journal separation science

Volume 1 / Issue 5

May 2009www.sepscience.com

A liquid chromatographer’s introduction to mass spectrometry

Analysing synthetic polymers with solvent enhanced light scattering

Minimizing decomposition of components during GC analysis

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contentsVolume 1 / Issue 5

May 2009www.sepscience.com


Analysing synthetic polymers with solvent enhanced light scattering




Michal Holčapek

18

features


Volume 1 / Issue 5May 2009

30

research round-up

Packing procedures for high e� ciency, short ion-exchange columns

Quantifying low levels of polymorphic impurity in clopidogrel bisulphate by vibrational spectroscopy and chemometrics

Carbon nanotubes as the sorbent for integrating μ-solid phase extraction within the needle of a syringe

Determination of dissociation constants between polyelectrolytes and proteins by a� nity capillary electrophoresis

Shell and small particles; Evaluation of new column technology

Separation of catechins and methylxanthines in tea samples by capillary electrochromatography

Direct analysis of valsartan or candesartan in human plasma and urines by on-line solid phase extraction coupled to electrospray tandem mass spectrometry

Rr

Cd chrom doctor Guest author Jaap de Zeeuw discusses how to minimize decomposition of components during GC analysis.

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

regulars

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06

08

10

11

12

14

16

An

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application notes

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

34

38

24 Analysing synthetic polymers with solvent enhanced light scattering

Jean-Luc Brousseau and Wei Sen Wong

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

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scienti� c advisory

councilPeter Myers

– Chief Scienti� c O� cer

[email protected]

David Barrow

University of Cardi� , UK

Zongwei Cai

Hong Kong Baptist University

Yi Chen

Chinese Academy of Sciences,

Beijing, China

Gert Desmet

Vrije Universiteit Brussel, Belgium

C. Bor Fuh

National Chi Nan University, Taiwan

Y.S. Fung

Hong Kong University

Xindu Geng

Northwest University, Xi’an, China

Luigi Mondello

University of Messina, Italy

Paul Haddad

University of Tasmania, Australia

Hian Kee Lee

National University of Singapore,

Singapore

Melissa Hanna-Brown

P� zer, UK

Tuulia Hyötyläinen

University of Helsinki, Finland

Gongke Li

Sun Yat-Sen University, Guangzhou,

China

Yong-Chien Ling

National Tsing Hua University,

Taiwan

Klara Valko,

GSK, UK

Jean-Luc Veuthey

University of Geneva, Switzerland

Claudio Villani

Universita’ degli Studi di Roma “La

Sapienza”, Italy

Cheing- Tong Yan

Center of Environmental Safety and

Hygene, Taiwan

Edward Browne

GSK, Singapore

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FoodEnviroDay One:

Pat SandraAdvances in Separation Sciences Deriven by the Metabolomics and Pro-teomics Quest for Biomarkers

Y.S. FungMicrofluidic Chip-Capillary Electrophoresis for Biomedical Applications

Eric Chun Yong ChanGC×GC/TOFMS Profiling of Human Bladder Cancer

Manfred RaidaMultidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes

Yi ChenNew Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples

Andrew JennerGC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism

Thomas WalczykElement Separation at the Microscale for High-Precision Isotopic Analysis of Biological Samples

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Day Two:

Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations

Phil NethercoteThe applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.

Sanjay GargThe Role of Analytical Science and Techniques in Early Phase Drug Discov-ery and Registration for Clinical Studies

Anne GohOnline Solid Phase Extraction-LC-MS in DMPK Applications

Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D

Shawn StanleyTBC

Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines

Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine

Day Three:

Alastair LewisTrace Pollutant Detection in Challenging Environments

Hian-Kee LeeSolvent-Minimized Sample Preparation for Separation Science

Siu Kwan SzeAn Advanced Proteomic Approach to the Discovery of Microbial Enzymes for Biorefining

Gongke LiMolecularly Imprinted Polymers for Trace Analysis of Complicated Samples

Paul HaddadDevelopment of Portable Separation Methods for the Identification of Terrorist Explosives by Analysis of Inorganic Residues

Philip MarriottHeadspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples

Jessie TongMultidimensional Gas Chromatographic Analyses of Flavours and Fragrances

Bahruddin SaadDetermination of Biogenic Amines in Food: Conventional and Nonconventional Approaches

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RrResearchround-up

Packing procedures for high efficiency, short ion-exchange columnsAustraliaAn optimized packing procedure for the production of high efficiency, short, particle-packed

ion-exchange columns was reported by Professor Paul Haddad from the Australian Centre

for Research on Separation Science at the University of Tasmania in Australia, in the Journal

of Chromatography A [1208 (1-2), 95-100 (2008)]. Professor Haddad and his colleagues are

involved in counter-terrorism studies and the development of methods for the identification

of inorganic improvised explosives, also known as ‘fertiliser bombs.’ Analytical methods

are required for preblast identification in situations such as airport screening, and also for

postblast identification of explosives using analysis of residues left after the explosion. “We

have undertaken extensive studies on postblast analysis and our current focus is on preblast

analysis. In this application we are developing a two-pronged screening procedure. First, a

rapid analysis (20 s) will confirm the presence of a range of indicator ions known to be present

in improvised explosives. This will be followed by a slightly longer (3 min) confirmatory test,

which will identify the particular explosive present. This confirmatory test will be conducted

using ion chromatography (IC) and we, therefore, needed a short column that would provide

the required resolution in the desired time frame. This led us to study the packing of short

(e.g., 30 mm) columns,” Haddad explained.

According to him, the study revealed that normal slurry-packing procedures were not

applicable to very short columns because of inhomegeneities in the packed bed, leading

to variable (and usually poor) efficiencies. “However, we found that by joining a number of

short column segments together and then packing this assembly as a whole, we could use

the middle segments as high-efficiency short columns. In fact, these columns showed similar

efficiency behaviour to that exhibited by longer columns. Using this approach we were

able to make short columns that provided the desired separation for the counter-terrorism

project,” he added.

The team is now in the process of incorporating these short columns into portable

instrumentation in order to apply them in routine screening operations, such as airports,

which involves the simplification of the technique so it can be used reliably by unskilled users.

6 research round-up www.sepscience.com

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Quantifying low levels of polymorphic impurity in clopidogrel bisulphate by vibrational spectroscopy and chemometricsHungaryVibrational spectroscopic methods were developed for quantitative analysis of clopidogrel bisulphate in Form I and

Form II polymorphic mixtures and published in the Journal of Pharmaceutical and Biomedical Analysis [49 (1), 32-41

(2009)]. Results showed that both IR and Raman spectroscopy combined with chemometrics are suitable to quantify

low levels of Form II in Form I, down to 2 and 3%, respectively, with less than 1% limit of detection.

Zoltán Német from the Drug Polymorphism Research Division at the Gedeon Richter PLC in Budapest, Hungary,

explained the aim in performing this research was twofold. “First, detection of the stable form of clopidogrel

bisulphate in the metastable form of the substance, which is the developed product of our company, is a constraint

from quality assurance point of view,” said Német. It needs quantitative solid state method development to meet

this requirement. “Second, there is little knowledge about the relative advantages of different methods suitable for

quantitative determination of polymorphic mixtures of pharmaceutical solids in general. We intended to perform

a comparative study about the possibilities of infrared and Raman spectroscopy combined with chemometrics,” he

added.

The key findings of the study were, on the one hand, the limits of detection and quantitation of the developed

methods, which is considered good compared to results from similar studies, and also to those obtained before

by x-ray powder diffraction for the same polymorphic system. “On the other hand, it was shown that common

multivariate data handling methods give similar results, provided quality of the dataset is high. It was also shown

that general problem of quantitative solid state Raman spectroscopy can be overcome by appropriate sampling

procedure, for which we have developed a special sample holder accessory,” he said.

He believes the idea of the mentioned sampling procedure for the Raman technique can be useful for other research

groups dealing with similar studies. The obtained low limits of detection and quantification may encourage others

to spend time on method development, even if it seems completely hopeless based on univariate data handling.

“As for ourselves, we continue gathering the knowledge about the potential and limitations of quantitative phase

analysis with new polymorphic systems (additional publication in press at JPBA: doi:10.1016/j.jpba.2008.11.033),” he

concluded.


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Carbon nanotubes as the sorbent for integrating μ-solid phase extraction within the needle of a syringeUSAProfessor Somenath Mitra, Chair of Chemistry and

Environmental Science at the New Jersey Institute

of Technology, USA, recently reported on a study

that looked at the implementation of micro-solid-

phase extraction (μ-SPE) in the needle of a syringe for

integrating sampling, analyte enrichment and sample

introduction into a single device. Published in the Journal

of Chromatography A [1216 (12), 2274-2274 (2009)] both

single- and multi-walled carbon nanotubes (CNTs) were

explored as high performance sorbents for μ-SPE in

packed and self assembled formats. The need for such a

sorbent was critical because the needle probe could hold

only a small amount of material (around 300 μg).

“Micro-extraction techniques have been developing

rapidly over the past few years to overcome some of

the limitations of conventional techniques such as

liquid-liquid extraction (LLE) and solid-phase extraction

(SPE). Both LLE and SPE involve multi-step sample

extraction and clean-up procedures that are tedious,

time consuming and result in high levels of dilution. In

addition, these techniques consume substantial amounts

of organic solvents. The development of relatively

simple, fast sampling techniques that require a reduced

amount of solvents is of great importance and will allow

widespread monitoring of trace level contamination,”

said Professor Mitra.

An example of a functionally simple, yet effective

sampling or sample preparation device is solid-phase

micro extraction (SPME), which is an alternative to the

abovementioned methods. SPME relies on passive

equilibrium between the two phases, which leads

to relatively higher detection limits. “The technique

developed here performs the equivalent of SPE. The

sampling is active, i.e., the sample is drawn through the

sorbent in the syringe. The trapping efficiency is relatively

high, leading to lower detection limits. The device itself

performs sample extraction, concentration and sample

introduction into a single procedure,” Mitra explained.

He feels the study showed that since the needle of

a syringe can only hold a small amount of sorbent, it

was important to use a sorbent that has a very high

capacity. The other important issue would be efficient

desorption from the high capacity sorbent. “CNTs were

effective in this application. The main advantage of CNTs

compared conventional carbon sorbents is that they

non-porous, and the solute is held on the surface by

van der Walls type forces,” he explained. This eliminates

the mass transfer resistance related to the diffusion into

the pore structures. The large specific capacity comes

from the nano-scale size of CNTs, while fast desorption is

facilitated by reduced diffusion resistance.

“Derivatization of the nanotube surface can offer not

only a more hydrophilic surface structure, but also a large

number of oxygen-containing polar functional group,

such as, -COOH, -OH, -NO2, and -HSO3 which increases

the ion-exchange and hydrogen bonding capability of

the CNTs. There are numerous unexplored possibilities,”

he added.

In summary, he believes it is possible to implement

sophisticated µ-SPE in the needle of a syringe for easy

sampling, enrichment and injection and that novel

materials offer some unique opportunities. “Our group

has extensive activities in the area of carbon nanotubes,

spanning chromatography to solar cells and we are

excited about these possibilities. Within the separations

area, our work has covered chromatography, microtrap

for air monitoring, and developing an understanding of

these materials as adsorbents. Another application has

been the development of membrane incorporating CNTs.

We plan to continue on the material science as well as

the analytical application of CNTs,” he concluded.


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atlantis_halfpage.indd 1 5/8/09 10:08:47 AM11research round-upseparation science — volume 1 issue 5

Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis

Sweden

A paper in the Journal of Chromatography B [877 (10), 892-896 (2009)] reports on the binding affinity between two

model proteins, human serum albumin (HSA) and ribonuclease A (RNase A), and negatively charged polyelectrolytes,

two different heparin fractions and dextran sulfate, by means of partial filling and affinity capillary electrophoresis.

Main author, Professor Roland Isaksson from the School of Pure and Applied Natural Sciences at the University of

Kalmar in Sweden, explains the main aim of the research investigates the use of polyelectrolytes such as heparin,

dextrane sulphate, etc, in pharmaceutics to formulate peptide and protein based drugs.

“This project included long-term studies of protein polyelectrolyte mixtures, determinations of protein

polyelectrolyte affinities, chemical purities of both proteins and polyelectrolytes as well as sustained release of the

protein from the protein polyelectrolyte complex by use capillary electrophoresis,” Professor Isaksson said.

He believes the key findings of the study are that CE by means of partial filling techniques which mimic the

physiological conditions is a useful complement to other methods to study protein polyelectrolyte interactions. “The

affinity determinations with this technique are relatively simple to perform with only small amounts of (expensive)

proteins,” he said.

“In the future we will combine or use this CE technique as a complement with other methods such as circular

dichroism (CD), FTIR and microcalorimetry, etc. to carefully characterise the protein polyelectyrolyte formulations. Our

technique will also be adopted to follow the release of the drug from its polyelectrolyte complex,” he concluded.

Shell and small particles; Evaluation of new column technologyHungary

The performance of 5 cm long columns packed with shell particles was compared to totally porous sub-2 μm

particles in gradient and isocratic elution separations of hormones (dienogest, fi nasteride, gestodene, levonorgestrel,

estradiol, ethinylestradiol, noretistherone acetate, bicalutamide and tibolone) in a study puiblished in the Journal of

Pharmaceutical and Biomedical Analysis [49 (1), 64-71 (2009)].

“The approach of applying shell type particles in small diameter (2.7 μm) was realized in 2006. We were curious to

know the real (not theoretical) performance of this superfi cial phase under both isocratic and gradient conditions, and

we also wanted to know whether UPLC can be substituted with other techniques,” explained main author, Dr Szabolcs

Fekete from Formulation Development at Gedeon Richter Plc in Budapest, Hungary.

According to Dr Fekete, there are many theoretical assessments about the kinetic effi ciency (plate heights) of both

sub-2 μm totally porous and small shell particles but in this case, his team intended to estimate the time required for

the separation. This is why kinetic plot methods were used to compare the two approaches. “In practice, we mostly

use gradient separations in pharmaceutical applications and in this case the peak capacity is a more suitable measure

for effi ciency,” he added. Peak capacity curves were measured and compared to evaluate the performance of sub-2

μm porous and 2.7 μm shell particles when steep/fast gradient elution (5 – 25 minutes) was applied. Furthermore the

overloading of the column is a critical factor in practical work. “We wanted to evaluate the new column technology

also in this respect. For biological samples, rapid methods are needed for screening purposes or obtaining samples for

high-resolution mass spectrometer,” he said.

The study showed that superfi cial (shell) stationary phase off ers a high separation power with modest operating

pressure. “The performance achieved under both gradient and isocratic condition, is comparable to those obtained

with totally porous sub-2 μm particles. But it is necessary to emphasize that the performance of columns packed

with shell particles is not as high as the theory predicted earlier when high linear velocity (u > 0.3 cm/s) is applied,” he

said. For him, both UPLC and superfi cial phases are adequate tools for screening purposes. Using gradient elution, an

increased injection volume can be applied for sample enrichment in the inlet of the column.

Columns packed with shell particles are worthy of rivaling to any other fast liquid chromatographic techniques

without the requirement and adverse eff ects of ultra-high pressure. Conventional HPLC systems with slight

modifi cations can be applied for fast separations. “In the future we are going to introduce superfi cial phases for

everyday routine applications to achieve fast separations and to save time in method development. We also intend

to apply these columns for environmental analysis. Our researches are focused on micro-pollutants in plastics or in

packing materials and also pharmaceutical residues in drinking and surface water,” he concluded.



Separation of catechins and methylxanthines in tea samples by capillary electrochromatographyItaly

A paper in the the Journal of Separation Science [32 (7), 1002-1010 (2009)] documents the simultaneous separation

of several polyphenols such as (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, theophylline, caff eine in green and

black teas by capillary electrochromatography (CEC). Several experimental parameters such as stationary phase

type, mobile phase composition, buff er and pH, inner diameter of the columns, sample injection, were evaluated

to obtain the complete separation of the analysed compounds by Dr Zeineb Aturki from the Institute of Chemical

Methodologies at the National Council of Research in Rome, Italy.

“The research concerning the analysis of catechins in tea was developed following the aim of our project to analyse

polyphenols in several food matrices with miniaturized techniques including capillary electrochromatography (CEC)

and nano-liquid chromatography (Nano-LC). Our interest in those compounds is due to their nutritional properties

and benefi cial implications for human health. In addition quantifi cation of polyphenols provides useful informations

for food quality control,” explained Dr Aturki.

For him, the key fi ndings of the study include the development of analytical methods using miniaturized

techniques. “They off er several advantages such as high precision, accuracy, sensitivity, short analysis time, low

consumption of samples and reagents, easy coupling to mass spectrometry. For all these purposes, CEC and

Nano-LC can be used as an alternative or complementary technique to high performance liquid chromatography,”

Aturki added.

He believes the results achieved in this research have proved the potential of CEC technique and their future goal is

to determine these compounds by coupling this analytical technique to the mass spectrometer.

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Direct analysis of valsartan or candesartan in human plasma and urines by on-line solid phase extraction coupled to electrospray tandem mass spectrometry

France

As documented in the Journal of Chromatography B

[877 (10), 919-926 (2009)], a direct on-line solid phase

extraction coupled to tandem mass spectrometry was

developed and validated to determine valsartan (5–2000

ng/mL) or candesartan (1–200 ng/mL) in human plasma

and urines.

Lead researcher, Dr Alain Pruvost from CEA, iBiTecS,

Service de Pharmacologie et d’Immunoanalyse in

Gif-sur-Yvette, France, explained this work was initiated

by a well known clinical team ‘Hôpital Européen Georges

Pompidou’ in Paris which investigated what eff ect a

dietary salt intake had on the pharmacokinetics (PK) and

the pharmacodynamic (PD) eff ects of diff erent blockers

of the renin-angiotensin system (RAS) in normotensive

subjects. “As it is known for orally administered drugs

such as verapamil and quinidine, low salt intake can

increase systemic drug availability and consequently

aff ect their PD eff ects. But it was not known whether

such a phenomenon existed with RAS blockers. For that

purpose, we were asked to develop and validate the

most robust, precise, accurate, reliable analytical method

in order to allow revelation of the weakest diff erence in

PK parameters and bioavailability. Indeed, a diff erence in

bioavailability around 15 to 20% may be covered by the

variability of the method if too high. So, we elected a

LC-MS/MS technique to measure drugs in human

plasma,” Dr Pruvost said.

His goals were to develop an analytical method

presenting fi rst, a very short analysis total run time in

order to process a large number of samples in the least

variable conditions and second the least sensitive to

matrix eff ect (large diff erence in samples; many subjects

and many PK time points). “We naturally turned to ‘

on-line’ methods to develop a very fast method,

including sample clean-up. Moreover, we chose not to

use an analytical column which would lower the total

run time resulting in the development of a SPE-MS/

MS method without true analytical chromatographic

separation. To do so and to satisfy the second point

cited above, we were compelled to eliminate the

largest number of endogenous compounds present

in the matrix. For this purpose and because the drugs

were ionisable compounds, we used mixed mode

sorbent packed in a very short column (20 mm),”

Pruvost elaborated. This kind of SPE sorbent allows the

researcher to use hydrophobic and ionic interactions to

retain compounds of interest and allow the use of strong

organic solvent to ‘wash off ’ the sample matrix thus

eliminating a large quantity of unwanted compounds.

Among the key fi ndings, the team cites good effi ciency

of the combination of mixed mode sorbent such as

OASIS MAX (anion exchange) and strong organic solvent

like tetrahydrofuran (THF) for cleaning up samples.

“Indeed, since target compounds are hydrophobic

(log Po/w around 5) and acidic compounds, they are

successfully retained on the stationary phase by ionic

interactions. Moreover, use of THF allows the clean-

up of samples by eliminating lipids, and especially

phospholipids which are known to reduce signals in

electrospray ionization when they are co-eluted with

analytes (matrix eff ect). In these conditions, the method

allows us not to use an analytical chromatographic

column and results in a high total recovery,” he said.

He believes that these kinds of on-line analytical

methods are very useful and successful. “They present

precision, robustness and a very moderate cost per

sample. They also present the advantage of a reduced

manual processing of biological samples and show

satisfactory result dispersion with high throughput. But

they require a very good knowledge of the diff erent

elements of the analytical system when home-build,

and may seem sometimes too complex to use. It is

why, now, to face up to the success of these methods,

some suppliers propose much evolved and ready to use

systems, he concluded.


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18 feature article — MS for chromatographers www.sepscience.com

19feature article — MS for chromatographersseparation science — volume 1 issue 5

mass spectrometer. The sample

introduction system introduces

the solid, liquid or gas sample into

the high vacuum required for ion

analysis and detection, while ion

optics transfer, focus and accelerate

ions once in vacuum. And no mass

spectrometer would be complete

without a noisy vacuum system and

a computer to control the instrument

and provide data handling and

reporting.

Mass spectrometry coupled to chromatographyMass spectrometry alone is best

suited for the analysis of pure

compounds, because the whole

sample is introduced to the ion

source at once. In practice, the

analysis of more complex mixtures is

often required, so we need to couple

the mass spectrometer to some

separation technique.

A liquid chromatographer’s introduction to mass spectrometryMichal Holčapek

Professor of Analytical Chemistry, University of Pardubice, Czech Republic.

Mass spectrometry (MS) is used for the structural elucidation or confirmation of organic, bioorganic and

organometallic compounds, and for quantitative analysis in environmental, pharmaceutical, forensic, food and

other sciences. The first step in MS measurement is the conversion of neutral molecules to charged species (i.e.,

ions), which are then separated according to their mass-to-charge (m/z) ratio in a mass analyser. The relative

abundances of individual m/z values are recorded by a suitable detector to produce what is known as a mass

spectrum. MS can be coupled to both gas-phase and liquid-phase separation techniques, enabling the structural

analysis of complex mixtures after their chromatographic separation without time-consuming off-line isolation.

This article will also include a glossary of relevant MS terminology.

experimental proof of the existence

of electrons and charged particles.

The field of mass spectrometry

has garnered four Nobel Prizes

for chemistry and physics so far.

Nowadays, mass spectrometry

influences and supports research in

many fields of chemistry, biology,

medicine and physics.

Generally speaking, the mass

spectrometer consists of 3 main parts:

Ion source: The role of the ion

source is the conversion (ionization)

of neutral species into charged

particles (ions).

Mass analyser: The mass analyser

then separates the ions according to

their m/z values

Detector: The detector records the

relative abundances of individual

m/z values.

In addition to these basic

parts, several other parts are

essential to the function of the

Actual mass spectra are records of

relative ion abundances (expressed

as per cent) vs mass-to-charge ratio

(x-axis). The most abundant ion in

the spectrum is called the base peak

and is assigned a relative intensity

of 100%. The relative abundances of

other ions in the spectrum are then

normalized to the base peak (Figure 1).

Most of the ions carry just one

charge, so the m/z values correspond

directly to the masses of the

particular ions. In the case of some

ionization techniques (especially

electrospray ionization), ions may be

produced with multiple charges. As

a result, the observed m/z values are

diminished by the factor 1/z.

The current mass spectrometric

nomenclature recommends use of

the Thomson (Th) as a unit for m/z

values, in honor of J. J. Thomson,

who was awarded the Nobel Prize for

physics in 1906 for the discovery and


suppression eff ects. Contamination

and suppression eff ects can also be

reduced by using orthogonal ion

source geometry, the standard for

current LC-MS systems.

How can ion suppression infl uence your spectrum?

The analyte may give a diff erent MS

response in a mobile phase without

ionic additives in comparison with

the identical system containing

ionic and/or non-volatile additives,

such as phosphate buff er. The

suppression occurs in the ionizer

when ionic species in the mobile

phase successfully compete with the

analyte for charges at the droplet

surface, thus reducing the ionization

of target analytes. In addition

to ionic additives in the mobile

phase, sample matrix components

can also result in the suppression,

or sometimes enhancement of

response when they co-elute with

target analytes.

What to do with non-volatile compounds?

LC-MS coupling has only one serious

In the past, the target compound

had to be isolated fi rst and then

analysed with a direct insertion

probe, a time-consuming process

that also made the analysis of trace

impurities diffi cult. During the

last several decades, the coupling

of gas chromatography (GC) to

electron ionization (EI) and chemical

ionization (CI) sources has made

MS a routine technique. GC-MS is

used for the analysis of complex

mixtures of gas-phase compounds,

which limits the range of analytes

to relatively volatile, non-polar

molecules .

To prevent extensive fragmentation,

soft ionization techniques are used.

Initially, LC-MS also relied on EI or CI,

but because of the limited sensitivity

and robustness of such devices,

intensive research brought new soft

ionization techniques, which are

ideally suited for LC-MS coupling

because they combine multiple

functions into one step, including:

the interface between the column

and MS system (sample transfer into

gas phase), and sample ionization.

Nowadays, the coupling of HPLC

and MS in analytical laboratories

is very common. MS is compatible

with the whole range of analytical

fl ow rates (from nL/min to 2 mL/min)

and the mobile phase composition

(except for normal phase eluents

without a polar modifi er), thanks to

the innovative ionizers developed

in the last few decades. The

main remaining limitation lies in

the choice of chromatographic

conditions, especially the selection

of buff ers and additives. Non-volatile

additives (e.g., phosphate buff ers,

tetraalkylammonium ion-pairing

agents, etc.) should be replaced

by more volatile analogues (e.g.,

ammonium acetate or formate,

formic or acetic acid, ammonia,

tri- or dialkylammonium acetate,

etc.), which should be used at the

lowest possible concentration

(usually 5-10 mmol/L at maximum)

in order to avoid contamination

of the mass spectrometer or ion

Figure 1

Figure 1: Electron ionization mass spectrum of benzophenone.

Figure 2

© CHROMEDIATo vacuum system

DetectorSource Mass

analyser(sanalyser(s)Inlet

Ions

Datasystem

Figure 2: General schematic of a mass spectrometer.


limitation concerning the choice of

mobile phase composition, which

is the use of non-volatile inorganic

buff ers and additives, such as

phosphate buff ers, inorganic acids,

non-volatile ion-pairing agents,

cyclodextrins, etc. When the HPLC

method containing such reagents

is converted to LC-MS, non-volatile

additives should be substituted by

more volatile additives.

How can this be achieved without the loss of chromatographic performance?

LC-MS coupling places only one

serious constraint on HPLC mobile

phase composition, ruling out the

use of non-volatile inorganic buff ers

and additives, including phosphate

buff ers, inorganic acids, non-volatile

ion-pairing agents, cyclodextrins, etc.

When an HPLC method containing

such reagents is adapted for LC-MS

coupling, non-volatile additives

should be substituted by more

volatile additives at the lowest

possible concentration, for example:

• Ammonium acetate or formate

(usually up to 5 mmol/l), formic

or acetic acid (up to 0.1%, in some

special cases a little bit more)

• Ammonium hydroxide for basic

pH values (up to 0.1%).

• For analyses employing ion-pairing

reagents, LC-MS is compatible

with di- or trialkylammonium

acetates or formates (for cationic

analytes) at or perfl uorocarboxylic

acids (for anionic analytes), all of

which can be used at

concentrations up to 3mmol/L.

Tandem mass spectrometry (MS/MS)The advantage of so called soft

ionization techniques arises from

the fact that they primarily produce

protonated or deprotonated

molecules and relatively few

fragment ions (thus the term ‘soft’),

which makes the molecular weight

(MW) determination relatively

simple. At the same time, the

absence of fragment ions may be

considered a disadvantage for

structure elucidation because MW

information alone is not suffi cient

to tease out molecular structure.

This drawback may be overcome by

using tandem mass spectrometry

(MS/MS), whereby the fi rst mass

analyser is used for the isolation of

a selected precursor ion (previously

called a ‘parent ion’), which is then

fragmented to give product ions

(originally knows as ‘daughter ions’)

for subsequent MS analysis (Figure 3).

In this manner, we can obtain

information on the sub-structure of

each precursor ion, which should

represent a portion of the molecule.

This process may be repeated for

several precursor ions, typically

using triple quadrupole or ion trap

analysers.

The ion trap analyser

allows repeated isolation and

fragmentation steps, producing

fragments of fragments of fragments,

a technique known as multistage

tandem mass spectrometry (MSn)

(Figure 4). This is valuable for

studying fragmentation paths,

as well as confi rming molecular

structures.

In-source collision induced dissociation (CID) and collision induced dissociation?In-source CID does not enable

precursor ion isolation, so all the

ions present in the ion source at

a given time are fragmented as a

group without prior isolation, which

is feasible with true tandem mass

spectrometry. Another diff erence

is that in-source CID occurs during

the ionization process in the ion

source, while CID in MS/MS occurs

in the ion trap or in the collision

cell, in the case of QqQ (triple

quadrupole) or QqTOF instruments.

The absence of the isolation step for

in-source CID may not be of concern

if chromatographic resolution is

adequate.

Glossary of basic termsAtmospheric pressure chemical

ionization (APCI) = soft ionization

technique typically used for LC-MS

coupling and the analysis of small

organic molecules with low to

medium polarity.

Atmospheric pressure

photoionization (APPI) = soft

Figure 3

© CHROMEDIA

To detectorFromsource

MS1 MS2Collisioncell

Figure 3: Tandem mass spectrometry principle.


ionization technique, nearly

identical applications as for APCI,

but it extends the polarity range

slightly towards non-polar or very

labile molecules.

Base peak = peak with the highest

abundance in the spectrum. Its

relative abundance is set to 100%,

relative abundances of other peaks

in the spectrum are related to the

base peak and fall in the range

0-100%.

Chemical ionization (CI) = fi rst soft

ionization technique, used mainly in

GC-MS.

Dalton = a unit of molecular

weight frequently used in mass

spectrometry.

Deprotonated molecule = even-

electron ion [M-H]- with an m/z

ratio one mass unit lower than the

molecular weight. This is typically

the base peak in negative-ion mass

spectra taken with soft ionization

techniques.

Electron ionization (EI) = fi rst

ionization technique generally used

for GC-MS coupling. It is sometimes

referred to as a ‘hard’ ionization

technique because ionized species

can obtain large amounts of internal

energy, which leads to extensive

fragmentation and the complete

absence of the molecular ion

for about 10% of all the volatile

compounds amenable to EI.

Electrospray ionization (ESI) =

the softest ionization technique,

especially useful for polar to ionic

compounds, biopolymers, non-

covalent complexes or any extremely

labile compounds.

Elemental composition = sum

of individual atoms present in

particular ion or molecule.

Fragmentation = process whereby

the ion is cleaved into smaller parts

called fragments.

Exact mass = precise mass of a

particular ion calculated to at least

four decimal places, taking into

account the number of electrons,

used to extract molecular formula

information from highly accurate

and precise mass spectra.

Ion cyclotron resonance (ICR) = very

precise mass analyser requiring

Fourier Transformation of its detector

signal to provide the highest

available resolution and mass

accuracy among mass analysers, but

at the expense of the most rigorous

vacuum requirements and high cost.

Ion trap (IT) = a relatively recent type

of mass analyser capable of iterative

tandem mass spectrometry.

Ionization = the process of

converting a neutral molecule into a

charged species (ion).

Magnetic sector analyser = the

oldest type of mass analyser. Ions

are separated because diff erent m/z

values result in diff erent trajectories

through the magnetic fi eld. Typically

used in series with an electrostatic

analyser to increases resolution.

Mass accuracy = the diff erence

between theoretical and measured

m/z values, reported as ppm.

Mass accuracy better than 5 ppm

is generally considered the

minimum necessary for exact mass

determination, which can allow

determination of the elemental mass

composition.

Mass-to-charge (m/z) = the

quantity graphed on the x-axis of

a mass spectrum. Determines the

interaction of the ion with magnetic

and electrical fi elds, a fact that is

exploited in ion analysers.

Matrix-assisted laser desorption/

ionization (MALDI) = soft desorption

ionization technique not frequently

coupled to separations. Very useful

for biopolymers and synthetic

polymers with high molecular

weights. Sometimes coupled to

HPLC off -line via fraction collection.

Molecular ion = odd-electron ion,

it may be M+. in positive-ion or –. in

negative-ion mode.

Molecular weight (MW) = sum

of masses of the most abundant

isotopes for each atom in

Figure 4

© CHROMEDIA

Ion source

Ionization

Mass spectrum MS/MS spectrum

Analyzer 1

Analyzer 2

Collision induced

dissociation

Fragmentation

Detector

848284

767472

20

40

60

78

Figure 4: Tandem MS allows repeat isolation and fragmentation.


the molecule. Note that MW

determination using the most

abundant isotopes (used in MS)

diff ers from MW determination

on the basis of averaged isotopic

masses (used in all fi elds of chemistry

except for MS. For example, a

mass spectrometrist should count

bromine as 79 (because 79Br is the

most abundant isotope) rather than

80 (average of 79Br and 81Br isotopes

in the ratio approximately 1:1)

Nominal mass = the integer value of

a particular ion calculated from the

most abundant natural isotopes.

Orbitrap = the newest type of FT

mass analyser introduced in 2005, it

provides high resolution and high

mass accuracy by detecting the

oscillation of ions in an electric fi eld.

Protonated molecule = even-

electron ion with m/z value higher

than the molecular weight by one

mass unit, the [M+H]+ ion is typically

the base peak in positive-ion

mass spectra generated with soft

ionization techniques.

Quadrupole analyser (Q) = low

resolution mass analyser commonly

coupled to chromatography.

Resolution = A measure of the

mass spectrometer’s ability to

distinguish (separate) two adjacent

spectral peaks. There are two basic

defi nitions: (1) the mass of the target

peak divided by the diff erence

between two neighbouring peaks

with the same heights and 10%

valley overlap (R10% valley); (2) the mass

of target peak is divided by the

peak width at the half height of this

peak (RFWHM). The second defi nition

is more widespread and is generally

accepted nowadays, although the

10% valley defi nition is still common

for magnetic sector instruments.

Roughly, RFWHM is approximately half

of R10% valley.

Soft ionization techniques = a group

of ionization techniques with the

common feature that the molecular

ion or deprotonated molecule

usually correspond to the base peak

of mass spectra with the lack or low

abundances of fragment ions.

Tandem mass spectrometry (MS/

MS) = coupling of two or more

analysers (both ion traps and ion

cyclotrons can actually achieve MS/

MS with at single analyser), used

for the isolation of precursor ion, its

subsequent fragmentation, and the

detection of their product ions.

Thomson (Th) = unit for m/z recently

proposed by mass spectrometric

nomenclature.

Time-of-fl ight (TOF) = mass analyser

based on the precise measurement

of the fl ight times of ions accelerated

by an electric fi eld.

Triple quadrupole (QqQ) = tandem

mass analyser consisting of

three quadrupole rods. The fi rst

quadrupole is used for precursor

ion selection, the second one serves

as a collision cell for fragmenting

precursors into product ions, which

are then analysed by the third

quadrupole.

This article was written by Michal

Holčapek, Professor of Analytical

Chemistry, University of Pardubice,

Czech Republic.

Publication of this article was made

possible through collaboration with

Chromedia.

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Recommended Further ReadingClick titles for more information

The Mass Spectrometry Topic Circle

Analysis of PAH’s in foods

LCMS of pesticides

LCMS of lipids

24 feature article — Solvent enhanced light scattering www.sepscience.com

Analysing synthetic polymers with solvent enhanced light scatteringJean-Luc Brousseau and Wei Sen Wong

Viscotek (A Malvern Company)

Solvent enhanced light scattering (SELS) is a gel permeation

chromatography (GPC) technique for the analysis of synthetic polymers.

With SELS two di� erent solvents are used – one to dissolve the polymer,

the other to act as the eluent – allowing users to select the most

appropriate solvent for each function. SELS is particularly valuable for

‘invisible’ polymers, materials with a refractive index close to that of the

solvent used for their analysis.

25feature article — Solvent enhanced light scatteringseparation science — volume 1 issue 5

GPC the polymer or polymer blend

is dissolved in a solvent and then

injected into a fl owing system.

Eluent carries the sample through

a column of porous material,

such as polystyrene gels or silica,

which separates on the basis of

hydrodynamic radius or volume.

Larger molecules fi t into fewer pores

in the packing material and therefore

elute more rapidly than smaller ones.

Detectors at the exit of the column

analyse the resultant size fractions.

temperatures, the melt temperature

of the polymer, for example, and

variables such as stiff ness, strength,

toughness and viscoelasticity, which

determine commercial usefulness

and value.

Gel permeation chromatography

(GPC) is a well-established technique

for the determination of both

molecular weight and molecular

weight distribution, and is widely

used for both macromolecular

research and quality control. With

Synthetic polymers are widely

manufactured to produce a vast

array of items. Containers for food

and pharmaceuticals, furniture, car

parts and clothing are just a few

examples of the many products

routinely made. For all polymers,

molecular weight and molecular

weight distribution are critical

parameters because they determine

the physical and mechanical

properties of the material. Molecular

weight infl uences transition


produce an overall improvement in

the analytical method. The technique

is particularly eff ective for addressing

the following issues:

• Lack of sensitivity with respect to

refractive index i.e. the refractive

index of the polymer and pure

solvent are similar.

• Cost and SHE concerns connected

with the use of a particular eluent.

• The need for high temperature

GPC.

When performing SELS the eluent

no longer needs to solubilize the

polymer; it must, however, support

the polymer solution. In terms of

detection, the polymer is measured

in the eluent and not in the solvent

in which it was initially dissolved,

thus simplifying application of

the technique. Figure 1 indicates

why this is the case, showing how

larger polymer molecules elute

fi rst, followed by smaller polymer

molecules, and then fi nally the

solvent used to solubilize the

polymer.

Column choice is particularly

important for

SELS because

the column must

tolerate diff erent

solvents and rapid

solvent change without

sustaining any damage.

Increasing measurement sensitivityAs ‘like dissolves like’, it is not

uncommon for the refractive index

of a polymer to be close to that of

the solvent used to dissolve it, which

with traditional GPC will also be the

eluent. When this is the case, dn/

dc (the rate of change of refractive

When a GPC system is calibrated

specifi cally with the same polymer

being analysed, then concentration

detection alone is suffi cient, because

the calibration process defi nes

the relationship between size and

molecular weight. Polymers that

elute at a certain time have a known

molecular weight. The simplest GPCs

have only diff erential refractometer

detectors. These determine the

concentration of polymer present

using the diff erence

in refractive index (RI)

between the eluting

fraction and pure

solvent. However, there

are standards for only a

handful of polymers, polystyrene

being the most widely used. Most

polymers are not available as

calibration standards so the GPC

reports relative rather than ‘absolute’

molecular weight, typically a

‘polystyrene equivalent molecular

weight’. With these systems the

relative molecular weight reported is

correct only if the calibration and the

unknown polymers have the same

density.

More sophisticated GPC systems

have triple or tetra detection

that includes UV, light scattering

and viscometry detectors. Such

systems off er an alternative option

for concentration determination

(UV) and direct absolute molecular

weight and viscosity measurement.

Column calibration is not required

for accurate molecular weight

determination.

The SELS conceptWith traditional GPC the solvent

used to solubilize the polymer is the

same solvent used as the eluent.

SELS, in contrast, involves the use of

diff erent solvents for each function.

Consequently the optimum solvent

can be selected for dissolution of the

polymer or elution of the sample, to

Figure 1

Figure 1: Schematic of SELS where a polymer is injected in solution (blue) but the polymer is measured in a di� erent liquid, the eluent (green).

“SELS involves the use of two di� erent solvents: one to solubilize

the polymer and another to act as the eluent”

27feature article — Solvent enhanced light scatteringseparation science — volume 1 issue 5

index (n) with concentration (c)) is

low, making it more diffi cult for a

RI detector to accurately determine

polymer concentration because the

detector signal is low and the signal-

to-noise ratio poor. This has a direct

impact on the quality of measured

data. With triple and tetra detection,

both light scattering and viscometry

detectors may use the concentration

data provided by the RI detector to

determine molecular weight. For a

light scattering detector, scattering

intensity is proportional to the

square of dn/dc, so a low value

will result in a very poor signal; an

increase in dn/dc from 0.01 to 0.03,

for instance, will increase signal

strength ninefold. So, a low dn/dc

compromises the performance of RI

detectors alone, and also advanced

detection systems, particularly those

incorporating a light scattering

detector.

With SELS it is possible to select an

eluent that will give a better contrast

dn/dc than the solvent, even if this

eluent is not a good solvent for the

polymer. It is important to re-iterate

at this point that the polymer will

be detected in the eluent, rather

than the solubilizing solvent,

which is why this approach works.

Figure 2 shows the eff ect of using

SELS in the analysis of polymethyl

trifl uoromethacrylate (PMTFMA).

PMTFMA is soluble in

tetrahydrofuran (THF) but this

system has a low dn/dc value.

PMTFMA is not soluble in acetone

but results show that THF and

acetone can be used successfully

in combination. Solubilizing the

PMTFMA in THF and using acetone

as the eluent triples dn/dc, from

0.03 to 0.09. For the triple detection

system used here this has the

expected benefi cial impact on light

scattering intensity, improving

both the precision and accuracy of

measurement.

SELS has also been used to

measure the molecular weight of

polyhydroxyalkanoate, a synthetic

bioplastic which is soluble in

chloroform. In this case chloroform

was retained as the solubilizing

solvent but THF was used as an

eluent, producing a fourfold increase

in dn/dc. A corresponding increase

in measurement sensitivity was

observed.

Figure 2

Figure 2: Low Angle Light Scattering (LALS) chromatogram for PMTFMA in THF. The black curve is the LALS signal when the solution is injected in a GPC running THF. The green curve is the LALS signal when the same solution is injected in a GPC system running with acetone as the eluent. Columns used were I-Series (Viscotek, a Malvern company), � ow rate 1.0 mL/min.

www.shodex.de

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instrument, columns and operator,

and is best avoided if alternative

options are available. Olefin rubbers,

for example, are typically analysed

in trichlorobenzene (TCB), a solvent

with recognized SHE issues, at

elevated temperature. Using SELS it

is possible to effect the same analysis

at a much lower temperature (45 oC)

by dissolving the polymer in xylene

and using THF as an eluent.

Conclusion

Solvent enhanced light scattering

is a GPC technique that decouples

the choice of solubilizing solvent

and eluent, by employing a different

solvent for each function. The

introduction of columns that tolerate

multiple solvents and/or a rapid

change in solvent will encourage its

use.

SELS is particularly valuable when

the refractive index of the polymer

and solvent are very similar, a

situation that can make both RI and

light scattering detection difficult.

With SELS, an eluent can be selected

to increase the contrast between

polymer and solvent refractive

index, enhancing signal quality, and

therefore measurement accuracy.

The tailoring of solvent systems is

also valuable for the development

of GPC methods that operate at

lower temperature or which use less

expensive or more benign solvents.

Jean-Luc Brousseau joined Malvern

Instruments in 2007 to work with the

Zetasizer Nano system and is now

a specialist for separation systems.

Jean-Luc received his PhD from

the Université du Québec à Trois-

Rivièeres in 1999 having conducted

research at the University of Miami

on macromolecules and sol-gel.

His post-doctoral work at Tulane

University was on light scattering of

polymers and novel characterization

of polymerization reactions.

Wei Sen Wong has been with

Viscotek, a Malvern company, since

1997 and is manager of analytical

services. He received his PhD in 1978

from Northeastern University under

Professor Barry Karger, and then joined

Shell Development Company where

he headed up the GPC laboratory for

more than 18 years.

Solvent benefitsWith traditional GPC, a solvent that

successfully dissolves the polymer

is used as the eluent, even if this

solvent is costly and/or potentially

hazardous. The amount of solvent

used to dissolve the polymer is just

a few millilitres whereas the eluent

in a GPC system can be several litres

per day. With SELS, the most suitable

solvent can be selected to dissolve

the polymer, and an alternative

solvent, less expensive or giving rise

to fewer SHE concerns, can be used

as the eluent. This is an effective way

of reducing the cost and/or hazard

associated with analysis and solvent

disposal.

For example, hexafluoroisopropanol

(HFIP) is a fluorinated solvent

commonly used to solubilize organic

polymers such as polyamides,

polyacrylonitriles, polyacetals,

polyesters, and polyketones. HFIP

is corrosive and gives rise to safety

concerns. It is also more expensive

than most commonly used GPC

solvents. Experimental work has

shown that with SELS it is possible to

dissolve certain polymers in HFIP, but

use THF or chloroform as the eluent.

The resulting minimization of HFIP

usage reduces potential hazard and

cost.

Reducing GPC running temperatureSome polymers are only sparingly

soluble at room temperature in

the solvents that can be used as an

eluent, in which case it becomes

necessary to operate the GPC

system at significantly elevated

temperature. High-temperature GPC

is not an easy analytical solution

because of higher demands on the

CdThe Chrom

Doctor


Gas chromatography is performed under

conditions where the components to

separated are in the gas phase. We use a

temperature-controlled oven to heat the

column to evaporate components with

higher boiling points. These ovens are

typically used up to 450 °C, which allows

analysis of components with boiling points

of up to 700 °C.

However, not all components are stable

when heated and decomposition can occur.

This can happen when the component

is evaporated during the injection step,

or it can happen when the component is

‘traveling’ through the capillary column.

Decomposition and peak shapeIf a component is thermally labile, or reactive,

we can expect an non-reproducible and

lower response for that component.

In most cases the component decomposes

into a ‘smaller’ product. If the decomposition

happens inside the injector, the response of

the component will be lower, and we will see

sharp decomposition peaks. We can change

injection conditions to minimize this effect.

If the decomposition happens while the

component is traveling through the column

we see a strong ‘leading’ peak (Figure 1).

Sometimes we see in our chromatogram a peak shape that we know is not ‘normal.’ Last month we discussed the overloading phenomena, which directly impacts peak shape. In this instalment we will again look at peak shape, but from a different perspective. If a component is not thermally stable, the peak shape and size may be a good indicator. There are several actions we can take if we observe the phenomena, but we need to recognize it first.

Figure 1

Figure 1: Example of decomposition during chromatographic separation.

The ‘lead’ of the peak is formed by the

decomposition products, as they elute faster.

As these products are formed during the

time the component is inside the column,

these products will not elute as a peak, but

as an elevated baseline.

Components that are known for

thermolability are pesticides (e.g., DDT,

carbamates etc.), and brominated diphenyl

ethers (e.g., flame retardants). Sometimes

unsaturated compounds, such as propadiene

and pentadienes decompose on activated

alumina surfaces.

30 chrom doctor www.sepscience.com

Reduction of component breakdownThe decomposition reaction is strongly

temperature-dependent. Practically, we need

to do the analysis at the lowest possible

thermal stress, meaning creating optimal

conditions for injection port temperature

and elution temperatures while performing

the GC separation.

Injection: Using evaporating injection

systems is always very challenging as the

component is exposed to high temperature

and will decompose. In splitted injection,

the injection takes a fraction of a second,

which usually is not a problem. With splitless

injection, the sample is initially exposed to

high injection port temperature. During

this time, interactions can take place and

components will decompose. Figure 2(a)

shows an example of what can happen

with carbamates when they are introduced

via splitless injection. The carbamates are

broken down into their phenolic esters.

These compounds will elute as sharp peaks

as they are focused on the column.

Figure 2(b) shows the same analysis

using on-column injection. Because of the

absence of thermal stress during injection,

the carbamates are injected onto the column

without decomposition.

If on-column is not an option and splitless

injection is to be used make sure that:

• the lowest possible injection port

temperature is used

• the highest possible flow rate (use

0.32 mm columns) is used

• a pressure pulse is used

• inert liners (siltek or siloxane-deactivated)

are used

• care is taken with glass wool packings as

these may initiate decomposition

This way we can minimize thermal

stress. An alternative injection technique

to consider is ‘programmed temperature

injection’ or PTV. Here the sample is

introduced into a cold liner, and flash

evaporated when the injector is heated.

PTV is not as good as the cold-on-column

method, but better than the splitless

technique.

The capillary separation column: Once

the sample is injected into the column, the

component must pass the whole column

and during this process decomposition

can occur. This decomposition is directly

dependent on temperature, but also on

column activity. If the column is not properly

deactivated, component breakdown will be

much higher.

Figure 2

Figure 2: Impact of injection technique on decomposition of carbamates: (a) = hot splitless, (b) = cold on-column; Peaks: 1 = bendiocarb, 2 = dimethoate, 3 = aminocarb, 4 = dioxacarb, 5 = carbaryl. (Ref: J. of HRC., Vol 13, nov.1990, p. 759.)

Figure 3

Figure 3 : Analysis of BDE according to EPA 1614 using the following EPA protocol: (a) using column as listed in method; (b) equivalent column, but with different deactivation. Both columns under exact similar conditions.

31chrom doctorseparation science — volume 1 issue 5

BDE or ‘flame-retardants’ are brominated

diphenyl ethers designed to be thermally

unstable, so they will act better as flame

retardants. GC analysis will be a challenge,

but it is possible.

Figure 3 shows the analysis of BDE-209

using the EPA 1614 methodology in which

the impact of deactivation on peak response

is shown. Using exactly similar conditions,

the well deactivated column produces less

degradation. Column inertness plays a role.

Even with well deactivated columns,

degradation still occurs as confirmed by

the ‘lead’ on DME-209. The key to setting

methods for thermolabile components is to

reduce the elution temperature.

Figure 4 shows the same column as in

Figure 3, but now the final temperature

does not exceed 295 °C. Consequently,

the decomposition of DBE-209 is greatly

reduced. Figure 5 shows an expansion of the

problem area.

Ways to reduce the elution temperatureThere are several ways to influence the

elution temperature. In the example of

Figure 5, the program did not exceed

295 °C. Typically, this will increase analysis

time as it will take longer to elute heavy

components.

Use higher flow rate, a flow program or a

pressure program: By doubling the optimal

flow rate, the elution temperatures will be

reduced by 20-25 °C. This is usually very

effective with non-MS detection systems. The

higher flow will cause some loss of efficiency,

Figure 5

Figure 5 : Expansion of problem area of BDE-209. Elution temperature has big impact on decomposition process.

Figure 4

Figure 4: Analysis of BDE using lower elution temperatures. Column: 30 m x 0.25 mm Rtx-1614, df = 0.1 μm; Oven: 120 °C (1 min) 295 °C (15 min) @ 15 °C/min; Injection: splitless; Carrier gas: He @ 2.5 mL/min constant flow

32 chrom doctor www.sepscience.com

so it may be a consideration to initiate the

pressure program after the key separations

are obtained.

Use a slower temperature program: By

using a slower temperature-programming

rate, components will elute at a lower

temperature. However, the downside of this

is longer analysis times and peak broadening

(lower response).

Use hydrogen, rather than helium, as the

carrier gas: Because of the higher optimal

flow rate, we can benefit from lower elution

temperatures, while working under optimal

conditions. Here, however, you must

deal with safety issues, which is another

discussion.

Use columns with thinner films: Elution

temperature is directly dependent on the

amount of stationary phase (film thickness).

Use a 0.10 μm film instead of a 0.25 μm one.

Use a 0.32 mm i.d. capillary: A 0.32 mm

capillary with 0.1 μm film will have higher

phase ratio, which results again in a lower

elution temperature. The 0.32 mm column,

however, will be lower in efficiency, so we

may lose some separation efficiency. If the

target components elute with sufficient

resolution from their neighbours, you can

also apply a pressure program. This is very

effective with 0.32 mm columns.

Use shorter columns: The absolute time

components are in the column should be

a short as possible. Shorter columns will,

therefore, give higher response, but will

have lower efficiency, which will impact on

resolution, similar to that discussed using

0.32 mm columns. If we take a 15 m column

instead of a 30 m one, resolution is only

impacted by a factor 1.4. Figure 6 shows

the separation of the BDE. The components

elute below 295 °C and the total time in

the column is now reduced by a factor of

2. To compensate for efficiency loss, one

can choose a smaller diameter column; for

example, a 20 m x 0.15 mm column will

generate the same efficiency as a 30 m x

0.25 mm one.

SummaryFor analysing thermally labile components,

the best injection technique is cold-on-

column. To minimize exposure to the high

temperature environment, we need to

use inert columns with a high phase ratio.

In addition, short columns are preferably

operated with high gas velocity and slow

temperature programming.

This article was written by Jaap de Zeeuw, a

specialist in gas chromatography working for

Restek Corp.

Figure 6

Figure 6: Fast analysis of DBEs using 15 m x 0.25 mm Rtx-1614 column. Shorter time at higher temperature will also result in reduction of decomposition.

33chrom doctorseparation science — volume 1 issue 5

AnApplication

notes

34 application notes www.sepscience.com

UHPLC – Resolution vs E� ciency

Company: Fortis Technologies

Summary: Fortis Technologies has published an application

note on the role of resolution vs e� ciency in UHPLC.

Previously, the increases that can be made from using

smaller particles in UHPLC, have been widely demonstrated,

including improved e� ciency leading to greater resolution,

sensitivity and speed of analysis. However the biggest gains

in resolution come from the use of selectivity. By having a range of phase chemistries

available the analyst can improve resolution which can then also lead to speed

increases. All of these factors are discussed in this current application note.

www.fortis-technologies.com Tel: +44 151 336 2266

Application Note

IntroductionThe current trend towards using high pres-sure in LC is well documented, high efficien-cies, good resolution and fast throughput being the goal that has driven the move towards the use of sub 2um particles.In previous work we have shown that for short fast gradients well packed 3um For-tis columns can provide equivalent, if not

more, peak capacity than other commer-cial sub 2um columns. In this poster we show that for those analysts already work-ing with ultra high pressure LC systems and 2um particle columns that it is im-portant to consider the role of stationary phase selectivity when trying to maximise resolution and not rely on efficiency alone.

Improving ResolutionApproaches to improving resolution involve making changes to one or more of three variables; efficiency, retention and selectiv-ity. The move towards using sub 2um par-ticles has been driven by the theory that

the resulting jump in efficiency will lead to significant improvements in resolution. As can be seen in the Carr Equation (figure 1) that efficiency (N) does play a significant part in improving resolution, however by far the greatest factor is column selectivity.

With new small particle columns being re-leased by column manufacturers it is important that a range of phase chemistries are offered to allow the analyst an opportunity to maxim-ise resolution rather than depending on effi-ciency alone. Also it should not be assumed that all commercial C18 products on the mar-ket have the same selectivity, therefore as well as evaluating new phase chemistries it might be wise to test some alternative C18’s.Figure 2 shows an example of where in-creasing efficiency by moving from 3um Fortis C18 to 2.1um Fortis C18 does not pro-vide the full resolution of two closely related

compounds. However by altering selectivity, using Fortis PhenylTM chemistry, at the same time as decreasing particle size we are able to obtain resolution whilst decreasing col-umn length and as a result analysis time.

Use of an alternative phase chemistry such as Phenyl can significantly reduce analysis time of a set of compounds whilst main-taining resolution of closely eluting peaks. By combining efficiency from small particles with selectivity from stationary phase chemis-tries much better resolving power is available, potentially in a much shorter period of time.

ConclusionThe use of small particles in Ultra High Pressure LC can provide the analyst with increased sensitivity and resolution. We have shown that an important considera-tion when trying to maximise resolution of closely eluting compounds is the role of selectivity. The application of alternative chemistries based on 2.1um particles can provide greater increases in resolution than the application of small particles alone.

mV

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3um Fortis C18 (150x2.1mm)

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SATIN

2.1um Fortis Phenyl (100x2.1mm)

Efficiency Selectivity

1. Isonicotinamide 2. Nicotinamide

3.5mins4mins

“it is important that a range of phase chemistries are of-fered to allow the analyst an opportunity to maximise res-olution rather than depending on efficiency alone”

UHPLC - Resolution vs Efficiency

Mobile Phase: 80:20 20mM NH4OAc Flow: 0.2ml/min Temp: 40°C Wavelength: 210nm

N

Efficiency SelectivityRetention

R= k’k’+1

-14N k’

k’+1k’

k’+1-1-

1.00 1.05 1.10 1.15 1.20 1.250.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5000 10000 15000 20000 25000

0 5 10 15 20 25

N

k

Nk

Res

olut

ion

(R)

1.00 1.05 1.10 1.15 1.20 1.250.0

0.5

1.0

1.5

2.0

2.5

3.0

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0 5 10 15 20 25 k

Automated SPE and Fast GC-ECD Analysis of PCBs in Waste Oil

Company: Gerstel

Summary: A fast SPE-GC-ECD method for the analysis of PCBs

in waste oil was developed. A complete pro� le was obtained

following SPE with a 12 minute GC run-time using a modular

accelerated column heater. Full automation of the sample

preparation and analysis (except sample weigh-in) enables

a daily throughput of 100 samples. A wide range of

concentrations can be determined using a dedicated column and electron capture

detection. This work describes the use of automated SPE with the GERSTEL MPS

3 autosampler with SPE option in combination with fast GC-ECD analysis for the

determination PCBs in waste oil.

Ap

pN

ote

6/2

008 Automated SPE and Fast GC-ECD

Analysis of PCBs in Waste Oil

Karine Jacq, Bart Tienpont, Frank DavidResearch Institute for Chromatography, Pres. Kennedypark 26, B-8500 Kortrijk, Belgium

KEYWORDSMACH, Fast GC-ECD, SPE, PCB, waste oil

ABSTRACTA fast SPE-GC-ECD method for the analysis of PCBs in waste oil was developed. A complete profi le was obtained following SPE with a 12 minute GC run-time using a modular accelerated column heater (MACH). Full automation of the sample preparation and analysis (except sample weigh-in) enables a daily throughput of 100 samples. A wide range of concentrations can be determined using a dedicated column and Electron Capture Detection (ECD).

INTRODUCTIONThe offi cial method for the analysis of PCBs in waste oil (DIN EN 61619) is time consuming and labor intensive (dilutions; extraction, column preparation and cleaning; manual solid phase extraction…) and it requires a long GC run (around 40 min).

Speed of analysis in capillary GC can be increased by using fast and ultra-fast temperature programming. In general, peak resolution will be reduced when the temperature gradient is very fast, but for several applications, some loss of resolution can be accepted. Recently, direct resistive heating of the capillary column resulting in very fast heating rates (> 1800 °C/min) has been introduced [1]. The system available via GERSTEL under the name Modular Accelarated Column Heater (MACH, GERSTEL GmbH, Mülheim an der Ruhr, Germany) is mounted onto the door of a standard GC holding up to four modules containing separate capillary columns

35application notes separation science — volume 1 issue 5

ZIC-HILIC Separation of Purines and Pyrimidines

Company: SeQuant

Summary: SeQuant o� ers an application note describing the hydrophilic

interaction liquid chromatography separation of the compounds thymine, uracil,

adenine, guanine and cytosine using a ZIC-HILIC, PEEK 150 x 2.1 mm, 5 μm, 200 Å

column. Chromatographic conditions are outlined, the resulting chromatogram

provided and retention factor and resolution calcualted for the purines and

pyrimidines.

Virus-Like Particle Characterization Using New AF4 Channel Technology

Company: Wyatt Technology

Summary: Virus-Like Particles (VLP) used for vaccination and immune

stimulation, are of growing interest in the pharmaceutical sciences. For quality

assurance there is a tremendous need for techniques that characterize di� erent

VLP fractions (fragments, monomers, dimers, trimers and aggregates). Wyatt has

previously demonstrated that the separation and subsequent quanti� cation of

di� erent VLP species is possible by AF4.

In this application note a stressed VLP sample was analysed by AF4 (equipped with multi-angle light

scattering and UV detection) by using either Wyatt’s standard channel (25 cm) or a smaller channel (18 cm)

with a spacer height of 350 μm.

DAWN®, miniDAWN®, ASTRA®, Optilab® and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation. ©2007 Wyatt Technology Corporation 9/12/07

Light Scattering for the Masses™

irus-Like Particles (VLP) used for vaccination and immune stimulation, are of growing interest in the pharmaceutical sciences. For quality assur-

ance there is a tremendous need for techniques that characterize different VLP fractions (fragments, mono-mers, dimers, trimers and aggregates). We have recently demonstrated that the separation and subsequent quan-tification of different VLP species is possible by AF4. However, some disadvantages, like long equilibration and analysis times, as well as the need for high sample amounts and large eluent volumes, been overcome by using new, shortened channel geometries.

A stressed VLP sample was analyzed by AF4 (equipped with multi-angle light scattering and UV detection) by using either Wyatt’s standard channel (25 cm) or a smaller channel (18 cm) with a spacer height of 350 µm.

Comparative AF4 measurements of VLPs with the standard channel (25 cm) and the new channel (18 cm) revealed almost similar peak heights when 20 µg VLP were injected in the standard channel or when 10 µg VLP were injected in the new channel, respectively (Figure 1). Increased peak heights obtained with the new channel are due to sharper peak resolutions. Thus, analysis is possible with significantly less sample amount (Figure 2). At the same time, analysis time and solvent volume were reduced (Table 1).

The standard channel technology has limitations concerning sample amount and separation time. By con-trast, applying Wyatt’s new channel technology analysis of far lower VLP amounts is possible in clearly shorter time and remarkably lower eluent volumes. Thus, it can be stated that the new channel technology is a clear improvement for VLP characterization as compared to the standard channel.

Virus-Like Particle Characterization Using New AF4 Channel Technology

Submitted June 21, 2007. This note graciously submitted by R. Lang and G. Winter,Ludwig Maximilians University, Department of Pharmacy, Pharma-ceutical Technology and Biopharmaceutics, 81377 Munich

Standard Channel (25 cm)

New Channel (18 cm)

time/run 56 mins 31 minseluent volume/run 159 mL 70 mLinjection amount 20 mg 2.5-10 mg

Table 1. Comparison between standard channel and shorter channel.

Figure 1. Comparison between standard channel and shorter channel.

Figure 2. Different injection amounts compared.

Rapid, High-Resolution, Normal-Phase Isocratic Chiral Separations

Company: Eksigent

Summary: Conventional carbon-centered enantiomericity has become a major

aspect of pharmaceutical drug development over the last twenty years. Although

enantiomeric drug forms have long been known to exist, attention to the relative

bioactivity of the enantiomers was often not addressed. More recently, drug

manufacturers have investigated the pharmacological pro� les of the individual

isomers, and in some cases, found that the bioactivity of the drug substance could

be wholly or substantially attributed to a single enantiomer.

0 1 2 3 4 5-100

0

100

200

300

400

500

600

700

800

900

Abs

orba

nce

(mA

U@

220n

m)

time (minutes)

fenoprofen

application note use of the expressLC™ system for chiral drug analysis

rapid, high resolution, normal phase isocratic chiral separations

introduction Conventional carbon-centered enantiomericity has become a major aspect of pharmaceutical drug development over the last twenty years. Although enantiomeric drug forms have long been known to exist, attention to the relative bioactivity of the enantiomers was often not addressed. More recently, drug manufacturers have investigated the pharmacological profiles of the individual isomers, and in some cases, found that the bioactivity of the drug substance could be wholly or substantially attributed to a single enantiomer.

figure 1. chromatogram of the two enantiomers of fenoprofen figure 2. chromatogram of the two enantiomers of thalidomide

0 2 4 6 8 10 12 14 16

0

50

100

150

200

250

300

350

Abo

rban

ce (m

AU

@22

0nm

)

time (minutes)

thalidomide

36 application notes www.sepscience.com

A Cryogen-free Method for Monitoring Trace Greenhouse Gases in Air

Company: Markes International

Summary: In response to the Kyoto Protocol, ‘Clean

Development Mechanism’ regulations are being enacted

in a number of countries to facilitate and control

greenhouse gas emission trading. Trace-level greenhouse

gases of interest include chloro� uorocarbons (CFCs) and

hydrochloro� uorocarbons (HCFCs). Improved methods for

monitoring many such compounds in air have recently been reported using sorbent

tube or canister-based air sampling methods together with thermal desorption (TD)-

GC/MS analysis per US EPA ‘air toxics’ Methods TO-17 and TO-15, respectively. This

note demonstrates detection limits below 100 ppt for all CFCs and HCFCs on the ‘air

toxic’ list using a Markes electrically cooled TD platform with GC/MS running in full

scan mode.

Introduction

In response to the Kyoto Protocol, ‘Clean

Development Mechanism’ (CDM) regulations

are being enacted in a number of countries to

facilitate and control greenhouse gas (GHG)

emission trading. Many of the new regulations

require the monitoring of bulk greenhouse

gases such as carbon dioxide and methane and

some require additional consideration of other

lower level and more analytically challenging

compounds. Examples of this include proposed

amendments to the European Emission Trading

Scheme Directive 2003/87/EC1 and Australia’s

recent government white paper on a low

pollution future2.

Trace-level greenhouse gases of interest

include chlorofluorocarbons (CFCs) and

hydrochlorofluorocarbons (HCFCs). Improved

methods for monitoring many such compounds

in air have recently been reported using

sorbent tube or canister-based air sampling

methods together with thermal desorption (TD)

- GC/MS analysis per US EPA ‘air toxics’

Methods TO-17 and TO-15 respectively (see

Markes TDTS Notes 81 and 86). This work

demonstrates detection limits below 100 ppt for

all CFCs and HCFCs on the ‘air toxic’ list using

a Markes electrically-cooled TD platform with

GC/MS running in full scan mode.

However, not all trace level green house gases

are included on the standard US EPA list of

target ‘air toxics’. Perfluorocarbons for example,

are a class of long lived greenhouse gases, the

most volatile of which, carbon tetrafluoride

(CF4), has a boiling point of -128°C. CF4 is

present in the atmosphere at very low

concentrations, but has more than 5,000 times

the ‘global warming potential’ (GWP) of CO2

and a half life in the atmosphere of many

thousands of years. The extreme volatility of

CF4 makes it very difficult to trap/concentrate

and measure at low levels. Similarly,

hexafluoroethane (C2F6), has a boiling point of

-78ºC and over 10,000 times the GWP of CO2.

Other analytically-challenging greenhouse

gases, which don’t appear on the air toxics list

include CF3Cl, nitrous oxide (N2O) and sulphur

hexafluoride (SF6) – see table 1.

Not all of these ultra-volatile GHGs are readily

available. CF3Cl, for example, is banned in

many countries and cannot be obtained as a

standard. It was therefore decided to evaluate

the applicability of the same cryogen-free TD-

GC/MS technology used for air toxics

monitoring (TDTS 81 and 86) for the most

challenging ultra-volatile GHG species (CF4,

C2F6, SF6 and N2O). If successful, this would

demonstrate that such a monitoring system

could be used for both ultra-volatile GHGs plus

higher boiling CFC & HCFC air toxics and, by

extrapolation, any compound in between.

T D T S Thermal Desorption Technical Support

Note 87: A cryogen-free method for monitoring trace

greenhouse gases in air

Key Words:

Trace green house gases, Kyoto Protocol, CFCs, HCFCs, UNITY 2, CIA 8, air toxics, GHG

ww

w.m

arkes.c

om

Markes International Ltd. T: +44 (0) 1443 230935 F: +44 (0) 1443 231531 E: [email protected]

CompoundBoiling point (°C)

GWP (100 year) 2001 IPCC

Estimatedatmospheric

lifetime (years)

CF4 -128 5700 50000

C2F6 -78 11900 50000

N2O -88 296 114

CF3Cl -81 14000 Info. not available

SF6 -64 23900 3200

Table 1: Greenhouse gases with high GWP, notfound in the regular list of US EPA ‘air toxics’

Edmass: Top-Down Sequence Validation on a Benchtop MALDI-TOF Mass Spectrometer

Company: Bruker Daltonics

Summary: This application note describes a concept called

Edmass, the Top-Down sequence analysis on a benchtop

linear MALDI-TOF (MALDI-TDS) to derive C- and N-terminal

protein sequence information directly in the mass

spectrometer – without initial protein digestion. MALDI-

TDS was applied to the research study 2009 for which the

ABRF-ESRG (Edman Sequencing Research Group of the Association of Biomolecular

Research Facilities) provided two samples and expected N-terminal sequence

assignments from both proteins.

Bruker Daltonics

This study describes the analysis of the 2 samples provided by ABRF-ESRG 2009 using Top-Down Sequencing on a benchtop MALDI-TOF [1]. It highlights how a benchtop MALDI-TOF can efficiently be applied to validate the N- and C-terminal sequences of proteins.

Introduction

The major application of Edman sequencing today is the validation of proper N-terminal sequence expression in recombinant protein production. Here, in fact, the availability of both N- and C-terminal sequences is the most important aspect that could not be addressed by Edman sequencing to date.We describe here a new concept called Edmass™, the Top-Down sequence analysis on a benchtop linear MALDI-TOF (MALDI-TDS) to derive C- and N-terminal protein sequence information directly in the mass spectrometer – without initial protein digestion [2, 3]. The technique is described in greater detail in our Application Note MT-96 [4].MALDI-TDS was applied to the research study 2009 for which the ABRF-ESRG (Edman Sequencing Research Group of the Association of Biomolecular Research Facili-ties) provided 2 samples and expected N-terminal sequence assignments from both proteins.

Experimental

Samples (20 pmol) were prepared using the sDHB matrix (#209813, Bruker) and analyzed on the microflex™ LT benchtop linear MALDI-TOF MS (Bruker) by in-source decay

Application Note # MT-95

Edmass™: Top-Down Sequence Validation on a Benchtop MALDI-TOF Mass Spectrometer

(ISD) as previously described [3, 4]. A method for peptide analysis in linear positive ion mode was optimized by mass range extension and detection gain enhancement. Several hundreds to thousands of shots were accumulated and processed. External calibration was performed using ISD c-fragment ions (average masses) of intact BSA.Linear mode ISD spectra (ISD) were peak picked in flexAnalysis™ 3.0, submitted to BioTools™ 3.2 (both Bruker software packages) and directly analyzed by database searching using a Mascot 2.2 (Matrix Science, UK) inhouse server. A new instrument type “MALDI-ISD” was created on Mascot Server with the following specification: 1+ ions only, a, c, z+2 and y-ions, as this reflects the typical ion types in ISD spectra that we used for MALDI-TDS. All protein sequencing work that was required in this study could be performed through straight MS/MS ion searches, where arbitrary strong ISD fragment ions were specified as “virtual” parent ions in the Mascot search dialog. SwissProt was used for Mascot searches. The N-terminus of sample 1 was identified by searching the NCBI database as it also contains recombinant protein constructs.

Results

Our results on the microflex [5] are summarized in the official ESRG documentation [6,7] as entry “ESRG-015” (Tab 1). Both samples (~ 40 kDa) provided sequence calls from the N-terminus and the C-terminus in the same dataset permitting their identification as ADH1_YEAST and G3P_RABIT. All samples were prepared with the 3 matrices

; ; ;

“If a protein aggregates, but there’s no Wyatt instrument to detect it, does it still aggregate?”

DAWN HELEOS. The most advanced multi-angle light scattering instrument for macromolecular characterization.

Optilab rEX. The refractometer with the greatest sensitivity and range.

ViscoStar. The viscometer with unparalleled signal-to-noise, stable baselines and a 21st-century interface.

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

DynaPro Plate Reader. Automated dynamic light sattering for 96 or 384 or 1536 well plate samples.

© 2008 Leo Cullum from cartoonbank.com. All Rights Reserved. DAWN, Optilab, DynaPro and the Wyatt Technology logo are registered trademarks, and ViscoStar and Eclipse are trademarks of Wyatt Technology Corporation.

CORPORATIONCORPORATIONCORPORA

That’s the problem with relying on elution times to characterize macromolecules. You don’t

really know if you’re right—you can only assume. Which is why every major pharmaceuti-

cal and biotechnology company, as well as most federal regulatory agencies are switch-

ing from relative methods to Wyatt Technology’s absolute measurements. Our DAWN®

multi-angle light scattering (MALS) instruments allow you to determine absolute

molecular weights and sizes without relying on so-called standards, or measurements made

in someone else’s lab. Wyatt instruments measure all of the quantities required for deter-

mining absolute molar masses directly. So call 805.681.9009 or visit wyatt.com and request

our free 28-page Ultimate Guide to Light Scattering. You’ll learn

how to end your dependence on reference standards forever,

and start detecting aggregates you never even knew were there.

TuTechnology

update

38 technology update www.sepscience.com

Key

Email the company

Product information

Applications

Additional information Agilent launches 1290 Infinity LC system into the UHPLC market

Manufacturer: Agilent

Manufacturer’s description: Agilent Technologies has introduced the 1290 Infinity Liquid

Chromatography System, designed to deliver greater power, speed and sensitivity for

enhanced performance in the high-end ultra high performance liquid chromatography

(UHPLC) market. Reported features of the system include:

• High separation power and flexibility: The company claims that the 1290 delivers the

industry’s largest analytical power range, enabling users to deploy any particle type, any

column dimensions or any mobile and stationary phases. In addition, it reportedly delivers

the foundations for method transferability from and to any vendor’s UHPLC and HPLC

systems.

• Complementary columns: Agilent has also introduced ZORBAX Rapid Resolution High

Definition (RRHD) columns. The 1.8 μm particle size delivers high resolution and peak

definition for both simple and complex separations.

• MS compatibility: The 1290 Infinity LC is designed to drive even higher levels of performance

from the company’s LC/MS systems. It is claimed that the lowest possible delay volume, low

sample carryover, integrated control and operation with MassHunter MS software, and the

ability to perform fast, ultrahigh resolution LC separations contribute to this performance.

39technology update separation science — volume 1 issue 5


www.sepscience.com

• Infi nity binary pump: The 1290 Infi nity binary pump module reduces background noise, contributing to the system’s

very high signal-to-noise ratio. Active Damping reduces ‘pump ripples’ and associated UV noise, and the company’s

proprietary Jet Weaver microfl uidic mixing technology, further enhances performance.

• UV Diode Array Detector: This contains a Max-Light Cartridge Cell with optofl uidic waveguides, providing very low

limits of detection and high signal-to-noise ratio. In addition, baseline drift is minimized for more reliable and precise

peak integration, because compromising refractive index and thermal eff ects are nearly eliminated, it is claimed.

• High throughput: The 1290 Infi nity Autosampler and Thermostatted Column Compartment modules contain a

number of usability and high-throughput features, including the ability to confi gure the system to run more than

2,000 samples per eight-hour shift. Alternating Column Regeneration (ACR) reduces cycle time by half compared

to single column confi guration, and throughput can be maximized further using automatic delay volume reduction,

overlapped injections, offl ine data analysis and external needle wash capabilities.

The company claims that the 1200 Series LC portfolio lets customers tailor the exact systems they need, from the

simplest manual isocratic LC through the world’s highest-performance, fastest, most sensitive UHPLC systems.

“Limits of detection for the pharmaceutical impurities were as low as 0.001% relative to the main compound using the

new diode array detector,” said Dr Pat Sandra of the Research Institute for Chromatography in Belgium, another early

access user. “This is more than one order of magnitude lower than required by US FDA.”


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Coupling capillary columns in gas

chromatography

Analytical trends in iso�avone

studies

Minimizing downtime in QA

pharmaceutical laboratories


Volume 1 / Issue 1

Febraury 2009

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Volume 1 / Issue 2

February 2009

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Exploiting particle size to reduce

acetonitrile consumption

Multiresidue analysis using SBSE

and GC-MS/MS

Chromatographic methods for

Con�rming biological activity

markers in fruit

Asia Paci�c


SampleGenie Improves HPLC Fraction Pooling Workflow

Manufacturer: Genevac

Manufacturer’s description: Genevac has

announced a technical study that illustrates

how its SampleGenie technology enables

removal of steps from a centrifugal evaporator/HPLC

fraction pooling protocol to improve workflow.

Traditionally HPLC fraction pooling protocols have involved drying multiple

fractions in a centrifugal evaporator, re-suspending pooled fractions into a single vial and then re-drying before

storage and analysis. Even with modern evaporators such processes typically take 2-3 days to complete. Offering the

ability to automatically pool (without robotics) multiple HPLC fractions into a single small sample vial, SampleGenie

has been designed to simplify the protocol to a single overnight drying step before storage and analysis, claims the

company. Improved speed of evaporation and a reduction in the number of sample transfer steps are highly desirable

in a sample preparation method in order to streamline workflow in busy laboratories.

SampleGenie enables samples in the company’s centrifugal evaporators to be concentrated, dried or fast freeze

dried directly into a single vial, eliminating the need for reformatting of samples after drying. The flasks act like a

funnel and permit multiple large volume samples to be concentrated directly into an HPLC (or GC) autosampler vial.

According to Genevac, the SampleGenie is available to cope with most solvent types.

41technology update separation science — volume 1 issue 5

GE Healthcare Launches Suite of 2-D Electrophoresis Products Manufacturer: GE Healthcare

Manufacturer’s description: GE Healthcare offers a suite of products to improve and simplify

2D electrophoresis workflow from sample preparation through to analysis, and further

enhance quantitation in protein expression studies.

The ready-to-use products offer significant improvement in data quality for 2D

electrophoresis experiments, and also reduce costs and time for protein expression

analysis (at least a threefold cost saving and sixfold time saving with 2-D DIGE), claims the

company. The products are designed to be used individually or as a complete solution to

maximize results from 2D analysis and are also fully compatible with 2D DIGE (Difference Gel

Electrophoresis).

“We have worked to identify and address the challenges that our customers face in 2D

electrophoresis. Most of them relate to unwanted sources of experimental variation,” said

Rita Marouga, Product Manager, GE Healthcare. “These products are designed specifically

to improve consistency and reduce heterogeneity throughout the 2D workflow, thereby

enabling identification of differences and changes in protein expression attributable to

biological variation with high confidence.”

The products include: precast low fluorescent DIGE gels and DIGE buffer kit; repackaged

CyDye DIGE Fluor saturation and minimal dyes that better suit experimental designs; and

reformulated IPG buffer and IPG strips (Immobiline DryStrip gels) that

improve spot resolution. These augment the previously

released 2D Protein Extraction Buffers, IPGbox and

DeCyder 2-D v7 software that contribute

to error reduction and optimization

of the results in the 2-D

experimental workflow.

www.sepscience.com

Low volume SPE assays enhanced by C18 sorbent

Manufacturer: Porvair Sciences

Manufacturer’s description: Porvair Sciences Ltd has announced the

availability of a range of BioVyon C18 silica columns and microplates

for use in low-volume solid phase extraction (SPE) assays.

Packed-bed SPE columns and microplates traditionally perform

relatively ineffi ciently when using the shallow sorbent beds necessary

to get good recovery from smaller sample volumes. By immobilizing

the C18 sorbent within the porous BioVyon polymer the company

claims to have created a novel, high surface area matrix that provides

excellent control of fl ow rate. Further, the immobilized C18 sorbent

cannot form liquid channels and does not require inert frits to

support it thereby minimizing hold-up volume. The combination of

these attributes has enabled Porvair to introduce a this range of SPE

columns and microplates designed to provide higher consistency

and greater recoveries for small sample volume assays.

BioVyon C18 is initially being off ered in 96-well microplates as a

10 mg per well loading suitable for low-volume bioassay preparation

and clean-ups. In the popular 1 mL cartridge format, BioVyon C18 is

available in a choice of 12.5, 25 and 50 mg loadings to suit individual

applications.

42 technology update

Register Now for your 20% Early Bird Discount

Conference Highlights

Singapore

www.sepscience.com

FoodEnviroDay One:

Pat SandraAdvances in Separation Sciences Deriven by the Metabolomics and Pro-teomics Quest for Biomarkers

Y.S. FungMicrofluidic Chip-Capillary Electrophoresis for Biomedical Applications

Eric Chun Yong ChanGC×GC/TOFMS Profiling of Human Bladder Cancer

Manfred RaidaMultidimensional Gel-free Protein Separation Approaches for In-depth Analysis of Complex Proteomes

Yi ChenNew Approaches to Online Anti-salt Stacking for Direct Capillary Electrophoresis of Biosamples

Andrew JennerGC-MS Analysis of Lipid Oxidation and Cholesterol Metabolism

Thomas WalczykElement Separation at the Microscale for High-Precision Isotopic Analysis of Biological Samples

Bioscience




sponsors:

For all delegate enquiries email [email protected]

26–28 AugustBiopolis Science Park, Singapore

Day Two:

Gert DesmetCurrent and Future Approaches to Speed Up HPLC Separations

Phil NethercoteThe applictaion of Quality by Design Principles to Analytical Method Development, Validation and Transfer.

Sanjay GargThe Role of Analytical Science and Techniques in Early Phase Drug Discov-ery and Registration for Clinical Studies

Anne GohOnline Solid Phase Extraction-LC-MS in DMPK Applications

Edward BrowneBiomarker Analysis for Preclinical Pharmaceutical R&D

Shawn StanleyTBC

Ping LiHPLC and Hyphenated Techniques for Analysing Ingedients in Herbal Medicines

Yizeng LiangSeparation Science for the Quality Control of Traditional Chinese Medicine

Day Three:

Alastair LewisTrace Pollutant Detection in Challenging Environments

Hian-Kee LeeSolvent-Minimized Sample Preparation for Separation Science

Siu Kwan SzeAn Advanced Proteomic Approach to the Discovery of Microbial Enzymes for Biorefining

Gongke LiMolecularly Imprinted Polymers for Trace Analysis of Complicated Samples

Paul HaddadDevelopment of Portable Separation Methods for the Identification of Terrorist Explosives by Analysis of Inorganic Residues

Philip MarriottHeadspace Analysis of Plant Materials by Using Comprehensive Two-Dimensional Gas Chromatography: Selected Examples

Jessie TongMultidimensional Gas Chromatographic Analyses of Flavours and Fragrances

Bahruddin SaadDetermination of Biogenic Amines in Food: Conventional and Nonconventional Approaches

Pharma TCM


Zebron ZB-XLB-HT Inferno GC columns for fast melamine analysis

Manufacturer: Phenomenex

Manufacturer’s description: Phenomenex has introduced the Zebron ZB-XLB-HT Inferno

high-temperature GC column designed to enhance routine GC/MS melamine analysis of

milk products. According to the company, the ZB-XLB-HT columns reduce total run time to

less than four minutes. Stable up to 400 ˚C, the high-temperature capability allows bake-

off of matrix contamination, present in milk and other food products, that would otherwise

decrease column lifetime.

Standard fused-silica columns are not engineered to withstand temperatures above 380˚C

and their coating begins to degrade, eventually becoming brittle

and inflexible. Phenomenex states that the Zebron Inferno

non-metal columns incorporate proprietary coating and

bonding technologies, providing stability at high

temperatures, low bleed and low activity.

The company’s Zebron ZB-5ms column is ideal

for routine analysis of milk products using the

FDA-recommended GC/MS method. Howevere, for

customers who need faster results, the Zebron ZB-

XLB HT Inferno GC column has been introduced. If a

laboratory prefers LC analysis, the Luna HILIC column

resolves cyanuric acid and melamine in less than three

minutes, claims the company. Phenomenex also offers complementary

Strata Melamine SPE cartridges.

“Our successful Zebron Inferno columns were the first non-metal columns to provide

stability at very high temperatures,” commented Sky Countryman, product manager for

Phenomenex. “With the addition of these new columns, our offering of products, methods

and expertise is the most comprehensive for melamine analysis.”

Automated dialysis as a sample preparation tool in ion chromatography

Manufacturer: Metrohm

Manufacturer’s description: Ion chromatography (IC) as an analytical technique has seen an impressive surge in

popularity. As for samples in a homogenous ionic form, hardly any preparation steps are required at all. According

to Metrohm, its patented stopped-flow dialysis paves the way for the convenient analysis of demanding samples

carrying high organic loads too.

In complex matrices carrying high organic loads such as waste water, soil eluates or dairy products, extensive

sample preparation is mandatory to prevent destruction of the column. Traditional preparation techniques such as

the Carrez precipitation do not provide a satisfying answer as they cannot be automated and are error-prone.

Metrohm claim that its compact stopped-flow dialysis is an elegant alternative. This fully automated sample

preparation setup is based on the selective diffusion of ions from one liquid (sample/donor solution) to another

(acceptor solution) through a membrane.

Contrary to dynamic dialysis, where two solutions continuously pass through the dialysis module, the acceptor

solution is stopped until its concentration is the same as that in the donor solution, the company states. This patented

stopped-flow procedure takes between 10 and 14 minutes and can be directly coupled to an IC setup. As the dialysis

is performed during the recording of the previous sample’s chromatogram, the overall analysis time is not significantly

prolonged.

45Technology update separation science — volume 1 issue 5

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Volume 1 / Issue 3

March 2009

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Coupling capillary columns in gas

chromatography

Analytical trends in iso�avone

studies

Minimizing downtime in QA

pharmaceutical laboratories


Volume 1 / Issue 1

Febraury 2009


液相色谱-质谱联用新方法的建立

微波辅助溶剂萃取与气相色谱-质谱

联用分析太子参中的挥发物

微芯片电泳用于生物医学

分析


Volume 1 / Issue 2

February 2009


Exploiting particle size to reduce

acetonitrile consumption

Multiresidue analysis using SBSE

and GC-MS/MS

Chromatographic methods for

Con�rming biological activity

markers in fruit

Asia Paci�c


Volume 1 / Issue 2

April 2009


固相微萃取-高效液相色谱分析羟烷基喹诺酮

固相萃取和气相色谱与三级四极杆质谱联用测

定熟食品中痕量食品衍生的有害物质

液相色谱填料尺寸对降低

溶剂消耗的影响

中国版

Volume 1 / Issue 5

May 2009

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A liquid chromatographer’s

introduction to mass spectrometry

Analysing synthetic polymers with

solvent enhanced light scattering

Minimizing decomposition of

components during GC analysis


Volume 1 / Issue 4

April 2009


A practical review of

multidimensional LC

How to deal with overloading in GC

Asia Pacific

Rapid determination of RRFs

for the quantitation of impurities

in pharmaceuticals

journal separation science

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