing pressure to reduce

or replace this solvent in their analytical

methods. One simple way to achieve a

reduction in solvent usage is to use a shorter

column. If the resolution of a separation

allows, then simply shortening the column

length from 250 mm to 150 mm will reduce

solvent usage by 40%.

A more dramatic saving in solvent usage

can be made by reducing the internal

diameter of a column, together with an

appropriate scaling down in the flow rate.

Separation efficiencies can be recovered

by also reducing particle size; for example,

columns packed with

sub-2 μm particles offer advantages over the

more traditional systems containing 3 μm

and 5 μm particles by allowing operation

at higher flow rates without compromising

efficiency. Consequently, this results in

shorter analysis times and a reduction

in solvent consumption, together with

associated improvements in resolving power,

sensitivity and peak capacity.

Experimental Conditions

HPLC conditions: Instrument, Accela U-HPLC

system; column, Hypersil GOLD 5 μm, 150 x

4.6 mm; mobile phase, A = 0.05% H3PO4 in

H2O/ACN (66.34), B = ACN; gradient, 0 mins

= 0% B, 25 mins = 0% B, 55 mins = 85%

B, 70 mins = 85% B; flow rate,1.0 mL/min;

injection volume, 10 μL; detection, UV at 214

nm (0.1 s rise time; 20 Hz); temperature, 30oC.

Fast U-HPLC conditions: Instrument, Accela

U-HPLC system; column, Hypersil GOLD 1.9

μm, 50 x 2.1 mm; mobile phase, A = 0.05%

H3PO4 in H2O/ACN (66.34), B = ACN; gradient,

0 mins = 0% B, 3.2 mins = 0% B, 7.1 mins =

85% B, 8.9 mins = 85% B; flow rate, 0.55 mL/

min; injection volume, 0.7 μL; detection, UV at

214 nm (0.1 s rise time; 20 Hz); temperature,

30oC.

Results and Discussion

When transferring methods, whether

from HPLC to HPLC or to U-HPLC, an

understanding of some practical calculations

can help to achieve the correct scaling and

maintain a consistent assay profile between

the original and transferred method. There

are two main considerations for isocratic

methods: scaling the flow rate and adjusting

the injection volume. When a gradient

method is transferred, it also becomes

necessary to adjust the gradient profile. This

is discussed in more detail below.

The global shortage of acetonitrile is causing concern for many HPLC operators. In this

article, the use of sub-2 µm particle packed columns as a strategy to help reduce or replace

acetonitrile consumption in analytical test methods is presented.

28 chrom doctor www.sepscience.com

1. Scale the flow rate: To maintain an

equivalent separation when transferring a

method it is important to keep the reduced

linear velocity constant between the original

and new method. The reduced linear velocity

is related to the flow rate, internal diameter

of the column and particle size. A simple

equation can be derived to calculate the flow

rate (F2) required for the new method. This is

shown below:

F2 = F1 x (dc22 / dc1

2) x (dp1 / dp2)

where F1 = original flow rate (mL/min), dc1

= original column internal diameter (mm),

dp1 = original column particle size (μm), dc2 =

new column internal diameter (mm), and dp2

= new column particle size (μm).

2. Adjust the injection volume: When a

method is transferred to smaller volume

columns, the same injection volume as used

in the original method will take up a larger

proportion of the new column, possibly

leading to band broadening or potentially

overloading the column. It is, therefore,

important to scale down the injection

volume to match the change in column

volume. Once again, a simple equation can

be used to calculate the injection volume

(Vi2) required for the new method.

Vi2 = Vi1 x (dc22 x L2 / dc1

2 x L1)

where Vi1 = original injection volume (μL),

dc1 = original column internal diameter (mm),

L1 = original column length (mm), Vi2 = new

injection volume (μL), dc2 = new column

internal diameter (mm), and L2 = new column

length (mm).

3. Adjust the gradient profile: Geometrical

transfer of the gradient requires calculation

of the number of column volumes of mobile

phase in each segment (time interval) of

the gradient in the original method to

ensure that the new calculated gradient

takes place over the same number of

column volumes, for the new column. The

following calculation should be performed

for each time segment of the gradient,

including column re-equilibration. It takes

into consideration the void volume of each

column (Vc, calculation described below), the

flow rate in the original method and the flow

rate in the new method (calculated in step 1

above) and the time segment in the original

method.

tg2 = tg1 x (Vc2/Vc1) x (F1/F2)

where tg1 = time segment in original

gradient (min), tg2 = time segment in new

gradient (min), Vc1 = original column void

volume (mL), Vc2 = new column void volume

Time (min)

Time (min)

(a)

(b)

Analytes: 1. Impurity K;2. Impurity D; 3. Impurity C; 4. Ibuprofen; 5. Impurity A; 6. Impurity B; 7 Impurity E.

1

2 3

4

67

1

2 3

4

6

7

5

5

Figure 1

29chrom doctorseparation science — volume 1 issue 2

(mL), F1 = original flow rate (mL/min), and F2

= new flow rate (mL/min).

The void volume of the column is the

volume that is not taken up by the stationary

phase (approximately 68% of the column

volume):

Vc = 0.68 x π x r2 x L

where Vc = column volume (mL), L =

column length (cm), and r = column radius

(cm).

To demonstrate the savings that can be

made, the method transfer process described

above has been applied to the separation

of ibuprofen and six impurities. The original

HPLC method, which uses a 150 x 4.6 mm, 5

μm column, is adapted from the European

Pharmacopoeia and the simple calculation

routines are used to transfer the method to

use a 50 x 2.1 mm column packed with 1.9 μm

particles.

The chromatographic profiles obtained

for the original HPLC method and the

geometrically scaled U-HPLC methods on

the smaller column packed with 1.9 μm

particles are shown in Figure 1(a) and 1(b),

respectively. When the method is transferred

to the 50 x 2.1 mm column the resolution of

peaks 5 and 6 is maintained while analysis

time is reduced approximately sevenfold.

The time and solvent savings that can

be gained by transferring HPLC methods

to U-HPLC are summarized in Table 3.

Once column re-equilibration is taken into

account, using the 50 mm column can result

in an eightfold reduction in time, saving 76

mins per sample. The analyst can now run

eight times as many samples. In terms of

solvent saving, only 6 mL of solvent is used

for the method utilizing the 50 mm column

packed with 1.9 µm particles compared with

87 mL for the method using the 150 mm

column packed with 5 µm particles. In such

a way 14 samples can be acquired for the

same solvent usage as one sample — saving

money.

System considerations

To obtain the best data using fast

chromatography it is critical that the liquid

chromatography (LC) instrument system is

optimized to operate under these conditions.

All system components for the assay should

be considered. System volume (connecting

tubing i.d. and length, injection volume,

flow cell volume in UV) must be minimized,

detector time constant and sampling rate

need to be carefully selected, and when

running fast gradients pump dwell volume

needs to be minimized.

Excess system volume gives rise to band

broadening, which has a detrimental effect

on the chromatographic performance. This

can arise from the column, the autosampler,

the tubing connecting the column to injector

and detector, and in the detector flow cell.

The extra-column effects become more

significant for scaled-down separations

because of the smaller column volumes and

HPLC

150 x 4.6 mm, 5 µm

U-HPLC

50 x 2.1 mm, 1.9 µm

Run time (min) 70 9

Total analysis time (min) 87 11

Time saved (min) 76

Flow rate (mL/min) 1.00 0.55

Solvent used (mL) 87 6

Solvent saved (mL) 81

30 chrom doctor www.sepscience.com

for less retained peaks, which have a lower

peak volume making it even more critical to

minimize extra column dispersion.

With sub-2 μm particles, operating

parameters can be optimized to give

fast analysis. This results in narrow

chromatographic peaks which may be in the

order of 1-2 s or less in width. It is important

to scan the detector (whether it is UV or

MS) fast enough to achieve optimum peak

definition, otherwise resolution, efficiency

and analytical accuracy will be compromised.

The HPLC pump dwell volume is particularly

important when running high-speed

applications using fast gradients, typical of

high-throughput separations on small particle

packed columns. This is because the pump

dwell volume affects the time it takes for the

gradient to reach the head of the column.

If we consider a method using a flow rate

of 0.4 mL/min and a fast gradient of 1 min,

the theoretical gradient reaches the column

immediately. A pump with a 65 μL dwell

volume will get the gradient onto the column

in 9.75 s. A traditional quaternary pump with

a dwell volume of

800 μL will take 2 mins to get the gradient to

the column. When running rapid gradients

this is too slow and it may become necessary

to introduce an isocratic hold at the end of

the gradient to allow elution of the analytes.

Conclusion

Transferring a method from a conventional

3 or 5 µm particle packed column to a sub-2

µm particle packed column can result in

significant time and solvent savings. In this

example, transfer of a method for the analysis

of ibuprofen and impurities was successfully

accomplished by geometrically scaling flow

rate, injection volume and gradient profile.

Analysis time was reduced from 87 mins to

11 mins (an eightfold saving), while at the

same time maintaining equivalent resolution

of the same critical pair. Solvent consumption

was reduced from 87 mL per sample to 6

mL per sample — a considerable saving per

analysis.

This article was written by Dafydd Milton

and Luisa Pereira, Thermo Fisher Scientific,

Runcorn, Cheshire, UK.

31chrom doctorseparation science — volume 1 issue 2

TuTechnology

update

32 technology update www.sepscience.com

Key

Email the company

Product information

Applications

Additional Information

Preparative HPLC systemManufacturer: Gilson

Manufacturer’s description: The Gilson Preparative HPLC systems’s wide flow rate range (from

the 333 and 334 Pumps) allows for both semi-preparative and preparative separations. High

pumping power accommodates a wide range of preparative column sizes. All injection

modules feature a continuous flow path design. The system can accommodate large injection

volumes with minimal sample waste and carryover. The new rinse station system incorporates

a flowing jet wash and allows the use of up to two different rinse solvents.

System features include low dwell volume, which allows for fast, reproducible gradients, off-

bed collection racks, which allow for true large-scale fraction collection into vessels of choice

without sacrificing bed space, and incorporate Gilson’s System Organizers for stacking to

conserve bench space. In addition it has injection of up to 15 g per run depending on column

dimensions, the 30X Pump Series is configured as injection pumps and can inject volumes

up to several litres to maximize sample loading on preparative HPLC columns and separate

injection and collection pathways eliminate contamination of fractions from the injection

process.

The system’s solvent valve allows for immediate access of up to five off-bed solvent

reservoirs. The GX Direct Injection Module features an innovative injection port design that

attaches directly to the injection valve reducing void volumes and minimizing carryover

associated with calibrated connection tubing and offers various sample loop sizes.

33technology update separation science — volume 1 issue 2


www.sepscience.com

Dawn Heleos II

Manufacturer: Wyatt Technology

Manufacturer’s description: The Dawn Heleos II is an 18-angle light scattering detector for the measurement of

absolute molecular weight, size and conformation of macromolecules in solution. It may be used in batch mode (off -

line) or connected on-line to an HPLC/HPSEC/AFFF. The Dawn Heleos II utilizes a 120 mW solid-state laser operating

at 658 nm and also has on-board digital signal processing hardware for up to four external devices such as RI and/or

UV detectors. It also contains an analogue output from the 90° detector for interfacing to strip chart recorders. The

Dawn Heleos II has a 64,000 colour LCD display, thermostatic control options, depolarization options and the ability to

interface to high-temperature GPC systems such as the PL210 and Waters Alliance 2000. In addition it features

on-board analogue-to-digital conversion with ethernet communication for data acquisition.


Professional IC

Manufacturer: Metrohm

Manufacturer’s description: The 850 Professional IC is the fi rst professional

ion chromatography system with intelligent components that are optimally

matched to one another, Metrohm states. Intelligence in the newly

developed hardware of the 850 Professional IC include iPump, iDetector,

iDosino, MagIC Net software and Metrosep iColumns.

These are the intelligent system components that monitor and optimize

all functions and, if required, will also document them in an FDA-compatible

form. The confi guration of the system is simplifi ed, as the components log

themselves in automatically and provide the MagIC Net software with all the relevant

information. The optimal operation of the 850 Professional IC and results are monitored. If a parameter leaves

the predefi ned range then the user is informed by e-mail or SMS.

The 850 Professional IC is a complex and effi cient system that can still be operated simply and intuitively

with the MagIC Net software. This applies for both power users with complex applications and complicated

calculations and users who value ‘one-button operation’. Any user level is possible. Innovation, fl exibility and

extremely simple operation result from the intelligence of the 850 Professional IC, the 858 Professional Sample

Processor and the MagIC Net software, claims the company.

Keep up to date with the latest analytical methods and chromatographic applications in the Pharmaceutical, Environmental or Food

science Industries by taking a FREE subscription to the Separation Science Reports.

THE NEW SEPARATION SCIENCE REPORTS ARE NOW AVAILABLE...

October 2008



WWEM 2008 Preview

Featured Books

draw to win

November 2008

Food ReportMSPD-GC-ECD analysis of lindane residues

A novel sample treatment for the LC-MS detection of peanut protein in foods

Evaluating chondroitin sulfate in dietary supplements

Screening for mycotoxins with LC-MS/MS

Please turn the pages using the control panel below



1of 5

iPod Touch





October 2008

October 2008



WWEM 2008 Preview

Featured Books






October 2008


Clarus 600 GC/Mass Spectrometers

Manufacturer: Perkin Elmer

Manufacturer’s description: The Clarus 600 Gas Chromatograph/Mass Spectrometer

(GC/MS) features a wide mass range (1-1200 u) and excellent detection limits for

a quadrupole MS, according to the company. The system’s high-speed scanning

delivers improved peak integrity and accuracy. Its gas chromatograph has a rapid heat-up and cool-down oven in a

conventional GC and the multiple pumping options offer an instrument for every laboratory’s needs, the company

states.

In addition, the system is driven by sample-centric TurboMass software for ease-of-use from data collection to

evaluation and reporting. The combination of the Clarus 600 GC/MS with TurboMatrix sample handling, user-friendly

software allows for an integrated, complete analytical solution from a single source.

According to PerkinElmer, the rugged Clarus 600 MS is the fastest quadrupole mass spectrometer available, acquiring

more spectra (up to 65 scans/s and 12,500 amu/s) across a GC peak than other quadrupole systems. This feature allows

users to easily define and quantify extremely narrow chromatographic peaks, generate more accurate and precise

results with better spectral fidelity, run ‘fast GC’ with the Clarus 600 GC/MS, obtain fast scan speeds that enable fast GC

applications and achieve high productivity using fast GC/MS techniques.

PL-GPC 220

Manufacturer: Polymer Laboratories

Manufacturer’s description: The PL-GPC 220 integrated GPC/SEC system has been developed from the PL-GPC210

instrument. The PL-GPC 220 provides full PC control and unbeatable reproducibility for any GPC/SEC application,

across the entire operating range, according to Polymer Laboratories. The flexible PL-GPC 220 is designed to run

almost all polymer, solvent and temperature combinations, with full automation, from 30 to 220 °C.

When analysing polymers by gel permeation chromatography, elevated temperatures are often required to either

reduce the viscosity of certain solvents (e.g., dimethyl formamide) or to maintain the solubility of the samples under

investigation (e.g., polyethylene). Using higher temperatures seriously complicates the GPC experiment and places

several new requirements on instrumentation that must be fulfilled for a successful analysis. To this end, Polymer

Laboratories has developed the PL-GPC 120 and PL-GPC 220 integrated GPC instruments for the analysis of polymers

across the temperature range. These fully integrated systems contain many key features which facilitate their use for

even the most trying of applications.


Prominence UFLC XR

Manufacturer: Shimadzu

Manufacturer’s description: Following on from the Prominence UFLC, which offered high speed as well as high analysis

accuracy and reliability, Shimadzu has developed the Prominence UFLC XR, which has been designed to achieve

greater data quality through higher separation performance.

By using a high-speed separation column and optimizing the system, the company states it has increased

separation performance to a level greater than that of conventional LC.

The Prominence UFLC XR offers greater separation performance through column efficiency, improved by optimizing

the balance between the particle diameter of the XR-ODS high-speed separation column and the column length. In

addition, the system has been optimized (pressure resistance: 66 MPa) to attain the highest possible level of column

performance.

The combination of the Prominence UFLC XR and a Shim-pack XR-ODS II column (length: 150 mm) achieves a level

of separation performance equivalent to that of at least a 250 mm column containing packing material with a particle

diameter of 5 µm. The wide dynamic range of the SPD-20A detector enables the high-separation, high-sensitivity

detection of fine peaks, such as those obtained for minute quantities of impurities in pharmaceuticals. In addition, the

low-carryover SIL-20AXR autosampler supports high-accuracy analysis.

Trizaic UPLC

Manufacturer: Waters

Manufacturer’s description: The TRIZAIC UPLC Proteomics System

with nanoTile Technology allows users to spend less time on setup

and more time on science, according to Waters. The TRIZAIC UPLC

with its easy-to-use nanoTile Technology utilizes sub-2 µm particle

chemistries, ensures optimized chromatographic performance

without compromising data.

The system combines novel nanofluidic separation technology with

Waters chemistries, unique solvent delivery and comprehensive data

management. Waters states it is a powerful platform for sustained

use in sample-limited analyses through direct, non-split nano-flow,

resulting in low solvent usage. In addition it reduces user interaction

to one step, allowing scientists to build on their knowledge, because

data intelligence is built right into the tile.

separation science — volume 1 issue 2

ZIC-pHILIC

Manufacturer: SeQuant

Manufacturer’s description: The ZIC-pHILIC hydrophilic

interaction liquid chromatography column is suitable for

the separation of polar and hydrophilic compounds as it

is orthogonal to reversed-phase chromatography (RPLC)

columns. For example, compounds such as amino acids,

peptides, carbohydrates, plant extracts and various other

polar compounds that might have little or no retention in

RPLC, generally have strong retention on the ZIC-pHILIC

column, SeQuant states.

The columns have a zwitterionic stationary phase on

porous polymer particles and the separation is achieved

by a hydrophilic interaction mechanism superimposed

on weak electrostatic interactions. As a result of the

polymeric support particle, the column can be operated

in a broad pH range, which can be used to improve

the retention and selectivity for many compounds. In

addition, the detection sensitivity in LC/MS applications

might benefi t from the use of eluents in the alkaline pH

range.

The zwitterionic ZIC-pHILIC stationary phase is

currently available in analytical column dimensions (2.1

and 4.6 mm i.d.) with column lengths of 50 to 150 mm.

Each column is individually tested and a protocol is

supplied in the delivery together with an Instruction for

Use and a Getting Started Guide.

The zwitterionic ZIC-pHILIC stationary phase is also

available on silica support (ZIC-HILIC) and as material

for fl ash chromatography separations and solid phase

extraction cartridges (ZIC-HILIC SPE).

rationdriving analytical chemistry forwardssciencationdriving analytical chemistry forwardsscienc






information on commercial productdevelopments?



Rxi GC Column Series

Manufacturer: Restek

Manufacturer’s description: The Ultimate High Performance Fused Silica Capillary Column by Restek features excellent

inertness, ultra-low bleed and guaranteed column-to-column reproducibility, which is ideal with retention time-

locking software.

While GC analytical methods have continued to evolve, capillary column technology has been largely unchanged

over the last several years. Using new techniques for deactivation, stationary phase synthesis and coating, and tight

controls over column manufacturing, Restek believe they have set the benchmark for column performance with the

Rxi series.

Rxi columns improve chromatography for many acidic or basic compounds. Surface activity in a column is revealed

by the sensitivity and peak shapes for analytes such as 2,4-dinitrophenol (acidic) and pyridine (basic). Sub-nanogram

quantities of these compounds are a stringent test of inertness. Rxi columns’ level of inertness allows analysis of acidic

or basic compounds under the same conditions.

According to Restek, bleed from Rxi columns is extremely low, simplifying trace-level analysis with mass

spectrometric detectors (MSD, ion trap, etc.), electron capture detection (ECD), nitrogen-phosphorus detection (NPD),

or other sensitive detection methods. Columns from each of three manufacturing batches show the reproducibility

assured by the new manufacturing process.

In developing Rxi columns, Restek’s first step was to work with its fused-silica tubing supplier to establish rigorous

controls on internal diameter, outer diameter, ovality and surface activity. These controls guarantee that the tubing

is a known starting point. This uniform tubing is then treated with unique deactivation chemistry, producing a

consistent, inert surface on which to apply the polymer. Restek also reformulated the polymers, taking steps to ensure

neutrality and to fine-tune selectivity for retention time locking. A neutral polymer and a neutral tubing surface are

important contributors toward excellent peak shape for both acidic and basic compounds.


Tempo Nano MDLC system

Manufacturer: Applied Biosystems

Manufacturer’s description: The Tempo nano multi-dimensional LC (MDLC) system provides

high performance, reliable, multidimensional liquid chromatography for proteomic

applications, according to Applied Biosystems. The main features of the system include its

ability to maintain precise nano-scale flow rate control with microfluidic flow control (MFC)

and continuous, independent flow rate feedback for each mobile phase. In addition, it has

a simplified Tempo LC system setup and control with wizard-driven software, clear user

interfaces and easy-to-read screens. Its simple mechanical design requires less maintenance

and features wide flow ranges: first dimension 1-20 µL/min; second dimension 20-1000 nL/

min, as well as a high flow rate precision of less than 0.5% variation at 500 nL/min.

The Tempo nano MDLC system offers the added capability of MDLC with two independent

binary gradient pumps for high-pressure gradient formation, a 6-port sample loading valve

and a 10-port column-switching valve. The company states the MFC delivers precise, accurate

flow rates without flow splitting. The system also includes a low-dispersion autosampler with

biocompatible flow paths and Peltier temperature control.

The Tempo nano MDLC system provides nano-scale LC flexibility by combining all the

functionality of the Tempo nano LC system with additional micro-flow mode. This system

offers three modes of chromatographic separation: one-dimension, two-dimension and

parallel separations. The 2-dimension mode accommodates a wide range of flow rates for

sample clean-up, column loading and equilibration. An integrated 10-port column switching

valve accommodates two trap columns plus an analytical column for a wide range of multi-

dimensional applications and total automation.

36 technology update www.sepscience.comsepa on scienc





information on commercial product developments?


... then get your FREE subscription to


dsencon scaration

driving analylyl ticticti al chemistcal chemistc ral chemistral chemist y ry r fofof rwawaw rdsrdsrscienceenal chemistrry rsepa atioatioaratio

iPod Touch



1of 5

cecece

journal separation science

Documents