Constantly Evolving -...

Constantly EvolvingLeon Blumberg reveals his role in the

development of separation science

2 Constantly Evolving Leon Blumberg has been at the cutting edge of chromatography since

the late 1980s. The Column spoke to him about his contribution to the past, present, and future of chromatography.

Cover Story

Features

21 Preview of Topics at HPLC 2016, Part 4: State-of-the-Art MS Methods for Structural Assessment of mAbs and ADCs: From the Research Lab to Routine Characterization Alain Beck1 and Arnaud Delobel2, 1Centre d’Immunologie Pierre Fabre (CIPF),

2Quality Assistance SA This is the fi nal instalment in a series of four articles exploring topics that will be

addressed at the HPLC 2016 conference in San Francisco, USA, from 19–24 June 2016.

18 Making Method Development Faster for the Analysis of Natural and Artificial Flavourings Philipp Jochems and Gesa Schad, Shimadzu Europa A simple, rapid, and robust ultrahigh-performance liquid chromatography (UHPLC)

method for the simultaneous determination of natural and artifi cial vanilla fl avouring substances as well as some precursors has been developed using an automated method scouting or method optimization workfl ow.

Regulars9 News The latest company news, peaks of the week, and news in brief are featured in this issue.

12 Tips & Tricks GPC/SEC: Branching Analysis Daniela Held, Peter Montag, and Wolfgang Radke, PSS Polymer Standards

Service GmbH Branching is one of the parameters chemists can adjust to produce polymer

materials with optimized physical properties. Chromatography and advanced detection can help to characterize branched molecules. This instalment of Tips & Tricks explains more.

25 The 31st International Symposium on Chromatography (ISC 2016) A preview of the upcoming 31st International Symposium on Chromatography

(ISC 2016), which is due to be held 28 August–1 September at University College

Cork, Ireland.

27 Training Courses and Events

28 Staff

7 June 2016 Volume 12 Issue 10

Constantly EvolvingLeon Blumberg reveals his role in the

development of separation science

2 Constantly Evolving Leon Blumberg has been at the cutting edge of chromatography since

the late 1980s. The Column spoke to him about his contribution to the past, present, and future of chromatography.

Cover Story

Features

21 Preview of Topics at HPLC 2016, Part 4: State-of-the-Art MS Methods for Structural Assessment of mAbs and ADCs: From the Research Lab to Routine Characterization Alain Beck1 and Arnaud Delobel2, 1Centre d’Immunologie Pierre Fabre (CIPF),

2Quality Assistance SA This is the fi nal instalment in a series of four articles exploring topics that will be

addressed at the HPLC 2016 conference in San Francisco, USA, from 19–24 June 2016.

18 Making Method Development Faster for the Analysis of Natural and Artificial Flavourings Philipp Jochems and Gesa Schad, Shimadzu Europa A simple, rapid, and robust ultrahigh-performance liquid chromatography (UHPLC)

method for the simultaneous determination of natural and artifi cial vanilla fl avouring substances as well as some precursors has been developed using an automated method scouting or method optimization workfl ow.

Regulars9 News The latest company news, peaks of the week, and news in brief are featured in this issue.

12 Tips & Tricks GPC/SEC: Branching Analysis Daniela Held, Peter Montag, and Wolfgang Radke, PSS Polymer Standards

Service GmbH Branching is one of the parameters chemists can adjust to produce polymer

materials with optimized physical properties. Chromatography and advanced detection can help to characterize branched molecules. This instalment of Tips & Tricks explains more.

25 The 31st International Symposium on Chromatography (ISC 2016) A preview of the upcoming 31st International Symposium on Chromatography

(ISC 2016), which is due to be held 28 August–1 September at University College

Cork, Ireland.

27 Training Courses and Events

28 Staff

7 June 2016 Volume 12 Issue 10

Constantly EvolvingLeon Blumberg has been at the cutting edge of chromatography since the late 1980s. The Column spoke with him about his contribution to the past, present, and future of chromatography.

— Interview by Lewis Botcherby

Q. You began your career as an

electrical engineer. How did you come

to work within the fi eld of analytical

chemistry and more specifi cally gas

chromatography?

A: I joined the Avondale Division of

Hewlett-Packard Co, which is now

Agilent Technologies, in 1977. HP

Avondale designed and manufactured gas

chromatography (GC) instrumentation. My

initial responsibilities were to design the

electronics and the software for digitizing

and analyzing the chromatograms. From that

grew the need for a better understanding

of the chromatographic limits to separation

performance and speed of analysis. I was

also asked to fi gure out the effect of peak

focusing by the velocity gradients within a

column on the column performance — a

controversial issue at the time.

Q. Why was this?

A: It is a fascinating story, but, first,

I would like to clarify that, in all

forthcoming answers, I distinguish the

peaks in chromatograms (their widths

are measured in the time units) from the

bands of analyte within a column (their

widths are measured in the distance

units). In the late 1980s, a group of

inventors approached HP with a proposal

to substantially improve the speed of GC

analysis by using the dynamic focusing

(negative temperature gradients moving

from the column inlet to the outlet). An

enormous increase in the speed of analysis

without losing resolution was expected

(120 min PONA analysis would be reduced

to a few minutes). The idea was that the

negative temperature gradients along

the column create the negative velocity

gradients causing the front of an analyte

band migrating along a column to move

slower than its tail. This compresses the

bands making them narrower in distance

along the column, and thus presumably

improves the separation-speed trade-offs.

Ray Dandeneau, a co-inventor of fused

silica capillary columns and R&D manager

at the time, asked me to contact the

inventors for a detailed exploration of the

potentials. After preliminary discussions, Ph

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Q&A: Blumberg2 News9 Tips and Tricks12 Jochems and Schad1899 122Beck and Delobel21 ISC Event Preview25 Training and Events27 Staff28252 272

The Column www.chromatographyonline.com

it was decided that HP would help the

inventors to construct the experiments,

and I began, with help from Terry Berger,

to search for theoretical answers. Soon

I found that the dynamic focusing was

an old idea proposed by Russian scientist

Zhukhovitskii1 in 1951 before the invention

of conventional temperature-programmed

GC.2 Since then, high expectations from

dynamic focusing had been periodically

flaring up and dying down again from time

to time with no definitive experimental

improvement and no theoretical solutions.

As others before them, our experiments

did not show any definitive improvement.

The explanation given by the inventors

was that the experiments were not good

enough and Terry and I continued the

search for definitive theoretical answers.

In 1992, we published a theoretical paper3

on the fundamentals of the effects of

the velocity gradients. I was lucky to have

the opportunity to have an encouraging

discussion of the paper with the late Prof.

Giddings during the 1992 International

Conference on Chromatography in

Aix-en-Province, France. Later, I published

a more general theory4,5 and then two

theoretical papers6,7 demonstrating that,

ideally, the velocity gradients can only

harm the separation-speed trade-offs in

chromatography, although the gradients

can reduce the harm from some non-ideal

conditions like poor sample introduction.

These general conclusions can be explained

this way. The same velocity gradients that

compress the analyte bands (make them

narrower in distance along the column)

also reduce the distance between the

bands and slow down the speed of their

migration and elution. The latter two

factors reduce the separation of the bands

and broaden the peaks in chromatograms.

Together, the three conflicting factors

(band compression, worsening of their

separation, and their slower elution) will at

best cancel each other out; at worst they

will reduce the separation-speed trade-offs.

After my involvement in the focusing study,

my expertise in GC theory became one of

my main responsibilities at HP.

Q. Could you briefl y talk about the

work you performed and what you

helped develop while you were at HP?

A: My first job assignment was to

develop an analog-to-digital converter

(ADC) for the first stand-alone digital

integrator in the industry, which

was under development at the time.

This was followed by development

of data-processing and presentation

software for another integrator that

the company introduced later, and then

Q&A: Blumberg

3


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by defining the speed-related aspects

for the next-generation high speed GC

instrument — the first in the industry with

fully integrated electronic pressure and

flow control. As part of that assignment,

I co-invented ADC for GC8 — the fastest

at the time for the needed resolution —

and a GC method translation concept.9,10

Later, I developed a GC method translation

software that became a popular method

development tool. I also co-invented the

retention time locking (RTL) concept11,12,13

— the basis for another useful software

tool introduced by HP. The tool enabled

an order of magnitude improvement in GC

retention time reproducibility.

Q. You continued to work on

the development of fast and

multi-dimensional GC. What do you

feel were some of your most important

publications on these topics?

A: 1D GC (one-dimensional GC): I

completed the publication of a four-part

series on the theory of fast GC,14–17 and

co-authored several papers on heating rate

optimization in GC.18,19

GC×GC (comprehensive two-dimensional

GC): In my first GC×GC lecture (25th

International Symposium on Capillary

Chromatography, Riva del Garda, Italy,

2002, published in 200320), I outlined

the first (as far as I know) theory of

optimization of GC×GC, and theoretically

demonstrated that the separation

performance of GC×GC can potentially be

orders of magnitude higher than that of

1D GC. However, I also demonstrated that,

because of insufficiently sharp modulation,

the GC×GC systems available at the time

were not better than their 1D counterparts.

This caused heated debates,21,22 lasting for

many years and summarized in Pat Sandra’s

2007 lecture23 at the 4th International

GC×GC Symposium (Dalian, China,

2007), published in 200824 (with several

co-authors including myself). Years later,

better modulators became available, and

the key problems of GC×GC configuration

were fixed (all as I was preaching for years).

Several years ago, Jack Cochran described

(International GC×GC Symposium, Riva

del Garda) the first GC×GC system known

to me at the time that performed near its

theoretical potential. The system evaluation

was presented by Matthew Klee at the

11th International GC×GC Symposium (Riva

del Garda, 2014) and published in a paper

that I initiated and co-authored.25 I am

also thankful to Luigi Mondello for inviting

me to contribute a chapter on theory and

optimization of GC×GC coupled to mass

spectrometry (MS) for his volume on the

same topic published in 2011.26

Q&A: Blumberg

4


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Q. In 2010 you published

Temperature-Programmed Gas

Chromatography, which is a very

comprehensive publication covering

many aspects of GC theory.27 How did

this book come about?

A: Although temperature programming

is a key factor of a column’s performance

in GC, no books on this topic had been

published since 1966.28 I had been thinking

about publishing such a book since the

mid-1990s. I began the writing in 2003. Up

until the book was published,27 I did not

work on much else. Unfortunately, I did not

include in the book everything I wanted

(performance metrics, optimization), but the

time to stop came. I believe, however, that

what’s covered is done reasonably well. In

addition to good published reviews, several

colleagues verbally expressed pleasant

comments. Later, Colin Poole gave me an

opportunity to publish a summary of the

performance metrics and the optimization in

a theoretical chapter of the volume on GC

that he edited.29

Q. Your most recent publication focused

on optimizing the mixing rate in

linear solvent strength gradient liquid

chromatography (LC),30 could you

briefl y explain what the mixing rate is

and its importance?

A: The mixing rate is the rate of the

temporal increase (the increase with time)

of the volume fraction of stronger solvent

in LC mobile phase. The mixer is the device

changing the solvent composition. From

that perspective, gradient LC can be viewed

as LC with programmed solvent mixing. It

is well known that the analysis time of this

technique can be substantially shorter than

that of isocratic LC (where the mobile phase

composition does not change during the

analysis). The term mixing rate, which was

recently introduced by Gert Desmet and

myself,30 is a synonym of the wider know

terms gradient slope and time steepness

of the gradient. Why the new term? The

solvent strength programming causes two

types of solvent strength change within

an LC column — the temporal change

and the spatial one (the change along the

column). It is important to recognize the

difference between these two types of

changes because they affect the column

performance differently (see below). Using

the term rate for the temporal changes,

and the term gradient only for the spatial

ones, helps to reinforce the distinction,

and to separately evaluate the effects of

each phenomenon. Using this distinction,

I was able to demonstrate theoretically31

that sharpness of chromatographic peaks

and the resulting speed improvement in

Q&A: Blumberg

5



gradient LC compared to isocratic conditions

comes from the temporal changes while

the effects of the gradients are practically

insignifi cant under typical conditions. It is

important for the clarity of terminology that

the term mixing rate that represents only

the temporal changes does not include the

words like “gradient” and “slope”, which

have spatial implications. From a broader

perspective, gradient LC is a special case

of chromatography with dynamic focusing,

and all conclusions regarding the general

case6,7 apply to LC. Interestingly, in line with

predictions of general theory of dynamic

focusing,6,7 the gradient band compression

typically broadens the peaks slightly30 rather

than focusing (narrowing) them as frequently

expected.

Q. When you optimize anything there is

a trade-off between parameters. These

changes are based on the optimization

goal. What exactly was the optimization

goal within this research?

A: The optimal mixing rate in LC (as

well as the optimal heating rate in GC)

is defi ned19,29,32 as the one at which a

required separation performance is obtained

in the shortest time.

Q. What practical recommendations

have come out of this research?

A: In essence, the optimal mixing rate

(OMR) mostly depends only on the void

time and on the molecular weights of the

sample components. What’s nice is that the

OMR does not directly depend on column

dimensions, its solid support structure, or the

solvent type, but only through their effect

on the void time. This makes it possible to

reduce the optimization results down to

specifi c numerical recommendations. Thus,

for small-molecule samples, the solvent

strength increase of about 5% per time

increment equal to the void time is optimal

or close to optimal for all LC analyses that

can be approximated by the LSS (linear

solvent strength model). The per-void-time

increment for proteins should be about 10

times smaller. This is similar to the optimal

heating rate of 10 ºC per void time in

GC.19,29

Q. What are you currently researching?

A: I am grateful for having the opportunity

to work with Gert Desmet — a leading

expert in theory of gradient LC. We are both

interested in optimization of the technique.

A key to the optimization is the simple and

transparent performance metrics. Together,

we published a paper on the metrics

of separation performance in gradient

LC30 and on the optimal mixing rate32

in a simple single-ramp solvent strength

Q&A: Blumberg

6


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program. Currently, we are working on

the optimization of more complex mixing

programs.

Q. What do you regard as the most

exciting areas of chromatography at the

moment?

A: In my view, comprehensive

multidimensional techniques are the

most promising developments in column

chromatography (LC, GC, etc.). The

GC×GC “is probably the most promising

invention in GC since discovery of capillary

columns.”24 The overall performance of

a chromatographic analysis is a trade-off

between three factors — the separation

performance, the time, and the detection

limit (DL). Here’s an example from GC with

which I am more familiar: Each twofold peak

capacity increase in 1D GC without changing

the DL costs eightfold longer analysis time

(1 h analysis becomes 8 h analysis). On the

other hand, adding the second dimension

can increase the peak capacity by more than

an order of magnitude without changing

the analysis time and DL. Currently, not all

potentials of GC×GC are exploited. Thus,

peak deconvolution substantially increases

the separation performance of 1D GC and

GC×GC. However, only the deconvolution

along the second dimension is currently used

in GC×GC. Adding the deconvolution to

the fi rst dimension of GC×GC can further

increase its overall separation performance

several times.20,26 Unfortunately this

approach remains mostly unknown.

Q. Will the evolution of mass

spectrometry eventually lead to the

extinction of chromatography?

A: I am not an expert in mass spectrometry

(MS) and in the multi-stage MS techniques.

However, the way I see this world, there

will always be the need for the separation

of more and more complex mixtures. The

improvement in the separation-time-DL

trade-offs in multi-dimensional chromatography

and in multi-stage MS will always complement

each other, but more separation power will

always be needed. Could you imagine a

day when there will be no practical need

for further substantial improvement in

computer speed or memory, or in the speed

of the Internet? I think the same is true for

the separation-time-DL performance of

analytical tools.

References

1. A.A. Zhukhovitskii, O.V. Zolotareva, V.A.

Sokolov, and N.M. Turkel’taub, Doklady

Akademii Nauk S.S.S.R. 77, 435–438

(1951).

2. J. Griffiths, D. James, and C.S.G. Phillips, The

Analyst 77, 897–904 (1952).

Q&A: Blumberg

7


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19. L.M. Blumberg and M.S. Klee, J. Microcolumn

Sep. 12, 508–514 (2000).

20. L.M. Blumberg, J. Chromatogr. A 985, 29–38

(2003).

21. C.M. Harris, Anal. Chem. 74, 410A (2002).

22. L.M. Blumberg, Anal. Chem. 74, 503A (2002).

23. P. Sandra, F. David, M.S. Klee, and L.M.

Blumberg, “Comparison of one-dimensional

and comprehensive two dimensional capillary

GC separations,” Dalian, China, 4–7 June

2007, (CD ROM).

24. L.M. Blumberg, F. David, M.S. Klee, and P.

Sandra, J. Chromatogr. A 1188, 2–16 (2008).

25. M.S. Klee, J.W. Cochran, M. Merrick, and L.M.

Blumberg, J. Chromatogr. A 1383, 151–159 (2015).

26. L.M. Blumberg, in Comprehensive

Chromatography in Combination with Mass

Spectrometry, L. Mondello, Ed. (Wiley,

Hoboken, NJ, USA, 2011), pp. 13–63.

27. L.M. Blumberg, Temperature-Programmed

Gas Chromatography (Wiley-VCH, Weinheim,

Germany, 2010).

28. W.E. Harris and H.W. Habgood, Programmed

Temperature Gas Chromatography (John Wiley

& Sons, Inc., New York, USA, 1966).

29. L.M. Blumberg, in Gas Chromatography, C.F.

Poole, Ed. (Elsevier, Amsterdam, 2012) pp.

19–78.

30. L.M. Blumberg and G. Desmet, J. Chromatogr.

A 1413, 9–21 (2015).

31. L.M. Blumberg, Chromatographia 77, 189–197

(2014).

32. L.M. Blumberg and G. Desmet, Anal. Chem.

88, 2281–2288 (2016).

Leon Blumberg

graduated from the

Leningrad Electrotechnical

Institute (Leningrad, USSR;

currently St Peterburg,

Russian Federation) in

1960 with a diploma

in electrical engineering and went on

to join a computer design company in

Leningrad. In 1961–1965, Leon completed

a full math course for professional

mathematicians from Leningrad University.

He obtained a PhD equivalent in electrical

engineering from Leningrad Electrotechnical

Institute of Telecommunications. In

1977, Leon immigrated to the US with

his family, joining the Avondale Division

of Hewlett-Packard Co. (now Agilent

Technologies). Currently, as part of his

consulting company Advachrom, Leon

provides consulting services mostly in

GC×GC. His main scientifi c interest

now is to develop a unifi ed theory of

temperature-programme GC and

gradient LC.

E-mail: [email protected]

3. L.M. Blumberg and T.A. Berger, J. Chromatogr.

596, 1–13 (1992).

4. L.M. Blumberg, J. Chromatogr. 637, 119–128

(1993).

5. L.M. Blumberg, J. High. Resolut. Chromatogr.

16, 31–38 (1993).

6. L.M. Blumberg, Anal. Chem. 64, 2459–2460

(1992).

7. L.M. Blumberg, Chromatographia 39, 719–728

(1994).

8. L.M. Blumberg, J. Bush, and R.P. Rhodes, U.S.

patent 5,448,239, 1995.

9. W.D. Snyder and L.M. Blumberg, U.S. patent

5,405,432, 1995.

10. L.M. Blumberg and M.S. Klee, Anal. Chem. 70,

3828–3839 (1998).

11. M.S. Klee, P.L. Wylie, B.D. Quimby, and L.M.

Blumberg, U.S. patent 5,827,946, 1998.

12. M.S. Klee, B.D. Quimby, and L.M. Blumberg,

U.S. patent 5,987,959, 1999.

13. L.M. Blumberg, B.D. Quimby, and M.S. Klee,

U.S. patent 6,153,438, 2000.


20, 597–604 (1997).


20, 679–687 (1997).


22, 403–413 (1999).


22, 501–508 (1999).

18. L.M. Blumberg and M.S. Klee, Anal. Chem. 72,

4080–4089 (2000).

Q&A: Blumberg

8


Changing the Landscape of Mass Detection in the

Chromatography Lab

EVENT OVERVIEW

Traditionally mass detection instruments have been for

the mass spec experts and not played a major role in

the majority of chromatography labs. With the advent of

smaller, more accessible mass detectors, the potential of

mass data is coming more and more within the reach of

the chromatographer. With this webcast we look to see

how the landscape of the chromatography lab is chang-

ing and how the value of mass data can be realized by

the chromatographer.

Key Learning Objectives

■ How the landscape of mass detection in the chromatog-raphy lab is changing

■ Value of mass data to a chromatographer

■ How a chromatography data system (CDS) should utilize the power of mass detection and mass data

Who Should Attend

■ Method development chemists

■ Development lab managers

■ QC lab managers

Sponsored by Presented by

All attendees will receive a FREE Executive Summary! For questions, contact Kristen Moore at [email protected]

ON-DEMAND WEBCASTOriginally aired 04/28/16

Presenter

David Wayland

Empower product

owner

Waters Informatics

Moderator

Laura Bush

Editorial Director

LCGC

Register free at www.chromatographyonline.com/lcgc/

landscape

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Waters Announces Additions to Leadership Team

Waters Corporation (Milford, Massachusetts, USA) has announced

the addition of Richard Chang and Xiao Ran Yu to its Asia Pacifi c

leadership team. Chang has been appointed as Vice President of

Waters’ Asia Pacifi c Operations and Yu has been appointed as

General Manager of Waters China.

“Mr. Chang’s and Mr. Yu’s impressive proven track records have

demonstrated an intimate understanding of the marketplaces in

the region,” said Dr. Mike Harrington, Senior Vice President, Global

Markets, Waters Corporation. “With their extensive experience and

strong commitment, our China business has become the single biggest

market for Waters outside the US. We strongly believe that they will

continue to drive growth in the Asia Pacifi c region,” he continued.

Beginning his career with Waters in Taiwan, Chang moved to

China following four successful years as General Manager to help

develop the Chinese market. Appointed as President of Greater

China operations in 2012, he has led Waters’ signifi cant growth

within the Asia Pacifi c region.

“It’s my great honour to lead the Asia Pacifi c team and I look

forward to working closely with our customers and partners in

meeting their needs, and bringing Waters innovation to them in the

most accessible way,” said Chang.

“I’m excited to join the leadership team in Asia Pacifi c and apply

my wealth of experience and passion for science and technology

to support customers in China,” enthused Yu, who has more than

22 years of experience in the analytical science industry. The former

operations manager for South China had led his team to signifi cant

sales revenues during his tenure.

Waters are hopeful these two new appointments can drive

growth within the region and capitalize on the emerging strength

of the Asia Pacifi c markets.

For more information please visit www.waters.com.

Thermo Fisher Partner with University of Birmingham’s New Phenome CentreThermo Fisher Scientifi c (San Jose, California, USA) has extended a technology collaboration with the University of

Birmingham in the UK. Thermo Fisher will supply a range of mass spectrometry instruments to be used at the University’s

new Phenome Centre.

The £8 million facility provides University of Birmingham scientists with the tools needed to conduct large-scale metabolic

phenotyping research, advancing the understanding of biochemical mechanisms, targets, and biomarkers associated with

ageing and disease.

“We are pleased to continue our long-standing collaboration with Thermo Fisher in these important areas of

metabolome research,” said Professor Mark Viant, Director of Phenome Centre Birmingham.

“Combined with our strong impact in technology and method development, we look forward to the benefi ts

we will translate to the human population through stratifi ed medicine approaches,” continued Viant.

Stratifi ed medicine, a method of predicting which treatments cancers are likely to respond to by

studying the cells and genetic make up of large groups of cancer patients, is part of the larger

strategy employed at the facility.

Iain Mylchreest, vice president of R&D for chromatography and mass spectrometry at Thermo

Fisher, congratulated the university on the opening of the Phenome Centre, adding, “These

scientifi c relationships are mutually benefi cial in helping us to advance our analytical

tools and technologies that, in turn, enable our customers to make the world healthier,

cleaner, and safer.”

This recent collaboration is just one of many between the University of Birmingham

and Thermo Fisher across 10 years. Most recently, the organizations have collaborated

to accelerate research in high-resolution accurate mass (HRAM) and triple quadrupole

liquid chromatography-mass spectrometry (LC–MS) for life sciences applications.— L.B.

For more information about Thermo Scientifi c,

please visit www.thermofi sher.com

For further information about the Phenome Centre Birmingham, please visit

http://www.birmingham.ac.uk/research/activity/phenome-centre

9



Shimadzu Lab4You AcceptingApplications

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Following a successful launch last year,

Shimadzu’s Lab4You program is once again

accepting applications from young and

enthusiastic researchers hoping to advance

their research.

Shimadzu is offering a few promising

scientists the chance to work at “Shimadzu

Laboratory World”. The winners will be

able to advance their research utilizing the

state-of-the-art equipment at the facility

including well-serviced analytical instruments

and on-hand product specialists.

“The work was very interesting and I

highly recommend other students apply for

the Lab4You student program,” said Carola

Schultz, University of Münster.

Schultz was one of two winners from last

year’s lab4you competition. Her research

investigated organic lithium-ion battery

electrolytes using liquid chromatography–

mass spectrometry (LC–MS).

“They [lithium-ion batteries] contain organic

carbonates and the lithium-ion conducting

salt LiPF6. But during cycling of the cell, the

electrolyte ages and the cell loses capacity.

The generated ageing products are built

up out of reactions from the salt with the

organic carbonates,” explained Schultz.

“So my aim was to do a full characterization

of the electrolyte with LC–MS, to investigate the

main components as well as ageing products

in order to facilitate the elucidation of ageing

mechanisms occurring inside the cell into the

electrolyte,” continued Schultz, who is currently

working towards publishing her results.

The Lab4You program emerged from

Shimadzu’s 2013 Laboratory World opening

in Duisburg, Germany. The 1500 m2

testing facility contains Shimadzu’s entire

product range and was used for customer

demonstrations and application training.

“We felt that there was still some more

capacity for the instruments to be used.

So we came up with the idea to invite students

with interesting research projects to come to

Duisburg and use the high-end equipment

that is not available at their University, in our

Laboratory Worlds,” said Dr. Gesa J. Schad,

HPLC Product Manager, Shimadzu.

Minimum requirements for candidates

include an undergraduate degree with a

science background, an approved topic of

research (MSc or PhD thesis or postdoc

research), and some practical experience in

analytical chemistry.

“We are also looking for good

interpersonal and communication skills.

The successful candidate will spend a

considerable amount of time working with

us, so they should fi t in well with the team.

Shimadzu offers to sponsor registration to

relevant conferences, where the obtained

data can be presented,” explained Schad.

Interested students can apply by submitting

a short abstract of their research at www.

shimadzu.eu/lab4you. Deadline for submission

is 31st October 2016. — L.B.

Carola Schultz during her time at Shimadzu’s Laboratory World.

From left to right: Anja Grüning, Product

Specialist LC–MS, Dr. Julia Sander, Product

Specialist Life Sciences; Carola Schultz,

lab4you participant; Uta Steeger, Manager

Marketing; Dr. Gesa Schad, Product Manager

HPLC; Robert Ludwig, Product Specialist HPLC;

and Philipp Jochems, Product Specialist HPLC.

News

10


News In Brief

Like us Join us Follow Us

LCGC magazine is pleased to announce the addition of Deirdre Cabooter and Debbie Mangelings to the editorial advisory boards of LCGC North America and LCGC Europe. Cabooter is an assistant professor in the Department of Pharmaceutical and Pharmacological Sciences at the University of Leuven in Belgium. Mangelings currently works as an associate professor in the Department of Analytical Chemistry and Pharmaceutical Technology.

W.R. Grace & Co. has agreed to sell product lines associated with its chromatography instruments, columns, and related laboratory products businesses to four separate buyers: Buchi, Dr. Maisch, Hichrom, and S*Pure. Grace will retain three growing service lines for the pharmaceutical and nutraceutical industries that are aligned with the company’s growth plans, while concentrating on its core materials science and manufacturing capabilities.https://grace.com/en-us/newsroom/Pages/

news-item.aspx?ItemID=498

A study has been conducted to evaluate the migration of monomers and plastic additives in microwaved solid or liquid packed and retailed food. Using GC–MS and QuEChERS recoveries ranged from 49 ± 16% (OP) to 130 ± 16% (BPA) and from 63 ± 22% (OP) to 127 ± 29% (NP) in solid and liquid foods. The QuEChERS method was able to determine the presence of these chemicals in packed food, thereby allowing the evaluation of compounds that can affect food quality.doi:10.1016/j.lwt.2015.06.066

LCGC TV HighlightsLCGC TV: Barbara Larsen on Replacing a

Mass Spectrometer: What to ConsiderReplacing an older mass spectrometer involves a reevaluation of the way the instrument is being used. Barbara Larsen from DuPont Central Research and Development discusses the checklist

you should follow.Watch Here>>

LCGC TV: The Potential for Ionic Liquid-Based Coatings in SPMEMore and more analytes are now being found in complicated matrices. Jared Anderson from the Iowa State University recently designed new SPME coatings based on polymeric ionic liquids suitable

for these matrices. He discusses the potential for these coatings to improve on SPME. Watch Here>>

Peaks of the WeekThe LCGC Blog: The Middle Ground on Unconventional Oil and Gas Development (aka

“Fracking”) is a Lonely Place — Kevin A. Schug discusses his research consortium’s work on the

quality of groundwater in close proximity to fracking sites, the disparity in the media’s interpretation of

the data, and addresses valid queries made by critics. Read Here>>

The Modulator in Comprehensive Two-Dimensional Liquid Chromatography —

This article illustrates the variety of commonly used modulators, paying particular attention to

focusing modulators.

Read Here>>

The SFC Renaissance? — Jean-Luc Veuthey and Alexandre Grand-Guillaume-Perrenoud reveal the

latest developments in supercritical fluid chromatography (SFC) that are bringing the technique

back to the limelight.

Read Here>>

The Column www.chromatographyonline.comPh

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News

11


Tips & Tricks GPC/SEC: Branching Analysis

Branching is one of the parameters chemists can adjust to produce polymer materials with optimized physical properties. Chromatography and advanced detection can help to characterize branched molecules. This instalment of Tips & Tricks explains more.

Daniela Held, Peter Montag, and Wolfgang Radke, PSS Polymer Standards Service GmbH, Mainz, Germany.

An advantage of polymer materials is that

their physical properties can be tailored

for a specific application by adjusting

many parameters. Besides composition,

average molar mass, and the width of

the molar mass distribution, another key

parameter to control application properties

is branching.

Branching requires at least a single

branch point where three or more chains

are connected. Branching can occur as an

undesired side reaction during synthesis

or it can be introduced deliberately to

optimize the physical properties of the

material. Different routes exist to synthesize

defined structures, such as star-shaped or

comb-shaped molecules.

The properties of branched polymers

differ significantly from linear ones with

respect to (melt) viscosity, glass transition

temperature, the coefficient of bulk

thermal expansion, solubility, and others.

The property change depends on the

parameters such as type of branching,

length of the branches, and branching

density.

The characterization of complex

mixtures comprising not only a molar mass

distribution but branching distribution

as well, represents a real challenge.

Depending on the type of branching

there are various detection and separation

options that provide deeper insight.

Gel permeation chromatography/

size-exclusion chromatography (GPC/SEC)

hyphenated with on-line viscometry1 (or

less accurate multi-angle light scattering)

can be used to characterize defined

structures, such as star or comb-shaped

polymers, or to investigate long chain

branching. High temperature GPC (HT-GPC)

with infrared (IR) detection can be used

to investigate short-chain branching in

polyolefins.2

For samples exhibiting broad molar mass

distributions for branches and backbone Ph

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12



and variations in branching density, the

resolution of the size-based separation

in GPC/SEC might not be sufficient to

fully resolve the structures. Therefore

alternative separation methods such as

interaction chromatography (for example

gradient polymer high performance liquid

chromatography [HPLC] or temperature

gradient interaction chromatography

[TGIC]) or two-dimensional (2D)

chromatography should be applied.3

GPC/SEC-Viscometry

An on-line viscometer is classifi ed as a

molar mass sensitive detector, however, the

signal intensity is dependent on the viscosity

rather than on molar mass. Viscometers

provide direct access to the density of the

molecules in solution. While the setup of

such instrumentation, in most cases in

combination with a light scattering detector,

is very common, understanding the results

and limitations requires more experience.

The Mark–Houwink plot is important for

branching analysis; the logarithm of the

intrinsic viscosity (obtained using on-line

viscometry) is plotted versus the logarithm

of the molar mass (obtained using universal

calibration or light scattering detection).

The slope of the Mark-Houwink plot

(Mark-Houwink coeffi cient, α) is dependent on

the shape of the molecule in solution. If the

intrinsic viscosity (solid sphere) has no molar

mass dependence a slope of 0 is expected,

however, the Mark–Houwink exponent of rigid

rods is 2. Typical random coil polymers exhibit

Mark-Houwink exponents in the range of 0.5

to 0.8, depending on solvent quality.

Branching analysis can be

straightforward, assuming that the data

can be compared to a linear chain of

identical chemical structure and molar

mass. Figure 1 shows Mark–Houwink

plots for different polyethylene samples.

A HT-GPC equipped with an on-line

viscometer has been used to generate this

plot. While the Mark–Houwink plot of the

linear low-density polyethylene (LLDPE),

which has only short chain branching,

nearly superimposes with the linear sample,

10 2

Vis

cosi

ty

PS

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ity,

Bu

ld 6

08

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Mark-Houwink-Plot

NBS-1475: lin. PE, pink, α = 0,68LLDPE-C6: black, α = 0,70

LDPE: blue, α = 0,42

10 1

1×10 4

Universal calibration (Da)

1×10 5 1×10 6

Figure 1: Mark-Houwink plot overlay of three different polyethylene samples. NBS-1475 (pink) is linear, linear low density polyethylene (LLDPE, black) has short-chain branching that does not infl uence the viscosity much, and low density polyethylene (LDPE, blue) exhibits long-chain branching, which leads to a reduction of viscosity.

Tips and Tricks

13


Proteins of biopharmaceutical interest are generally hetero-geneous mixtures of proteoforms comprised of modifications. Accurate knowledge of the proteoform profile is critical for assessing the safety and stability of the drug. Current methods of analyzing post-translational modifications (PTMs) and glycan structures require proteolysis or glycan profiling that can result in lost information on correlated PTMs. Sample handling can also introduce artifacts and therefore, minimal sample preparation is desirable. Top-down mass spectrometry (MS) analysis of highly heterogeneous biopharmaceuticals is challenging due to the limited ability of current separation techniques in resolving proteoforms with small structural changes. In this work, we describe the top-down analysis of interferon-β1 (Avonex) in detail and preliminary data on middle-down (reduced) and intact mAbs.

Key Learning Objectives:

■ Find out how CESI-MS enables high resolution intact separation and on-line top-down MS identification of PTMs with minimal sample preparation

■ Learn how glycan isomers (such as the two isomers G2F and 1 NANA) and deamidations can be resolved by intact analysis

■ Learn how a similar approach was applied for intact and reduced analysis of mAbs

Who Should Attend

■ Principal Investigators, department chairs, senior scientists, R&D directors, post-doctoral fellows, post-graduate researchers, and medical researchers.

Sponsored by

Presented by

For questions, contact Kristen Moore at [email protected]

High-Resolution Quantitative Characterization of Intact Biopharmaceuticals and Their ProteoformsON-DEMAND WEBCAST Aired May 25, 2016

Register for free at www.chromatographyonline.com/lcgc/sciex_series2

Presenter:

David R. Bush, Ph.D.Scientific ManagerGenedata, Inc.

Moderator:

Laura Bush

Editorial Director LCGC

30 MinuteFormat


the low-density polyethylene (LDPE), which

comprises long-chain branching, deviates

significantly. At the same molecular weight

the LDPE chains reveal a significantly lower

intrinsic viscosity compared to the linear

sample. This is a consequence of branches

being present. The deviation increases

with increasing branching density. By

extrapolating the Mark–Houwink plots

of the branched and linear polymer to

a common intercept, it is possible to

detect the molar mass at which branching

first occurs. By taking the ratio of the

intrinsic viscosity of the branched and

linear polymer at the same molar mass, it

is possible to determine the contraction

factor, g’, from which conclusions on the

number of branches can be deduced.

Figure 2 shows the results of GPC/

SEC-viscometry of a poly(tert-butyl acrylate),

PtBuA, star polymer. Star polymers are

relatively simple branched polymers because

they consist of several arms (linear chains)

connected to a central core.

The star polymer was synthesized using

the arm-first approach. PtBuA arms of

narrow molar mass distribution have

been coupled using a small amount of

a bifunctional cross-linker to form the

core. This means that the molar mass

increase of the star was obtained by

coupling an increasing number of arms

of approximately the same length to the

core. The Mark–Houwink plot reveals a

maximum for the intrinsic viscosity, which

has also been observed for dendrimers.

Starting with a linear precursor, the

coupling of two linear chains forms a still

linear molecule, the dimer. Further reaction

of precursor molecules with the core leads

to three-arm and higher arm star polymers.

Here the increase of the intrinsic viscosity

with molar mass is counterbalanced by

the decreasing intrinsic viscosity resulting

from an increasing segment density

with increasing number of arms for the

branched structures.

This variation in molecular structure

can be nicely monitored from the viscosity

measured using an on-line viscometer.

0.010

102

0.009

0.008

PSS W

inG

PC

Un

ity

Sp

eci

fic

vis

cosi

ty

Intr

inis

c V

isco

sity

Number of ArmsDegree of Branching

Intr. Visc.Linear Counterpart

Star Polymer withaverage arm number 5

4.5% Dimer

12.1% Residual Arm

Molar mass from universal calibration curve (D)

4.0% CoupledStar Polymer(H-shaped)

0.007

0.006

0.005

0.004

0.003

0.002

0.001

5*10 4

1*10 5

5*10 5

1*10 6

(still linear)

Figure 2: Mark-Houwink for a star polymer using the arm-fi rst approach. Arms with a narrow molar mass distribution are coupled to a core; the molar mass increase is a result of the addition of more arms to the core. The structure change (linear coil to a more and more dense sphere) is refl ected by a maximum in intrinsic viscosity.

Tips and Tricks

14


LIVE WEBCAST: Wednesday, June 15, 20168 am PDT | 10 am CDT | 11 am EDT | 4 pm BST | 5 pm CESTRegister free at: www.chromatographyonline.com/lcgc/quantitation

EVENT OVERVIEW

The screening and routine quantitation of pesticide residues

in food products is one of the most important and demanding

applications in food safety. Despite the recent technological

advancements in LC-MS, it is still challenging to quantify

hundreds of LC-amenable pesticides with a robust, sensitive

workflow solution.

This presentation describes the development and implemen-

tation of complete workflow solutions based on LC-MS/MS

and LC-HRAM-MS/MS. These ready-to-go solutions have been

validated in three matrices across four different laboratories.

In addition, customized software used for data acquisition

and processing allows the users to rapidly implement these

methods and enhance productivity.



■ Address critical challenges in targeted or untargeted quanti-

tation of pesticides in food laboratories using either triple

quadrupole MS or high-resolution accurate mass (HRAM)

MS instrumentation

■ Learn about robust, routine workflows that can increase

laboratory and organizational productivity

Who Should Attend

■ Researchers and analysts in need of fast and cost-effective

solutions for the analysis of pesticides in food

Sponsored by

Presented by

Presenters

Ed George

Senior Applications Scientist,

Environmental and Food Safety,

Chromatography and Mass Spectrometry

Thermo Fisher Scientific, Inc.

Debadeep Bhattacharyya

Senior Marketing Manager,

Triple Quadrupole MS

Thermo Fisher Scientific, Inc.

Moderator:

Laura Bush, Editorial Director, LCGC

Pesticide Residues Analysis Webinar Comprehensive Pesticide Quantitation Workflow with LC-MS


A very interesting observation is the

sudden change of intrinsic viscosity

occurring at approximately twice the

molecular weight of the peak maximum.

The drastic increase in viscosity is most

probably a result of the formation of

H-shaped molecules as coupling of two

stars occurs via an arm.

In contrast to the application above,

star polymers can also be synthesized

using the core first approach, for

example, using a multifunctional initiator.

In this case the observations and results

would be different. There would be no

structural change because the molar mass

increase of the star would result from the

3,0

2,5

2,0

1,5

1,0

0,5

0,0

-0,5

-1,03 4 5

Linear

3 arm star

4 arm star

7 arm star

log M

log

(η

)

6

Figure 3: Schematic Mark-Houwink plots for three star polymers synthesized via core-fi rst approach compared to a linear molecule. Arms are started from a multi-functional initiator core — molar mass increase is a result of the addition of monomer units to the arms. The Mark-Houwink plots are parallel to one of the linear polymers but shifted to lower intrinsic viscosities.

Tips and Tricks

15


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Bioanalysis: LC–MS-MS, Sample Prep, and Dried Blood Spot Analysis Bioanalysis uses a variety of separation techniques to analyze samples ranging from plasma and urine to dried blood spots. Participants in this Technology Forum are Ling Bei, Patrik Appelblad, and Dave Lentz of EMD Millipore; Nadine Boudreau of PharmaNet Canada; Diab Elmashni, Jeff Zonderman, and Simon Szwandt of Thermo Fisher Scientific; and Debadeep Bhattacharya of Waters Corporation. More...

Name your budget or application—the Agilent 1200 Infinity Series has you covered. From our affordable 1220 Infinity LC starting at just $15,000 to our cutting-edge 1290 Infinity LC, we have a solution that’s right for you. Plus, our most popular 1200 Infinity Series LC configurations are now available with a 3-5 year up-and-running guarantee. Read more.

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KNAUER is now offering its PLATINblue UHPLC systems with the MSQ Plus mass detector in a special package deal. PLATINblue systems and the MSQ Plus are an ideal combination for high-throughput applications. For a limited-time only, the UHPLC-MS package is being offered at a very special price. Don’t miss out!

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and Tandem Mass Spectrometry Barbora Brazdova and Marta Kozak, Thermo Fisher Scientific

Learn about a 9-min, sensitive (LOQ 1—50 ng/mL) method to quantitate 30 immunosuppressant

drugs using TurboFlow technology and LC–MS-MS.

Evaluation of the Ultra Inert Liner Deactivation for Active Compounds Analysis by GCLimian Zhao, David Mao, and Allen Vickers, Agilent Technologies

Endrin and DDT breakdown and active semivolatiles tests were used for the Ultra Inert liner deactivation performance evaluation. The results indicate that the Ultra Inert deactivated liners provide superior inertness for analysis of active compounds.

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Food Analysis of PAHs Using GCxGC-TOFMS and QuEChERSLECO CorporationThe combination of QuEChERS extraction and GCxGC-TOFMS is a fast and accurate method for

detecting and identifying PAH contaminants in complicated foodstuff matrices such as liquid infant formula and blended blueberries.

��

Screening and Identification with High Confidence Based on High Resolution and Accurate Mass LC–MS-MSAndre Schreiber and David Cox, AB SciexThis note describes a workflow and tools to identify targeted and nontargeted pesticides in fruits and vegetables. High resolution, accurate mass LC–MS-MS data is mined using advanced

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� Highly Sensitive UV Analysis with the Agilent 1290 Infinity LC System for Fast and Reliable Cleaning Validation – Part 1Edgar Naegele and Katja Kornetzky, Agilent TechnologiesThis application note demonstrates high sensitivity measurement of pharmaceutical compounds

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Pesticides in Fatty MatricesDon Shelly, UCT"Fat is where it's at" when it comes to finding most pesticides. Extracting the pesticides and not

the lipids can be a challenge! This months featured application is easy, quick, effective, rugged, and inexpensive.

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Which GPC Column First? Bruce Kempf, Tosoh BioscienceMost manufacturers recommend the installation of SEC columns in order of decreasing pore size when running columns in series. Scientists at Tosoh Bioscience tested the validity of this recommendation.

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Ultra-Fast Analytical Method for the Sample Cleanup and LC–MS-MS Analysis of

Chloramphenicol in Shrimp and Other Marine Food ProductsPhilip J. Koerner, Matthew Trass, Liming Peng, and Jeff Layne, PhenomenexA method for the analysis of chloramphenicol in shrimp has been developed with a limit of quantitation (LOQ) of 0.001 ng/g in shrimp (0.001 ppb) based on the calibration standards. This is 300 times lower than the current U. S. Food and Drug Administration (USFDA) method. The method described uses Strata-X solid phase extraction (SPE) cartridges for sample cleanup and

concentration, followed by ultra-fast LC–MS-MS analysis (<5 min) using a Kinetex core-shell column.

��

Topics and categories include: )1-$�t�($�t�4BNQMF�1SFQ�t�-$�.4�BOE�

($�.4�t�&NFSHJOH�UFDIOJRVFT

www.chromatographyonline.com/enews


growth of the arms. For such star

polymers the Mark–Houwink plots

are expected to be as shown in Figure

3. For each star polymer the Mark-

Houwink plot will be shifted in parallel

to lower viscosities at the same molar

mass. Analysis would yield the same

Mark-Houwink, α, but a reduced

Mark-Houwink, K (intercept). The shift to

lower intrinsic viscosities increases with

increasing number of arms.

Advanced Separation Techniques

One limitation of GPC/SEC is that it

separates only based on the size of the

molecule in solution. This has the following

consequences for branched samples:

t��$POWFOUJPOBM�DBMJCSBUJPO�XJUI�SFGFSFODF�

materials will underestimate the molar

mass of the branched samples. A

solution here is the use of molar mass

sensitive detectors, such as on-line

viscometers or light scattering detectors.

100

90

80

70

60

50

40

30

20

10

0

0 5 10 15

Elution volume (mL)

No

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co

nce

ntr

ati

on

Star with 3 Arms

2 arms/Dimer

Arm/Precursor

4 5 6 7 8 910

20 25 30 35 40

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Figure 4: Separation of a star branched polymer by interaction chromatography. The resolution of GPC/SEC for such samples is lower, because GPC/SEC separates based on size, which for this application increases only slightly for stars with more arms.

The viscometer can then be used for

structure analysis and for molar mass

determination based on universal

calibration.

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increases only slightly with molar mass,

such as arm-first star polymers, the

resolution of size-based separations

is limited. In this case interaction

chromatography can be used as a

complementary technique. Figure 4

shows the chromatogram of a gradient

separation of an arm-first star polymer

with a very high resolution even for stars

with higher arm numbers.

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distributions besides structural

heterogeneity (for example, branched

and linear chains present) the risk

of co-elution increases. In that case

branched molecules of higher molar

mass with the same hydrodynamic size

as linear chains of lower molar mass

elute at the same retention volume.

Consequently, the GPC/SEC fractions

eluting from the column cannot be

regarded to be monodisperse any longer.

Comprehensive characterization of

branched polymers might be possible when

2D separations are applied. Two-dimensional

combines two independent separation

Tips and Tricks

16


ULTRA-FAST GCUseful Tool or Just Another Gimmick?

EVENT OVERVIEW

The fundamental principle of ultra-fast gas chromatogra-

phy is based on rapid temperature programming of the

GC analytical column at rates generally between 60 and

200 °C per minute. At these ramp rates, the dynamic tem-

perature range of most columns is used up in less than 2

minutes, and this is insufficient time to fully elute high-

boiling-point compounds, so the technique lends itself

to short columns. Shorter columns have less resolving

power, therefore it’s important to know the techniques to

optimize the parameters that give speed and resolution

without sacrificing column capacity.


■ Where to use ultra-fast GC

■ How to optimize parameters to get the best resolution without sacrificing column capacity

■ Types of ultra-fast GC systems

Who Should Attend

■ GC Users with large numbers of samples looking to increase sample throughput

■ GC Users with long analysis cycle times looking for faster cycle times

■ GC Users looking to reduce energy usage and environmental impact of GC analysis

ON-DEMAND WEBCAST Aired May 31, 2016

Register free at

www.chromatographyonline.com/lcgc/useful_tool

Sponsored by Presented by

FREE executive summary for all webcast attendees.


Presenter

Phillip JamesManaging DirectorEllutia Ltd

Moderator

Meg L’HeureuxManaging EditorLCGC


two-dimensional chromatography can be

applied.

References

1. D. Held, The Column 8(2), 12–16 (2012).

2. P. Montag, The Column 12, 14–17 (2008).

3. J. Gerber and W. Radke, Polymer 46,

9224–9229 (2005) doi: 10.1016/j.

polymer.2005.07.038.

Daniela Held studied polymer chemistry

in Mainz, Germany. She currently works

at the PSS software and instrument

department and is responsible for

education and customer training.

Peter Montag studied chemistry at

the University of Duesseldorf, Germany,

achieving his PhD at the Max Planck

Institute for Coal Research. He is the head

of the PSS contract analysis department

and responsible for hyphenated techniques.

Wolfgang Radke studied polymer

chemistry in Mainz, Germany, and

Amherst, Massachusetts, USA. He is

head of the PSS application development

department and is also responsible for

instrument evaluation and for customized

training courses.

techniques to generate contour plots, which

can also be used to quantify the different

species. Figure 5 shows an example where

linear and comb shaped molecules of

different composition have been successfully

separated.

Summary

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polymerizations or can be introduced to

tailor application properties.

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density of a molecule and can help to

characterize branched molecules as they

monitor structural changes with molar

mass.

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can be analyzed by on-line viscometry,

while information on short-chain branching

can be gained by Fourier-transform infrared

spectroscopy (FT-IR) detection.

t�� $P�FMVUJPO�PG�CSBODIFE�BOE�MJOFBS�

molecules can be a problem. In such

cases advanced separation techniques or

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

8

7

6

5

4

3

2

1

5

1

2

3

4

5

6

7

8

6 7 8 9

SEC elution volume (mL)

Gra

die

nt

elu

tio

n v

olu

me (

mL)

10 11 12

20

40

60

80

100

Figure 5: Separation of linear and comb shaped copolymers, which co-elute in GPC/SEC alone.

Tips and Tricks

17


Making Method Development Faster for the Analysis of Natural and Artificial FlavouringsPhilipp Jochems and Gesa Schad, Shimadzu Europa, Duisburg, Germany.

Vanilla is one of the most important

flavours worldwide and is widely used

in foods, beverages, and perfumes.

Natural vanilla extract contains up to

several hundred substances with vanillin,

vanillic acid, 4-hydroxybenzoic acid,

and 4-hydroxybenzaldehyde the major

components. As a result of continuously

increasing demand and the resulting high

costs of natural vanilla extracts, artificial

flavourings are often used instead.

Vanillin can be obtained through various

methods such as chemical synthesis,

biotransformation, or degradation of

waste sulphite liquors, as well as extraction

of natural vanilla pods. These artificial

flavours can contain synthetic vanillin,

ethyl vanillin, eugenol, guaiacol, vanillin

mandelic acid, and others.

As authenticity criteria for vanilla, the

ratios of the major components vanillin,

4-hydroxybenzaldehyde, vanillic acid,

and 4-hydroxybenzoic acid are frequently

used. In order to monitor the composition

and therefore quality of vanilla flavours

contained in food, an analytical method

needs to enable individual quantification of

any of these ingredients as well as possible

ingredients like the precursors from the

synthesis of vanillin.

A simple, rapid, and robust ultrahigh-performance liquid chromatography (UHPLC) method for the simultaneous determination of natural and artifi cial vanilla fl avouring substances as well as some precursors has been developed using an automated method scouting or method optimization workfl ow. The most suitable mobile phase and stationary phase combination was identifi ed in a scouting run. These conditions were used to create a two-dimensional model in computer simulation software. Temperature and gradient time were varied to establish the optimum fast and robust separation conditions. This approach resulted in a 5.5 min gradient method that allowed for fast screening of 11 compounds of interest.

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18



The article describes the computer-assisted development and optimization of a rapid ultrahigh-performance liquid chromatography (UHPLC) screening test for the separation and quanti� cation of natural and arti� cial vanilla � avouring substances as well as some precursors for the quality control

of vanilla products. This article illustrates the effectiveness and speed of software-assisted method development tools.

ExperimentalEquipment and Chromatographic Methods: For UHPLC method scouting,

a Nexera X2 Method Scouting System (Shimadzu) was used, consisting of two quaternary solvent pumps, autosampler, and column oven including a six-column switching valve. The system was also equipped with a high resolution photo diode array detector. Acetonitrile and water + 10 mM HCOONH4 (pH 2.8) were used as mobile phase. The different stationary phases applied for method scouting for the separation of 11 analytes relevant to vanilla products are displayed in Table 1.

Method scouting was performed in a sequence using 7.5 min gradient runs at 40 °C with combinations of organic and aqueous mobile phases (acetonitrile/H2O + 10 mM HCOONH4; gradient: 5–95%) on the six different columns, which cover a wide spectra of diverse stationary phase materials for reversed phase chromatography.

The phenyl-hexyl-column showed the most promising results with all analytes of interest at least partly separated using a mobile phase consisting of A: 10 mM ammonium formate in H2O (pH 2.8) and B: acetonitrile. These conditions were used to create a two-dimensional DryLab model (Molnár Institute) using 2 min and 6 min gradient runs at 25 °C and 50 °C as input data. These experiments resulted in

Table 1: Stationary phases used in method scouting.

Column

10 × 2.1 mm, 3-µm C18 column

10 × 2.1 mm, 2.5-µm phenyl-hexyl column

10 × 2.1 mm, 3-µm C18-column with aromatic selectivity

10 × 2.1 mm, 3-µm RP-cyano column

10 × 2.1 mm, 3-µm RP-amide column

10 × 2.1 mm, 3-µm pentafluorophenyl bonded column

6.50

T (o C

)

50

45

40

35

30

25

5 10

6.005.505.004.504.003.503.002.502.001.501.000.500.00

tG (min)

Figure 1: Colour-coded resolution map for UHPLC method development.

Jochems and Schad

1919

Q&A: Blumberg2 Q&A: Blumberg2 News9 Tips and Tricks12 Jochems and Schad18 Jochems and Schad181899 News99 1212 Tips and Tricks12Beck and Delobel21 Beck and Delobel21 ISC Event Preview25 Training and Events27 Staff28 Staff28282525 ISC Event Preview2525 2727 Training and Events27

OCTOBER 5 - 7, 2016

I N V I E N N A , A U S T R I A

SFC 2016

CALL FOR

PAPERSABSTRACT SUBMISSION

DEADLINES

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ORAL PRESENTATIONS:

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The software predicted an optimum

separation with a minimum resolution of

the critical peak pair of 2.1 in a gradient run

from 5% to 70% B in 5.5 min at a fl ow rate

of 0.5 mL/min at 50 °C. A comparison of

the predicted chromatogram with an actual

sample run is displayed in Figure 2.

The advantage of a computer-assisted

method development approach compared

to a more traditional one lies in the time

savings because four gradient runs (see

above) are enough input for the software

to calculate the optimal conditions

(gradient, oven temperature) for the

method. This can mean lower costs

because of fewer working hours and

smaller solvent consumption. However,

there are some limitations such as when

developing a method with a two- (or

more) step gradient. In such cases the

simulations are not that precise as those

with a linear gradient. This is because the

data for the calculation are from a linear

gradient run and a computer simulation

is more precise the more similar the

conditions from the input data and the

simulation are.

Conclusion

A robust, fast, and sensitive UHPLC method

for the simultaneous separation of natural

and artifi cial vanilla fl avouring substances as

well as some precursors has been developed.

The method scouting experiment and further

optimization using computer simulation

software was able to save time and offered

visualization of the design space in a

resolution map to establish the most robust

separation method.

E-mail: [email protected]: www.shimadzu.eu

Philipp Jochems graduated in 2013 with

a master of science in applied chemistry

from the University of Applied Science in

Krefeld, Germany. In 2014 he was a scientifi c

assistant at the University Medical Center of

the Johannes Gutenberg University Mainz,

Germany, and since 2016 has worked as

a HPLC product specialist in the analytical

business unit of Shimadzu Europa in

Duisburg, Germany.

Gesa Schad graduated with a diploma in

chemical engineering from the Technical

University, NTA in Isny, Germany, in 2004

and as a master of science in pharmaceutical

analysis from the University of Strathclyde

in Glasgow, UK, in 2005. Until 2006 she

worked as a consultant in HPLC method

development and validation in an analytical

laboratory of the FAO/IAEA in Vienna,

Austria. She gained her doctorate for

research in pharmaceutical sciences at the

University of Strathclyde in 2010 and was

employed as an HPLC specialist in the R&D

department at Hichrom Ltd. in Reading, UK,

from 2009. Since 2013, she has worked as

a HPLC product specialist in the analytical

business unit of Shimadzu Europa in

Duisburg, Germany.

a colour-coded resolution map for simple

identification of the optimum separation

conditions (Figure 1). The figure shows

the calculated resolution (colour-coded)

for the different simulated combinations

of retention time (x-axis) and oven

temperature (y-axis).

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

1.2

Van

illic

man

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aci

d

Van

illic

aci

d

Van

illin

Feru

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illin

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4-hy

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1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

DryLab Model

Actual RunTime (min)

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

Time (min)

Figure 2: Comparison of predicted and actual chromatogram of the UHPLC analysis of natural and artifi cial vanilla fl avourings.

Jochems and Schad

20


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Preview of Topics at HPLC 2016, Part 4: State-of-the-Art MS Methods for Structural Assessment of mAbs and ADCs: From the Research Lab to Routine Characterization

This is the fi nal instalment in a series of four articles exploring topics that will be addressed at the HPLC 2016 conference in San Francisco, USA, from 19–24 June 2016.

Alain Beck1 and Arnaud Delobel2, 1Centre d’Immunologie Pierre Fabre (CIPF), Saint-Julien-en-Genevois, France, 2Quality Assistance SA, Donstiennes, Belgium.

Monoclonal antibodies (mAbs) are

highly complex tetrameric glycoproteins

that require extensive analytical and

structural characterization to become

drug candidates. This is also true for

antibody–drug conjugates (ADCs). These

immunoconjugates are based on highly

cytotoxic small-molecule drugs covalently

attached via conditionally stable linkers to

mAbs and are among the most promising

next-generation empowered biologics

for cancer treatment. ADCs are more

complex than naked mAbs, because the

heterogeneity of the conjugates adds

to the inherent microvariability of the

biomolecules. The development and

optimization of ADCs rely on improving

their analytical and bioanalytical

characterization by assessing several

critical quality attributes, namely the

distribution and position of the drug, the

amount of naked antibody, the average

drug-to-antibody ratio (DAR), and the

proportions of residual small-molecule

drug and drug-linker).1

As a result of advances in multilevel

(top, middle, bottom) state-of-the-art

mass spectrometry (MS) methods,

including native MS, ion mobility MS,2

capillary electrophoresis–electrospray

ionization–MS,3,4 two-dimensional

liquid chromatography–MS (2D LC–

MS),5 extended bottom-up,6 and

top-down sequencing,7 combined with

chromatographic and electrophoretic

techniques8 very precise characterization

21



of biotherapeutics is now possible. Until

recently, however, these techniques

were considered suitable only for research

use. With the advent of robust and

user-friendly solutions (both hardware

and software), these techniques are

now amenable for routine use.

Examples of their application to the

characterization of mAbs and ADCs

are discussed below.

Middle-Level Characterization of mAbs

During their biosynthesis or during their

shelf life, mAbs can undergo many

modifications, such as glycosylation,

oxidation, deamidation, and C-terminal

lysine clipping, to name a few. In most

cases, these variants cannot be easily

identified at the intact antibody level,

because of limitations in chromatographic

separation and MS resolution.

A middle-up approach using IdeS

digestion (to yield Fab, Fc/2, and

light-chain fragments) combined with

a super macroporous reversed-phase

column enables quick and efficient

characterization of mAb variants.9 The

relatively low molecular mass of the

subunits (~25 kDa) allows accurate mass

determination by high-resolution MS as

well as top-down sequencing by electron

transfer dissociation (ETD). Using the

same sample preparation, the glycoforms

can also be separated and characterized

using an approach such as hydrophilic

interaction chromatography (HILIC) with

MS detection.10 An example of this

approach is presented in Figure 1 for

adalimumab.

Determination of Drug-to-Antibody

Ratio on Intact ADCs

The number of cytotoxic molecules

attached to an antibody (the

drug-to-antibody ratio, or DAR) is a critical

quality attribute of an ADC. It can be

determined by UV spectrophotometry or

hydrophobic interaction chromatography

(HIC) with UV detection,11–13 but these

methods are not universal and have some

drawbacks. Mass spectrometry can be a

universal tool to determine the DAR value,

whatever the coupling chemistry or the

Fc/2

Fd’

Fc/2

LC

Fd’0.008

(a)

(b)

0.001

750

300

200

100

0

500

250

0

9.43

12.51

16.28

16.28

16.3911.14

11.65

15.4117.34

8 9 10

10

16 16.5 17 17.5 18 1918.5

8 12 14 16 18 20 22

11 12 13 14 15 16 17 18 19 20 21

Time (min)

Time (min)

Time (min)

Inte

nsi

ty (

cou

nts

)Sig

nal (E

U)

Sig

nal (E

U)

LC

Oxidized species Pyroglutamic acid

Figure 1: HPLC–UV spectra obtained for adalimumab sample digested with IdeS and analyzed on (a) a reversed-phase or (b) a HILIC column.

Beck and Delobel

22


Data integrity problems in pharmaceutical quality control

laboratories are driving more regulatory action than ever

before. It is obvious that something has changed to drive

all this activity. There is plenty of information available,

but much of it seems to confuse or frustrate rather than

clarify or help. In this webinar, we will provide clarity,

dispelling confusion by looking at the facts, based on a

study of available resources and direct interactions with

FDA staff and their consultants.

Key questions that indicate you should tune in to this web seminar

■ Do I understand what the new wave of data integrity enforcement means?

■ Are my laboratory software and processes ready for the increased scrutiny?

■ Do I understand what my responsibilities are for ensuring that both my vendor’s software and my organization’s processes will ensure data integrity?

After this webinar, you should be able to■ Articulate the drivers behind the “new” data integrity

regulatory enforcement actions

■ Communicate the current interpretations of existing regulations

■ Understand a methodology to evaluate your laboratory software

Who should attend■ Quality control laboratory managers, compliance and quality

managers, IT managers

For questions, contact Kristen Moore

at [email protected]

Presenter:

LOREN SMITH

Software Compliance

Program Manager

Agilent Technologies

Moderator:

LAURA BUSH

Editorial Director

LCGC

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Register for free at:

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size-exclusion chromatography (SEC)14

and detected by high-resolution

electrospray ionization MS. The DAR

can be automatically calculated by

software after deconvolution of the

multicharged electrospray spectrum.

When working with cysteine-linked

ADCs, native conditions must be used

to avoid disruption of noncovalent

interactions. In these methods, native

SEC–MS is commonly used, which requires

optimization of mobile phases and MS

conditions.

Characterization of mAbs and

ADCs by UHPLC–MS and HPLC–MS

Peptide Mapping

Peptide mapping with MS detection

is a common methodology for protein

characterization. It can be used for the

confirmation of the primary sequence,

the quantification of post-translational

modifications (PTMs), and the study of

disulphide-bond scrambling. Thanks to

commercially available software packages

dedicated to biopharmaceutical analysis,

the analysis of these MS data can be fully

automated.

Applied to ADC characterization,

peptide mapping is also a valuable tool

to localize conjugation sites and

determine site occupancy. ETD

fragmentation can be used to localize

conjugation sites for lysine-conjugated

ADCs on peptides containing several

lysine residues.

nature of the drugs.

After N-deglycosylation, the ADC is

desalted online using reversed-phase

high performance LC (HPLC) or

0 1 2 4 6 8

(5.1%) (0.8%) (1.9%) (38.4%-45.2%)(15.4%) (15.8%) (12.4%)

2e5

2.5e7

1.5e8

1e8

5e7

3e1

2e1

1e1

0

2e7

1.5e7

1e7

5e6

0

(a)

(b)

Max A

bso

rban

ce (

AU

)

Inte

nsi

ty (

cou

nts

)In

ten

sity

(co

un

ts)

Inte

nsi

ty (

cou

nts

)

Retention time (min)

1412108642

25041.00

25017.00

24800

76400 76600 76800 77000 77200 77400 77600

24500 25000 25100 25200 25300 25400

25095.00 25139.00

103437.00

76678.00

75840.00

76531.00

76473.00

77002.00

77158.00

Isomer 4a

x2

Isomer 4b

103277.00

105558.00

102756.00103104.00

104063.00

1.03e51.03e5 1.03e5 1.04e5 1.04e51.03e5

Mass (Da)

Mass (Da)

Figure 2: (a) HIC–UV chromatogram obtained for brentuximab vedotin and (b) deconvoluted mass spectra obtained by HIC–reversed-phase LC–MS for the two isomers of DAR4.

Beck and Delobel

23


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9. S. Sjögren, F. Olsson, and A. Beck, unpublished

data.

10. A. Peria, S. Fekete, A. Cusumano, J.L. Veuthey,

A. Beck, M. Lauber, and D. Guillarme,

unpublished data.

11. M. Rodriguez-Aller, D. Guillarme, A. Beck,

and S. Fekete, J. Pharm. Biomed. Bioanal. 118,

393–403 (2016).

12. A. Cusumano, D. Guillarme, A. Beck, and S. Fekete,

J. Pharm. Biomed. Bioanal. 121, 161–173 (2016).

13. S Fekete, J.L. Veuthey, A Beck, and D. Guillarme,

J Chromatogr. B, in press.

14. M. Rodriguez-Aller, A. Cusumano, A Beck, D.

Guillarme, and S. Fekete, J Chromatogr. B, in press.

15. M. Sarrut, A. Corgier, S. Fekete, D. Guillarme,

D. Lascoux, M.C. Janin-Bussat, A. Beck, and H.

Heinisch, unpublished data.

16. M. Sarru, M.C. Janin-Bussat, S. Fekete,

D. Guillarme, A. Beck, and H. Heinisch,

unpublished data.

17. R.E. Birdsall, S.M. McCarthy, M.C. Janin-Bussat,

M. Perez, J.F. Haeuw, C. Weibin, and A. Beck,

mAbs 8(2), 306–317 (2016).

Alain Beck is the Senior Director of the

Physico-Chemistry Department at the

Centre d’Immunologie Pierre Fabre (CIPF)

in Saint-Julien-en-Genevois, France.

Arnaud Delobel is the scientific manager

for biologics and the R&D team manager

at Quality Assistance SA, in Donstiennes,

Belgium.

E-mail: [email protected] / [email protected]: www.cipf.com/en / www.quality-assistance.com

quantification of small-molecule drugs.17

The application of this methodology

to brentuximab vedotin is presented in

Figure 2.

All these new techniques and case

studies will be discussed at the HPLC 2016

meeting in San Francisco in June.

References

1. A. Beck, G. Terral, F. Debaene, E. Wagner-

Rousset, J. Marcoux, MC Janin-Bussat, O. Colas,

A. Van Dorsselaer, and S. Cianferani, Exp. Rev.

Proteomics 13(2), 157 –183 (2016).

2. G. Terral, A. Beck, and S. Cianferani, J

Chromatogr. B, in press.

3. R. Gahoual, A. Beck, Y.N. François, and E.

Leize-Wagner, J. Mass Spectrom. 51(2), 150–158

(2016).

4. Y.N. François, M. Biacchi, N.Said, C. Renard, A.

Beck, R. Gahoual, and E. Leize-Wagner, Anal.

Chim. Acta 908, 168–176 (2016).

5. D. Stoll, J. Danforth, K. Zhang, and A. Beck,

unpublished data.

6. N. Gasilova, K. Srzentic, L. Qiao, B. Liu, A. Beck,

Y.O. Tsybin, and H.H. Girault, Anal. Chem. 88,

1775–1784 (2016).

7. R Gahoual, A. Beck, E. Leize-Wagner, and Y.N.

François, unpublished data.

8. A. Resenman, W. Jabs, A. Wiechmann, E

Wagner-Rouset, O. Colas, W. Evers, E. Belau,

L. Vorwerg, C. Evans, A. Beck, and D. Suckau,

mAbs 8(2), 318–330 (2016).

2D LC–MS Analysis of mAbs and

ADCs

Two-dimensional LC with MS detection

is widely used for the identification of

proteins from complex proteome samples

in many laboratories. Robust 2D–HPLC and

2D–UHPLC systems are now commercially

available, thus enabling the routine analysis

of biopharmaceuticals with this technology.

Two-dimensional LC can be used

to hyphenate MS-incompatible

chromatographic separations to mass

spectrometry detection: After a first

dimension using mobile phases containing

nonvolatile salts, the peak of interest is

sent to a second dimension consisting of

a reversed-phase column to desalt the

sample and separate potentially coeluted

species. This methodology (heart-cutting

2D LC–MS) can be routinely applied to

identify the different species detected

in size-exclusion and ion-exchange

chromatography of monoclonal

antibodies.

Another main application of 2D LC–MS

is the rapid on-line structural elucidation

of species observed in HIC distribution

profiles of cysteine-conjugated ADCs.15,16

The identification of the different species

is required to be able to determine the

DAR value based on the HIC–UV profile

as well as for the detection and the

Beck and Delobel

24


The 31st International Symposium on Chromatography (ISC 2016)A preview of the upcoming 31st International Symposium on Chromatography (ISC 2016), which is due to be held 28 August–1 September at University College Cork, Ireland.

The 31st International Symposium on

Chromatography (ISC 2016) will be held from

28 August–1 September 2016 at University

College Cork, Cork, Ireland. This major

chromatography conference will be hosted

in the Emerald Isle for the fi rst time in its

history and will attract chromatographers

from around the world. The greater Cork

area is home to nine of the top 10 global

pharmaceutical companies in the world, and

seven out of 10 of the world’s best-selling

drugs are produced there. As a major

European centre for the life science industry,

Cork is an ideal choice to host ISC 2016.

Furthering the international appeal

of the ISC series, The Chromatographic

Society (ChromSoc) will be sponsoring key

presentations at ISC 2016 in celebration of

its 60th anniversary. ISC 2016 will also host

the prestigious “Award for Outstanding

Achievements in Separation Science”,

which is awarded to a preeminent

separation scientist by the California

Separation Science Society (CaSSS).

ISC 2016 provides the perfect forum for

scientific exchange between attendees

from academia, industry, and government

research institutions, as well as excellent

networking opportunities with up to 800

international delegates expected to attend.

The major focus of the symposium will be

on the impact and continuing contribution

25


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of chromatography and separation science

to the pharmaceutical industry, food,

health, science, and medicine.

The major theme of ISC 2016 will be

the Innovation and Impact of

Chromatographic Separations on Science,

Industry, and Life. The symposium

programme reflects these themes, and

aims to highlight new challenges and

emerging opportunities in separation

science detection systems, methods,

and marketing solutions. The scientific

programme is set to be wide ranging and

diverse with topics including:

t� Pharmaceutical

t� Biomedical

t� Forensics

t� Environmental analysis

t� Process chromatography

t� PAT

t� Biomarkers

t� Diagnostics and clinical analysis

t� “Omics” technologies

t� New material science

t� Characterization, miniaturization, and

on-chip devices

t� Mass spectrometry

t� Food and health

t� Separation and sensing

t� Trace elements speciation

t� Supercritical fluid applications

t� Biopharmaceutical

International leaders in each of

these areas will provide inspiring and

thought-provoking presentations

to stimulate researchers. While an

international exhibition and vendor lecture

series on instrumentation and services

for chromatography, separation science,

and mass spectrometry will add another

integral part to the scientific programme.

ISC organizers look forward to

introducing attendees to the famous Irish

hospitality and the charms of Cork with

the nearby coastlines, beaches, hiking

routes, cycling routes, and world-class golf

courses offering exceptional scenery. While

the city’s wide array of hotels, restaurants,

traditional music and dancing, and, of

course, exceptional scientific conferences

complete the package.

On-line registration closes 25 August

2016.

Co-Chairs : Apryll Stalcup and Jeremy D. GlennonTel. : +353 1 280 2641E-mail : [email protected] : http://www.isc2016.ie/

ISC Event Preview

26


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Training CoursesGC

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Complete GC and GC–MS

13–17 June 2016

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The Technique of GC in Three

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The Theory of HPLC

On-line training from CHROMacademy

Website: http://www.chromacademy.

com/hplc-training.html

HPLC Troubleshooting and

Maintenance

6 July 2016

Kings College London, London, UK

Website: http://www.crawfordscientific.

com/training-online-calendar.asp

HPLC Masterclass2–24 August 2016

Laserchrom HPLC Laboratories Ltd,

Rochester, Kent, UK

Website: http://www.hplccourses.com/

index.htm (please mention The Column

when you make an enquiry)

SAMPLE PREPARATIONSampling Techniques for GC & GC–MS12 October 2016

The Open University, Milton Keynes, UK

Website: http://www.anthias.co.uk/

training-courses/sampling

GPC/SECLight Scattering and Viscometry Hands-On Training23–24 June 2016

Mainz, Germany

Website: www.pss-polymer.com

Please send your event and training course information to Kate Mosford [email protected]

27–30 June 2016

International Network of Environental Forensics Conference

Örebro Castle, Örebro, Sweden

Tel: +49 19 301 209


Website: www.inef2016.com

26–29 September 2016

International Symposium on GPC/SEC and Related Techniques

Novotel, Amsterdam, Netherlands

Tel: +1 508 482 3129


Website: www.gpcevent.com

13–16 November 2016

27th International Symposium on Pharmaceutical and Biomedical Analysis

Guangzhou, P.R. China


Website: www.PBA2016.org

30 November–2 December 2016

2nd ACROSS International Symposium on Advances in Separation

Science (ASASS 2)

Hobart, Tasmania, Australia

Chairman: Brett Paull


Website: http://www.utas.edu.au/across

Event News

The Column www.chromatographyonline.com Training & Events

27


Mission StatementThe Column (ISSN 2050-280X) is the analytical chemist’s companion within the dynamic world of chromatography. Interactive and accessible, it provides a broad understanding of technical applications and products while engaging, stimulating and challenging the global community with thought-provoking commentary that connects its members to each other and the industries they serve.Whilst every effort is made to ensure the accuracy of the information supplied, UBM EMEA accepts no responsibility for the opinions and statements expressed.Custom Reprints: Contact Brian Kolb at Wright’s Media, 2407 Timberloch Place, The Woodlands, TX 77380. Telephone: 877-652-5295 ext. 121. Email: [email protected].

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Editor-in-ChiefAlasdair [email protected]

Managing EditorKate [email protected]

Assistant EditorLewis [email protected]

UBM EMEAHinderton Point, Lloyd Drive, Ellesmere Port, CH65 9HQ, UKTel: +44 (0)151 353 3621Fax: +44 (0)151 353 3601

Vice President/Group PublisherMichael J. [email protected]

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