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Enhancing Environmental AnalysisModern sample preparation

methods for POPs

2 Modern Sample Preparation Methods for POPs Persistent organic pollutants (POPs) pose an on-going threat to human health, but are often trapped within environmental samples, thereby making analysis challenging. Bethany Degg of The Column spoke to Lourdes Ramos from the Department of Instrumental Analysis and Environmental Chemistry Institute of Organic Chemistry of the CSIC (Madrid, Spain) about her innovative research on new sample preparation methods for POPs.

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19 43rd Symposium of HPLC and Related Techniques (HPLC 2015 Beijing)

The 43rd Symposium of HPLC and Related Techniques (HPLC 2015 Beijing Conference) will take place at the Beijing International Conference Centre, Beijing, People’s Republic of China, from 21–25 September 2015.

17 Developing HPLC Methods for Biomolecule Analysis An excerpt from LCGC’s e-learning tutorial on high performance liquid chromatography (HPLC) methods for biomolecule analysis at CHROMacademy.com

Regulars8 News Investigating yeast products in sparkling wine, diagnosing infection using MS, and the latest company news and on-line highlights are featured in this issue.

12 Tips & Tricks GPC/SEC Strategies to Save Solvent

Jasmin Preis and Daniela Held, PSS Polymer Standards Service GmbH Tetrahydrofuran (THF) is set to be classif ed as carcinogenic as proposed by the Committee for Risk Assessment (RAC). Analytical laboratories therefore need to f nd ways to reduce THF solvent use and waste, far beyond the demands of green chemistry. This instalment of Tips & Tricks presents different strategies to meet this goal.

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7 August 2015 Volume 11 Issue 14

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Modern Sample Preparation Methods for POPsPersistent organic pollutants (POPs) pose an on-going threat to human health, but are often trapped within environmental samples, thereby making analysis challenging. Bethany Degg of The Column spoke to Lourdes Ramos from the Department of Instrumental Analysis and Environmental Chemistry Institute of Organic Chemistry of the CSIC (Madrid, Spain) about her innovative research on new sample preparation methods for POPs.

Q. Why are persistent organic

pollutants (POPs) a concern? When

did scientists recognize the threat of

exposure to human health?

A: Persistent organic pollutants (POPs)

are mainly halogenated organic chemicals

that are toxic; highly resistant to any type

of chemical, biological, and photolytic

degradation; and bioaccumulative. In

addition, they can be transported long

distances from the emission point by

wind and water. These characteristics give

POPs, also classed as PBTs (persistent,

bioaccumulative, and toxic), the potential

to adversely affect human health and

the environment around the world — as

recognized by the international agreement

signed in May 2001 known as the

Stockholm Convention.

POP classes include intentionally produced

chemicals currently or once used in

agriculture, disease control, manufacturing,

or industrial processes, and by-products of

industrial and combustion processes. Under

the Stockholm Convention, countries agreed

to reduce or eliminate the production, use,

and release of legacy chemicals and to

conduct a constant revision process, which

has led to the gradual incorporation of other

POPs of global concern to the list over the

years.

Q. In your view, what are the main

challenges associated with the analysis

of POPs in environmental samples?

A: The biggest challenge from an analytical

point of view is the complexity of the

environmental matrices in which POPs are Ph

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Q&A: Ramos2 News8 Tips and Tricks12 The Essentials1788 122HPLC Beijing Event Preview19 CHROMacademy21 Training & Events22 Staff23212 2222


The Column www.chromatographyonline.com

sorbed or entrapped, combined with the

very low levels of detection required. This

is particularly true in the case of (semi-)

solid matrices and for those chemicals

and by-products that have already been

subjected to specif c regulations and

controls, which are typically found at

ultratrace levels in environmental samples.

This combination of low concentration levels

and matrix complexity has led to the use

of labourious multi-step sample treatment

protocols followed by highly sophisticated

instruments for f nal instrumental

determination. The aim is to ultimately

ensure the quantitative extraction of target

compounds and subsequently exhaustive

purif cation and fractionation of co-

extracted matrix components.

Generally speaking, the lower the

concentration of the analyte to be

determined, the more demanding the sample

treatment. However, advances achieved

over the years in the f eld of analytical

instrumentation — in particular of mass

spectrometry (MS) — allow highly sensitive

and accurate analyses to be performed

with instruments that are available in most

academic and commercial laboratories today.

Unfortunately, the potential offered by

advanced instrumentation has not always

been used to simplify the previous sample

preparation protocol.

Q. Your research interests include the

development of new miniaturized

sample preparation methods for

determining trace organic pollutants

with chromatographic techniques. What

led you to begin this work and why is

it important to develop miniaturized

sample preparation methods?

A: In a typical gas chromatography

(GC)-based analysis of POPs, the purif ed

extract is usually diluted to a f nal volume

of ca. 50–100 µL, of which only 1–2 µL

are injected in the splitless mode in the

analytical instrument. In practice, this

means that only a small fraction of the

initial amount of sample is used for f nal

instrumental determination. Consequently,

a simple reduction of the solvent used to

dissolve this f nal extract to an acceptable

volume of 10–20 µL could promote an

in-line reduction of the initial sample

amount without affecting the detectability

of the investigated analytes. Nowadays,

using any of the large volume injection

(LVI) techniques available can contribute to

further reducing the initial sample amount

while maintaining the total amount of

analyte injected in the GC system. Today,

it is therefore possible to signif cantly

reduce the amount of sample use for POP

determination in environmental samples

without affecting the quality of the analysis.

Q&A: Ramos

3




In addition to being the best alternative

when dealing with the analysis of

size-limited samples, miniaturization has

many other advantages including faster

sample preparation times, minimal sample

manipulation, reduced reagents consumption,

and reduced waste generation.

Q. You recently developed a

miniaturized pressurized-liquid

extraction (PLE) with in-cell purif cation

method for the simultaneous extraction

of endogenous PCBs in feedstuffs. Can

you tell us how you developed this

method?

A: This method can be considered a logical

continuation of my previous research in

the f eld of miniaturized sample treatment

of biological tissues and fat-containing

foodstuffs for the analysis of legacy PCBs.

These former studies were based on the

dispersion of freeze-dried tissue on the

surface of an appropriated sorbent. As

well as providing a sorbent-like mixture

that can easily be packed in a solid-phase

extraction (SPE) cartridge, this process

contributed to the preliminary purif cation

of the matrix extract. Packing of this

homogeneous matrix solid-phase dispersion

(MSPD) mixture on top of an appropriate

co-sorbent (in this case, silica modif ed

with sulphuric acid, SiO2-HSO4) allowed

complete sample treatment to be performed,

in this instance, quantitative extraction of

the target compounds plus fat removal in

a single step with minimum reagents and

time consumption. Despite its simplicity, this

sample treatment procedure resulted in ready

for analysis extracts and, when combined

with LVI–GC–ITD (ion trap detection)

(MS–MS), the methodology provided

results similar to those obtained using the

conventional large-scale procedure involving

GC–high-resolution mass spectrometry

(HRMS) in use in our laboratory, even for the

less abundant dioxin-like PCB congeners.

Q. What were the challenges you faced

and how did you overcome them? What

are the advantages of this approach

compared to other methods?

A: The analysis of more sorptive analytes,

like polybrominated diphenyl ethers

(PBDEs), in a more sorptive material, like

feedstuffs, made it necessary to adopt

a different analytical strategy. Direct

application of the previous method would

have resulted in an undesirable increase

of the f nal extraction solvent volume and

of the analytical time. The use of one of

the modern enhanced solvent extraction

techniques, and pressurized-liquid extraction

(PLE) in particular, was the most evident

and advantageous alternative. However,

Q&A: Ramos

4




commercially available PLE instruments were

not really adapted for the treatment of small

size samples under the principles of the

minimum reagent consumption and waste

generation we were interested in.

We consequently decided to develop

our own miniaturized-PLE system. This

homemade instrument had technical

and operational specif cations similar to

commercial systems but was ready to hold

tailored extraction cells that would f t the

investigated sample. The latter provided

an additional f exibility when setting up

methods that were impossible to attain with

the commercial instruments. In practice, that

meant that we were ready to develop a new

miniaturized PLE-based methodology for

the simultaneous and exhaustive extraction

of PCBs and PBDEs from highly sorptive

matrices, such as feedstuffs.

The f nal optimized method was based

on the MSPD of the water-normalized

feedstuff with a mixture of anhydrous

Na2SO4 and SiO2-HSO4. The homogenous

and dried MSPD mixture was then packed

in the extraction cell on top of SiO2-HSO4,

which acted as co-sorbent and allowed the

in-cell purif cation of the extracts. When

combined with either GC–ITD(MS–MS) or

GC coupled to negative chemical ionization

(NCI)–MS, the proposed method allowed the

accurate determination of the endogenous

PCBs and PBDEs, respectively, at the levels

typically found in commercial (that is,

non-contaminated according to the legislation

then in force) feeds using only 250 mg of

sample, 8 mL of organic solvent, and 3.5 g

of sorbent. Complete sample preparation

was done in less than 45 min, which sharply

contrasted with the several hours of work

required by more conventional large-scale (in

this instance, off-line) approaches for these

types of determinations.

Q. In another study, you developed an

approach using ultrasound-assisted

extraction followed by disposable

pipette purif cation for the

determination of PCBs in small

biological tissue samples. Why did you

choose ultrasound-assisted extraction

over other methods?

A: The determination of trace organic

compounds in samples of very small

size (less than 100 mg) represents an

analytical challenge but, in my view, also an

opportunity to explore the feasibility of new

analytical approaches and conf gurations.

With such a small sample size, the challenge

starts before the analysis to ensure the

representativeness of the sub-sample to

be analyzed. In precedent application

studies, freeze-drying of the investigated

heterogeneous sample followed by its

Q&A: Ramos

5


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grinding, thorough homogenization and

pre-treatment of a representative (2–5-times

larger than required) amount of sample,

contributed to minimize this problem. This

approach was also followed in the present

study. However, it should be noted that

the idea of analyzing such small samples

came from another research study carried

out in our group.1 In that case, samples

were actually that size (in fact, they were

below 10 mg), but they were individual

samples, thus eliminating the problem of its

representativeness.

Manual sample handling can also become

a problem when dealing with the analysis

of very small-size samples. Apart from

the (possible) diff culty associated with

manipulation and quantitative transfer through

the sample preparation procedure, the risk

of contamination increases at this level, at

which residual background levels can easily

become apparent in analytical and laboratory

blanks and ruin the analysis. At such a

level, rigorous QC procedures should be

adopted to prevent (and detect) any possible

sample contamination through storage and

processing. The use of appropriate (clean)

laboratory material, recovery surrogates, and

control samples becomes not only advisable

but, in most instances, mandatory. In this

context, the lower the manipulation of the

sample, the better.

For this reason, we decided to develop a

sample treatment that could be performed

entirely in the Eppendorf in which such

a type of sample could be collected and

stored until analysis. With this idea in

mind, treatment with a 2-mm ultrasonic

tip probe appeared as the best alternative

to ensure an exhaustive extraction of

the target microcontaminants from the

investigated biological tissues. After only

20 pulses of 2 s with the ultrasonic tip, the

matrix was completely desegregated and

PCBs were quantitatively extracted in the

150 µL of n-hexane used as extractant.

The supernatant was then separated

by centrifugation for 2 min and slowly

aspirated with a micropipette into a 5-mL

polypropylene tip modif ed to contain the

clean-up sorbent. After 10 s of contact,

the purif ed extract was ejected into a

chromatographic vial or, in the case of very

fatty samples, into a new (clean) Eppendorf

to perform a second clean-up step. In all

cases, ready-for-analysis extracts were

obtained and, as demonstrated by the

analysis of appropriate reference materials,

when combined with GC–ITD(MS–MS),

the proposed method allowed accurate

determination of most of the investigated

PCBs — even for such a small amount as

50 mg. In addition, and because of the

simplicity of the operations carried out

Q&A: Ramos

6


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during sample treatment, the proposed

method exhibited potential for (at least

partial) automation.

Q. Have you been involved in other

projects involving POPs analysis?

A: My PhD, completed in the mid-1990s,

dealt with the development of analytical

methods for the determination of PCBs

and PCDD/Fs in fatty foodstuffs, a type

of analysis considered a challenge at that

time. I then undertook a postdoctoral stay

in Amsterdam, at Vrije University, under the

supervision of Prof. Dr. Udo A.Th. Brinkman.

My goal was to learn about the systems

and procedures used for miniaturized SPE of

liquid samples and to subsequently explore

the possibility of developing (as much as

possible) equivalent set-ups and systems

for the treatment of semi-solid and solid

matrices. Whilst there, I had the opportunity

to start working in that line of novel research

in the late-1990s and developed some

miniaturized methods for the analysis of

pesticides and PAHs in non-fatty matrices,

such as fruits, soils, and sediments. Back

in Madrid in 2000, I continued with that

type of investigation and I rapidly expanded

to the analysis of POPs and other trace

organic compounds in fat-containing

samples, from serum to biological tissues

and foodstuffs among others. Today, the

development of novel miniaturized sample

preparation procedures for the analysis of

trace microcontaminants and components in

(semi-)solid samples can be considered a well

established and active research line in my

department.

Q. Is sample preparation still the main

bottleneck in environmental analysis?

A: Sample preparation is still recognized

as the bottleneck of many analytical

procedures. The efforts carried out during

the last two to three decades have resulted

in the development of a number of novel

analytical approaches and techniques

that have contributed to solve some

of the most pressing shortcomings of

conventional (large-scale) sample treatment

procedures, namely long analytical times,

large consumption of sample and reagents,

high risk of extract contamination because

of their continuous manual manipulation,

and generation of large amounts of waste.

Today, on-line coupling (with or without

automation) is a recognized and accepted

approach in many application areas dealing

with the analysis of gases or volatile

compounds and with the treatment of liquid

samples. Attempts to develop equivalent

procedures for the treatment of (semi-)

solid samples have been much more limited,

probably because of the diff culty of the

European Journal of Chemistry 10, 480–520

(2012).

Lourdes Ramos currently

holds the position

of Senior Scientif c

Researcher of the Spanish

Scientif c Research

Council (CSIC, Madrid),

at the Department

of Instrumental

Analysis and Environmental Chemistry of

the Institute of Organic Chemistry. Her

research interests include the development

of new miniaturized sample preparation

methods for the fast determination of trace

organic pollutants in environmental and

food samples and the evaluation of new

chromatographic techniques, especially

GCxGC-based approaches, for unravelling the

composition of complex mixtures of organic

microcontaminants. Member of the editorial

board of various journals and invited editor of

several special issues, she has published over

80 peer-reviewed scientif c papers, 12 book

chapters, and has edited a multi-authored

book on comprehensive two-dimensional

gas chromatography.

initial extraction step, for which large-scale

(off-line) approaches are mainly used.

Results reported on the development

of hyphenated systems for gaseous and

liquid samples have demonstrated that

miniaturization of the techniques and

approaches are probably a key aspect when

attempting (at least partial) integration

of the different analytical steps. Some of

the modern simplif ed, faster, cheaper,

and greener techniques designed for the

treatment of solid matrices (in particular

MSPD and PLE) have demonstrated their

potential in this f eld through a number

of illustrative examples.2,3 However, to

achieve a level of development similar to

that shown at present for other solvent- and

sorbent-based techniques used in coupled

systems, more work is still required from

both academia and companies, who should

support and promote the development of

appropriate analytical instrumentation.

References

1. J. Sanz-Landaluze, M. Pena-Abaurrea, R.

Muñ oz-Olivas, C. Cá mara, and L. Ramos. Enviro.

Sci.Technol. 49, 1860–1869 (2015).

2. J. Escobar-Arnanz, L. Ramos. TRAC Trends

Anal. Chem. (2015) http://dx.doi.org/10.1016/j.

trac.2015.02.023

3. J.L. Tadeo, C. Sanchez-Brunete, B. Albero,

A. Garcia-Valcarcel, and R.A. Pérez, Central

E-mail: [email protected]: http://www.iqog.csic.es/ iqog/es/node/18689

Q&A: Ramos

7



Agilent Technologies Presents Thought Leader Award

to Dr Lawrence Lesko

Agilent Technologies Inc. (Santa Clara, California, USA) has

announced that Dr. Lawrence J. Lesko, of the Center for

Pharmacometrics and Systems Pharmacology at the University

of Florida’s College of Pharmacy in Orlando, USA, has

received an Agilent Thought Leader Award for his research

into preclinical toxicological assessments of new medicines.

Dr. Lesko and his team are working to identify novel

biomarkers that can help drug makers better assess new drug

candidates and can also potentially enable doctors to better

manage treatments.

Monty Benefiel, Agilent vice president and general manager

of the Mass Spectrometry Division, said: “We are pleased to

support Dr. Lesko’s research, which is addressing important

needs in the rapidly evolving field of systems toxicology.”

“I am very pleased and appreciative of Agilent’s Thought

Leader Award,” said Dr. Lesko. “It enables our team to

identify metabolomic safety biomarkers of drug-induced renal

toxicity and the mechanisms by which these toxicities occur.

This research will lead us to identifying at-risk patients and

exploring the use of concomitantly administered medicines

as protectants against drug-induced renal damage. It could

have a major effect on improving drug development and

clinical care.”

www.agilent.com

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Investigating Yeast Products in Sparkling Wine ProductionA team of researchers in Spain has examined the use of commercial yeast products in the production of white and rosé

sparkling wines.1 Using a range of analytical techniques, including gas chromatography–mass spectrometry (GC–MS) and

high performance liquid chromatography (HPLC), the team examined four yeast autolysates to f nd out how they affect

the chemical composition, foam, and sensory properties of sparkling wines aged on lees for 9 months.

During the production of wine, mannoproteins — highly glycosylated proteoglycans made up of mannose, glucose,

and proteins — are released via yeast autolysis. These mannoproteins can have positive effects on the wine, such as an

improvement in aroma and a reduction in bitterness. Traditionally the best sparkling wines can take many months to

ferment to the perfect quality. The longer the process takes, the higher the associated production costs and the greater

the risk of microbiological and organoleptic alterations. To speed up this process commercial yeast products rich in

mannoproteins have been developed. The effect of these products on still wines has been extensively studied, and they

have been found to improve the quality characteristics of the wine. However, very few studies have examined the effect

on sparkling wines.

All of the wine used in the study was produced following the traditional

(champenoise) method. The team used GC–MS to determine the monosaccharide

composition of the dry yeast products. The percentage of mannose in the four

yeast products varied between 53% and 86%; the percentage of glucose was

between 14% and 47%. HPLC was used to analyze phenolic compounds

and amino acid and biogenic amine content. The foaming properties of

the sparkling wine were evaluated according to the Mosalux procedure. By

comparing the control and the sparkling wines, the team found that the

addition of the yeast products did not affect the foam quality of the wines

or the phenolic compound content in any signif cant way. In fact, the yeast

product with the highest mannoprotein content actually enhanced the

volatile composition and the subsequent fruity aroma in some of the sparkling

wines. — K.M.

Reference1. Silvia Pérez-Magariño, Leticia Martínez-Lapuente, Marta Bueno-Herrera,

Miriam Ortega-Heras, Zenaida Guadalupe, and Belén Ayestarán, Journal of

Agricultural and Food Chemistry 63(23), 5670–5681 (2015).

8




New MS-Based Method “Fingerprints” Air to Diagnose Infection

A team of chemists and microbiologists from

the University of Leicester has developed

a new method of rapidly diagnosing the

infection Clostridium diff cile (C. diff cile).1

This bacterium causes diarrhoea and is

a particular problem in hospitals, where

immune-compromised patients are

particularly susceptible to it. The bacterium

is resilient and can live for weeks or longer

on surfaces; thus, if it is not rapidly identif ed

and contained, it can spread quickly.

The new method uses the fact that these

microorganisms produce metabolic volatile

organic compounds (VOCs) during growth

and in other processes. By analyzing the

headspace above a C. diff cile sample,

the organism can be identif ed. This

test is carried out using proton transfer

reaction-time of f ight-mass spectrometry

(PTR–TOF–MS). In this technique, the

additional proton in a hydronium ion has

a higher aff nity for the VOCs than it does

for water, which results in the organic

compound becoming charged, after which

it can be detected in the mass spectrometer.

The ionization technique, which uses H3O+,

is relatively gentle, so most peaks in the

mass spectrum are assumed to be from

protonated molecular ions.

In essence, this method is “f ngerprinting”

air, by identifying and measuring the

quantity of the volatile organic compounds

present. Thus, the diarrhoea of a person

suffering from C. diff cile would give off

different VOCs than a sample from an

uninfected person. Standard immunoassay

tests currently in use for C. diff cile diagnosis

under national guidelines in the UK usually

take 24–48 h. However, this new test can

take as little as 5 minutes. This test can also

differentiate between strains of the bacteria

(not all of which are pathogenic) because

different types of C. diff cile produce

different mass spectra. This differentiation

information is not provided by standard tests

currently in use.

Professor Paul Monks, a professor of

analytical chemistry at Leicester and who is

a member of the research team at Leicester,

said that further development is necessary

before the method can be applied in a

clinical setting. “It will require some time

and investment before a cheap, simple, and

practical sensor can be developed that will

give a simple yes-no answer on a sample,”

he said. “The good news is that this may

only take a couple of years, a very short time

in the context of clinical advances.”

The fundamental principle of this

technique can also be applied to a wide

range of areas, such as to track air pollution,

or to test breath in order to determine

the length of time since someone last had

an alcoholic drink, or even to tell when

mangoes are ripe. Over the next few years

we could see a rise in the development of

a variety of applications for this method of

“f ngerprinting air”.

Reference

1. S. Kuppusami, M.R.J. Clokie, T. Panayi, A.M. Ellis,

and P.S. Monks, Metabolomics 11, 251–260 (2014).

Dominic Clearkin, Leicester, UK.

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News In Brief

Like us Join us Follow Us

Relationship Between VOCs and Pollination in Onion PlantsA rapid and solvent-free method using headspace

SPME–GC–MS has been used to analyze volatile

compounds emitted from onion plants. Of

particular note was the substantial difference

between the number of bee visits and the seed

yield among the onion lines analyzed. Repellent

compounds, such as dioxolanes, piperidines, and

organosulphurs, were found to result in less bee

visits and a lower seed production yield, negatively

affecting pollination.

DOI:10.1016/j.microc.2015.04.017

Novel Method for Lithium QuantitationResearchers have published a novel method for the

quantitation of lithium using mixed-mode HPLC

with charged aerosol detection (CAD). The paper

published in the Journal of Chromatography A states

that the method is capable of separating lithium

from drug matrices and other ions in one run.

DOI: 10.1016/j.chroma.2015.06.063

David S. Bell Joins LCGC’s Editorial Advisory BoardLCGC Magazine is pleased to announce the addition

of David S. Bell, research and development manager

in the HPLC Surface Chemistry and Health Sciences

Research department at Sigma-Aldrich/Supleco, to

its editorial advisory board. Over the past 20 years,

Bell has worked directly in the chromatography

industry, focusing his efforts on the design,

development, and application of chromatographic

stationary phases to advance liquid chromatography

and related hyphenated techniques.

LCGC TV HighlightsLCGC TV: Should You be Using GC×GC

in Your Routine Analysis?Two-dimensional gas chromatography (GC×GC) started out as a specialized technique reserved for the domain of academic researchers. But the technique, and the instrumentation, have evolved

signif cantly in the past decade. Does it make sense to use GC×GC in routine analysis? Frank Dorman weighs in.Watch Here>>

LCGC TV: Michal Holcapek on Trends in LC–MSIn this video from LCGC TV, Michal Holcapek from the University of Pardubice in the Czech Republic describes the requirements of “fast LC–MS” and areas where the technique can be

applied in industry.Watch Here>>

Peaks of the WeekThe LCGC Blog: Paired Ion Electrospray Ionization for Trace Anion Analysis — Using electrospray

ionization mass spectrometry (ESI–MS) to perform quantitative binding analyses and determine association

constants depends on the ability of the ionization process to preserve the system equilibrium. If association

or dissociation kinetics are relatively fast, then the shrinking-droplet ESI process can alter equilibria. This is not

good for studying noncovalent complexation in solution, but it is central to the success of a technique called

paired ion electrospray ionization (PIESI). Kevin Schug explains more. Read Here>>

The Top 10 HPLC and UHPLC Column Myths: Slideshow — Webster’s New Collegiate Dictionary

def nes a myth as “an ill-founded belief held uncritically, especially by an interested group”. Could that

group be misinformed chromatographers? Find out by clicking through the slideshow here.

Interview: A Spotlight on SFC — Supercritical f uid chromatography (SFC) has experienced a recent

increase in popularity. Gilles Goetz, a Principal Scientist at Pf zer in Groton, Connecticut, USA, is doing

some interesting work in non-traditional uses of SFC. He recently spoke to us about this research.

Read Here>>

TOP 10

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Platforms: iPhone, iPad, and iPod touch (requires iOS 3.0 or later).

What it does: The LC Calculator app is designed for quick calculation of f ow rate and back pressure under a variety of conditions and column dimensions, allowing users to explore “what if” scenarios. According to the company, the app’s Back Pressure Calculator can be used to determine what column conf gurations will work within a system’s pressure range, and its Flow Rate Calculator can be used to f nd the column f ow rate generated at the desired system back pressure.

COST: Free

News

11



Tips & Tricks GPC/SEC: Strategies to Save Solvent

Tetrahydrofuran (THF) — a widely used solvent in gel permeation

chromatography/size-exclusion chromatography (GPC/SEC) — is set to

be classif ed as carcinogenic as proposed by the Committee for Risk

Assessment (RAC). Analytical laboratories therefore need to f nd ways

to reduce THF solvent use and waste, far beyond the demands of green

chemistry. This instalment of Tips & Tricks presents different strategies

to meet this goal.

Jasmin Preis and Daniela Held, PSS Polymer Standards Service GmbH, Mainz, Germany.

Resolution in gel permeation chromatography/

size-exclusion chromatography (GPC/SEC)

depends on two parameters — the

available pore volume and the dispersion

in the system as indicated by the plate

count. It is common practice in GPC/SEC

to combine several columns in a column

bank to increase resolution and molar

mass separation range; however,

this increases analysis time, solvent

consumption, and waste. Here, we

present a number of ways to perform

environmentally friendly GPC/SEC.

Overlaid Injection

Overlaid injection introduces a sample

onto the column while the previous

sample is still eluting from the column.

The maximum amount that the samples

can be overlaid is equivalent to the

interstitial volume of the column. Using

overlaid injection can reduce solvent

consumption by 25–35% and is an elegant

way to save time. The big advantage of

this is that it does not sacrifice resolution

and can be performed with columns from

all manufacturers.

Figure 1 shows an example of an overlaid

injection with UV and light scattering

data for two samples. The first sample

elutes with the internal flow marker

around 12.5 mL (min); however, the next

sample PS 1124a is already injected after

approximately 8.25 mL (min). If software is

used correctly, overlaid injection does not

affect the data evaluation of results.

Column Concepts

Reducing particle size can increase

resolution in GPC/SEC as in Ph

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ultrahigh-pressure liquid chromatography

(UHPLC), saving time and solvent, but it

is still a concept under investigation. In

the case of macromolecules, for example,

forcing high molar mass or stiff polymer

chains through an LC system at very high

pressure can result in chain degradation

and will generate results only for the

fragments.1 Despite this, many applications

for oligomers or spherical and stable

macromolecules can be transferred to newer

solutions. Depending on the GPC/SEC

equipment two approaches can be applied:

a) Continue to use traditional analytical

Figure 1: Overlaid injection example. Before the system peaks and the internal standard (light green triangle) of “PS 280 br” are eluted, the next sample “PS 1124 a” is already injected at 8.5 mL (blue triangle). The analysis and data evaluation of sample “PS 280 br” is not affected by that; baseline and integration limits can be set as required by national and international GPC/SEC guidelines, for example, ISO 13885/DIN 55672.

Tips and Tricks

13


Ascentis Express UHPLC and HPLC ColumnsFaster HPLC on Any System

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columns with an internal diameter of

approximately 8 mm and f lled with

particles of the same chemistry and pore

size, but a different particle size. Figure 2

compares the resolution of a polystyrene

oligomer on styrene-divinylbenzene

material with different particle sizes.

Using 3-µm particles allows separations

with the same or better resolution on just

one or two analytical columns instead

of three. This decreases the amount of

solvent and waste signif cantly.

b) Even more solvent can be saved when

the internal diameter of the columns is

Figure 2: Resulting molar mass distribution for a polystyrene mixture of four different molar mass calibrants including an oligomer mixture. For one styrene-divinylbenzene column (8 × 300 mm, 3-µm) a better resolution can be achieved in less time and using less solvent than when using a combination of three 5-µm styrene-divinylbenzene columns with a comparable pore size distribution.

Tips and Tricks

14




reduced from 8 mm to around 4.6 mm;

however, the cell volume of a lot of

traditional GPC/SEC detector cells is

too large to profit from the enhanced

separation using semi-microcolumns.

While UV–vis detectors are, in general,

ready for these applications, either

upgrade of existing refractive index

detectors (RI) or purchase of new,

optimized µRIs, light scatterings

detectors, or viscometers is required.

Figure 3 shows how the excellent

resolution of microcolumns gets lost

when analytical RI detectors are used

for detection. Optimized µRIs provide

much better results.

Intelligent Calibration

Another way that solvent can be saved

is by using an intelligent approach to

calibration. GPC/SEC calibration involves

measuring the elution volume of several

molar mass calibration standards. It is

normally possible to inject a combination

of three to four standards in the same

injection if the standards differ significantly

Figure 3: Comparison of the chromatogram of a polystyrene oligomer separated in 5.5 mL using micro columns and a standard RI detector (blue trace) versus an optimized µRI detector with smaller cell volume (green trace).

Tips and Tricks

15


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Derivatization,

Addition of Standards,

Dilution, Calibration

Extraction, Filtration,

SPE, Evaporation

(mVAP)

Thermal Desorption,

Dynamic Headspace

(DHS) and PYRO

MAESTRO PrepAhead

Productivity



in molar mass. If the separation is

sufficient, even more standards can be

added and analyzed within the same

run. A 10- to 12-point calibration curve

can therefore be created with three

injections or less, but care is required in

choosing the correct concentration for the

different molar masses.2 Mixtures of high

quality reference materials with precise

concentrations and molar masses are

available as polymer cocktails pre-weighed

into autosampler vials, eliminating the

need to weigh in calibrants.

Detector Concepts

Multi-detection is one of the strengths

of GPC/SEC — molar mass sensitive

detectors, such as on-line viscometers

(stand-alone or as part of a triple

detection system), provide valuable

information for many applications. All

commercially available detectors apply the

same major principle: they split the sample

eluting from the column in a bridge

design and compare the viscosity (or, more

precisely, the pressure in the tubing) of

the pure mobile phase with that of the

sample.

The viscosity of the pure solvent is

measured using either hold-up columns

or a reservoir filled with solvent. If a

detector has a reservoir, the split part of

the sample is diluted to zero concentration

in that reservoir. If a detector uses hold-up

columns, the split part of the sample is

delayed while travelling through these

hold-up columns. The delayed sample

parts then elute after the system peaks of

the sample. They are visible as negative

peaks in the delta pressure signals of

the viscosity detector. Therefore, the

amount of solvent required per sample

is much higher for viscometers with

hold-up columns than for viscometers with

reservoirs.

Summary

There are several ways to save solvent and

to avoid waste in GPC/SEC.

• Overlaid injection reduces solvent

consumption by 25–35%, without

sacrificing the resolution; however,

dedicated software is required.

• Reducing particle size to reduce the

number of columns required for a

good resolution is applicable for many

applications of compact and low molar

mass macromolecules. For example,

applications in THF or other organic

solvents can be transferred from

5 µm styrene-divinylbenzene to 3 µm.

Aqueous separations can be run on

5 µm for typical water-soluble polymers

or biopolymers, or 2–3 µm for proteins.

• The use of microcolumns with a

reduced internal diameter requires an

adjustment of the cell dimensions of

typical GPC/SEC detectors. For some

RI detectors a detector modification is

available.

• For on-line viscometry/triple detection

the use of viscometers with a

reservoir instead of hold-up columns

halves the amount of solvent required

because there is no need to wait for a

negative peak.

References

1. A. Striegel, W.W.Yau, J.J.Kirkland, and

D.D. Bly, Modern Size-Exclusion Liquid

Chromatography: Practice of Gel Permeation

and Gel Filtration Chromatography

(John Wiley & Sons, New York,

USA, 2009).

2. D. Held, The Column 9(6), 9–12 (2013).

Daniela Held studied polymer chemistry

in Mainz, Germany, and works in the PSS

software and instrument department.

She is also responsible for education and

customer training.

Jasmin Preis studied polymer chemistry

in Mainz, Germany, and works in the PSS

production department. She is responsible

for custom synthesis of specialty polymers

and particles.

E-mail: [email protected]: www.pss-polymer.com

Tips and Tricks

16



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Developing HPLC Methods for Biomolecule Analysis An excerpt from LCGC’s e-learning tutorial on high performance liquid chromatography

(HPLC) methods for biomolecule analysis at CHROMacademy.com

Biopharmaceuticals offer great hope

in treating medical conditions that are

currently poorly served by traditional

pharmaceuticals.

Figure 1 is a schematic representation

of trastuzumab, a monoclonocal antibody

(mAb) therapeutic agent used in the

treatment of HER2 positive metastatic

breast cancer and recombinantly produced

in Chinese hamster ovarian (CHO) cells.

Various regions and features of the protein

are outlined in the figure caption and

represent some of the important features

of the drug that need to be characterized.

Protein Titer (Assay)

During manufacture, the amount of

protein in the reaction mixture needs to

be determined and this is achieved using

columns onto which a protein A or G

ligand is immobilized. The monocolonal

antibody is retained via conjugation with

the Fc region of the mAb while all other

species are unretained. The antibody

is then released using a low-pH eluent

(pH 2.6 typically). This form of separation

is known as affinity chromatography.

Charged Variant Analysis

Ion-exchange chromatography is

typically used to monitor for enzymatic

or chemically induced post-translational

modifications (PTMs), which result in

charge differences between proteins

and can affect efficacy, stability,

and toxicity. Monoclonal antibodies

are typically net positive (basic) and

cation-exchange chromatography is used

for characterization and release testing,

with the eluent pH adjusted to promote

the charged form of both the analyte and

stationary phase surface. Elution of the

intact protein is typically facilitated using

salt or pH gradients, the latter being more

amenable to mass spectrometry (MS)

detection; small particle size columns have

been introduced to facilitate rapid, high

efficiency separations.

17




Size Analysis

Size-exclusion chromatography (SEC) is

used to separate proteins, fragments,

and aggregates according to their

hydrodynamic volume, which is a product

of analyte mass, folding, and size of

the hydration shell, and is used for both

characterization and release testing.

Silica or polymer stationary phases are

used to physically “filter” the species

via the pore structure, and the careful

selection of the correct pore size range is

important. Aggregation and fragmentation

of mAbs affect the efficacy and toxicity

of the species, and this testing can

reveal not only macro changes, such as

fragmentation at the hinge region (region

1 in Figure 1), but also subtle differences in

linkages between light and heavy chains.

Structural Characterization

Intact and partially fragmented proteins

can be analyzed using reversed-phase

chromatography with small particles

possessing relative large pore diameter

(volume) (300 Å being typical) and using

shorter ligands such as C4. High-efficiency

particles such as the sub-2-µm or

core–shell particles combined with short,

narrow column geometries are sometimes

used when high throughput is necessary.

Typically, digestion techniques are used

to obtain the Fc and Fab regions (using

dithiothreitol) or light and heavy chains

(papain) where differences in sequence

or modification of the constituent amino

acids can be determined.

Digestion using trypsin will produce the

constituent peptides that are typically

analyzed using high-efficiency C18 or C8

reversed-phase columns of standard pore

size and the highly complex chromatograms

obtained via MS detection (peptide maps)

are used to determine protein sequence.

Glycan Analysis

Region 3 in Figure 1 shows the glycan

(sugar) region of the mAb, which

determines efficacy of the protein. The

sugar subunits of glycans are vitally

important and are used to optimize

efficacy and to produce “biosimilar

or biobetter” molecules that are the

“generics” of the biopharmaceutical

Get the full tutorial at www.CHROMacademy.com/Essentials (free until 20 September).

world. After enzymatic cleavage and

labelling, hydrophilic interaction liquid

chromatography (HILIC) is typically used

to separate the various glycoforms, which

are highly hydrophilic; fluorescence or

MS detection are used depending on the

labelling reagent used.

Amino Acid Analysis

Amino acid analysis is a regulatory

requirement and following acid hydrolysis

and labelling with OPA or FMOC reagents,

separation is achieved using C18 columns,

with core–shell and sub-2-µm particles

becoming increasingly dominant. The

stability of the phase at high pH is

important, and on-line derivatization

followed by fluorescence or UV detection

is growing in popularity.

For a full treatment of the topics included

here please visit: www.chromacademy.com/

HPLC-Techniques-in-Biopharmaceutical-

Analysis.html or see reference 1.

Reference

1. K. Sandra, I. Vandenheede, and P. Sandra, J.

Chromatogr. A 1335, 81–103 (2014).

N-ter

Fab

Fc3

21

(C00–) C-ter C-ter (C00–)

C-ter

N-ter (NH3+)(NH

3+)

C-ter

Lc

Hc

Figure 1: Schematic representation of trastuzumab, a monoclonal antibody protein. Hc = heavy chain, Lc = light chain, N-ter = N-terminus, C-ter = C-terminus, Fc = fragment crystallizable, Fab = fragment antibody binding, 1 = hinge region, 2 = disulphide bridge, 3 = glycan moiety.

The Essentials

18



43rd Symposium of HPLC and Related Techniques (HPLC 2015 Beijing)The 43rd Symposium of HPLC and Related Techniques (HPLC 2015 Beijing Conference) will take place at the Beijing

International Conference Centre, Beijing, People’s Republic of China, from 21–25 September 2015.

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The whole word faces a number of

challenges — such as quality control of

drugs, the production and packaging

of food, disinfection of drinking water,

air pollution in large cities, and disease

(including tumours and diabetes

mellitus). High performance liquid-phase

separations and related techniques

are the core and paramount means

to understand and to seek solutions

to these issues. It is these and other

topics that will be the focus of the HPLC

2015 Beijing Conference. Scientists

from around the world — Canada, the

United States, Netherlands, Switzerland,

Germany, Turkey, Norway, Australia,

Japan, South Korea, and China — will

come together to present research

highlighting recent advances in HPLC

and related techniques. The programme

includes the following highlights:

• Capillary Electrophoresis:

Professor Dovichi from the University

of Notre Dame (Indiana, USA) will

present a study of deep bottom-up

proteomics using capillary zone

electrophoresis (CZE). Professor

Sweedle from the University of

Illinois at Urbana-Champaign (Illinois,

USA) will show single-cell analysis

via high-throughput single cell

mass spectrometry (MS) profiling

and capillary-based separations.

Professor Kennedy of the University

of Michigan (Michigan, USA) will

present the development and

application of rapid electrophoretic

and mass spectrometric assays as

novel approaches to high-throughput

screening.

• Two-Dimensional LC: Professor

Schoenmakers, from the University

of Amsterdam (Amsterdam, The

Netherlands), will review the “why,

when, and how” of comprehensive

2D LC.

19




• Column Design: Professor Svec, at the

E.O. Lawrence Berkeley National Laboratory

(California, USA), will report the less

common techniques using porous polymer

monoliths. Professor Yeung from Iowa

State University (Iowa, USA) will deepen

our understanding of chromatography at

fundamental levels using single molecule

imaging and analysis.

• UHPLC: Professor Mary Wirth at the

Purdue University (Indiana, USA) and

Dr. Michael Dong from Genentech

(San Fransisco, USA) will put emphasis

on the understanding and applications

of ultrahigh-performance liquid

chromatography.

• Proteomics: Professor Yukui Zhang, an

academician of the Chinese Academy

of Sciences (Beijing, China), will talk

about the latest progress in quantitative

proteomics, and Professor Hanfa Zou, at

the Dalian Institute of Chemical Physics

at the Chinese Academy of Sciences, will

present the recent work of his group on

proteomics, but with an emphasis on

protein modif cation. Professor Andy Tao,

from Purdue University (Indiana, USA), will

report specif c isolation and detection of

phosphorylation for proteomic analysis.

• Environmental Analysis: Professor X.

Chris Le and Professor Xingfang Li, both

from the University of Alberta (Alberta,

Canada), will show their extraordinary

contributions to the development of

advanced analytical techniques and

methods for solving issues in environmental

health. Professor Le will emphasize the

chemical species of heavily polluted metals

and their fate in the environment and in

organisms; Professor Li will discuss the

disinfection of drinking water, an issue

faced by global populations.

In addition to the general and parallel

sessions packed with plenary, keynote,

invited, and contributed talks, is the

exposition where companies are invited to

launch new products and showcase the latest

instrumentation, software, and tools related

to all types of liquid-phase separations.

E-mail: [email protected] Website: www.hplc2015-beijing.org

HPLC Beijing Event Preview

20


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GCGC–MS Interpretation12 August 2015Hilton Glasgow Grosvenor, Glasgow, UKWebsite: http://www.crawfordscientific.com/training-online-calendar.asp

Complete Hands-on GC and GC–MS 28 September–2 October 2015 Open University, Milton Keynes, UK Website: www.hichrom.co.uk

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3–4 November 2015/5 November 2015

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3–6 November 2015

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Cover Story - Informa Marketsimages2.advanstar.com/PixelMags/lctc/pdf/2015-08-07-uk.pdf · 8/7/2015 · Investigating yeast products in sparkling wine, diagnosing infection using